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Chapter IV
Real-time qPCR Monitoring of Genes Involved in Water Stress Tolerance in Tea
Chapter IV
89
41 Introduction
Water stress is one of the most important environmental factor that limits
crop productivity and quality throughout the world resulting in substantial
loss every year (Tan et al 2008) Efficient irrigation is one of the key factor
for sustainable agriculture as pointed out by many researchers from time to
time (Hillel and Vlek 2005 Khan et al 2006 Hsiao et al 2007 Pannunzio
et al 2008 Pannunzio 2008) However due to continuous depletion and
uneven distribution of water resources in many regions of the world it has
become necessary to genetically improve the performance of crop plants in
such water stress environments for a sustainable and economically viable
solution
Successful cultivation of tea is highly dependent on availability of rain water
There are reports of yield losses every year due to recurring prolong dry
spell in the tea growing areas (Barua 1989 Jain 1999) of India and other
countries like Kenya and Sri Lanka Keeping in view the continuous increase
of drought hit regions due to decreased rainfall the present need of the tea
industry is a good quality high yielding tea genotype that can withstand and
perform well during the prolonged dry spells of the year One of the ways to
achieve this goal is to study the drought responsive mechanism in tea at
molecular level to identify the different drought responsive pathways and
their corresponding genes which would potentially contribute towards
development of drought tolerant tea plants However the development of
drought tolerant tea genotypes has been hindered by lack of knowledge of
more precise physiological and molecular parameters that reflect genetic
potential for improved productivity under water stress environment
Comparative study of the transcriptome of a drought tolerant tea genotype
with a susceptible one is one of the potential ways to decipher the
mechanism of drought tolerance in tea plant We have identified a large
numbers of differentially expressed transcripts in a drought tolerant cultivar
TV23 by comparative transcriptome analysis with a susceptible cultivar
S3A3 (Chapter III) from an induced water stress experiment by SSH
approach Some of these differentially expressed transcripts are highly
represented in TV23 compared to S3A3 which may potentially be
Chapter IV
90
contributing for contrasting drought tolerance behaviour of TV23 However
the expression pattern of these transcripts has to be studied at various
degrees of water stress as well as in unstressed conditions to correlate their
potential role that they might play during water stress We have identified
(Chapter III) a total of 49 drought responsive transcripts represented by 3 or
more than 3 ESTs in the drought tolerant cultivar TV23 In the present study
we have selected 29 transcripts that are highly represented in TV23
compared to S3A3 and studied their expression pattern at different stages
of drought in both the cultivars using qRT-PCR
Water stress response in plants involves expression of a large number of
genes encoding proteins with an adaptive role These complex set of genes
are thought to function not only in protecting cells from water deficit by
production of important metabolic proteins but also in regulation of genes for
signal transduction in water stress response (Shinozaki et al 2003) The
products of these genes can be classified into two groups The first group
includes genes encoding proteins that function directly in the protection of
plant cells against stresses such as heat stress proteins LEA proteins
osmoprotectants detoxification enzymes and free radical scavengers
whereas the second group includes those that control gene expression and
signal transduction (Wang et al 2003) Monitoring the expression of these
genes at mRNA level in different tissues or in tissues under different degrees
of stress by qRT-PCR is a well established technique for study of differential
expression pattern in real time during water stress environment Real-time
RT-PCR is the most sensitive method for detection of low abundance mRNA
(Bustin 2000) Here we have used qRT-PCR to monitor the expression
pattern of selected genes at three stages of drought induction The qRT-
PCR analysis of these genes at BWS will further validate our SSH library
constructed in Chapter III Therefore the present study was carried out with
the objective to validate the SSH library by monitoring the expression pattern
of highly induced drought responsive genes from library BWE3E4 at BWS
and also to monitor their expression pattern at WS and AWS in both
cultivars
Chapter IV
91
42 Materials and Methods
421 Plant materials and RNA Isolation
The plant material used in the present study for isolation of RNA was a bud
along with 1st and 2nd leaf collected during induced water stress experiment
(Chapter II) Total RNA was isolated from tissues of TV23 and S3A3
collected at three stages of drought induction ie before wilting stage
(TV23BWS and S3A3BWS) wilting stage (TV23WS and S3A3WS) and
after wilting stage (TV23AWS and S3A3AWS) using RNAqueous kit
(Ambion USA Cat No Am1912) following the manufacturers protocol Total
RNA was also isolated from TV23 grown under well watered condition
(TV23C) The purity and concentration of RNA was checked using a
spectrophotometer (BioPhotometer Eppendorf Germany) and integrity was
ensured by running a 1 denaturing agarose gel
422 Selection of Internal Control Genes for Normalization
For internal standard we have selected four housekeeping genes viz 26S
rRNA 18S rRNA Camellia tubulin and ribulose-1 5-bisphosphate
carboxylaseoxygenase and tested their variability of expression at three
stages of drought induction (BWS WS and AWS) in both the cultivars under
consideration The gene sequences were retrieved from public database and
primer was designed using Primer3 [(httpfrodowimiteduprimer3)
(Rozen et al 2000)] The details of gene accession numbers and designed
primers are given in Table 41
Table 41 Housekeeping gene and primers used for normalization
Genes Gene Bank acc number
Primer sequence (5ʹ - 3ʹ) Length (nt)
26S rRNA AY283368 TCAAATTCCGAAGGTCTAAAG CGGAAACGGCAAAAGTG
21 17
18S rRNA AY563528 GGCCGGCTCCGTTACTTTG GTTTCAGCCTTGCGACCATACTC
19 23
Camellia tubulin DQ444294 AGCGTGCGGTTTGCATGA GCCCAAAGGTTTGGCATCA
18 19
Ribulose-1 5-bisphosphate carboxylaseoxygenase
EF0110751 AAGCACAATTGGGAAAAGAAG AAAGTGAAAATGAAAAGCGACAAT
21 24
Chapter IV
92
423 Selection of Drought Induced Genes for qRT-PCR Assays
Among highly represented drought induced ESTs in BWE3E4 library (Table
32 Chapter III) we have selected 29 genes (Table 42) for expression
studies based on their existing report of induction under drought and other
environmental stresses and also on the role they might play in giving drought
tolerance to plants (Table 43)
4231 Primer Design
ESTs with high quality value representing the above genes were considered
for primer designing using Primer3 A total of 29 primer pairs were designed
whose length varies from 20 nt (nucleotide) to 22 nt and Tm from 60oC to
64oCThe details of selected genes and corresponding primer sequence and
product size are given in Table 42
424 Standardization of PCR Parameters and Product size Verification
The PCR parameters for all the housekeeping genes and the selected
drought induced genes were standardized in a gradient thermal cycler
(Mastercycler gradient Eppendorf Germany) before going in to the qRT-
PCR analysis The product sizes were verified by running an agarose gel
These standardised PCR profiles were used during qRT-PCR analysis
425 Two Step qRT-PCR
4251 Reverse Transcription
1 microg total RNA was reverse transcribed using Transcriptor First Strand
cDNA Synthesis kit (Roche Germany Cat No 04379012001) following the
manufacturers protocol Briefly 1 microg of total RNA and 1 microl of anchored-
oligo(dT)18 primer and required volume of nuclease free water to make the
final volume of 13 microl were gently mixed in a 02 ml nuclease free PCR tube
and incubated at 65ordmC for 10 minutes in a thermal cycler (ABI 2720) to
denature the template-primer mixture The tube was immediately chilled on
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Chapter IV
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Chapter IV
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Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
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Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
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Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
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users and for biologist programmers Plant Mol Biol 5 69 ndash 76
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
89
41 Introduction
Water stress is one of the most important environmental factor that limits
crop productivity and quality throughout the world resulting in substantial
loss every year (Tan et al 2008) Efficient irrigation is one of the key factor
for sustainable agriculture as pointed out by many researchers from time to
time (Hillel and Vlek 2005 Khan et al 2006 Hsiao et al 2007 Pannunzio
et al 2008 Pannunzio 2008) However due to continuous depletion and
uneven distribution of water resources in many regions of the world it has
become necessary to genetically improve the performance of crop plants in
such water stress environments for a sustainable and economically viable
solution
Successful cultivation of tea is highly dependent on availability of rain water
There are reports of yield losses every year due to recurring prolong dry
spell in the tea growing areas (Barua 1989 Jain 1999) of India and other
countries like Kenya and Sri Lanka Keeping in view the continuous increase
of drought hit regions due to decreased rainfall the present need of the tea
industry is a good quality high yielding tea genotype that can withstand and
perform well during the prolonged dry spells of the year One of the ways to
achieve this goal is to study the drought responsive mechanism in tea at
molecular level to identify the different drought responsive pathways and
their corresponding genes which would potentially contribute towards
development of drought tolerant tea plants However the development of
drought tolerant tea genotypes has been hindered by lack of knowledge of
more precise physiological and molecular parameters that reflect genetic
potential for improved productivity under water stress environment
Comparative study of the transcriptome of a drought tolerant tea genotype
with a susceptible one is one of the potential ways to decipher the
mechanism of drought tolerance in tea plant We have identified a large
numbers of differentially expressed transcripts in a drought tolerant cultivar
TV23 by comparative transcriptome analysis with a susceptible cultivar
S3A3 (Chapter III) from an induced water stress experiment by SSH
approach Some of these differentially expressed transcripts are highly
represented in TV23 compared to S3A3 which may potentially be
Chapter IV
90
contributing for contrasting drought tolerance behaviour of TV23 However
the expression pattern of these transcripts has to be studied at various
degrees of water stress as well as in unstressed conditions to correlate their
potential role that they might play during water stress We have identified
(Chapter III) a total of 49 drought responsive transcripts represented by 3 or
more than 3 ESTs in the drought tolerant cultivar TV23 In the present study
we have selected 29 transcripts that are highly represented in TV23
compared to S3A3 and studied their expression pattern at different stages
of drought in both the cultivars using qRT-PCR
Water stress response in plants involves expression of a large number of
genes encoding proteins with an adaptive role These complex set of genes
are thought to function not only in protecting cells from water deficit by
production of important metabolic proteins but also in regulation of genes for
signal transduction in water stress response (Shinozaki et al 2003) The
products of these genes can be classified into two groups The first group
includes genes encoding proteins that function directly in the protection of
plant cells against stresses such as heat stress proteins LEA proteins
osmoprotectants detoxification enzymes and free radical scavengers
whereas the second group includes those that control gene expression and
signal transduction (Wang et al 2003) Monitoring the expression of these
genes at mRNA level in different tissues or in tissues under different degrees
of stress by qRT-PCR is a well established technique for study of differential
expression pattern in real time during water stress environment Real-time
RT-PCR is the most sensitive method for detection of low abundance mRNA
(Bustin 2000) Here we have used qRT-PCR to monitor the expression
pattern of selected genes at three stages of drought induction The qRT-
PCR analysis of these genes at BWS will further validate our SSH library
constructed in Chapter III Therefore the present study was carried out with
the objective to validate the SSH library by monitoring the expression pattern
of highly induced drought responsive genes from library BWE3E4 at BWS
and also to monitor their expression pattern at WS and AWS in both
cultivars
Chapter IV
91
42 Materials and Methods
421 Plant materials and RNA Isolation
The plant material used in the present study for isolation of RNA was a bud
along with 1st and 2nd leaf collected during induced water stress experiment
(Chapter II) Total RNA was isolated from tissues of TV23 and S3A3
collected at three stages of drought induction ie before wilting stage
(TV23BWS and S3A3BWS) wilting stage (TV23WS and S3A3WS) and
after wilting stage (TV23AWS and S3A3AWS) using RNAqueous kit
(Ambion USA Cat No Am1912) following the manufacturers protocol Total
RNA was also isolated from TV23 grown under well watered condition
(TV23C) The purity and concentration of RNA was checked using a
spectrophotometer (BioPhotometer Eppendorf Germany) and integrity was
ensured by running a 1 denaturing agarose gel
422 Selection of Internal Control Genes for Normalization
For internal standard we have selected four housekeeping genes viz 26S
rRNA 18S rRNA Camellia tubulin and ribulose-1 5-bisphosphate
carboxylaseoxygenase and tested their variability of expression at three
stages of drought induction (BWS WS and AWS) in both the cultivars under
consideration The gene sequences were retrieved from public database and
primer was designed using Primer3 [(httpfrodowimiteduprimer3)
(Rozen et al 2000)] The details of gene accession numbers and designed
primers are given in Table 41
Table 41 Housekeeping gene and primers used for normalization
Genes Gene Bank acc number
Primer sequence (5ʹ - 3ʹ) Length (nt)
26S rRNA AY283368 TCAAATTCCGAAGGTCTAAAG CGGAAACGGCAAAAGTG
21 17
18S rRNA AY563528 GGCCGGCTCCGTTACTTTG GTTTCAGCCTTGCGACCATACTC
19 23
Camellia tubulin DQ444294 AGCGTGCGGTTTGCATGA GCCCAAAGGTTTGGCATCA
18 19
Ribulose-1 5-bisphosphate carboxylaseoxygenase
EF0110751 AAGCACAATTGGGAAAAGAAG AAAGTGAAAATGAAAAGCGACAAT
21 24
Chapter IV
92
423 Selection of Drought Induced Genes for qRT-PCR Assays
Among highly represented drought induced ESTs in BWE3E4 library (Table
32 Chapter III) we have selected 29 genes (Table 42) for expression
studies based on their existing report of induction under drought and other
environmental stresses and also on the role they might play in giving drought
tolerance to plants (Table 43)
4231 Primer Design
ESTs with high quality value representing the above genes were considered
for primer designing using Primer3 A total of 29 primer pairs were designed
whose length varies from 20 nt (nucleotide) to 22 nt and Tm from 60oC to
64oCThe details of selected genes and corresponding primer sequence and
product size are given in Table 42
424 Standardization of PCR Parameters and Product size Verification
The PCR parameters for all the housekeeping genes and the selected
drought induced genes were standardized in a gradient thermal cycler
(Mastercycler gradient Eppendorf Germany) before going in to the qRT-
PCR analysis The product sizes were verified by running an agarose gel
These standardised PCR profiles were used during qRT-PCR analysis
425 Two Step qRT-PCR
4251 Reverse Transcription
1 microg total RNA was reverse transcribed using Transcriptor First Strand
cDNA Synthesis kit (Roche Germany Cat No 04379012001) following the
manufacturers protocol Briefly 1 microg of total RNA and 1 microl of anchored-
oligo(dT)18 primer and required volume of nuclease free water to make the
final volume of 13 microl were gently mixed in a 02 ml nuclease free PCR tube
and incubated at 65ordmC for 10 minutes in a thermal cycler (ABI 2720) to
denature the template-primer mixture The tube was immediately chilled on
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Bray Eds Plant responses to cellular dehydration during
Environmental Stress American Society of Plant Physiologists
Rockville MD pp 91 ndash 103
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Thomas T L Sung Z R 1989 Common amino acid sequence
domains among the LEA proteins of higher plants Plant Mol Biol 12
475 ndash 486
47 Ecker J R 1995 The ethylene signal transduction pathway in plants
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extracellular invertase and a glucose transporter in Chenopodium
rubrum by cytokinins Plant J 11 539 ndash 548
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drought stress and ethylene production in Norway spruce Physiol
Plant 86 297 ndash 300
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contributes to drought-stress tolerance in plants Plant Cell Rep 25
349 ndash 358
51 Feng H Chen Q Feng J Zhang J Yang X Zuo J 2007
Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
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Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
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signalling A metabolic interface between stress perception and
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
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stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
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65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
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66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
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67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
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68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
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403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
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expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
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80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
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83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
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Identification of Arabidopsis genes regulated by high light-stress using
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Plant Physiol 131 309 ndash 316
Chapter IV
133
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of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
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Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
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91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
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L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
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synthesis of heat shock proteins and increased thermotolerance in
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95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
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Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
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genes under drought stress in perennial ryegrass Physiologia
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100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
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102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
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Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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Chapter IV
136
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
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121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
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Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
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126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
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Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
90
contributing for contrasting drought tolerance behaviour of TV23 However
the expression pattern of these transcripts has to be studied at various
degrees of water stress as well as in unstressed conditions to correlate their
potential role that they might play during water stress We have identified
(Chapter III) a total of 49 drought responsive transcripts represented by 3 or
more than 3 ESTs in the drought tolerant cultivar TV23 In the present study
we have selected 29 transcripts that are highly represented in TV23
compared to S3A3 and studied their expression pattern at different stages
of drought in both the cultivars using qRT-PCR
Water stress response in plants involves expression of a large number of
genes encoding proteins with an adaptive role These complex set of genes
are thought to function not only in protecting cells from water deficit by
production of important metabolic proteins but also in regulation of genes for
signal transduction in water stress response (Shinozaki et al 2003) The
products of these genes can be classified into two groups The first group
includes genes encoding proteins that function directly in the protection of
plant cells against stresses such as heat stress proteins LEA proteins
osmoprotectants detoxification enzymes and free radical scavengers
whereas the second group includes those that control gene expression and
signal transduction (Wang et al 2003) Monitoring the expression of these
genes at mRNA level in different tissues or in tissues under different degrees
of stress by qRT-PCR is a well established technique for study of differential
expression pattern in real time during water stress environment Real-time
RT-PCR is the most sensitive method for detection of low abundance mRNA
(Bustin 2000) Here we have used qRT-PCR to monitor the expression
pattern of selected genes at three stages of drought induction The qRT-
PCR analysis of these genes at BWS will further validate our SSH library
constructed in Chapter III Therefore the present study was carried out with
the objective to validate the SSH library by monitoring the expression pattern
of highly induced drought responsive genes from library BWE3E4 at BWS
and also to monitor their expression pattern at WS and AWS in both
cultivars
Chapter IV
91
42 Materials and Methods
421 Plant materials and RNA Isolation
The plant material used in the present study for isolation of RNA was a bud
along with 1st and 2nd leaf collected during induced water stress experiment
(Chapter II) Total RNA was isolated from tissues of TV23 and S3A3
collected at three stages of drought induction ie before wilting stage
(TV23BWS and S3A3BWS) wilting stage (TV23WS and S3A3WS) and
after wilting stage (TV23AWS and S3A3AWS) using RNAqueous kit
(Ambion USA Cat No Am1912) following the manufacturers protocol Total
RNA was also isolated from TV23 grown under well watered condition
(TV23C) The purity and concentration of RNA was checked using a
spectrophotometer (BioPhotometer Eppendorf Germany) and integrity was
ensured by running a 1 denaturing agarose gel
422 Selection of Internal Control Genes for Normalization
For internal standard we have selected four housekeeping genes viz 26S
rRNA 18S rRNA Camellia tubulin and ribulose-1 5-bisphosphate
carboxylaseoxygenase and tested their variability of expression at three
