MOLECULAR, BIOCHEMICAL & PHYSIOLOGICAL ANALYSIS OF …shodhganga.inflibnet.ac.in/bitstream/10603/33751/5/... · 2018-07-02 · CHAPTER 5 MOLECULAR, BIOCHEMICAL & PHYSIOLOGICAL ANALYSIS

CHAPTER 5

MOLECULAR BIOCHEMICAL amp PHYSIOLOGICAL ANALYSIS OF

TRANSGENIC TOBACCO AND TOMATO PLANTS

50 ABSTRACT

The transgenic tobacco and tomato plants harbouring carrot AFP were analysed

The integration of AFP in tobacco and tomato plants was confirmed by Southern blot

All further analysis was carried out in T1 generation AFP expression level in plant

organs after cold stress was studied in tobacco by sq RT-PCR The expression analysis

revealed that AFP is stably expressed in all the transgenic lines of tobacco and tomato

The expression of AFP in vegetative and reproductive tissues was also confirmed

The transcript accumulation of various antioxidant enzymes and signaling molecules after

cold stress in transgenic tobacco plants revealed that the expression of all the genes were

high in WT plants during cold stress while the transgenic lines showed a steady

expression level Transgenic plants subjected to the chilling stress showed a significant

decrease in membrane injury index compared to the WT which was determined by

electrolytic leakage assay The growth of transgenic plants was normal as that of WT

tobacco plants in normal conditions but inhibited during cold stress

201

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51 INTRODUCTION

Plants have a multi-facetted response to low temperature stress and they combat

the stress by bringing a myriad of molecular biochemical cellular and physiological

changes (Singh et al 2002 Thomashow 1999) These changes are acquired by the

change in photosynthetic rates calcium influxes activation of kinasephosphatise

cascades (Singh et al 2002) alteration in the membrane lipid composition (Kung 1998)

accumulation of osmolytes like proline glycine betaine and soluble sugars (Chen amp

Murata 2002) and increasing the levels of various antioxidants (Prasad et al 1994)

A notable upregulation or down regulation in the specific gene transcripts of the above

mentioned process occurs when a low temperature stress is thrusted on the plants

With the onset of low temperature stress putative thermo sensors like Calcium

messengers at the cell membrane generates a series of stress signals that are further

transmitted and amplified through a cascade that include Ca2++ signaling and a stepwise

kinasephosphatase chain This cascade ultimately reaches the nucleus and transcription

factors which act as ―master switch to activate the gene expression Low temperature

activates transcription of many genes which are either up or downregulated (Shinozaki amp

Yamaguchi-Shinozaki 2000) The upregulated genes encodes mainly 2 groups of

proteins the first group includes proteins playing active role in stress tolerance LEA

proteins antifreeze proteins water channel proteins enzymes involved in sugar and

proline synthesis detoxification enzymes etc The second group includes those involved

in regulation of signal transduction and gene expression which are activated during the

stress that includes protein kinases transcription factors and the enzymes in phospholipid

metabolism like phospholipases This alteration in gene expression and accumulation of

202


other organic molecules like osmolytes protects the cell from low temperature induced

damages However some plants adapt to the low temperature stress which is very

complex and the exact molecular biochemical and physiological changes are poorly

understood

512 Antioxidant Enzymes- It is widely reported that the injury to the plant is caused

by stress and it is related to oxidative damage at the cellular level In plants most of the

degenerative reactions are allied with various abiotic biotic stresses that are due to toxic

Reactive Oxygen Species (ROS) Free radical scavenging antioxidant enzymes either

catalyzes the reactions by quenching ROS (without being transformed into a destructive

radical itself) or process directly Although they are all often induced in similar stress

situations they mostly show differential expression in response to specific stress

(Adam et al 1995) Organelles like chloroplasts and mitochondria are major source of

ROS production in plant cells because of high oxidizing metabolic activity or due to the

high rate of electron flow In plants plastids are the main source of ROS which produces

high amounts of superoxide radicals and hydrogen peroxide especially during reduced rate

of carbon fixation which are common during abiotic stresses (Takahashi amp Murata 2005)