stages of drought induction (BWS WS and AWS) in both the cultivars under
consideration The gene sequences were retrieved from public database and
primer was designed using Primer3 [(httpfrodowimiteduprimer3)
(Rozen et al 2000)] The details of gene accession numbers and designed
primers are given in Table 41
Table 41 Housekeeping gene and primers used for normalization
Genes Gene Bank acc number
Primer sequence (5ʹ - 3ʹ) Length (nt)
26S rRNA AY283368 TCAAATTCCGAAGGTCTAAAG CGGAAACGGCAAAAGTG
21 17
18S rRNA AY563528 GGCCGGCTCCGTTACTTTG GTTTCAGCCTTGCGACCATACTC
19 23
Camellia tubulin DQ444294 AGCGTGCGGTTTGCATGA GCCCAAAGGTTTGGCATCA
18 19
Ribulose-1 5-bisphosphate carboxylaseoxygenase
EF0110751 AAGCACAATTGGGAAAAGAAG AAAGTGAAAATGAAAAGCGACAAT
21 24
Chapter IV
92
423 Selection of Drought Induced Genes for qRT-PCR Assays
Among highly represented drought induced ESTs in BWE3E4 library (Table
32 Chapter III) we have selected 29 genes (Table 42) for expression
studies based on their existing report of induction under drought and other
environmental stresses and also on the role they might play in giving drought
tolerance to plants (Table 43)
4231 Primer Design
ESTs with high quality value representing the above genes were considered
for primer designing using Primer3 A total of 29 primer pairs were designed
whose length varies from 20 nt (nucleotide) to 22 nt and Tm from 60oC to
64oCThe details of selected genes and corresponding primer sequence and
product size are given in Table 42
424 Standardization of PCR Parameters and Product size Verification
The PCR parameters for all the housekeeping genes and the selected
drought induced genes were standardized in a gradient thermal cycler
(Mastercycler gradient Eppendorf Germany) before going in to the qRT-
PCR analysis The product sizes were verified by running an agarose gel
These standardised PCR profiles were used during qRT-PCR analysis
425 Two Step qRT-PCR
4251 Reverse Transcription
1 microg total RNA was reverse transcribed using Transcriptor First Strand
cDNA Synthesis kit (Roche Germany Cat No 04379012001) following the
manufacturers protocol Briefly 1 microg of total RNA and 1 microl of anchored-
oligo(dT)18 primer and required volume of nuclease free water to make the
final volume of 13 microl were gently mixed in a 02 ml nuclease free PCR tube
and incubated at 65ordmC for 10 minutes in a thermal cycler (ABI 2720) to
denature the template-primer mixture The tube was immediately chilled on
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
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n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
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25
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35
BWS WS AWS
Re
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xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
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xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
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Chapter IV
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Chapter IV
134
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Chapter IV
135
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LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
91
42 Materials and Methods
421 Plant materials and RNA Isolation
The plant material used in the present study for isolation of RNA was a bud
along with 1st and 2nd leaf collected during induced water stress experiment
(Chapter II) Total RNA was isolated from tissues of TV23 and S3A3
collected at three stages of drought induction ie before wilting stage
(TV23BWS and S3A3BWS) wilting stage (TV23WS and S3A3WS) and
after wilting stage (TV23AWS and S3A3AWS) using RNAqueous kit
(Ambion USA Cat No Am1912) following the manufacturers protocol Total
RNA was also isolated from TV23 grown under well watered condition
(TV23C) The purity and concentration of RNA was checked using a
spectrophotometer (BioPhotometer Eppendorf Germany) and integrity was
ensured by running a 1 denaturing agarose gel
422 Selection of Internal Control Genes for Normalization
For internal standard we have selected four housekeeping genes viz 26S
rRNA 18S rRNA Camellia tubulin and ribulose-1 5-bisphosphate
carboxylaseoxygenase and tested their variability of expression at three
stages of drought induction (BWS WS and AWS) in both the cultivars under
consideration The gene sequences were retrieved from public database and
primer was designed using Primer3 [(httpfrodowimiteduprimer3)
(Rozen et al 2000)] The details of gene accession numbers and designed
primers are given in Table 41
Table 41 Housekeeping gene and primers used for normalization
Genes Gene Bank acc number
Primer sequence (5ʹ - 3ʹ) Length (nt)
26S rRNA AY283368 TCAAATTCCGAAGGTCTAAAG CGGAAACGGCAAAAGTG
21 17
18S rRNA AY563528 GGCCGGCTCCGTTACTTTG GTTTCAGCCTTGCGACCATACTC
19 23
Camellia tubulin DQ444294 AGCGTGCGGTTTGCATGA GCCCAAAGGTTTGGCATCA
18 19
Ribulose-1 5-bisphosphate carboxylaseoxygenase
EF0110751 AAGCACAATTGGGAAAAGAAG AAAGTGAAAATGAAAAGCGACAAT
21 24
Chapter IV
92
423 Selection of Drought Induced Genes for qRT-PCR Assays
Among highly represented drought induced ESTs in BWE3E4 library (Table
32 Chapter III) we have selected 29 genes (Table 42) for expression
studies based on their existing report of induction under drought and other
environmental stresses and also on the role they might play in giving drought
tolerance to plants (Table 43)
4231 Primer Design
ESTs with high quality value representing the above genes were considered
for primer designing using Primer3 A total of 29 primer pairs were designed
whose length varies from 20 nt (nucleotide) to 22 nt and Tm from 60oC to
64oCThe details of selected genes and corresponding primer sequence and
product size are given in Table 42
424 Standardization of PCR Parameters and Product size Verification
The PCR parameters for all the housekeeping genes and the selected
drought induced genes were standardized in a gradient thermal cycler
(Mastercycler gradient Eppendorf Germany) before going in to the qRT-
PCR analysis The product sizes were verified by running an agarose gel
These standardised PCR profiles were used during qRT-PCR analysis
425 Two Step qRT-PCR
4251 Reverse Transcription
1 microg total RNA was reverse transcribed using Transcriptor First Strand
cDNA Synthesis kit (Roche Germany Cat No 04379012001) following the
manufacturers protocol Briefly 1 microg of total RNA and 1 microl of anchored-
oligo(dT)18 primer and required volume of nuclease free water to make the
final volume of 13 microl were gently mixed in a 02 ml nuclease free PCR tube
and incubated at 65ordmC for 10 minutes in a thermal cycler (ABI 2720) to
denature the template-primer mixture The tube was immediately chilled on
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Chapter IV
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
92
423 Selection of Drought Induced Genes for qRT-PCR Assays
Among highly represented drought induced ESTs in BWE3E4 library (Table
32 Chapter III) we have selected 29 genes (Table 42) for expression
studies based on their existing report of induction under drought and other
environmental stresses and also on the role they might play in giving drought
tolerance to plants (Table 43)
4231 Primer Design
ESTs with high quality value representing the above genes were considered
for primer designing using Primer3 A total of 29 primer pairs were designed
whose length varies from 20 nt (nucleotide) to 22 nt and Tm from 60oC to
64oCThe details of selected genes and corresponding primer sequence and
product size are given in Table 42
424 Standardization of PCR Parameters and Product size Verification
The PCR parameters for all the housekeeping genes and the selected
drought induced genes were standardized in a gradient thermal cycler
(Mastercycler gradient Eppendorf Germany) before going in to the qRT-
PCR analysis The product sizes were verified by running an agarose gel
These standardised PCR profiles were used during qRT-PCR analysis
425 Two Step qRT-PCR
4251 Reverse Transcription
1 microg total RNA was reverse transcribed using Transcriptor First Strand
cDNA Synthesis kit (Roche Germany Cat No 04379012001) following the
manufacturers protocol Briefly 1 microg of total RNA and 1 microl of anchored-
oligo(dT)18 primer and required volume of nuclease free water to make the
final volume of 13 microl were gently mixed in a 02 ml nuclease free PCR tube
and incubated at 65ordmC for 10 minutes in a thermal cycler (ABI 2720) to
denature the template-primer mixture The tube was immediately chilled on
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
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78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
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ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
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277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
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protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
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Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
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Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
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Plant Biol 12 690 ndash 698
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
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osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
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of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
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155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Table 42 Details of drought induced genes selected for expression studies
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product size (bp)
N23E3E4_A176 HS393998 Abscisic stress ripening protein (ASR1) 3e-19
ACCACCAACGCCTATGGAAG CTGCAGCCACAGCTGCTATC
6220 6224
268
N23E3E4_A482 HS394253 Ascorbate peroxidase 6e-109
CACGATGCTGGGACGTATGA CTGCAACAACACCAGCAAGC
6306 6298
120
N23E3E4_A682 HS395169 APE1 2e-49 GGGAAATGTGCCACTCCAAT TCGCGAATCATTTGCAGAAC
6205 6223
164
SSH2_269 HS396211 Copper-containing amine oxidase 5e-21 GCTTCGATCTAAAACCGGTCA AGCTGCAAACAACACCCAAG
6147 6126
211
N3E4E3_A388 HS393556 Clp G3PD 5e-28 TGGTCAAGGTAGTGGCATGG TCCTTCTCAGCAGGGCTTGT
6192 6241
157
N23E3E4_A691 HS394527 Glutathione S-transferase 2e-06 GGTCCTCCAACTACCGACTCC AAAAGGGCCAGCATTCAGAC
6190 6000
191
N23E3E4_A261 HS394072 Loss of the timing of ET and JA biosynthesis 2
1e-26 AATGGGCAAGTGGTTGGAGA TTGATTTCGCTAGCACGTTTCA
6276 6297
206
N23E3E4_A552 HS394303 ACC oxidase 1e-15
TATGCCACCGTGAAGTTCCA TGACCCACTCTTAAGCTGTTGC
6243 6171
106
N23E3E4_A233 HS394047 Lipase 2e-20
CGTGCAGAATTGCAATGGAT AGACAAACCGCAACCCAACT
6198 6186
158
23E3E4_A139 HS393777 Dehydrin 4e-14 GAGGCCATCATCAGCAGCAT CTGGTCTTTGTGCCCACCTG
6319 6289
155
SSH1_226 HS395745 DNA J protein 4e-38 CAGTGCAAGGCTCTTGAAGG ACCACCACCGTGCATATCAT
6110 6108
180
N23E3E4_A147 HS393974 Ethylene responsive transcription factor 4e-46
TGACTTTGGGTGGGGAGAAT TCATTTGGGACTCGAAAGCA
6165 6169
206
N23E3E4_A680 HS394396 Aquaporin 1 MIP family 6e-59 GAGTGCTGCACCGATGAATG GTGCCAGAGACTCCCACGTC
6282 6328
203
N23E3E4_A157 HS393983 Ehylene- induced esterase 8e-35 GTCTCATGGCCACAAGGTCA TTTTGTGGGGGAACTTGTCC
6213 6204
209
N23E3E4_A244 HS394057 Hydrogen peroxide induced protein 1 5e-20 ACCTGGACGAACAGGTCCAT AAGGCCTTCTGCTCCAACAC
6177 6172
184
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Environmental Stress American Society of Plant Physiologists
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domains among the LEA proteins of higher plants Plant Mol Biol 12
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drought stress and ethylene production in Norway spruce Physiol
Plant 86 297 ndash 300
50 Eun K and Choo B 2006 Over-expression of tobacco NtHSP70-1
contributes to drought-stress tolerance in plants Plant Cell Rep 25
349 ndash 358
51 Feng H Chen Q Feng J Zhang J Yang X Zuo J 2007
Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
II J Plant Nutr 15 2471 ndash 2490
Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
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Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
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acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
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68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
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1321 ndash 1332
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
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Saccharomyces cerevisiae Gene 170 243 ndash 248
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tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
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oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
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Phytochemistry 29(7) 2119 ndash 2123
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Chapter IV
136
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
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Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
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Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
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Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
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146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
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World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
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151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
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155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
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Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
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163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
94
Table 42 Contd
EST ID dbEST acc no
BlastX homology with E-value Designed primer sequence 5ʹ - 3ʹ (20 ndash 22 nt)
Tm (ordmC) Product Size (bp)
N23E3E4_A169 HS393992 NO SIMILARITY -- CCAGGAGGCTAAGGAGCAACTA GCCACCATCAAGGTTAAGGCTA
6206 6250
235
N23E3E4_A139 HS393967 Hydroxyproline-rich glycoprotein 3e-59 AATGCCGAAGCTTGCTGTGT3 TTCAGGAAAGCCCTCAGCAG
6319 6289
162
3E4_A16 HS394695 Glutathion peroxidase 5e-50 ATCAGTTTGGCGAGCAGGAG GCTGTCCCCAAGAAGTCCAC
6279 6202
170
N23E3E4_612 HS394342 Elicitor responsive gene 3 8e-33 TCCATTCCAATACCGTCGAA GCATGGCGACAGCTTCATAC
6123 6176
217
N23E3E4_A17 HS393845 Proline-rich protein 7e-53
TACCCTGTTGGCGTTCCATC CGCCATCTCCAGGTCAAGTC
6314 6313
217
SSH1_415 HS395830 Cinnamoyl-CoA reductase 3e-50 ACTGCTATGGGCCCAATTCT AAGTGCCTTCCAGTTGCTGA
6085 6098
179
N23E3E4_A164 HS393988 Zn finger protein 2e-72
TGGAAGCTTTGCCACCACTT CGATGGTTGGCACAGTCAAA
6303 6305
222
SSH2_304 HS396243 Diaminopimelate decarboxylase 2e-29 ATAGGGGGCTTCCAACTCCA TCTCTGCATGACGGATCTTGG
6296 6362
159
N23E3E4_A182 HS394003 TRAPP complex 2e-29 TCCCATGTGGAATCATACGG AGCATGGTTCATTCAGGGAGA
6153 6194
299
N23E3E4_A301 HS394100 Jasmonate ZIM domain protein 6e-06
AAAAAGCGTTTCGGGTGAGA CCCCAAAAACAAACCAAACA
6201 6060
280
N23E3E4_A338 HS394126 Eukaryotic translation initiation factor 5A4 6e-32 AGGATGGATTCGGTGAAGGA AGATTGCTGCACACCCACAT
6176 6156
196
N23E3E4_A317 HS394113 TCP transcription factor 1e-28 AAAGATGGGCACAGCAAGGT CGCTCAATTGCTGAGGAGTG
6482 6403
220
N23E3E4_A460 HS394234 Drm3-like protein 3e-32
ATCAGCTCTGGGACGACACC GTCGGCGGTGGTTTCAGTAT
6264 6222
169
N23E3E4_A693 HS395085 Hexose transporter 7e-33
TTCCCACTCGAAATCCGTTC GGTCATCACCACCACGAAGA
6228 6198
153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
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Chapter IV
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missing link in jasmonate signalling Nature 448 666 ndash 671
38 Chow W S Melis A Anderson J M 1990 Adjustments of
photosystem stoichiometry in chloroplasts improve the quantum
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40 Chrispeels M J and Maurel C 1994b Aquaporins the molecular
basis of facilitated water movement through living plant cells Plant
Physiol 105 9 ndash 13
41 Close T J 1997 Dehydrins a commonality in the response of plants
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Chapter IV
129
43 Dat J Vandenabeele S Vranova E Van Montagu M Inze D Van
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786 ndash 790
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Bray Eds Plant responses to cellular dehydration during
Environmental Stress American Society of Plant Physiologists
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475 ndash 486
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349 ndash 358
51 Feng H Chen Q Feng J Zhang J Yang X Zuo J 2007
Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
II J Plant Nutr 15 2471 ndash 2490
Chapter IV
130
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
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stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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dependent plant defense responses Plant Cell 8 1773 ndash 1791
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physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
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encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
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68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
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82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
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94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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111 Nam H G 1997 The molecular genetic analysis of leaf senescence
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ethylene production in wheat a fact or artefact Plant Physiol 96 406
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113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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Chapter IV
136
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
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Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
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136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
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Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
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cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
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146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
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World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
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151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
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155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
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163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
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166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
95
Table 43 Functions of selected genes reported in the literature
Selected genes showing homology with
Stress regulation or putative function References
Abscisic stress ripening protein (ASR1)
Induced under water stress enhance drought tolerance ABA signalling
Silhavy et al 1995 Yang et al 2005
Ascorbate peroxidase Drought and salt tolerance Badawi et al 2004
APE1 Involved in acclimatization of photosynthesis during stress Drought responsive
Walters et al 2003
Copper-containing amine oxidase ABA induced stomatal closure during stress An et al 2008
Clp G3PD Heat shock responsive ABA mediated growth and stomatal closure
Yang et al 1993 Munoz-Bertomeu et al 2011
Glutathione S-transferase Improved drought and salt tolerance Ji et al 2010
Loss of the timing of ET and JA biosynthesis 2
Function unknown in plants Kushwaha et al 2009
ACC oxidase Ethylene biosynthesis during stress
Dehydrin Drought cold and salinity responsive drought tolerance
Battaglia et al 2008 Rinne et al 1998 Zhu et al2000 Nylander et al 2001 Kimura et al 2003 kim et al 2010
DNA J protein Induced under drought Alamillo et al 1995 Coca et al 1996 Campalans et al 2001
Ethylene responsive transcription factor
Transcription factors that mediate exoression of defers related ethylene responsive genes
Ecker 1995
Aquaporin 1 MIP family Key gene for plant water balance and water use efficiency
Knepper 1994 Tyerman et al 2002 Aharon et al 2003 Kaldenhoff and Fischer 2006 Maurel 2007
Hydrogen peroxide induced protein 1
functions in plant development growth and stress responses
Neill et al 2002 Foyer and Noctor 2005
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
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Chapter IV
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PMA80 and PMA enhance dehydration tolerance of transgenic rice
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O Garcia-Casado G Lopez-Vidriero I Lozano F M Ponce M R
Micol J L Solano R 2007 The JAZ family of repressors is the
missing link in jasmonate signalling Nature 448 666 ndash 671
38 Chow W S Melis A Anderson J M 1990 Adjustments of
photosystem stoichiometry in chloroplasts improve the quantum
efficiency of photosynthesis PNAS USA 87 7502 ndash 7506
39 Chrispeels M J and Agre P 1994a Aquaporins water channel
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40 Chrispeels M J and Maurel C 1994b Aquaporins the molecular
basis of facilitated water movement through living plant cells Plant
Physiol 105 9 ndash 13
41 Close T J 1997 Dehydrins a commonality in the response of plants
to dehydration and low temperature Physiol Plant 100 291 ndash 296
42 Coca M Almoguera C Thomas T Jordano J 1996 Differential
regulation of small heat-shock genes in plants Analysis of a water
stress-inducible and developmentally activated sunflower promoter
Plant Mol Biol 31 863 ndash 876
Chapter IV
129
43 Dat J Vandenabeele S Vranova E Van Montagu M Inze D Van
Breusegem F 2000 Dual action of the active oxygen species during
plant stress responses Cell Mol Life Sci 57 779 ndash 795
44 Downton W J S Berry J A Seemann J R 1984 Tolerance of
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786 ndash 790
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Environmental Stress American Society of Plant Physiologists
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475 ndash 486
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349 ndash 358
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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accumulation by tomato plants under water and salinity stresses Part-
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Chapter IV
130
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
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Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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physiology J Expt Bot 60 2971 ndash 2985
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87 55 ndash 84
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Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
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gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
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tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
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96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
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ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
96
Table 43 Contd
Selected proteins Stress regulation or putative function References
LEA Drought tolerance Imai et al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al 2000
Hydroxyproline-rich glycoprotein
functions in defense against plant pathogen Lamport 1970
Glutathion peroxidase Drought tolerance Miao et al 2006
Elicitor responsive gene 3 Defence related Day et al 2002
Proline-rich protein Drought responsive Harrak et al 1999
Cinnamoyl-CoA reductase Defense signalling Umemura et al 2006
Zn finger protein Functions in growth differentiation transcription signal transduction and oncogenesis
Reddy et al 1992
Diaminopimelate decarboxylase
Lysine metabolism during stres Less and Galali 2008
TRAPP complex Functions in vesicular transport Sacher et al 2001
Jasmonate ZIM domain protein
Functions in Jasmonate (JA) signalling during plant growth development and defense
Chini et al 2007 Melotto et al 2008
Eukaryotic translation initiation factor 5A
Functions in plant growth and development by regulating cell division cell growth and cell death
Feng et al 2007
TCP transcription factor Involved in flower development regulation of growth and cell division shoot branching
Broholm et al 2008 Li et al 2005 Poza-Carrion et al 2007
Drm3-like protein Dehydration responsive Zhang et al 2009
Hexose transporter Induced during leaf senescence Ehness et al 1997
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
the AtCCR1 gene in Arabidopsis thaliana effects on phenotype lignins
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58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
59 Grbic V and Bleecker A B 1995 Ethylene regulates the timing of
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and Medicine 3rd Edn Oxford University Press New York
61 Hammond-Kosack K E and Jones J D G 1996 Resistance gene-
dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
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and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
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109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
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Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
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249 ndash 279
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aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
ice To the above mix 4 microl of 5X transcriptor reverse transcriptase reaction
buffer 05 microl protector RNase inhibitor (40Umicrol) 2 microl deoxynucleotide mix
(10 mM each) and 2 microl transcriptor reverse transcriptase (20Umicrol) were
added mixed briefly centrifuged and incubated at 50ordmC for 1 hour The
reverse transcriptase enzyme was deactivated by heating the tube at 85ordmC
for 5 min The first strand was stored at -20ordmC for downstream application
4252 Quantitative Real Time PCR (qRT-PCR)
LightCycler 480 SYBR green I Master kit (Cat No 04707516001 Roche
Germany) was used to carry out the expression assays in a LightCycler 480
II real time machine (Roche Germany) The reverse transcribed first strand
cDNA was used as template All the reactions were carried out in a 20 microl
volume prepared according to the protocol mentioned in the kit manual
Three technical replicates of each reaction were used to reduce the error
rate The following amplification programme was used pre incubation at
95ordmC for 5 min followed by 45 cycles of amplification with 10 sec
denaturation at 95ordmC annealing for 5 - 20 sec (primer dependent) and
extension at 72ordmC for 30 sec This was followed by one cycle of melting
curve analysis to check specificity of amplified product The temperature for
melting curve analysis was 95ordmC for 5 sec 65ordmC for 1 min and 97ordmC for
continuous acquisition This was followed by one cycle of final cooling at
40ordmC
4253 Data Acquisition
Data on the expressions were obtained in the form of crossing point (Cp) the
point where the samplersquos fluorescence curve turns sharply upward The data
acquisition was done employing the 2nd derivative maximum method
(Tichopad et al 2003 2004) as computed by the software of LightCycler
(Roche Diagnostics) Carousel-based system
426 Computation of Normalization Factor
We have used geNorm (httpmedgenugentbe~jvdesompgenorm)
software to find out the most stable genes among the set of housekeeping
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
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L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
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98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Press San Diego 391 ndash 439
Chapter IV
136
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induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
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Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
98
genes at three stages of drought geNorm calculates a gene expression
normalization factor for each tissue sample based on the geometric mean of
a user defined number of reference genes (Vandesompele et al 2002) It
eliminates the most unstable genes from the group of housekeeping genes
step by step and ends up with two most stable reference genes In the actual
process the raw Cp values of all the housekeeping genes are transformed
into relative quantities using delta Ct method These values are than used for
computation of normalization factor in geNorm for all the biological
replicates
427 Relative Quantification
In relative quantification assays the expression of the target gene is
expressed as ratio of target-to-reference gene in the same sample Here the
mean CP values (obtained from three technical replicates) of all the target
genes of all the biological replicates (seven biological replicates in our case)
under consideration were transformed into relative quantities using delta Ct
method The normalized expression level was calculated by dividing the
quantities by respective normalization factor This normalised expression
value of a gene in different biological replicates was compared to know the
relative expression level
428 Hierarchical Clustering of Drought Induced Genes
The hierarchical clustering was performed to study the expression profile of
genes across the seven biological replicates to identify groups of genes that
share similar expression profile This was performed using GenePattern
(Reich et al 2006) on line server
(httpgenepatternbroadinstituteorggppagesindexjsf)
43 Results
431 Expression of Housekeeping Genes
Four genes were tested using geNorm software in order to find out the most
stable housekeeping genegenes under water stress in the seven biological
replicates of the two cultivars Step wise exclusion of most unstable genes
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Revival of resurrection plant correlates with its antioxidant status Plant
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binding member of the late embryogenesis abundant protein family
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protein interactions Biol Res 33 21 ndash 30
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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response Cell 79 583 ndash 593
Chapter IV
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subjected to water stress Plant Growth Regul 25 81 ndash 87
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genes under drought stress in perennial ryegrass Physiologia
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Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
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expressing proteins of unknown function Plant Physiol 148 280 ndash 92
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W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
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and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
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109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
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New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
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Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
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Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
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osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
99
by geNorm ended up with two most stable housekeeping genes viz
ribulose-1 5-bisphosphate carboxylaseoxygenase and 18S rRNA The
normalization factors thus calculated for these stable housekeeping genes in
different samples are given in Table 44
432 Relative Expression of Drought Induced Genes
The relative expression studies of selected genes by qRT-PCR in both the
tolerant and susceptible cultivars have shown diverse expression pattern
(Appendix 41) Expression of some genes was found to be continuously up-
regulated in both the cultivars while some were found to be down-regulated
Some of the genes were found to be either up-regulated or down-regulated
transiently in either of the cultivars in three stages of drought However the
expression levels of these genes were different in both the cultivars To get a
clear picture we compared the expression pattern of these genes in both the
cultivars separately at three stages of drought ie BWS WS and AWS
(Figure 41 and 42) The relative expression values of all the genes
considered at three stages of drought in both the cultivar is given in
Appendix 41
4321 Relative Expression in TV23
The relative expression of these genes in tolerant cultivar TV23 has shown
four distinct patterns (Figure 41) The group A (12 genes) have been found
to be continuously up-regulated while group B (8 genes) continuously down
Table 44 Normalization factor of housekeeping genes calculated using geNorm
Biological replicates 18S Rubisco Normalization Factor
TV23(BWS) 206E-01 216E-01 05655
S3A3(BWS) 847E-01 735E-01 21140
TV23(WS) 296E-01 193E-01 06406
S3A3(WS) 818E-01 339E-01 14118
TV23(AWS) 415E-02 791E-02 01536
S3A3(AWS) 963E-01 730E-01 22462
TV23C 100E+00 100E+00 26805
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
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142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
100
regulated in three stages with progression of drought Two genes in group B
(elicitor-responsive gene 3 and cinnamoyl-CoA reductase) with very high
expression values (110821 and 142724) at BWS are not shown in Figure
41 as their inclusion in the graph drastically makes the drowning of other
bars The genes in group C (6 genes) have shown up-regulation at WS
followed by down-regulation at AWS The group D consists of only one gene
showing down-regulation at WS followed by up-regulation at AWS
D
0 50 100 150 200 250
Copper-containing amine oxidase
Loss of the timing of ET and JA hellip
Hydrogen peroxide induced protein 1
Aquaporin 1 PIP subfamily
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
clp G3PD
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase theta
Ethylene-induced esterase
Expression level
Gen
es
TV23 (AWS)
TV23 (WS)
TV23(BWS)
A
B
C
Figure 41 Expression pattern of drought induced genes in TV23 at three stages
of drought
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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447
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Chapter IV
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29
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Voinikov V K 2002 Accumulation of dehydrin-like proteins in the
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19 Boudet J Buitink J Hoekstra F A Rogniaux H Larre C Satour
P Leprince O 2006 Comparative analysis of the heat stable
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salt and osmotic stress in Arabidopsis thaliana Plant Cell Rep 26
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22 Broholm S K Tahtiharju S Laitinen R A E Albert V A Teeri T
H Elomaa P 2008 A TCP domain transcription factor controls flower
Chapter IV
127
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24 Buchanan-Wollaston V Earl S Harrison E Mathas E Navabpour
S Page T and Pink D 2003 The molecular analysis of leaf
senescence ndash a genomics approach Plant Biotech J 1 3 ndash 22
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Endo-1 4-beta-glucanase gene expression and cell wall hydrolase
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reverse transcription polymerase chain reaction assays J Mol
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27 Cakir B Agasse A Gaillard C Saumonneau A Delrot S
Atanassova R 2003 A grape ASR protein involved in sugar and
abscisic acid signalling Plant Cell 15(9) 2165 ndash 2180
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response to periodic drying events in leaves of tree tobacco Plant
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29 Campalans A Pages M Messeguer R 2001 Identification of
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30 Caramelo J J and Iusem N D 2008 When cells lose water lessons
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ndash 6
31 Carpita N C and Gibeau D M 1993 Structural models of primary
cell walls in flowering plants consistency of molecular structure with the
physical properties of the walls during growth Plant J 3 1 ndash 30
32 Cascardo J Almeida R Buzeli R Carolino S Otoni W Fontes
E 2000 The phosphorylation state and expression of soybean BiP
Chapter IV
128
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basis of facilitated water movement through living plant cells Plant
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Chapter IV
129
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786 ndash 790
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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Chapter IV
130
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physiology J Expt Bot 60 2971 ndash 2985
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acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
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Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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quantitative approach to improve water use efficiency in agriculture
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Saccharomyces cerevisiae Gene 170 243 ndash 248
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403
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
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Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
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76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
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95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
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genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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Chapter IV
136
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
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121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
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Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
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in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
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users and for biologist programmers Plant Mol Biol 5 69 ndash 76
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
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Curr Opin Plant Biol 6 410 ndash 417
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
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characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
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Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
101
4322 Relative Expression in S3A3
The expression pattern of the genes in S3A3 at three stages of drought can
be divided into three distinct groups as shown in figure 42 Group A
represents five genes with continuous up-regulation at three stages of
drought while group B represent 20 genes with up-regulation at WS followed
by down-regulation at AWS The group C represent only four genes that are
down-regulated at WS followed by up-regulation at AWS A total of 19 genes
out of 29 considered for study have shown up-regulation at BWS
A
B
C
0 20 40 60 80 100 120 140 160
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
DNA J protein
Elicitor responsive protein
Cinnamoyl CoA reductase
Copper-containing amine oxidase
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger
Abscisic stress ripening protein
LEA
TRAPP complex
TCP24 transcription factor
Ascorbate peroxidase
ACC oxidase
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione-S transferase theta
Hexose transporter
Lipase
Eukaryotic translation initiation factor 5A
Hydroxyproline-rich glycoprotein
APE1
Drm3-like protein
Clp G3PD
Expression level
Gen
es
S3A3 (AWS)
S3A3(WS)
S3A3(BWS)
Figure 42 Expression pattern of drought induced genes in S3A3 at three stages of drought
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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sustainable Agric Water Manage 80 87 ndash 99
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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channels PNAS USA 91 6255 ndash 6258
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RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
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of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
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94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
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96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
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98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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111 Nam H G 1997 The molecular genetic analysis of leaf senescence
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ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
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Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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aging in plants (Nooden L D and Leopold A C Eds) Academic
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Chapter IV
136
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
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Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
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17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
102
433 Comparative Expression Pattern of Drought Induced Genes
The water stress experiment (Chapter II) has indicated that the drought
tolerance of TV23 is much higher compared to S3A3 therefore we were
more interested to find out the genes that are up-regulated in TV23 at three
stages of drought in comparison to S3A3 We have divided the genes into
three categories based on their highest expression level in TV23 at each
stage of drought and compared them with the S3A3 (Figure 43 44 and
45) at that stage There are 11 genes (elicitor responsive gene 3 and
cinnamoyl-CoA reductase were not shown to avoid drowning of other bars in
the graph) whose expression was found to be highest at BWS of TV23
compared to WS and AWS We compared the expression of these genes
with S3A3 at that stage (Figure 43) However the expression of
choloroplast glyceraldehydes-3 phosphate dehydrogenase in this group was
found to be down-regulated when compared to control plant There were 6
genes highly expressed at WS in TV23 as compared to other two stages of
drought Of these only three were found to be highly expressed compared
to WS of S3A3 (Figure 44) These three genes are glutathione S-
transferase ethylene-responsive transcription factor and ACC oxidase
Likewise there were 12 genes that showed highest expression in TV23 at
AWS compared to WS and BWS Nine genes out of these 12 were found to
be highly expressed at AWS of TV23 when compared with the AWS of
S3A3 (Figure 45) Therefore of these 29 genes 21 (9 in BWS 3 in WS
and 9 in AWS) have shown higher relative expression in TV23 compared to
S3A3 in three stages of drought There were two genes viz loss of the
timing of ET and JA biosynthesis 2 and hydrogen peroxide induced protein 1
which showed similar pattern of expression with continuous up-regulation
with the progression of drought in both the cultivars at three stages of
drought (relative expression values are represented by bold numbers in
Appendix 41 Other two genes having similar expression pattern with up-
regulation at WS followed by down-regulation at AWS in both the cultivars
are ethylene-responsive transcription factor and glutathione-S transferase
whose relative expression values are indicated as shaded box in Appendix
41 Genes that showed completely opposite pattern of relative expression in
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
128
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Chapter IV
129
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World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
103
0 50 100 150 200
DNA J protein
Ethylene-induced esterase
Glutathion peroxidase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A
Drm3-like protein
Clp G3PD
Expression level
Ge
ne
s
TV23C
S3A3(BWS)
TV23(BWS)
Figure 43 Comparison of expression of genes having highest expression at
BWS in TV23 with BWS of S3A3
0 10 20 30 40 50 60 70 80 90
Dehydrin
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
ACC oxidase
Ethylene-responsive transcription factor
Glutathione-S transferase
Expression level
Ge
ne
s
TV23C
S3A3(WS)
TV23(WS)
Figure 44 Comparison of expression of genes having highest expression at
WS in TV23 with WS of S3A3
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
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oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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111 Nam H G 1997 The molecular genetic analysis of leaf senescence
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and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
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Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
104
0 20 40 60 80 100 120 140 160 180
Copper-containing amine oxidase
Loss of the timing of ET and JA biosynthesis 2
Hydrogen peroxide induced protein 1
Aquaporin MIP family
Dehydrin
Proline-rich protein
Diaminopimelate decarboxylase
Zinc finger
Hydroxyproline-rich glycoprotein
Abscisic stress ripening protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
DNA J protein
ACC oxidase
Clp G3PD
Ethylene-induced esterase
Ethylene-responsive transcription factor
Glutathion peroxidase
Glutathione S-transferase
Hexose transporter
Lipase
APE1
Eukaryotic translation initiation factor 5A4
Drm3-like protein
Expression level
Ge
ne
s
S3A3(BWS)
TV23(BWS)
Figure 46 Comparison of relative expression of drought induced genes in TV23 and S3A3 at BWS
0 100 200 300
Coper-containing amine oxidase
Loss of the timing of ET and hellip
Hydrogen peroxide induced hellip
Aquaporin MIP family
Proline-rich protein
Diaminopimelate decarboxylase
Zinc f inger protein
LEA
TRAPP complex
TCP transcription factor
Ascorbate peroxidase
Jasmonate ZIM-domain protein
Expression level
Gen
es
TV23C
S3A3(AWS)
TV23(AWS)
Figure 45 Comparison of expression of genes having highest expression at AWS in
TV23 with AWS of S3A3
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
128
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Chapter IV
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141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
105
both the cultivars at three stages of drought are the DNA J elicitor-
responsive gene 3 and cinnamoyl-CoA reductase The expression of these
three genes (their relative expression values are indicated by bold and
underline in Appendix 41) were found to be decreased in TV23 with the
increase of drought while in S3A3 they showed a completely opposite
pattern of expression with continuous increase There is another group
represented by 9 genes (their relative expression values are indicated as
bold italics and underlined in Appendix 41) whose expression followed a
similar pattern in both the cultivars with continuous up-regulation at BWS
and WS However all these genes were found to be further up-regulated in
TV23 at AWS while they are down-regulated at AWS in S3A3 The gene
chloroplast glyceraldehyde-3 phosphate dehydrogenase (indicated as bold
italics in Appendix 41) was found to be down-regulated in TV23 compared
to control The comparison of expression at BWS (column indicated by super