In addition to the superoxide and hydrogen peroxide chloroplasts can also produce

singlet oxygen through chlorophyll excitation after the absorption of light by the pigment

systems Mitochondria are the major source of ROS in non-photosynthetic tissues

(Navrot et al 2007) The third intracellular source of ROS is the peroxisomes They

contain many oxidases that produce H2O2 and O2-

as byproducts of the metabolic

reactions which they catalyze The glycolate oxidase is one of the important enzymes

located in peroxisome specifically relevant during abiotic stresses In addition to the

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above ROS sources in the presence of redox-active metals hydroxyl radicals (OHbull) can

be formed from H2O2 through the Fentonlsquos reaction or from H2O2 and O2- through the

Haber-Weiss reaction The OHbull radicals are highly reactive that can cause extensive

oxidative damage in the cell (Moller et al 2007) Apart from the general metabolic

reactions various biotic and abiotic stresses increase ROS formation in plants

An increase in the free radicals in the cells can initiate severe oxidation of various cellular

components and thereby changing the cells redox status (Mittler et al 2004) hence

continuous control of ROS is very essential under stress conditions (Meyer et al 2007)

There exists equilibrium between the generation of ROS and their scavenging by various

antioxidative enzymes (Foyer et al 2005) This equilibrium may be perturbed by

different biotic and abiotic stress factors and the disturbances in equilibrium that lead to

increased levels of ROS which can cause significant damage to cell membranes

The general reaction catalysed by various antioxidant enzymes is as follows

Enzyme Reaction catalyzed

SOD O2 -

+ O2 - + 2H

+ harr 2H2O2 + O2

CAT 2H2O2 harr O2 + 2H2O

GPX 2GSH + PUFA-OOH harr GSSG + PUFA + 2H2O

GST RX + GSH harr HX + R-S-GSH

APX AA + H2O2 harr DHA + 2H2O

GR NADPH + GSSG harr NADP+ + 2GSH

(R may be aliphatic aromatic or heterocyclic group Ali amp Alqurainy 2006)

A variety of defence mechanisms have been suggested on the basis of the

biochemical and physiological responses against cold injuries Acclimation to low

temperature may be related to an enhanced antioxidant system by the activity of different

204


scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which

prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved

different mechanisms to cope with the low temperature related oxidative stress Low

temperature induced accumulation of glutathione (GSH) has been observed in Picea abies

and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was

also induced in response to low temperature in soybean summer squash and wheat under

experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above

responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-

soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase

(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and

glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in

detoxification of ROS (fig 51)

Plants with high level of antioxidants or antioxidant enzymes are reported to have a

relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp

Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase

(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976

Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen

(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that

are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively

less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate

peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to

ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the

monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid

205


which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an

electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and

NADPH were reported by Bowler et al (1992)

Figure 51- ROS Scavenging Pathways in Different Plant Organelles

(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)

(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)

APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate

DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase

GR glutathione reductase GSH glutathione GSSG oxidized glutathione

MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox

NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)

513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites

which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon

(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells


during stress without interfering in normal metabolic processes and their accumulation

confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main

osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)

and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as

an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in

the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in

an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically

expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress

tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals

and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline

thus performs various functions in plants and also acts as a rescue molecule which is

accumulated under the adverse conditions

514 Signaling during Stress Responses- The mechanism by which plants perceive

various stress signals and transmit them to cellular machinery to activate stress responses

are necessary for developing strategies to improve stress tolerance A signal transduction

pathway is initiated with the perception of stress signal followed by the production of

second messengers such as inositol phosphates and ROS The drought cold and salinity

stresses have been reported to induce a transient calcium influx into the cytoplasm