scribing with in Appendix 41 Figure 46) of both the cultivar has shown 19
genes that are up-regulated in TV23 compared to S3A3 Two highly
expressed genes (elicitor responsive gene 3 and cinnamoyl-CoA reductase)
are not shown in Figure 46 to avoid drowning of others Among these the
maximum expression was observed for cinnamoyl-CoA reductase and
elicitor responsive gene 3 (their expression values are indicted by super
scribing with yen in Appendix 41)
434 Hierarchical Cluster Analysis of Drought Induced Genes
The hierarchical cluster analysis of the expression profiles of the drought
induced genes showed differences between drought tolerant and susceptible
cultivar (Figure 47) Clustering separated the seven biological replicates into
two groups (A and B Figure 47) indicating tolerant and susceptible cultivars
clustered separately However the biological replicate TV23 (AWS) is
included in susceptible group B The results also showed that there were
four distinct expression patterns (cluster I to IV Figure 47) of the genes in
three stages of drought in both the cultivars The cluster I is represented by
11 genes whose expression is higher at WS and AWS of TV23 and WS of
S3A3 among all three stages considered The gene for ethylene-
responsive transcription factor is however very weakly induced compared to
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
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82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
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control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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ndash 410
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and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
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Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
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126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
106
other genes in that group although its expression is slightly higher than
TV23C in all the biological replicates The cluster II includes 5 genes whose
expression is higher in two stages (WS and AWS) of S3A3 and also in
AWS of TV23 The cluster III includes two genes whose expression is down-
regulated in comparison to TV23C in both the cultivars with the progression
of drought The cluster IV is represented by 11 genes that are highly
Figure 47 Hierarchical cluster analysis of drought induced genes in TV23 and
S3A3 at three stages of drought
Hydrogen peroxide induced protein 1
Aquaporin 1 MIP family
Copper-containing amine oxidase
Diaminopimelate decarboxylase
Ascorbate peroxidase
LEA
Zinc finger
Proline-rich protein
TCP transcription factor
Dehydrin
Ethylene-responsive transcription factor
Hydroxyproline-rich glycoprotein
Loss of the timing of ET and JA biosynthesis 2
Jasmonate ZIM-domain protein
Abscisic stress ripening protein
TRAPP complex
Clp G3PD
APE1
ACC oxidase
Glutathione S-transferase
Hexose transporter
Glutathione peroxidase
Eukaryotic translation initiation factor 5A4
Ethylene-induced esterase
DNA J protein
Lipase
Cinnamoyl CoA reductase
Elicitor responsive gene 3
Drm3 like protein
A B
I
II
III
IV
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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York pp178 ndash 190
Chapter IV
125
2 Agre P Sasaki S Chrispeels M J 1993 Aquaporins a family of
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
G 2003 Over-expression of a plasma membrane aquaporin in
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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ndash 1099
5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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Biochemistry (Mosc) 68 945 ndash 951
6 Amiard T Guenzi A C Martin B Cushman J C 2003 Tolerance
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Plant Physiol 131 1748 ndash 1755
7 An Z Jing W Liu Y Zhang W 2008 Hydrogen peroxide generated
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8 Apel K and Hirt H 2004 Reactive oxygen species metabolism
oxidative stress and signal transduction Annu Rev Plant Biol 55 373
ndash 399
9 Asada K and Takahashi M 1987 Production and scavenging of
active oxygen in photosynthesis In Photoinhibition (Kyle DJ et al
eds) pp 227 ndash 287 Elsevier
10 Badawi G H Kawanoa N Yamauchi Y Shimada E Sasaki R
Kubo A Tanaka K 2004 Over-expression of ascorbate peroxidase in
tobacco chloroplasts enhances the tolerance to salt stress and water
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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Chapter IV
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Chapter IV
131
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Chapter IV
133
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Revival of resurrection plant correlates with its antioxidant status Plant
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Chapter IV
134
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135
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Chapter IV
138
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Chapter IV
140
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plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
107
expressed in BWS and WS of TV23 among seven biological replicates Of
these 9 genes were found to be highly expressed in BWS and remaining 2
genes in WS Among these 29 drought induced genes three most highly
expressed genes in all the biological replicates are cinnamoyl-CoA
reductase followed by elicitor-responsive gene 3 and lipase (class 3) with
relative expression values of 1427244 110821 and 171732 respectively
435 Validation of Subtractive Library
To validate our subtractive library we have compared the relative expression
of drought induced genes mostly selected from the library BWE3E4
Nineteen genes (65) out of 29 selected were found to be highly expressed
(Figure 46) in TV23 compared to S3A3 at BWS This clearly showed that
the efficiency of subtraction was as high as 65
44 Discussion
When plants experience environmental stresses such as drought salinity
etc they activate a diverse set of physiological metabolic and defence
processes to survive and to sustain growth Tolerance and susceptibility to
these stresses are very complex Drought tolerance or susceptibility is a
multigenic trait and therefore difficult to control Transcriptomics proteomics
and gene expression approaches are commonly used to identify the
activation and regulation of several stress-related transcripts and proteins
which are generally classified into two major groups One group is involved
in signalling cascades and in transcriptional control whereas members of
the other group functions in membrane protection as osmoprotectants as
antioxidants and as ROS scavengers (Shinozaki and Yamaguchi-Shinozaki
1997) Manipulation of genes that protects and maintains cellular functions
or that maintains the structure of cellular components has been the major
target of attempts to produce plants having enhanced stress tolerance The
study of expression pattern of such genes at different level of water stress
will pave the way to understand the drought responsive mechanisms in tea
In the present study we have studied the expression pattern of 29 genes
under induced drought condition in two tea cultivars with contrasting drought
tolerance behaviour in field as well as in controlled condition Among these
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Chapter IV
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Chapter IV
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Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
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water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
108
there are genes that codes for proteins involved in membrane protection
eg LEA protein which has been reported to play roles in membrane and
protein stabilization and cellular homeostasis in several species (Amiard et
al 2003 Wang et al 2004 Houston et al 2005 Boudet et al 2006
Cameron et al 2006) aquaporin that facilitates transport of water across
cell membranes (Agre et al 1993 Chrispeels et al 1994a Chrispeels et
al 1994b) dehydrins involved in osmotic adjustment (Taiz and Zeiger
2002 Brini et al 2007) antioxidant enzymes such as ascorbate peroxidase
(Yamaguchi et al 1995) glutathione transferase (Noctor et al 1998)
glutathione peroxidase (Miao et al 2006) involved in scavenging ROS The
other group includes genes encoding hydrogen peroxide induced protein
that functions in plant development growth and stress responses
particularly those induced by environmental stimuli (Neill et al 2002 Foyer
and Noctor 2005) transcription factors like Zn finger protein (Leon and
Roth 2000) abscisic acid stress ripening protein (Cakir et al 2003) and
TCP transcription factor Besides we have monitored the expression of
other genes encoding proteins as listed in Table 43 that are reported to be
induced during water and other environmental stresses in planta It has been
found that the expression pattern of all these genes (as evident from the
Figure 48a-c) are either continuously or transiently up-regulated or down-
regulated during progressive drought which also implies that these genes
are not constitutive but are drought responsive
We monitored the expression of some of the transcription factors that are
found to be up-regulated or down-regulated during the induced drought
experiment The Zinc finger protein is a C3HC4-type RING finger whose
expression was found to be induced by drought (Figure 48a) This protein
has been reported to have crucial roles in the growth differentiation
transcription signal transduction and oncogenesis (Reddy et al 1992) and
also involved in the ubiquitin-mediated protein degradation pathway (Lorick
et al 1999) The transgenic Arabidopsis over-expressing the gene has been
found to have increased cellular ABA and drought tolerance compared to
wild type plant (Ko et al 2006) We have observed higher induction of this
gene in S3A3 compared to TV23 at BWS and WS However its expression
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
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277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
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96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
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102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
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Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
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122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
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Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
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126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
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osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
109
Figure 48a Relative Expression of drought induced gene at three stages of
drought
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Coper-containing amine oxidase
TV23
S3A3
0
20
40
60
80
100
120
140
160
180
BWS WS AWS
Rel
ativ
e E
xp
ress
ion
Hydrogen peroxide induced
protein1
TV23S3A3
0
50
100
150
200
250
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Aquaporin MIP family
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Proline-rich protein
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Diaminopimelate decarboxylase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Zinc finger protein
TV23S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xp
ress
ion
LEA
TV23S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
TRAPP complex
TV23
S3A3
0
10
20
30
40
50
60
70
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
TCP transcription factor
TV23
S3A3
0
20
40
60
80
100
120
140
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ascorbate peroxidase
TV23S3Ahellip
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
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H Elomaa P 2008 A TCP domain transcription factor controls flower
Chapter IV
127
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Chapter IV
128
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basis of facilitated water movement through living plant cells Plant
Physiol 105 9 ndash 13
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Chapter IV
129
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Breusegem F 2000 Dual action of the active oxygen species during
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786 ndash 790
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Environmental Stress American Society of Plant Physiologists
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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Chapter IV
130
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Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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403
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
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expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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diacylglycerol acyltransferase during leaf senescence Plant Physiol
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80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
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ndash 209
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Identification of Arabidopsis genes regulated by high light-stress using
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abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
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of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
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Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
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91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
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10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
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synthesis of heat shock proteins and increased thermotolerance in
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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Chapter IV
134
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11369
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Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
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Chapter IV
135
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Chapter IV
136
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137
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Cell Environ 21 601 ndash 11
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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Chapter IV
138
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
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143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
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145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
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148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
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World J Agri Sci 4(3) 307 ndash 313
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151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
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159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
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197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
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162 Wang W Vinocur B Altman A 2003 Plant responses to drought
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163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
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166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
110
0
2
4
6
8
10
12
14
16
18
20
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Ethylene-responsive
transcription factor
TV23
S3A30
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
DNA J protein
TV23
S3A3
0
200
400
600
800
1000
1200
BWS WS AWS
Re
lati
ve
Ex
pre
ssio
n
Elicitor responsive gene 3
TV23
S3A3
0
5
10
15
20
25
30
35
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Ethylene-induced esterase
TV23
S3A3
0
200
400
600
800
1000
1200
1400
1600
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Cinnamoyl CoA reductase
TV23
S3A3
0
10
20
30
40
50
60
70
80
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Dehydrin
TV23
S3A3
0
10
20
30
40
50
60
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Abscisic stress ripening protein
TV23
S3A3
0
10
20
30
40
50
60
70
80
90
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
ACC oxidase
TV23
S3A3
0
5
10
15
20
25
30
35
40
45
50
BWS WS AWS
Re
lati
ve E
xpre
ssio
n
Glutathione S-transferase
TV23S3A3
0
2
4
6
8
10
12
14
BWS WS AWS
Re
lati
ve
Exp
ress
ion
Clp G3PD
TV23
S3A3
Figure 48b Relative Expression of drought induced gene at three stages of
drought
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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York pp178 ndash 190
Chapter IV
125
2 Agre P Sasaki S Chrispeels M J 1993 Aquaporins a family of
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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6 Amiard T Guenzi A C Martin B Cushman J C 2003 Tolerance
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9 Asada K and Takahashi M 1987 Production and scavenging of
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10 Badawi G H Kawanoa N Yamauchi Y Shimada E Sasaki R
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11 Bais H P Vepachedu R Gilroy S Callaway R M Vivanco J M
2003 Allelopathy and exotic plant invasion from molecules and genes
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Chapter IV
126
12 Bartels D 2001 Targeting detoxification pathways an efficient
approach to obtain plants with multiple stress tolerence Trends Plant
Sci 6 284 ndash286
13 Barua D N 1989 Science and practice in Tea culture (Calcutta Tea
Research Association) pp 509
14 Battaglia M Olvera-Carrillo Y Garciarrubio A Campos F
Covarrubias A A 2008 The enigmatic LEA proteins and other
hydrophilins Plant Physiol 148(1) 6 ndash 24
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29
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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Chapter IV
130
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Chapter IV
131
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Identification of Arabidopsis genes regulated by high light-stress using
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Plant Physiol 131 309 ndash 316
Chapter IV
133
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Physiol 35 821 ndash 827
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Revival of resurrection plant correlates with its antioxidant status Plant
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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Chapter IV
134
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Chapter IV
135
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Cell Environ 21 601 ndash 11
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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Chapter IV
138
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139
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Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
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Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
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160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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165 Wood A J and Goldsbrough P B 1997 Characterization and
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Chapter IV
141
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170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
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836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
111
is down-regulated at AWS in S3A3 where as in TV23 it continues to
increase with progressive drought
Another gene whose expression was induced by drought is ethylene-
responsive transcription factor which is a well known transcription factor
having DNA binding domain that mediates the expression of defense related
ethylene responsive genes (Ecker 1995) by binding with the 11 bp
consensus sequence (TAAGAGCCGCC) called GCC box (Ohme-Takagi
and Shinshi 1995 Shinshi et al 1995) of ethylene responsive element
(ERE) in the promoter region Over-expression of ethylene-responsive
transcription factor gene (JERF3) in tobacco has been reported to increase
adaptation to drought salt and freezing by activating the expression of
oxidative stress related genes such as superoxide dismutase and by
decreasing the accumulation of ROS Therefore higher expression of this
gene in TV23 may be playing a positive role for giving drought tolerance by
reducing the accumulation of ROS and enhancing the production of ROS
scavengers like ascorbate peroxidase (Figure 48a) and glutathione
peroxidase (Figure 48c) Another transcription factor gene the eukaryotic
translation initiation factor 5A4 (eIF5A4) was also highly induced by drought
in TV23 compared to S3A3 This gene has been widely reported to be
involved in plant growth and development by regulating cell division cell
growth and cell death (Feng et al 2007) The partial inactivation of eIF 5A
gene by over-expressing antisense DHS (Deoxyhypusine synthase) cDNA
gene in transgenic Arabidopsis has been reported to delay senescence and
resistance to drought stress (Wang et al 2003) But in our study we have
observed a high expression of the gene in the tolerant tea cultivar TV23
(Figure 48c) which contradicts the above result Moreover its expression
level decreased further at WS and AWS at which we observed maximum
senescence of lower mature leaves during the induced drought experiment
Therefore this contrasting expression pattern of eIF5A in tea needs to be
considered for further investigation that will help to identify its role in plant
adaptation to stress This may be due to interaction of (genotype x
environment) factor as because drought is a multigenic trait and we did our
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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ndash 1099
5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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6 Amiard T Guenzi A C Martin B Cushman J C 2003 Tolerance
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Plant Physiol 131 1748 ndash 1755
7 An Z Jing W Liu Y Zhang W 2008 Hydrogen peroxide generated
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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Chapter IV
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Chapter IV
131
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Chapter IV
133
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Revival of resurrection plant correlates with its antioxidant status Plant
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Chapter IV
134
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135
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Chapter IV
138
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Cell Environ 25 173 ndash 194
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Chapter IV
140
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
112
drought induced experiment in pot condition which seems to be different
from natural field condition
TCP transcription factor whose up-regulation was found to be low in TV23
compared to S3A3 during the induced water stress experiment It is a
member of a large group of transcription factor family called TCP family
which in Arabidopsis is represented by 24 genes This family has been
reported to be involved in flower development (Broholm et al 2008)
regulation of growth and cell division (Li et al 2005) shoot branching (Poza-
Carrion et al 2007) etc There is no report of induction of this transcription
factor under water stress
The LEA gene found to be highly expressed in the present study (Figure
48a) belongs to group 3 or D-29 family (classification adapted by Dure et
al 1989 Dure 1993) The role of LEA proteins towards improving drought
tolerance in plants and yeast has been reported in several studies (Imai et
al 1996 Xu et al 1996 Swire-Clark and Marcotte 1999 Zhang et al
2000) The expression profile of LEA gene under stress supports its role as
protective molecule that enables cells to survive protoplasmic water
depletion (Ingram and Bartels 1996) Studies on over-expression of LEA
gene also support its protective roles by improving the stress tolerance of
plants For example the expression of Barley gene HVA1 in wheat and rice
increases drought tolerance (Sivamani et al 2000) and also over-
expression of LEA genes PMA80 and PMA1959 (Cheng et al 1995) and
OsLEA3-1 (Xiao et al 2007) increases dehydration tolerance in transgenic
rice These results are consistent with our findings that the expression of
LEA in drought tolerant cultivar TV23 is much higher compared to
susceptible cultivar (Figure 48a) at BWS The expression was found to
increase further in TV23 with increase of water stress in WS and AWS
compared to S3A3 This contrasting expression pattern of the gene might
be contributing towards higher drought tolerance of TV23
Another gene whose expression was found to be highly induced is
aquaporin 1 that code for protein of MIP (Membrane Intrinsic protein) family
and PIP (Plasma membrane intrinsic protein) subfamily which is well
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
G 2003 Over-expression of a plasma membrane aquaporin in
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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ndash 1099
5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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6 Amiard T Guenzi A C Martin B Cushman J C 2003 Tolerance
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8 Apel K and Hirt H 2004 Reactive oxygen species metabolism
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ndash 399
9 Asada K and Takahashi M 1987 Production and scavenging of
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10 Badawi G H Kawanoa N Yamauchi Y Shimada E Sasaki R
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11 Bais H P Vepachedu R Gilroy S Callaway R M Vivanco J M
2003 Allelopathy and exotic plant invasion from molecules and genes
to species interactions Science 3011377 ndash 1380
Chapter IV
126
12 Bartels D 2001 Targeting detoxification pathways an efficient
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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Chapter IV
130
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Chapter IV
131
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Chapter IV
133
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Revival of resurrection plant correlates with its antioxidant status Plant
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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Chapter IV
134
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135
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Chapter IV
138
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139
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Cell Environ 25 173 ndash 194
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
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Chapter IV
140
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160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
113
documented to play a key role in plant water balance and water use
efficiency (Knepper 1994 Tyerman et al 2002 Aharon et al 2003
Kaldenhoff and Fischer 2006 Maurel 2007) by mediating movements of
small solutes and water across membranes (Maurel et al 2009) However
the role of aquaporin in leaf water transport under drought conditions
remains unclear (Heinen et al 2009) It has been reported that transgenic
tomato over-expressing tobacco aquaporin 1 showed higher stomatal
conductance transpiration rate and net photosynthesis (Sade et al 2010)
which are very much essential for a plant to maintain growth In present
study we have found higher expression of the gene in TV23 compared to
S3A3 (Figure 48a) This higher expression of the gene may be responsible
for efficient movement of water to growing tissues in TV23 compared to
S3A3 to maintain a water balance under induced drought condition The
higher drought tolerance of TV23 has also been supported by its higher
photosynthesis and transpiration rate observed during the induced drought
experiment compared to S3A3 at a given level of water stress (Chapter II)
Moreover there are reports of reduction in drought tolerance and hydraulic
conductivity in Arabidopsis with reduced aquaporin 1 gene expression
(Siefritz et al 2002) This finding is also consistent with our findings that
expression of aquaporin 1 gene in susceptible is less compared to the
tolerant cultivar
Dehydrin is another gene that was found to be highly induced in the present
study in both the cultivars However its expression goes down as drought
became more severe at AWS (Figure 48b) The higher expression of
dehydrin in our experiment is consistent with the findings that it accumulates
under drought and other stresses like low temperature and or salt-stress in
vegetative tissues of plant (Ingram et al 1996rsquo Allagulova et al 2003
Close 1997) Dehydrin is a type of LEA group protein originally designated
as D-11(Battaglia et al 2008) Studies have suggested many functions of
dehydrin in buffering water sequestering ions stabilizing membranes or
acting as chaperones or molecular shields (Rorat et al 2006 Tunnacliffe et
al 2007 Koag et al 2003 Goyal et al 2005 Kovacs et al 2008 Kruger
et al 2002) Although constitutively expressed the accumulation of dehyrin
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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Chapter IV
126
12 Bartels D 2001 Targeting detoxification pathways an efficient
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14 Battaglia M Olvera-Carrillo Y Garciarrubio A Campos F
Covarrubias A A 2008 The enigmatic LEA proteins and other
hydrophilins Plant Physiol 148(1) 6 ndash 24
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29
18 Borovskii G B Stupnikova I V Antipina A I Vladimirova S V
Voinikov V K 2002 Accumulation of dehydrin-like proteins in the
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treatment BMC Plant Biol 2 5
19 Boudet J Buitink J Hoekstra F A Rogniaux H Larre C Satour
P Leprince O 2006 Comparative analysis of the heat stable
proteome of radicles of Medicago truncatula seeds during germination
identifies late embryogenesis abundant proteins associated with
desiccation tolerance Plant Physiol 140 1418 ndash 1436
20 Bowler C Montagu M V Inze D 1992 Superoxide dismutase and
stress tolerance Ann Rev Plant Physiol Plant Mol Biol 43 83 ndash 116
21 Brini F Hanin M Lumbreras V Irar S Pages M Masmoudi K
2007 Over-expression of wheat dehydrin DHN-5 enhances tolerance to
salt and osmotic stress in Arabidopsis thaliana Plant Cell Rep 26
2017 ndash 2026
22 Broholm S K Tahtiharju S Laitinen R A E Albert V A Teeri T
H Elomaa P 2008 A TCP domain transcription factor controls flower
Chapter IV
127
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23 Buchanan-Wollaston V 1997 The molecular biology of leaf
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24 Buchanan-Wollaston V Earl S Harrison E Mathas E Navabpour
S Page T and Pink D 2003 The molecular analysis of leaf
senescence ndash a genomics approach Plant Biotech J 1 3 ndash 22
25 Burns J K Lewandowski D J Nairn C J Brown G E 1998
Endo-1 4-beta-glucanase gene expression and cell wall hydrolase
activities during abscission in Valencia orange Physiol Plant 102 217
ndash 225
26 Bustin S A 2000 Absolute quantification of mRNA using real-time
reverse transcription polymerase chain reaction assays J Mol
Endocrinology 25 169 ndash 193
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643
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ndash 6
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Chapter IV
128
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Chapter IV
129
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Environmental Stress American Society of Plant Physiologists
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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Chapter IV
130
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Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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physiology J Expt Bot 60 2971 ndash 2985
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Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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403
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
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expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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characterization of irregular xylem4 (irx4) a severely lignin-deficient
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
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80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
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Identification of Arabidopsis genes regulated by high light-stress using
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abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
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Plant Physiol 131 309 ndash 316
Chapter IV
133
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Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
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Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
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transports iron in the phloem of Ricinus communis L J Biol Chem
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91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
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10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
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genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
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95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
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oxidative burst orchestrates the plant hypersensitive disease resistance
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Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
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Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
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11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
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W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
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Chapter IV
135
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Chapter IV
136
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
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Chapter IV
137
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Cell Environ 21 601 ndash 11
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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Chapter IV
138
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A Sinauer Associates Publishers
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Chapter IV
139
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World J Agri Sci 4(3) 307 ndash 313
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Nucl Acids Res 31(20) e122
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multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
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Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
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162 Wang W Vinocur B Altman A 2003 Plant responses to drought
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
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LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
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166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
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611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
114
was found to be increased in many studies involving stresses like drought
cold salinity and ABA (Battaglia et al 2008 Rinne et al 1998 Houde et
al 1992 Wood and Goldsbrough 1997 Ismail et al 1999 Zhu et al2000
Nylander et al 2001 Kimura et al 2003 Kim et al 2010 Borovskii et al
2002) which also corroborates our findings Higher expression of this gene
was observed in TV23 compared to susceptible cultivar S3A3 This higher
expression level of dehydrin in both the cultivars compared to control plants
seems to be involved in the dehydrationosmotic stress response in tea and
might contribute to the drought tolerance behaviour This assumption is
supported by data showing that under conditions of water deficit
transcription of dehydrin gene is significantly higher in drought-tolerant than
in drought-sensitive species A correlation between plant drought tolerance
and dehydrin accumulation was also found in Sorghum and in Sunflower
(Wood et al 1997 Cellier et al 1997) Therefore the higher expression of
dehydrin may be responsible for high drought tolerance behaviour of TV23
compared to S3A3
DNA J a heat shock protein reported to involve in a variety of cellular
processes including protein folding protein transport across membranes
regulation of protein degradation modulation of protein activity and
prevention of irreversible protein aggregation (Sun et al 2001 Lee et al
1995 Mohd et al 2006) Induction of this protein under water stress has
been well reported in plants (Alamillo et al 1995 Coca et al 1996
Campalans et al 2001) The higher expression of this gene in TV23 at BWS
(Figure 48b) may be attributed for higher drought tolerance of TV23 Our
observation is consistent with the findings that the higher expression of heat
shock proteins enhance drought tolerance in tobacco and other plants
(Cascardo et al 2000 Eun and Choo 2006) However its expression
sharply decreased at WS and AWS in both the cultivars (Figure 48b)
A secondary effect of dehydration in plant is the production of ROS like
singlet oxygen superoxide anion radicals hydroxyl radicals and hydrogen
peroxide (Smirnoff 1998 Bartels 2001 Apel and Hirt 2004) in chloroplast
as well as in mitochondria Higher accumulation of ROS is found to be toxic
as it can oxidize proteins lipids and DNA (Halliwell and Gutteridge 1999)
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Bray Eds Plant responses to cellular dehydration during
Environmental Stress American Society of Plant Physiologists
Rockville MD pp 91 ndash 103
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Thomas T L Sung Z R 1989 Common amino acid sequence
domains among the LEA proteins of higher plants Plant Mol Biol 12
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rubrum by cytokinins Plant J 11 539 ndash 548
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Plant 86 297 ndash 300
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contributes to drought-stress tolerance in plants Plant Cell Rep 25
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development by regulating cell division cell growth and cell death
Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
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Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
semiarid zones Ann Rev of Plant Physiol 29 277 ndash 317
54 Foyer C H and Noctor G 2005 Redox homeostasis and antioxidant
signalling A metabolic interface between stress perception and
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
Calle I M 1994 Inhibition of polyamine synthesis by cyclohexylamine
stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
57 Goujon T Ferret V Mila I Pollet B Ruel K Burlat V Joseleau
J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
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58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
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and Medicine 3rd Edn Oxford University Press New York
61 Hammond-Kosack K E and Jones J D G 1996 Resistance gene-
dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
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encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
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68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
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1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
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gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
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73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
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76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
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77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
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80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
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ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
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Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
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Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
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salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
115
leading to cell death (Asada 1987 Dat et al 2000 Hammond-Kosack et al
1996) Therefore production of ROS has to be fine tuned to prevent
oxidative damage In fact plant has developed mechanisms to keep the
concentrations of ROS under tight control by producing antioxidants which
may be enzyme or non-enzyme proteins Therefore alleviation of oxidative
damage and increase resistance is correlated with an efficient antioxidative
system (Smirnoff 1998 Shalata et al 2001 Kranner et al 2002) In the
present study we have monitored the expression of such genes coding for
antioxidative proteins like ascorbate peroxidase glutathione peroxidase and
glutathione S-transferase The expression of these genes has been found to
be highly up-regulated in TV23 compared to S3A3 at BWS (Figure 48a-c)
The higher expression of these genes indicates the accumulation of ROS
during drought stress For example the accumulation of ROS like H2O2 is
substantiated by higher expression of H2O2 induced protein (Figure 48a) in
the present study Hydrogen peroxide is an interesting molecule which can
play dual role during stress it induces a defense response at lower
concentration while at higher concentration it results into programmed cell
death (PCD) (Vandenabeele et al 2003) which is essential for a number of
developmental processes and environmental responses including aleurone
cell death the hypersensitive response to pathogens and allelopathic
plantndashplant interactions (Bethke and Jones 2001 Bais et al 2003 Apel and
Hirt 2004) We have observed starting of senescence browning and drying
of lower leaves (cell death) in both the cultivars at WS and AWS during the
water stress experiment This may be due to higher accumulation of H2O2 as
indicated by higher expression of hydrogen peroxide induced protein in WS
and AWS The higher accumulation of H2O2 is also supported by higher
expression of ascorbate peroxidase a scavenging enzyme for H2O2 during
the experiment (Figure 48a) This molecule has also been reported to
increase the level of glutathione S-transferase (Levine et al 1994 Chen et
al 1996 Polidoros and Scandalios 1999) an enzyme responsible for the
detoxification of highly reactive lipid peroxidation products generated from
oxidative stress damaged membranes (Polidoros and Scandalios 1999)
during drought In fact the expression of glutathione S-transferase was
found to be highly up-regulated in both the cultivars during the experiment
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
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RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
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of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
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jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
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109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
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249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
116
(Figure 48b) Transgenic plants over-expressing these antioxidant enzymes
have been reported to have improved drought tolerance characters For
example transgenic Tobacco and Arabidopsis over-expressing ascorbate
peroxidase (Badawi et al 2004) and glutathione peroxidase (Miao et al
2006) respectively were found to have higher drought tolerance compared to
wild type plant Likewise over-expression of glutathione S-transferase gene
was found to improve drought and salt tolerance in transgenic Tobacco (Ji et
al 2010) Therefore the higher expression of these ROS scavenging
enzymes in our study might be responsible for higher drought tolerance of
TV23 as reported in other plants (Li et al 1998 Bowler et al 1992)
The gene encoding Copper-containing amine oxidase was found to be
induced during progressive drought in both the cultivars to varying degrees
(Figure 48a) This enzyme catalyses the catabolism of polyamine putrescine
to produce H2O2 ammonia and ∆1-pyrroline The H2O2 thus produced is
reported to be involved in ABA induced stomatal closure during stress (An et
al 2008) ∆1-pyrroline is further reduced by pyrroline-5-carboxylate
reductase to produce proline a well known osmoprotectant involved in
protection of cells from damage due to water stress (Shinozaki et al 1997)
in plants The high expression of this gene in S3A3 (15 days) compared to
TV23 (22 days) at BWS may be correlated with the earlier closing of
stomata in S3A3 than TV23 which was actually observed during the
induced drought experiment
During severe drought stress plant has to depend on reserve food for
carbon source for survival When drought becomes so severe that it results
in drought induced leaf senescence the reserved food in the form of
triacylglycerol (TAG) from dying leaves and other tissues are converted to
fatty acid and glycerol by lipase class 3 enzyme (Trigacylglycerol lipase or
class 3 lipase) which are further converted to sugars to support growth
Although seeds are the main storage for TAG its synthesis and
accumulation has also been reported in chloroplast as a means for
sequestering free fatty acids that are rapidly de-esterified from galactolipids
in the event of stress (Sakaki et al 1990a and b Navari-Izzo et al 1990)
and during natural senescence (Kaup et al 2002) In our study we have
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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131
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Chapter IV
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Chapter IV
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
117
observed a very high up-regulation of lipase (Class 3) in TV23 compared to
S3A3 at BWS followed by sharp decrease in WS and AWS (Figure 48c)
This higher expression of lipase may be contributing for higher efficiency of
TV23 to convert the stored and senescence induced TAG into fatty acids
and glycerol These two compounds are further metabolised to produce
sugars required to maintain metabolic processes to sustain during water
stress period Therefore the high expression of Lipase in TV23 might be
responsible for supplying carbon source for maintaining metabolic processes
to sustain during water stress This source of energy might be contributing in
TV23 to withstand prolonged drought up to 25 days (WS) compared to 18
days in S3A3 in the present study
The gene encoding cinnamoyl-CoA reductase that catalyzes the synthesis of
lignin via phenyl propanoid pathway (Piquemal et al 1998 Jones et al
2001 Goujon et al 2003 Kawasaki et al 2006 Leple et al 2007
Wadenback et al 2008 Zhou et al 2010) have been found to be highly
expressed in the tolerant TV23 at BWS This high induction of the gene