(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal

calcium ion release and the importance is the presence of repetitive transient bursts The first

round of transient calcium generation leads to generation of secondary signaling molecules

like ABA and ROS which further stimulate calcium release These multiple rounds of bursts

in calcium signaling from different sources results in different signaling consequences


and therefore different physiological responses in plants during cold stress During

stress phospholipids serve as a precursor for the generation of molecules which acts as

secondary messengers (Munnik et al 1998) Drought cold and salinity stresses

upregulates the expression of phospholipase C (PLC) which is a major enzyme

hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase

in expression of PLC results in the increase of cleavage of phosphotidylinositol

45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as

key secondary messenger activating the protein kinase C by triggering Ca2+

release

The phospholipase D (PLD) has also proved to be involved in transducing the stress

signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus

formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)

The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to

specific phosphorylation cascade under stress has been reported (Urao et al 1994

Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased

tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-

mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for

transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-

MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in

alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)

208


52 MATERIALS AND METHODS

521 Screening of Putative Transformants (T0)-Tobacco and Tomato

5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the

putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as

mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP

internal primers The PCR products were resolved in 14 agarose gel and stained with

ethidium bromide The bands were visualized and documented The positive plants were

used for Southern analysis

5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot

Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines

(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of

young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle

The powdered samples were transferred to a 50mL sterile oak ridge tube containing

15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH

80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)

The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation

of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC

The supernatant was carefully transferred to a new tube and equal volume of Chloroform

Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again

centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with

chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase

was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC

overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for

209


10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash

solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at

14000g for 10 min at 20degC The washing step was repeated twice The supernatant was

removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A

(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the

samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241

Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several

times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was

transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of

25 M sodium acetate were added and mixed well The tubes were incubated overnight at

-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet

was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was

resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1

agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu

Tokyo Japan)

52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel

Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac

I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used

for the transgenic lines used for the Southern blot are shown in the table 51


Sl No Sample Line Number

01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3

Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot

The restriction enzyme digestion reaction mix was made as mentioned the table 52

Sl No Components Volume

01 Genomic DNA 20 microg (10 microL)

02 Digestion buffer 10X 5microl

03 Sterile water 21microL

04 Enzyme (10UμL) 2 + 2 microL

Total 50 microL

Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot

The samples were briefly centrifuged and incubated at 37degC in a waterbath

overnight for complete digestion The digestion product (5microL) was loaded and analysed

for the digestion in the agarose gel The digested genomic DNA from all transgenic lines

along with negative control (digested genomic DNA from WT plants) and positive

control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)

containing ethidium bromide was used The samples were resolved at 20V for overnight

The gel was photographed with a UV scale for determining the size


52122 Agarose Gel Treatments prior to Southern Hybridisation

521221 Depurination of Agarose Gel- The agarose gel was washed in depurination

solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed

The gel was washed twice with deionised water after depurination

521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M

NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water

521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation

solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was

washed with deionised water and was once again treated with neutralisation solution for

another 15 min The gel was finally washed with deionised water

521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the

positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by

capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)

blotting buffer The transfer was left for overnight After the transfer the membrane was

briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane

and the membrane was dried at room temperature The membrane was crosslinked by

exposing to UV light (120mJ) for 3 min

521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane

was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution

[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]

50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The

membrane was incubated at 42degC in a rotating hybridisation chamber


521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc

Maryland USA) was used for labelling the probe and detected using chemiluminsecent

method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly

1μL of biotin labelling mix was added along with the PCR reaction using AFP specific

primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)

The probe was denatured at 95degC for 10 min and immediately placed in ice

521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was

added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of

denatured probe was added to the hybridisation solution The hybridisation was carried

out for 12h at 42degC in a rotating hybridisation chamber

521228 Detection- After the hybridisation the membrane was washed twice in 2X

SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01

SDS at 65degC for 10 min The membrane was partially dried by placing between the 2