under drought is consistent with the findings of So et al (2010) A strong
correlation between leaf lignin content and drought tolerance has been
reported in maize that can be used as an index for evaluation of drought
tolerance (Hu et al 2009) Assuming that high expression of cinnamoyl-CoA
reductase observed (Figure 48b) will eventually result in to higher
accumulation of lignin in TV23 which may be contributing for its higher
drought tolerance
Diaminopimelate decarboxylase is an enzyme that catalyzes the
decarboxylation of diaminopimelate to produce lysine We have observed
up-regulation of this gene under drought in both the cultivars (Figure 48a)
However its expression was found higher in TV23 and continues to increase
at WS and AWS Probably this is the first report of induction of this gene
under drought stress in plants
APE1 (Acclimation of photosynthesis to the environment) is another gene
highly expressed in tolerant cultivar in the present study at BWS (Figure
48c) The differential expression of the gene under drought has been
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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22 Broholm S K Tahtiharju S Laitinen R A E Albert V A Teeri T
H Elomaa P 2008 A TCP domain transcription factor controls flower
Chapter IV
127
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24 Buchanan-Wollaston V Earl S Harrison E Mathas E Navabpour
S Page T and Pink D 2003 The molecular analysis of leaf
senescence ndash a genomics approach Plant Biotech J 1 3 ndash 22
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Endo-1 4-beta-glucanase gene expression and cell wall hydrolase
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26 Bustin S A 2000 Absolute quantification of mRNA using real-time
reverse transcription polymerase chain reaction assays J Mol
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27 Cakir B Agasse A Gaillard C Saumonneau A Delrot S
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abscisic acid signalling Plant Cell 15(9) 2165 ndash 2180
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accumulation of cuticular wax and expression of lipid transfer protein in
response to periodic drying events in leaves of tree tobacco Plant
Physiol 140 176 ndash 183
29 Campalans A Pages M Messeguer R 2001 Identification of
differentially expressed genes by the cDNA-AFLP technique during
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30 Caramelo J J and Iusem N D 2008 When cells lose water lessons
from biophysics and molecular biology Prog Biophys Mol Biol 99(1) 1
ndash 6
31 Carpita N C and Gibeau D M 1993 Structural models of primary
cell walls in flowering plants consistency of molecular structure with the
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32 Cascardo J Almeida R Buzeli R Carolino S Otoni W Fontes
E 2000 The phosphorylation state and expression of soybean BiP
Chapter IV
128
isoforms are differentially regulated following abiotic stresses J Biol
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34 Chang S Puryear J D Dias M A D L Funkhouser E A Newton
R J Cairney J 1995 Gene expression under water deficit in loblolly
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Physiol Plant 95 1 ndash 10
35 Chen W Chao G Singh K B 1996 The promoter of a H2O2-
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PMA80 and PMA enhance dehydration tolerance of transgenic rice
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37 Chini A Fonseca S Fernandez G Adie B Chico J M Lorenzo
O Garcia-Casado G Lopez-Vidriero I Lozano F M Ponce M R
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basis of facilitated water movement through living plant cells Plant
Physiol 105 9 ndash 13
41 Close T J 1997 Dehydrins a commonality in the response of plants
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Chapter IV
129
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786 ndash 790
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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Chapter IV
130
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Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
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physiology J Expt Bot 60 2971 ndash 2985
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87 55 ndash 84
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acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
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68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
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Saccharomyces cerevisiae Gene 170 243 ndash 248
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403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
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Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
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76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
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(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
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80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
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103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
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82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
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ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
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84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
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103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
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Plant Science 173 510 ndash 520
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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Chapter IV
136
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
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121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
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Chapter IV
137
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ndash 411
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osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
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136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
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Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
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constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
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158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
118
reported in ryegrass (Liu and Jiang 2010) Plants subjected to variation in
environmental temperature has the capacity to acclimate the process of
photosynthesis (for review see Berry and Bjorkman 1980 and Down ton et
al 1984) that improves the efficiency with which light energy is used in
photosynthesis (Chow et al 1990 Walters and Horton 1995) Study on
ape1 gene in Arabidopsis mutants showed acclimation-defective phenotype
(Walters et al 2003) The higher expression of this gene in TV23 under
induced drought (Figure 48c) may therefore be helping the plant to
acclimatize their photosynthetic apparatus to survive under drought
condition However the expression of the gene was found to be decreased
at WS
Chloroplast glyceraldehyde-3-phosphate dehydrogenase is the gene among
all the genes considered in present study whose expression has been found
to be continuously down-regulated compared to the control plant with the
progression of drought (Figure 48b) A similar result of down regulation of
the gene has also been reported in Arabidopsis under heat shock treatment
(Yang et al 1993) and in water-deceit tolerance in C4 perennial grass
species (Zhao et al 2011) Recently the mutant studies in Arabidopsis has
also indicated that it play a positive role in ABA mediated growth and
stomatal closure (Munoz-Bertomeu et al 2011) Therefore down regulation
of the gene in the present study might be associated with the down
regulation of carbon metabolism and ABA signalling during drought stress
One of the responses of plants to environmental stress is establishing a
temporary growth arrest that allows adaptation to adverse conditions
Inhibited or reduced growth is considered to be an adaptive feature for
survival allowing plants to employ multiple resources to combat stress
(Xiong and Zhu 2001) This was also observed in our study by down-
regulation of Drm3 or Auxin repressed gene (Figure 48c) This down
regulation of the gene during drought stress may be associated in arresting
the growth of the plant to overcome the drought situation
Elicitors are molecules that can trigger accumulation of phytoalexin in plants
They are often pathogen derived and can trigger the biosynthesis of one or a
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
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109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
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Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
119
combination of the hormone-like compounds jasmonic acid (JA) salicylic
acid and ethylene (Reymond and Farmer 1998) These hormones are
widely reported to involve in different signal transduction pathways in plants
under stress condition Therefore the higher expression of elicitor
responsive gene 3 in the present study may be associated in stress related
signal transduction pathways under drought The hydroxyproline-rich
glycoprotein is a structural cell wall protein (Lamport 1970) that functions in
defense against plant pathogen The expression of gene encoding this
protein was found to be up-regulated due to drought in the present study at
BWS Its expression pattern in the two cultivars under consideration was
found opposite to each other (Figure 48c) The down-regulation of the
protein under drought has been reported for example repression of cell wall
extensions (a hydroxyproline-rich glycoprotein) whose function is to cross
link cell walls after elongation (Carpita and Gibeau 1993) was found to
reduce cross linking of cell walls in drought-stressed P acutifolius compared
to wild type non-stressed plant (Micheletto et al 2007) Down-regulation of
this gene in the present study therefore may be involved in suppression of
growth by suppressing the cell wall cross linking under drought stress
Jasmonate ZIM-domain protein (JAZ) whose expression was found to be
down-regulated or nil at BWS in the present study (Figure 48c) has been
reported to act as a repressors (Chini et al 2007 Melotto et al 2008) of
Jasmonate (JA) signalling during plant growth development and defense
signalling However expression of the gene was found to increase
continuously with the advancement of drought after BWS (Figure 48c) LEJ2
(Loss of the timing of ET and JA biosynthesis 2) whose expression was
found to be continuously up-regulated at all the stages of drought (Figure
48c) in the present study encodes proteins that belongs to a group
containing CBS (cystathionine β-synthase) domain whose function are
largely unknown in plants (Kushwaha et al 2009) Some of these proteins
have been reported to enhance oxidative stress tolerance in transgenic
Arabidopsis (Luhua et al 2008) Therefore high expression of the gene
may also play a role during drought stress which needs further investigation
Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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York pp178 ndash 190
Chapter IV
125
2 Agre P Sasaki S Chrispeels M J 1993 Aquaporins a family of
water channel proteins Am J Physiol 261 F461
3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
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transgenic tobacco improves plant vigor under favorable growth
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447
4 Alamillo J Almogura C Bartels D Jordano J 1995 Constitutive
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5 Allagulova C R Gimalor F R Shakirova F M Vakhitov V A
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Chapter IV
126
12 Bartels D 2001 Targeting detoxification pathways an efficient
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13 Barua D N 1989 Science and practice in Tea culture (Calcutta Tea
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14 Battaglia M Olvera-Carrillo Y Garciarrubio A Campos F
Covarrubias A A 2008 The enigmatic LEA proteins and other
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29
18 Borovskii G B Stupnikova I V Antipina A I Vladimirova S V
Voinikov V K 2002 Accumulation of dehydrin-like proteins in the
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treatment BMC Plant Biol 2 5
19 Boudet J Buitink J Hoekstra F A Rogniaux H Larre C Satour
P Leprince O 2006 Comparative analysis of the heat stable
proteome of radicles of Medicago truncatula seeds during germination
identifies late embryogenesis abundant proteins associated with
desiccation tolerance Plant Physiol 140 1418 ndash 1436
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21 Brini F Hanin M Lumbreras V Irar S Pages M Masmoudi K
2007 Over-expression of wheat dehydrin DHN-5 enhances tolerance to
salt and osmotic stress in Arabidopsis thaliana Plant Cell Rep 26
2017 ndash 2026
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Chapter IV
127
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643
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Chapter IV
128
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Chapter IV
129
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Environmental Stress American Society of Plant Physiologists
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
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Chapter IV
130
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Chapter IV
131
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403
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Chapter IV
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Soybean (Glycine soja) enhances drought and salt tolerance in
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Identification of Arabidopsis genes regulated by high light-stress using
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Plant Physiol 131 309 ndash 316
Chapter IV
133
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Physiol 35 821 ndash 827
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Plant Physiol 147 381 ndash 390
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Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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Chapter IV
134
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11369
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Chapter IV
135
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136
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Cell Environ 21 601 ndash 11
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Chapter IV
138
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142 Smart C M 1994 Gene expression during leaf senescence New
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Chapter IV
139
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Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
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157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
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Chapter IV
140
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165 Wood A J and Goldsbrough P B 1997 Characterization and
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Chapter IV
141
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170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
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171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
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209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
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Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
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17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
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Chapter IV
120
The Asr1 gene belongs to the Asr gene family (named after abscisic acid
stress and ripening) classified as a new group of LEA (Caramelo et al
2008 Battaglia et al 2008) has been reported to be induced under water
stress (Silhavy et al 1995 Chang et al 1995) and involved in adaptation to
dry climates (Frankel et al 2003) It is also reported to involve in abscisic
acid signalling and used to develop transgenic Arabidopsis with enhance
drought and salt tolerance (Yang et al 2005) It can also act as a
transcription factor targeting the enhancer of a hexose transporter gene
(Cakir et al 2003) It was proposed that Asr1 is a link between sugar and
ABA metabolism and signalling Therefore Asr1 might act as non-histone
chromosomal protein involved in protection against a range of stress signals
by modulating cell sugar traffic The localization of this protein has been
reported in both nucleus and cytoplasm (Wang et al 2005) Its function in
the nucleus has been proposed in protecting DNA structure during water
loss and in gene regulation upon stress by changing DNA topology (Iusem et
al 1993 Silhavy et al 1995) The role of Asr1 as transcription factor for
Hexose transporter gene is also consistent with our findings that the hexose
transporter gene under water stress is highly induced During the drought
induced experiment we have observed senescence at WS in the lower
mature leaves This senescence may results in the coordinated
degradation of macromolecules and the mobilization of regained
nutrients like nitrogen carbon and minerals from senescing tissues into
other growing tissue (in our case to the buds along with 1st and 2nd leaf)
parts of the plant (Buchanan 1997 Nooden 1988) The induction of
hexose transporter has also been reported during leaf senescence (Ehness
et al 1997) This mobilization is further facilitated by higher expression of
hexose transporter gene as observed in the present study (Figure 48c) The
over expression of hexose transporter in TV23 is further supported by the
over expression of ASr1 gene that act as a transcription factor of the former
Therefore the higher induction of hexose transporter and Asr1 gene in TV23
may be responsible for its higher drought tolerance Its higher expression in
TV23 at wilting stage may be contributing towards efficient mobilization of
carbon source from the senescence leaf to the growing leaves to cope with
the drought stress The low level expression of the gene in the susceptible
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Chapter IV
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110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
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Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
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Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
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Plant Signalling amp Behavior 2(6) 551 ndash 552
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coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
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Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
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pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
121
cultivar at BWS might have resulted in inefficient mobilization of the carbon
source to the growing parts of the plants which is further supported by the
low level expression of Asr1 gene at BWS of S3A3
Drought avoidance in plants include closing of stomata to minimize water
loss adjustment of sinksource allocation by increasing root growth and
decrease canopy by reducing growth and shedding of older leaves (Fischer
et al 1978) Accelerated leaf senescence and leaf abscission are
associated with drought in nature as a means to decrease canopy size In
perennial plant like tea this strategy may contribute to the survival of the
plant under drought stress In the present induced drought experiment we
have observed that the senescence of lower mature leaves in case of
susceptible cultivar started much earlier (18 days) compared to the tolerant
cultivar (25 days) This delayed leaf senescence in TV23 might be
responsible for its higher drought tolerance compared to S3A3 which is
consistent with the findings that delayed leaf senescence induces extreme
drought tolerance in Sunflower (Rivero et al 2007) Transgenic Tobacco
plants were generated expressing an isopentenyltransferase (enzyme that
catalyzes the rate-limiting step in cytokinin synthesis) gene from
Agrobacterium driven by promoter of senescence associated receptor
protein kinase (SARK) of bean Remarkably the suppression of drought-
induced leaf senescence resulted in outstanding drought tolerance as shown
by vigorous growth after a long drought period that killed the control plants
Such transgenic plants were also found to maintain high water contents and
retained photosynthetic activity during drought
ACC oxidase is the enzyme that catalyses one of the rate-limiting steps for
ethylene biosynthesis in plants (Wang et al 2003) Ethylene is a plant
hormone that involved in a wide range o f physiological processes in plants
including germination (Gallardo et al 1994) abscission (Abeles 1973
Jacobs1968 Osborne 1973) senescence (Grbic et al 1995) flowering
and fruit ripening (Theologis 1993) Ethylene is also involved in plant
responses to external signals for example responses to wounding
pathogens and stress caused by environmental pollutants such as ozone
(Zarembinski et al 1994) In the present study we have observed higher
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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Chapter IV
125
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Functional characterization of the Arabidopsis eukaryotic translation
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Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
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Chapter IV
130
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
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stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
57 Goujon T Ferret V Mila I Pollet B Ruel K Burlat V Joseleau
J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
the AtCCR1 gene in Arabidopsis thaliana effects on phenotype lignins
and cell wall degradability Planta 217 218 ndash 228
58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
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and Medicine 3rd Edn Oxford University Press New York
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dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
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IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
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motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
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For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
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systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
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111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
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ndash 410
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and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
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Curr Opin Plant Biol 5 388 ndash 395
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249 ndash 279
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Chapter IV
136
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Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
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Plant Cell 7 173 ndash 182
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
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124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
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Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
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17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
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ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
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136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
122
expression of of ACC oxidase gene in TV23 at BWS compared to S3A3
Therefore we can speculate that this higher expression of the gene also
resulted in to the higher production of ethylene at BWS Although increased
production of ethylene has been reported to induce leaf senescence in
plants (Smart 1994) in our experiment we did not observe any leaf
senescence in both the cultivars at BWS At this stage both the plants have
shown wilting phenotype but there was no visible symptoms of leaf
senescence Therefore higher production of ethylene in the present study
may not be related to leaf senescence Moreover similar results has also
been obtained in different studies of ethylene-insensitive mutants where it
was shown that ethylene is neither necessary nor on its own sufficient
to induce leaf senescence Leaves should reach a certain age to be
susceptible to the ethylene signal (Buchanan-Wollaston et al 2003)
Ethylenersquos involvement in senescence appears to be related to the timing of
the process (Nam 1997 Grbic and Bleecker 1995) Ethylene has been
reported to inhibit the ABA mediated leaf stomatal closure (Tanaka et al
2005) which is a very important mechanism for a plant to survive in a water
stress environment Assuming that the high expression of ACC oxidase
gene in drought tolerant cultivar also resulted in higher production of
ethylene compared to susceptible cultivar present expression studies have
shown an opposite picture with higher ethylene production in TV23
compared to S3A3 It has been reported (Yang et al 2005) that stomata
gets closed much later in tolerant Arabidopsis thaliana developed by over
expressing LLA23 gene (an ortholog of Asr1 from Lily) compared to wild
type at same level of drought The higher production of ethylene in the
tolerant cultivar may therefore be attributed to inhibition of stomatal closure
mediated by ABA during water stress to maintain the normal physiological
events Moreover we have observed that closing of stomata started much
earlier in S3A3 compared to TV23 (data not shown) Therefore higher
expression of ACC oxidase might be responsible for giving drought tolerance
in TV23 by inhibiting the ABA mediated stomatal closure through higher
production of ethylene However there are reports of both decrease
(Morgan et al 1990 Feng and Barker 1992 Narayana et al 1991 Eklund
et al 1992) and increase (Bergner and Teichmann 1993 Huberman et al
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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York pp178 ndash 190
Chapter IV
125
2 Agre P Sasaki S Chrispeels M J 1993 Aquaporins a family of
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3 Aharon R Shahak Y Wininger S Bendov R Kapulnik Y Galili
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Chapter IV
127
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Chapter IV
128
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Chapter IV
129
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Chapter IV
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Chapter IV
131
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Chapter IV
133
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Revival of resurrection plant correlates with its antioxidant status Plant
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Chapter IV
134
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135
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Chapter IV
138
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Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
123
1993 Michelozzi et al 1995 Tudela and Primo Millo 1992) of ethylene
production during water stress Therefore role of ethylene during water
stress has to be further investigated Ethylene has been reported to induce
the expression of a complex set of genes for example ethylene
biosynthesis genes (Whittaker et al 1997) ethylene receptor genes (Hua et
al 1998) hydrolase genes (Burns et al 1998) and pathogenesis-related
genes (Koiwa et al 1994) Among these we have observed high expression
of an ethylene induced esterase gene in TV23 compared to S3A3 at BWS
during the drought experiment (Figure 48b)
45 Conclusion
In the present study we have monitored the expression of 29 genes at three
stages of drought in two tea cultivars with contrasting drought tolerance
character Transient or continuous up- or down-regulation of these genes
clearly indicates that these genes are true drought responsive and not
constitutive Out of 29 genes selected for expression studies from library
BWE3E4 19 genes were found to be up-regulated in TV23 compared to
S3A3 at BWS which accounts for a subtraction efficiency as high as 65
The relative expression of all the genes (except chloroplast glyceraldehydes-
3-phosphate dehydrogenase) considered in the present study have shown
up-regulation in both the cultivars compared to control plant However the
expression pattern varies between the two cultivars at different level of
induced drought stress We were more interested in those genes whose
relative expression at BWS was higher in tolerant cultivar TV23 compared to
susceptible cultivar S3A3 Because we presume that these are the
potential group of genes (early responsive) that could be responsible for
extending drought tolerance period of TV23 to 22 days (BWS) as compared
to 15 days (BWS) for S3A3 during the induced drought experiment
Therefore high expression of these genes in TV23 might contribute for its
higher drought tolerance However there are few genes (as discussed
above) whose relative expression was found higher in S3A3 The down-
regulation of these genes in TV23 may also play a key role that may
probably contribute for higher drought tolerance We have identified two
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
References
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Chapter IV
125
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Chapter IV
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Chapter IV
128
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Chapter IV
129
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131
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Chapter IV
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Chapter IV
135
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17(9) 344 ndash 345
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Chapter IV
137
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Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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130 Rorat T 2006 Plant dehydrins-tissue location structure and function
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
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characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
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A Sinauer Associates Publishers
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Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
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of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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Nucl Acids Res 31(20) e122
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Cell Environ 25 173 ndash 194
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Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
124
genes (elicitor-responsive gene 3 and cinnamoyl-CoA reductase) whose
expression was exceptionally higher in the tolerant cultivar compared to
susceptible (Appendix 41 Figure 46) at BWS It is interesting to note that
the expression patterns of these two genes are completely opposite
(continuously increasing in S3A3 and continuously decreasing in TV23 with
the progressive drought Appendix 41) in the two cultivars at all the stages
of drought (Figure 48b) These genes therefore may involve in higher
drought tolerance behaviour of TV23 Further studies are required to explore
the role of these two genes along with others during drought stress in tea
We have studied the expression of these genes under controlled
environmental variables in pot condition and there is always a genotype and
environment interaction factor (Genotype x Environment) that affects the
gene expression pattern in plants Therefore the expression patterns in the
present study may differ from field drought condition One has to consider
these factors to further substantiate the findings of present study that might
deviate in field condition Therefore from the present study we can conclude
that we have identified a set of gene whose expression was highly affected
by drought and these might be the potential group of genes responsible for
contrasting drought tolerance in tea Further studies are required to know the
functional role of these genes during drought stress that may pave the way
in understanding the drought tolerance mechanism in tea and help to devise
future strategies for improvement of drought tolerance There are several
ways to further substantiate the role of these genes in drought tolerance
mechanism for example full length cloning and heterologous expression to
study the structure (which is the subject matter of next chapter of this thesis)
and putative functions of these proteins study of drought tolerance of
transgenic plants over-expressing these proteins
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135
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137
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Cell Environ 21 601 ndash 11
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138
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139
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140
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Chapter IV
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dependent plant defense responses Plant Cell 8 1773 ndash 1791
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disulphide isomerase family in plants including single-domain protein
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Identification of Arabidopsis genes regulated by high light-stress using
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Chapter IV
133
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Physiol 35 821 ndash 827
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Revival of resurrection plant correlates with its antioxidant status Plant
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Downregulation of cinnamoyl-coenzyme A reductase in poplar
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Chapter IV
134
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Chapter IV
135
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136
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Cell Environ 21 601 ndash 11
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Chapter IV
138
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140
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Chapter IV
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J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
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and Medicine 3rd Edn Oxford University Press New York
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dependent plant defense responses Plant Cell 8 1773 ndash 1791
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physiology J Expt Bot 60 2971 ndash 2985
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encoding a 50-kilodalton protein specifically induced by cold
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Chapter IV
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disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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Chapter IV
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Chapter IV
133
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Revival of resurrection plant correlates with its antioxidant status Plant
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Chapter IV
134
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Chapter IV
135
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Chapter IV
136
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137
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Chapter IV
138
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Chapter IV
139
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Chapter IV
140
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Chapter IV
141
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Chapter IV
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Chapter IV
128
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Chapter IV
129
43 Dat J Vandenabeele S Vranova E Van Montagu M Inze D Van
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Chapter IV
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and Medicine 3rd Edn Oxford University Press New York
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dependent plant defense responses Plant Cell 8 1773 ndash 1791
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physiology J Expt Bot 60 2971 ndash 2985
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Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
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quantitative approach to improve water use efficiency in agriculture
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67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
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stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
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91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
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L reveals their developmental and stress regulation BMC Genomics
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92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
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synthesis of heat shock proteins and increased thermotolerance in
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protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
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subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
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W Thines B Staswick P E Browse J Howe G A He S Y
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105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
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106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
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Chapter IV
135
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Chapter IV
136
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121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
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Chapter IV
137
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Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
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Conversion of monogalactosyldiacylglycerols to triacylglycerols in
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Chapter IV
138
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Chapter IV
139
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Chapter IV
140
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159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
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Chapter IV
141
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170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
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Stress response and metabolic regulation of glyceraldehyde-3-
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172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
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175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
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Chapter IV
128
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34 Chang S Puryear J D Dias M A D L Funkhouser E A Newton
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35 Chen W Chao G Singh K B 1996 The promoter of a H2O2-
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36 Cheng Z Targolli J Huang X Wu R 1995 Wheat LEA genes
PMA80 and PMA enhance dehydration tolerance of transgenic rice
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37 Chini A Fonseca S Fernandez G Adie B Chico J M Lorenzo
O Garcia-Casado G Lopez-Vidriero I Lozano F M Ponce M R
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38 Chow W S Melis A Anderson J M 1990 Adjustments of
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basis of facilitated water movement through living plant cells Plant
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41 Close T J 1997 Dehydrins a commonality in the response of plants
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42 Coca M Almoguera C Thomas T Jordano J 1996 Differential
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Plant Mol Biol 31 863 ndash 876
Chapter IV
129
43 Dat J Vandenabeele S Vranova E Van Montagu M Inze D Van
Breusegem F 2000 Dual action of the active oxygen species during
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45 Dure L 1993 Structural motifs in LEA proteins In T J Close E A
Bray Eds Plant responses to cellular dehydration during
Environmental Stress American Society of Plant Physiologists
Rockville MD pp 91 ndash 103
46 Dure L Crouch M Harada J J Ho T Mundy J Quatrano R S
Thomas T L Sung Z R 1989 Common amino acid sequence
domains among the LEA proteins of higher plants Plant Mol Biol 12
475 ndash 486
47 Ecker J R 1995 The ethylene signal transduction pathway in plants
Science 268 667 ndash 675
48 Ehness R and Roitsch T 1997 Co-ordinated induction of mRNAs for
extracellular invertase and a glucose transporter in Chenopodium
rubrum by cytokinins Plant J 11 539 ndash 548
49 Eklund L Gieociala E and Hallgren J E 1992 No relation between
drought stress and ethylene production in Norway spruce Physiol
Plant 86 297 ndash 300
50 Eun K and Choo B 2006 Over-expression of tobacco NtHSP70-1
contributes to drought-stress tolerance in plants Plant Cell Rep 25
349 ndash 358
51 Feng H Chen Q Feng J Zhang J Yang X Zuo J 2007
Functional characterization of the Arabidopsis eukaryotic translation
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development by regulating cell division cell growth and cell death
Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
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Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
semiarid zones Ann Rev of Plant Physiol 29 277 ndash 317
54 Foyer C H and Noctor G 2005 Redox homeostasis and antioxidant
signalling A metabolic interface between stress perception and
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55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
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56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
Calle I M 1994 Inhibition of polyamine synthesis by cyclohexylamine
stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
57 Goujon T Ferret V Mila I Pollet B Ruel K Burlat V Joseleau
J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
the AtCCR1 gene in Arabidopsis thaliana effects on phenotype lignins
and cell wall degradability Planta 217 218 ndash 228
58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
59 Grbic V and Bleecker A B 1995 Ethylene regulates the timing of
leaf senescence in Arabidopsis Plant J 8 595 ndash 602
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and Medicine 3rd Edn Oxford University Press New York
61 Hammond-Kosack K E and Jones J D G 1996 Resistance gene-
dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
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Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
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Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
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123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
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mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
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132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
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133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
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spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
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139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
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effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
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142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
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145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
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147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
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Chapter IV
139
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World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
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150 Theologis A 1993 One rotten apple spoils the whole bushel the role
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151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
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152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
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153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
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154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
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155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
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16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
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158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
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photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
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162 Wang W Vinocur B Altman A 2003 Plant responses to drought
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163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
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164 Whittaker D J Smith G S Gardner R C 1997 Expression of
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34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
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Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
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167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
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166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
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Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
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membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
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confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
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611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
129
43 Dat J Vandenabeele S Vranova E Van Montagu M Inze D Van
Breusegem F 2000 Dual action of the active oxygen species during
plant stress responses Cell Mol Life Sci 57 779 ndash 795
44 Downton W J S Berry J A Seemann J R 1984 Tolerance of
photosynthesis to high temperature in desert plants Plant Physiol 74
786 ndash 790
45 Dure L 1993 Structural motifs in LEA proteins In T J Close E A
Bray Eds Plant responses to cellular dehydration during
Environmental Stress American Society of Plant Physiologists
Rockville MD pp 91 ndash 103
46 Dure L Crouch M Harada J J Ho T Mundy J Quatrano R S
Thomas T L Sung Z R 1989 Common amino acid sequence
domains among the LEA proteins of higher plants Plant Mol Biol 12
475 ndash 486