Whatmann filter papers The detection was carried out according to the manufacturerlsquos

instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at

room temperature with moderate shaking The membrane was then incubated in blocking

solution for 30 min at room temperature with moderate shaking The membrane was then

washed twice with detection buffer for 10 min each After the second wash 20mL of

detection buffer was added and freshly prepared substrate solution was added over the

membrane The membrane was incubated at room temperature until a very specific blue

to pink insoluble precipitate forms on the membrane (colour development) representing

the probe specific bands


522 Analysis of T1 Transgenic Plants

5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT

tobacco plants were inoculated in half strength MS media supplemented with 75mgL

Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was

determined to check the segregation

5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato

were sowed (in commercially available soil mix in pots) The germinated plants were

maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished

with 110 strength sterile MS media daily The T1 plants were used for subsequent studies

52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1

transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was

used as the template for PCR using AFP and nptII specific primers The nptII specific

primers are as follows

nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo

nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo

The PCR programme for nptII are as follows an initial denaturation at 94degC for

5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension

at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min

5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13

AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for

48h Young leaf samples were collected every 24h and all the samples were frozen in


liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants

were reverted to the normal condition and samples were collected after 24h (recovery

period)

The expression of AFP in tomato transgenic AFP plants was studied Three

transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress

for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further

process

In order to determine the expression of AFP in different tissues of transgenic tobacco

plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h

Different tissues like root stem mature leaves young leaves and flowers were harvested at

every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression

of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse

Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation

The plant phenotype under stress was monitored carefully Comparative analyses

of the various molecular biochemical physiological and phenotypic parameters related

to cold stress were assayed in both transgenic and non-transformed plants under normal

(non-stress) and stressed conditions

5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two

weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold

stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of

stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until

further process


52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy

RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction

(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase

treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according

to manufacturerlsquos instruction

52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA

was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280

ratio was also determined The integrity of RNA was checked in denaturing agarose gel

The same protocol was followed as mentioned in Chapter 3 - 3231

52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using

RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as

mentioned in Chapter III- 3232

52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling

Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted

110 times in nuclease free water The PCR was performed using ―Ready to Go PCR

beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was

same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the

reference for the PCR reaction to normalize the gene expression The samples were

resolved using 14 agarose gel and the gel was stained for 30 min with ethidium

bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and

documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT

from different treatment times (0h 3 days and 7 days stress) were used for the expression

analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as


mentioned above and PCR was performed using PCR beads The primer sequence and

PCR conditions for the antioxidant enzymes and signaling molecules are shown in the

table 53

523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT

plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3

sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of

glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water

bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene

(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene

layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The

experiment was repeated thrice with different samples under the same conditions

524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato

tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)

were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated

overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for

5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for

1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment

was repeated thrice with different samples under the same conditions

525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was

performed according to Xu et al (2005) The control and transgenic plants were given cold

stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs

were equilibrated in deionised water for 4h and initial conductivity was measured The

217


samples were autoclaved to get all the solutes from cells and the final conductivity was

measured

The relative conductivity () was calculated using the formula

Relative Conductivity () =

The experiment was repeated thrice with different samples under the same conditions

Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo

Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo

94ordmC 5 min 94ordmC 30 s

58ordmC 30 s 72ordmC 30 s

29 cycles 72ordmC 10 min

02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo

Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo




03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo

Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo


60ordmC 30 s 72ordmC 30 sec


04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo

Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo




05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo

Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo




06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo

Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo




07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo

Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo




08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo

Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo




09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo

Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo




10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo

Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo




11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo

Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo




Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes

Signaling Molecules

X 100

Initial conductivity

Final conductivity


526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT

plants were germinated in sterile soil under controlled conditions mentioned above

Ten days old seedling was transferred to cooling incubator at 4degC The growths of the

plants were measured for three weeks The ability of WT and transgenic lines to recovery

(after stress) was also studied The experiment was repeated twice under the same

conditions

219


53 RESULTS AND DISCUSSION

531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an

attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco

and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers

Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of

tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot

analysis was performed using the AFP PCR product as a probe for two PCR positive

plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP

integration (not copy number) the genomic DNA was digested with Xba I and SacI

which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal

corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]

This shows that the AFP was integrated to the plant genome in all the 4 plants analysed

Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants

Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants

Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants

WT Tomato DNA Tobacco DNA

250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220


Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants

Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants

Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco

Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)