47 Ecker J R 1995 The ethylene signal transduction pathway in plants
Science 268 667 ndash 675
48 Ehness R and Roitsch T 1997 Co-ordinated induction of mRNAs for
extracellular invertase and a glucose transporter in Chenopodium
rubrum by cytokinins Plant J 11 539 ndash 548
49 Eklund L Gieociala E and Hallgren J E 1992 No relation between
drought stress and ethylene production in Norway spruce Physiol
Plant 86 297 ndash 300
50 Eun K and Choo B 2006 Over-expression of tobacco NtHSP70-1
contributes to drought-stress tolerance in plants Plant Cell Rep 25
349 ndash 358
51 Feng H Chen Q Feng J Zhang J Yang X Zuo J 2007
Functional characterization of the Arabidopsis eukaryotic translation
initiation factor 5A-2 that plays a crucial role in plant growth and
development by regulating cell division cell growth and cell death
Plant Physiol 144 1531 ndash 1545
52 Feng J and Barker A V 1992 Ethylene evolution and ammonium
accumulation by tomato plants under water and salinity stresses Part-
II J Plant Nutr 15 2471 ndash 2490
Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
semiarid zones Ann Rev of Plant Physiol 29 277 ndash 317
54 Foyer C H and Noctor G 2005 Redox homeostasis and antioxidant
signalling A metabolic interface between stress perception and
physiological responses Plant Cell 17 1866 ndash 1875
55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
species dwelling in arid habitats Mol Biol Evol 20(12) 1955 ndash 1962
56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
Calle I M 1994 Inhibition of polyamine synthesis by cyclohexylamine
stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
57 Goujon T Ferret V Mila I Pollet B Ruel K Burlat V Joseleau
J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
the AtCCR1 gene in Arabidopsis thaliana effects on phenotype lignins
and cell wall degradability Planta 217 218 ndash 228
58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
59 Grbic V and Bleecker A B 1995 Ethylene regulates the timing of
leaf senescence in Arabidopsis Plant J 8 595 ndash 602
60 Halliwell B and Gutteridge J M C 1999 Free Radicals in Biology
and Medicine 3rd Edn Oxford University Press New York
61 Hammond-Kosack K E and Jones J D G 1996 Resistance gene-
dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
130
53 Fischer R A and Tuner N C 1978 Plant productivity in arid and
semiarid zones Ann Rev of Plant Physiol 29 277 ndash 317
54 Foyer C H and Noctor G 2005 Redox homeostasis and antioxidant
signalling A metabolic interface between stress perception and
physiological responses Plant Cell 17 1866 ndash 1875
55 Frankel N Hasson E Iusem N D Rossi M S 2003 Adaptive
evolution of the water stress-induced gene Asr2 in Lycopersicon
species dwelling in arid habitats Mol Biol Evol 20(12) 1955 ndash 1962
56 Gallardo M Gallardo M E Matilla A J De Rueda P M Sanchez-
Calle I M 1994 Inhibition of polyamine synthesis by cyclohexylamine
stimulates the ethylene pathway and accelerates the germination of
Cicer arietinum seeds Physiologia Plantarum 91 9 ndash 16
57 Goujon T Ferret V Mila I Pollet B Ruel K Burlat V Joseleau
J P Barriere Y Lapierre C Jouanin L 2003 Down-regulation of
the AtCCR1 gene in Arabidopsis thaliana effects on phenotype lignins
and cell wall degradability Planta 217 218 ndash 228
58 Goyal K Walton L J Tunnacliffe A 2005 LEA proteins prevent
protein aggregation due to water stress Biochem J 388 151 ndash 157
59 Grbic V and Bleecker A B 1995 Ethylene regulates the timing of
leaf senescence in Arabidopsis Plant J 8 595 ndash 602
60 Halliwell B and Gutteridge J M C 1999 Free Radicals in Biology
and Medicine 3rd Edn Oxford University Press New York
61 Hammond-Kosack K E and Jones J D G 1996 Resistance gene-
dependent plant defense responses Plant Cell 8 1773 ndash 1791
62 Heinen R B Ye Q Chaumont F 2009 Role of aquaporins in leaf
physiology J Expt Bot 60 2971 ndash 2985
63 Hillel D and Vlek P 2005 The sustainability of irrigation Adv Agron
87 55 ndash 84
64 Houde M Danyluk J Laliberte J F Rassart E Dhindsa R S
Sarhan F 1992 Cloning characterization and expression of a cDNA
encoding a 50-kilodalton protein specifically induced by cold
acclimation in wheat Plant Physiol 99(4) 1381 ndash 1387
65 Houston N L Fan C Xiang J Q Schulze J M Jung R Boston
R S 2005 Phylogenetic analyses identify 10 classes of the protein
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
131
disulphide isomerase family in plants including single-domain protein
disulphide isomerase related proteins Plant Physiol 137 762 ndash 778
66 Hsiao T C Steduto P Fereres E 2007 A systematic and
quantitative approach to improve water use efficiency in agriculture
Irrig Sci 25 209 ndash 231
67 Hu Y Li W-Ch Xu Y-Q Li G-J Liao Y Fu F-L 2009 Differential
expression of candidate genes for lignin biosynthesis under drought
stress in maize leaves J Appl Genet 50(3) 213 ndash 223
68 Hua J Sakai H Nourizadeh S Chen Q G Bleecker A B Ecker
J R Meyerowitz E M 1998 EIN4 and ERS2 are members of the
putative ethylene receptor gene family in Arabidopsis Plant Cell 10
1321 ndash 1332
69 Huberman M Pressman E Jaffe M J 1993 Pith autolysis in plants
IV The activity of polygalacturonase and cellulase during drought
stress-induced pith autolysis Plant Cell Physiol 34 795 ndash 801
70 Imai R Chang L Ohta A Bray E A Takagi M 1996 A Lea-class
gene of tomato confers salt and freezing tolerance when expressed in
Saccharomyces cerevisiae Gene 170 243 ndash 248
71 Ingram J and Bartels D 1996 The molecular basis of dehydration
tolerance in plants Annu Rev Plant Physiol Plant Mol Biol 47 377 ndash
403
72 Ismail A M Hall A E Close T J 1999 Allelic variation of a
dehydrin gene cosegregates with chilling tolerance during seedling
emergence PNAS USA 96 13566 ndash 13570
73 Iusem N D Bartholomew D M Hitz W D Scolnik P A 1993
Tomato (Lycopersicon esculentum) transcript induced by water deficit
and ripening Plant Physiol 102 1353 ndash 1364
74 Jacobs W P 1968 Hormonal regulation of leaf abscission Plant
Physiol 43 1480 ndash 1495
75 Jain N K 1999 Global advances in tea science (New Delhi Aravali
Books International) pp 882
76 Ji W Zhu Y Li Y Yang L Zhao X Cai H Bai X 2010 Over-
expressing of a glutathione S-transferase gene GsGST from wild type
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
132
Soybean (Glycine soja) enhances drought and salt tolerance in
transgenic tobacco Biotechnol Lett 32(8) 1173 ndash 1179
77 Jones L Ennos A R Turner S R 2001 Cloning and
characterization of irregular xylem4 (irx4) a severely lignin-deficient
mutant of Arabidopsis The Plant J 26 205 ndash 216
78 Kaldenhoff R and Fischer M 2006 Aquaporins in plants Acta Physiol
(Oxf) 187 169 ndash 176
79 Kaup M T Froese C D Thompson J E 2002 A role for
diacylglycerol acyltransferase during leaf senescence Plant Physiol
129 1616 ndash 1626
80 Kawasaki T Koita H Nakatsubo T Hasegawa K Wakabayashi
K Takahashi H Umemura K Umezawa T Shimamoto K 2006
cinnamoyl-CoA reductase a key enzyme in lignin biosynthesis is an
effector of small GTPase Rac in defense signalling in rice PNAS USA
103 230 ndash 235
81 Khan S Tariq R Yuanlai C Blackwell J 2006 Can irrigation be
sustainable Agric Water Manage 80 87 ndash 99
82 Kim S Y and Nam K H 2010 Physological roles of ERD10 in abiotic
stresses and seed germination in Arabidopsis Plant Cell Rep 29 203
ndash 209
83 Kimura M Yamamoto Y Y Seki M Sakurai T Sato M Abe
T Yoshida S Manabe K Shinozaki K Matsui M 2003
Identification of Arabidopsis genes regulated by high light-stress using
cDNA microarray Photochem Photobiol 77(2) 226 ndash 233
84 Knepper M A 1994 The aquaporin family of molecular water
channels PNAS USA 91 6255 ndash 6258
85 Ko J H Yang S H Han K H 2006 Upregulation of an Arabidopsis
RING-H2 gene XERICO confers drought tolerance through increased
abscisic acid biosynthesis Plant J 47 343 ndash 355
86 Koag M C Fenton D R Wilkens S Close T J 2003 The binding
of maize DHN1 to lipid vesicles gain of structure and lipid specificity
Plant Physiol 131 309 ndash 316
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
133
87 Koiwa H Sato F Yamada Y 1994 Characterization of accumulation
of tobacco PR-5 proteins by IEF-immunoblot analysis Plant Cell
Physiol 35 821 ndash 827
88 Kovacs D Kalmar E Torok Z Tompa P 2008 Chaperone activity
of ERD10 and ERD14 two disordered stress-related plant proteins
Plant Physiol 147 381 ndash 390
89 Kranner I Beckett R P Wornik S Zorn M Pfeifhofer H W 2002
Revival of resurrection plant correlates with its antioxidant status Plant
J 31 13 ndash 24
90 Kruger C Berkowitz O Stephan U W Hell R 2002 A metal-
binding member of the late embryogenesis abundant protein family
transports iron in the phloem of Ricinus communis L J Biol Chem
277(28) 25062 ndash 25069
91 Kushwaha H R Singh A K Sopory S K Singla-Pareek S L
Pareek A 2009 Genome wide expression analysis of CBS domain
containing proteins in Arabidopsis thaliana (L) Heynh and Oryza sativa
L reveals their developmental and stress regulation BMC Genomics
10 200
92 Lamport D T A 1970 Cell wall metabolism Annu Rev Plant Physiol
21 235 ndash 270
93 Lee J Hubel A Schoffl F 1995 Derepression of the activity of
genetically engineered heat shock factor causes constitutive
synthesis of heat shock proteins and increased thermotolerance in
transgenic Arabidopsis Plant J 8 603 ndash 612
94 Leon O and Roth M 2000 Zinc fngers DNA binding and protein-
protein interactions Biol Res 33 21 ndash 30
95 Leple J C Dauwe R Morreel K Storme V et al 2007
Downregulation of cinnamoyl-coenzyme A reductase in poplar
multiple-level phenotyping reveals effects on cell wall polymer
metabolism and structure The Plant Cell 19 3669 ndash 3691
96 Levine A Tenhaken R Dixon R Lamb C 1994 H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease resistance
response Cell 79 583 ndash 593
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
134
97 Li C Potuschak T Colon-Carmona A Gutierrez R A Doerner P
2005 Arabidopsis TCP20 links regulation of growth and cell division
control pathways PNAS USA 102(36) 12978 ndash 12983
98 Li L Staden J V Jager A K 1998 Effects of plant growth
regulators on the antioxidant system in seedlings of two maize cultivars
subjected to water stress Plant Growth Regul 25 81 ndash 87
99 Liu S and Jiang Y 2010 Identification of differentially expressed
genes under drought stress in perennial ryegrass Physiologia
Plantarum 139 375 ndash 387
100 Lorick K L Jensen J P Fang S Ong A M Hatakeyama S
Weissman A M 1999 RING fingers mediate ubiquitin-conjugating
enzyme (E2)-dependent ubiquitination PNAS USA 96(20) 11364 ndash
11369
101 Luhua S Ciftci-Yilmaz S Harper J Cushman J Mittler R 2008
Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants
expressing proteins of unknown function Plant Physiol 148 280 ndash 92
102 Maurel C 2007 Plant aquaporins novel functions and regulation
properties FEBS Lett 581 2227 ndash 2236
103 Maurel C Santoni V Luu D-T Wudick M M Verdoucq L 2009
The cellular dynamics of plant aquaporin expression and functions
Plant Biol 12 690 ndash 698
104 Melotto M Mecey C Niu Y Chung H S Katsir L Yao J Zeng
W Thines B Staswick P E Browse J Howe G A He S Y
2008 A critical role of two positively charged amino acids in the Jas
motif of Arabidopsis JAZ proteins in mediating coronatine- and
jasmonoyl isoleucine-dependent interactions with the COI1 F-box
protein Plant J 55 979 ndash 988
105 Miao Y Lv D Wang P Wang Xue-Chen Chen J Miao C
Songa Chun-Peng 2006 An Arabidopsis glutathione peroxidase
functions as both a redox transducer and a scavenger in abscisic acid
and drought stress responses The Plant Cell 18 2749 ndash 2766
106 Micheletto S Rodriguez-Uribe L Hernandez R Richins R D
Curry J OrsquoConnell M A 2007 Comparative transcript profiling in
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
135
roots of Phaseolus acutifolius and P vulgaris under water deficit stress
Plant Science 173 510 ndash 520
107 Michelozzi M Johnson J D Warrag E I 1995 Responses of
ethylene and chlorophyll in two eucalyptus clones during drought New
For 9(3) 197 ndash 204
108 Mohd N Anju B Dhananjay S 2006 Immunogenicity and
protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae
against lethal infection in mice Vaccine 24 6225 ndash 6231
109 Morgan P W 1990 Effects of abiotic stresses on plant hormone
systems ndash In stress responses In plants adaptation mechanisms (R
Alscher and J Gumming eds) pp 313 ndash 314 Wiley-Liss Inc Publ
New York ISBN0-471-56810-4
110 Munoz-Bertomeu J Bermudez M A Segura J Ros R 2011
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate
dehydrogenase show alterations in abscisic acid (ABA) signal
transduction interaction between ABA and primary metabolism J Expt
Bot 62(3) 1229 ndash 1239
111 Nam H G 1997 The molecular genetic analysis of leaf senescence
Curr Opin Biotech 8 200 ndash 207
112 Narayana L Lalonde S Saini H S 1991 Water-stress-induced
ethylene production in wheat a fact or artefact Plant Physiol 96 406
ndash 410
113 Navari-Izzo F Vangioni N Quartacci M F 1990 Lipids of soybean
and sunflower seedlings grown under drought conditions
Phytochemistry 29(7) 2119 ndash 2123
114 Neill N Desikan R Hancock J 2002 Hydrogen peroxide signalling
Curr Opin Plant Biol 5 388 ndash 395
115 Noctor G and Foyer C H 1998 Ascorbate and glutathione keeping
active oxygen under control Annu Rev Plant Physiol Plant Mol Biol 49
249 ndash 279
116 Nooden L D 1988 Whole plant senescence In Senescence and
aging in plants (Nooden L D and Leopold A C Eds) Academic
Press San Diego 391 ndash 439
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
136
117 Nylander M Svensson J Palva E T Welin B V 2001 Stress-
induced accumulation and tissue-specific localization of dehydrins in
Arabidopsis thaliana Plant Mol Biol 45(3) 263 ndash 279
118 Ohme-Takagi M and Shinshi H 1995 Ethylene-inducible DNA
binding proteins that interact with an ethylene-responsive element
Plant Cell 7 173 ndash 182
119 Osborne D J 1973 Shedding of Plant Parts Academic Press New
York pp 125 ndash 147
120 Pannunzio A 2008 Efectos de sustentabilidad de los sistemas de
riego por goteo en arandanos 113 p Tesis MgSc Universidad de
Buenos Aires Facultad de Ciencias Veterinarias Buenos Aires
Argentina
121 Pannunzio A Texeira P Perez D Sbarra G Grondona y A 2008
Efectos de sustentabilidad de los sistemas de riego de araacutendanos en la
Pampa Huacutemeda p 24-25 Libro de Resuacutemenes del I Congreso
Latinoamericano de Arandanos y otros berries Agosto 2008
Universidad de Buenos Aires Facultad de Agronomiacutea (FAUBA)
Buenos Aires Argentina
122 Piquemal J Lapierre C Myton K OrsquoConnell A Schuch W
Grima-Pettenati J Boudet A M 1998 Down-regulation of cinnamoyl-
CoA reductase induces significant changes of lignin profiles in
transgenic tobacco plants The Plant J 13 71 ndash 83
123 Polidoros A N and Scandalios J G 1999 Role of hydrogen
peroxide and different classes of antioxidants in the regulation of
catalase and glutathione S-transferase gene expression in maize (Zea
mays L) Physiol Plant 106 112 ndash 120
124 Poza-Carrion C Aguilar-Martinez J A Cubas P 2007 Role of TCP
gene BRANCHED1 in the control of shoot branching in Arabidopsis
Plant Signalling amp Behavior 2(6) 551 ndash 552
125 Reddy B A Etkin L D Freemont P S 1992 A novel zinc finger
coiled-coil domain in a family of nuclear proteins Trends Biochem Sci
17(9) 344 ndash 345
126 Reich M Liefeld T Gould J Lerner J Tamayo P Mesirov J P
2006 GenePattern 20 Nature Genetics 38 no 5 pp 500 ndash 501
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
137
127 Reymond P and Farmer E E 1998 Jasmonate and salicylate as
global signals for defense gene expression Curr Opin Plant Biol 1 404
ndash 411
128 Rinne P Willing A Kaikuranta P 1998 Onset of freezing tolerance
in birch (Betula pubescens Ehrh) involves LEA proteins and
osmoregulation and is impaired in an ABA-deficient genotype Plant
Cell Environ 21 601 ndash 11
129 Rivero R M Kojima M Gepstei A Sakakibara H Milter R
Gepstein S Blumwald E 2007 Delayed leaf senescence induces
extreme drought tolerance in a flowering plant PNAS USA 104(49)
19631 ndash 19636
130 Rorat T 2006 Plant dehydrins-tissue location structure and function
Cell Mol Biol Lett 11 536 ndash 556
131 Rozen S and Skaletsky H J 2000 Primer3 on the WWW for general
users and for biologist programmers Plant Mol Biol 5 69 ndash 76
132 Sade N Gebretsadik M Seligmann R Schwartz A Wallach R
Moshelion M 2010 The role of Tobacco aquaporin1 in improving
water use efficiency hydraulic conductivity and yield production under
salt stress Plant Physiol 152 245 ndash 254
133 Sakaki T Kondo N Yamada M 1990a Pathway for the synthesis of
triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated
spinach leaves Plant Physiol 94 773 ndash 780
134 Sakaki T Saito K Kawaguchi A Kondo N Yamada M 1990b
Conversion of monogalactosyldiacylglycerols to triacylglycerols in
ozone-fumigated spinach leaves Plant Physiol 94 766 ndash 772
135 Shalata A Mittova V Volokita M Guy M Tal M 2001 Response
of the cultivated tomato and its wild salt-tolerant relative Lycopersicon
pennellii to salt-dependent oxidative stress The root antioxidative
system Physiologia Plantarum 112 487 ndash 494
136 Shinozaki K and Yamaguchi-Shinozaki K 1997 Gene expression
and signal transduction in water stress response Plant Physiol 115
327 ndash 334
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
138
137 Shinozaki K Yamaguchi- Shinozaki K Seki M 2003 Regulatory
network of gene expression in the drought and cold stress responses
Curr Opin Plant Biol 6 410 ndash 417
138 Shinshi H Usami S Ohme-Takagi M 1995 Identification of an
ethylene-responsive region in the promoter of a tobacco class I
chitinase gene Plant Mol Biol 27 923 ndash 932
139 Siefritz F Tyree M T Lovisolo C Schubert A Kaldenhoff R
2002 PIP1 plasma membrane aquaporins in tobacco From cellular
effects to function in plants Plant Cell 14 869 ndash 876
140 Silhavy D Hutvagner G Barta E Banfalvi Z 1995 Isolation and
characterization of a water-tress-inducible cDNA clone from Solanum
chacoense Plant Mol Biol 27 587 ndash 595
141 Sivamani E Bahieldin A Wraith J M Al-Niemi T Dyer W E Ho
T-HD Qu R 2000 Improved biomass productivity and water use
efficiency under water deficit conditions in transgenic wheat
constitutively expressing the barley HVA1 gene Plant Sci 155 1 ndash 9
142 Smart C M 1994 Gene expression during leaf senescence New
Phytol 126 419-448
143 Smirnoff N 1998 Plant resistance to environmental stress Curr Opin
Plant Biol 9 214 ndash 219
144 So Hyun-Ah Chung E Cho Chang-Woo Kim Kee-Young Lee
Jai-Heon 2010 Molecular cloning and characterization of soybean
cinnamoyl-CoA reductase induced by abiotic stresses Plant Pathol J
26(4) 380 ndash 385
145 Sun W Bernard C Van D Van M Verbruggen N 2001 At-
HSP17 6A encoding a small heat-shock protein in Arabidopsis can
enhance osmotolerance upon over-expression Plant J 27 407 ndash 415
146 Swire-Clark G A and Marcotte W R Jr 1999 The wheat LEA
protein Em functions as an osmoprotective molecule in Saccharomyces
cerevisiae Plant Mol Biol 39 117 ndash 128
147 Taiz L and Zeiger E 2002 Plant physiology 3rd edn Sunderland M
A Sinauer Associates Publishers
148 Tan J Zhao H Hong J Han Y Li H Zhao W 2008 Effects of
exogenous nitrick oxide on photosynthesis antioxidant capacity and
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
139
proline accumulation in wheat seedlings subjected to osmotic stress
World J Agri Sci 4(3) 307 ndash 313
149 Tanaka Y Sano T Tamaoki M Nakajima N Kondo N
Hasezawa S 2005 Ethylene inhibits abscisic acid-Induced stomatal
closure in Arabidopsis Plant Physiol 138 2337 ndash 2343
150 Theologis A 1993 One rotten apple spoils the whole bushel the role
of ethylene in fruit ripening Cell 70 181 ndash 184
151 Tichopad A Didier A Pfaffl M W 2004 Inhibition of real-time RT-
PCR quantification due to tissue-specific contaminants Mol Cell
Probes 18 45 ndash 50
152 Tichopad A Dilger M Schwarz G Pfaffl M W 2003 Standardized
determination of real-time PCR efficiency from a single reaction set-up
Nucl Acids Res 31(20) e122
153 Tudela D and Primo Millo E 1992 1-Aminocyclopropane-1-
carboxylic acid transported from roots to shoots promotes leaf
abscission in Cleopatra mandrin (Citrus rashni Hort ex tan) seedlings
rehydrated after water stress Plant Physiol 100 131 ndash 137
154 Tunnacliffe A and Wise M J 2007 The continuing conundrum of the
LEA proteins Naturwissenschaften 94(10) 791 ndash 812
155 Tyerman S D Niemietz C M Bramley H 2002 Plant aquaporins
multifunctional water and solute channels with expanding roles Plant
Cell Environ 25 173 ndash 194
156 Vandenabeele S Kelen K V D Dat J Gadjev I Boonefaes T
Morsa S Rottiers P Slooten L Montagu M V Zabeau M Inze
D Breusegem F V 2003 A comprehensive analysis of hydrogen
peroxide- induced gene expression in tobacco PNAS USA 100(26)
16113 ndash 16118
157 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N
De Paepe A Speleman F 2002 Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biology 3 341 ndash 3411
158 Wadenback J Von Arnold S Egertsdotter U Walter M H Grima-
Pettenati J Goffner D Gellerstedt G Gullion T Clapham D 2008
Lignin biosynthesis in transgenic Norway spruce plants harbouring an
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
140
antisense construct for cinnamoyl- CoA reductase (CCR) Transgenic
Research 17 379 ndash 392
159 Walters R G and Horton P 1995 Acclimation of Arabidopsis thaliana
to the light environment changes in photosynthetic function Planta
197 306 ndash 312
160 Walters R G Shephard F Rogers J J Rolfe S A Horton P
2003 Identification of mutants of Arabidopsis defective in acclimation of
photosynthesis to the light environment Plant Physiol 131 472 ndash 481
161 Wang H J Hsu C M Jauh G Y Wang C S 2005 A Lilly pollen
ASR protein localizes to both cytoplasm and nuclei requiring a nuclear
localization signal Physiol Plant 123 314 ndash 320
162 Wang W Vinocur B Altman A 2003 Plant responses to drought
salinity and extreme temperatures towards genetic engineering for
stress tolerance Planta 218 1 ndash 14
163 Wang W Vinocur B Shoseyov O Altman A 2004 Role of plant
heat-shock proteins and molecular chaperones in the abiotic stress
response Trends in Plant Sci 9 244 ndash 252
164 Whittaker D J Smith G S Gardner R C 1997 Expression of
ethylene biosynthetic genes in Actinidia chinensis fruit Plant Mol Biol
34 45 ndash 55
165 Wood A J and Goldsbrough P B 1997 Characterization and
expression of dehydrins in water stressed Sorghum bicolor Physiol
Plant 99 144 ndash 152
166 Xiao B Huang Y Tang N Xiong L 2007 Over-expression of a
LEA gene in rice improves drought resistance under the field
conditions Theor Appl Genet 115 35 ndash 46
167 Xiong L and Zhu J K 2001 Abiotic stress signal transduction in
plants molecular and genetic perspectives Physiol Plant 112 152 ndash
166
168 Xu D Duan X Wang B Hong B Ho T Wu R 1996 Expression
of a late embryogenesis abundant protein gene HVA1 from barley
confers tolerance to water deficit and salt stress in transgenic rice
Plant Physiol 110 249 ndash 257
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153
Chapter IV
141
169 Yamaguchi K Mori H Nishimura M 1995 A novel isoenzyme of
ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal
membranes in pumpkin Plant Cell Physiol 36(6) 1157 ndash 1162
170 Yang Chin-Ying Chen Yu-Chuan Jauh Guang Yuh Wang Co-
Shine 2005 A Lily ASR protein involves abscisic acid signalling and
confers drought and salt resistance in Arabidopsis Plant Physiol 139
836 ndash 846
171 Yang Y Kwon Hawk-Bin Peng Hsiao-Ping Shih Ming-Che 1993
Stress response and metabolic regulation of glyceraldehyde-3-
phosphate dehydrogenase genes in Arabidopsis Plant Physiol 101
209 ndash 216
172 Zarembinski T I and Theologis A 1994 Ethylene biosynthesis and
action a case of observation Plant Mol Biol 26 579 ndash 597
173 Zhang L Ohta A Takagi M Imai R 2000 Expression of plant
group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed
functional divergence among LEA proteins J Biochem (Tokyo) 127
611 ndash 616
174 Zhao Y Du H Wang Z Huang B 2011 Identification of proteins
associated with water-deficit tolerance in C4 perennial grass species
Cynodon dactylon times Cynodon transvaalensis and Cynodon dactylon
Physiologia Plantarum 141 40 ndash 55
175 Zhou R Jackson L Shadle G Nakashima J Temple S Chen F
Dixon R A 2010 Distinct cinnamoyl-CoA reductases involved in
parallel routes to lignin in Medicago truncatula PNAS USA 107
17803 ndash 17808
176 Zhu B Choi D W Fenton R Close T J 2000 Expression of the
barley dehydrin multigene family and the development of freezing
tolerance Mol Gen Genet 264 145 ndash 153