10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221


Figure 54- Southern Blot Analysis of Transgenic Plants with AFP

Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe

Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting

Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco

DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-

Tomato AFP 1 amp AFP 3

Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR

product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-

WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3

AFP nptII CaMV 35S LB RB

SacI XbaI

11kb

54 (a)

54 (b)

Tomato Tomato Tobacco Tobacco

54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7


532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found

that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds

inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few

WT seeds germinated initially in the selection media however they failed to grow later

Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media

with Kanamycin to Study the Segregation Analysis

533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was

performed for T1 transgenic tomato and tobacco plants with AFP primers The expected

size bands (11kb) were observed in both tobacco and tomato transgenic plants However

the number of PCR positive lines was less for tomato The tobacco plants were also

confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)

Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines

Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines

Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223


Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers

Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP

amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA

Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers

Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1

tobacco lines Lane 7- WT DNA

600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224



Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines


Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato

lines Lane 6- PCR amplicon from Carrot DNA

10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225


534 AFP Expression Analysis by sq RT-PCR

5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all

the samples and the integrity of the RNA was checked in denaturing gel Equal amount of

RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA

and the purity is given in the following tables (54 55 56)

Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio

01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212

19 WT root (24 hour recovery) 4877 212

20 AFP 5 (24 hour recovery) 5192 215





Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during

Cold Stress


Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco

and (3)- T1 lines of tomato that had been exposed to low temperature stress

Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines

studied in T1 generation However the expression of gene was same throughout the stress

period (fig 510 511) There was also no change in the expression levels of AFP during the

recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis

Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in

conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants

are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP

with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence

it prevents the plant cells from the cold damage Among the different transgenic lines tested

Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR

analysis which may be due to the different integration sites in the tobacco genome Hence

these two transgenic lines were chosen for detailed analysis in following works

Figure 511- sq RT- PCR in T1 Tomato Lines

Three different tomato lines and WT plants were given cold stress at 4degC for different time

intervals β-tubulin was used as the reference gene for sq RT-PCR

C 48h 48 hour stress (48h) Control (C)

AFP

β- Tubulin

RNA

AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT


Figure 512- sq RT- PCR in T1 Tobacco Lines

Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different

time intervals β-tubulin was used as the reference gene

It was also found in tobacco that the AFP gene is ubiquitously expressed in

different vegetative tissues and reproductive organ like flower of the transgenic plants

The expression levels of AFP appeared to be almost same in all the tissues examined

Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was

high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze

24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA

WT AFP5 AFP14 AFP32 AFP37 AFP41

AFP


activity was recently characterised from Chimonanthus praecox and its expression levels

were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)

This ubiquitous expression level of AFP in transgenic plants can be attributed to the

activity of constitutive CaMV 35S promoter With the increase in isolation and

characterisation of inducible and tissue specific promoter it may be possible in future to

tune the expression of AFP to obtain the desired level of cold tolerance avoiding any

undesirable consequences of constitutive expression In addition to this further studies in

the field conditions after getting all the biosafety approval


01 WT root (Control) 1230 216

02 WT mature leaves (Control) 6656 216

03 WT young leaves (Control) 12004 215

04 WT stem (Control) 4801 21

05 WT flower (Control) 6866 215

06 AFP 41 root (Control) 1484 208

07 AFP 41 mature leaves (Control) 4562 217

08 AFP 41 young leaves (Control) 9087 204

09 AFP 41 stem (Control) 6641 219

10 AFP 41 flower (Control) 4523 213

11 WT root (24 hour stress) 2720 211

12 WT mature leaves (24 hour stress) 5321 214

13 WT young leaves (24 hour stress) 13721 215

14 WT stem (24 hour stress) 5546 199



15 WT flower (24 hour stress) 6230 215

16 AFP 41 root (24 hour stress) 2840 209

17 AFP 41 mature leaves (24 hour stress) 4232 212

18 AFP 41 young leaves (24 hour stress) 9286 211

19 AFP 41 stem (24 hour stress) 5992 218

20 AFP 41 flower (24 hour stress) 5440 214











Table 55- Quantification of RNA from Different Organs in WT and Transgenic

Plants After Cold Stress


Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines

Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from

different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)

cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to

determine the transcript accumulation of AFP in different organs β-tubulin was used as the

reference gene

Freezing occurs first in the extracellular space in a plant where the fluids in the

cell have a higher freezing point than the fluids present inside the cells (Atici amp

Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing

β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control

R S ML YL F R S ML YL F

WT AFP 32


cell dehydration All known AFPs from the plant have shown limited effect on thermal

hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization

inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities

(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical

role in conferring freezing tolerance in plants

The T1 transgenic lines of tobacco and tomato were germinated and studied for

response to cold stress by a number of important abiotic stress related molecular

biochemical and physiological parameters which may be regulated due to the integration

of the AFP gene into the genome of tobacco and tomato lines

The expression levels of critical genes implicated in oxidative metabolism and

signaling enzymes were studied by sq RT-PCR in order to gain insights into the

regulation of the antioxidant and signaling pathways in transgenic plants with AFP

The transcript levels of endogenous genes in tobacco were monitored before and after

cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT

NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and

delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis

of proline (fig 514 515) These genes or homologues have been often shown to be

involved in cold stress response No significant variation in transcript levels were

observed in both the transgenic lines (at all time points) however the transcript level of

all the antioxidant enzymes were high (except catalase) at all time points in the WT

plants The expression of signaling candidates MAPKK AOX were also high in WT

plants The transcript abundance of PLA and PLC were high in one of the transgenic line

(AFP 32) studied during stress This change in expression level between different


transgenic lines can be attributed to the difference in the site of integration of the AFP in

the plant genome The expression of P5CS was also high in the WT plants Higher

expression levels of these genes in WT plants may probably provide more chaperones for

various substrates the up-regulation of antioxidant enzymes for scavenging free radicals

increase in synthesis of osmolytes signaling for activation of transcription factors for

induction of cold responsive genes etc which are known mechanisms in plants to

sustain growth during low temperature stress though in our studies it is not the case


01 WT (Control) 10393 211

02 WT (3 days stress) 3097 213

03 WT young leaves (7 days stress) 5507 212

04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214

09 AFP 41 stem (7 days stress) 7568 212

Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines

during Cold Stress


Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines

Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time

intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for

PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used

as the reference gene

Exposure to low temperature may result in the increased generation of free radicals in

higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants

cellular components but also deliver signals for perceiving the changed environment

(Fridovich 1991) The relationship between antioxidant capacity and low temperature

tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter

cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities

of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba

et al 1999) These data suggests that increase in tolerance to such stress is always

accompanied by increased expression of specific genes encoding different antioxidant

enzymes The expression of SOD were high in WT plants in our studies where as the

AFP 32 AFP 41 WT

C 3days 7days C 3days 7days C 3days 7days

SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA


expression level was stable in both the transgenic lines tested during all time points

The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria

The up-regulation of gene in WT plants observed in this study suggests a protective

mechanism targeted to the mitochondria As the electron transport chain in mitochondria has

been shown to be very sensitive to the change in environmental temperatures the electrons

that are not transported to the final electron acceptor ie oxygen to produce water might be

used for the formation of superoxide ion (O2minus

) The operation of the mitochondrial

alternative pathway involving the expression of AOX at low temperature may also produce

more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes

are targeted to chloroplast suggesting that the constant expression of these genes may protect

chloroplasts from damage induced by superoxide during cold acclimation Apart from this

the APX also play a key role in chloroplasts in capturing the free radicals APX quickly

scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is

located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing

peroxides from the cell The transcript level of APX were high in WT type plants during

stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has

not affected in similar manner the transgenic lines on the exposure to cold temperature

The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression

of GPX were little higher in WT plants after one week of stress once again in transgenic

lines we could not find any change These observations suggests GPX (along with GR

which we have not studied) might be involved in the production of NAD(P)+ which may be

incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the

pentose phosphate pathway or glycolytic pathway These processes contribute to the net


carbon assimilation and relative growth rates especially in WT plants The assimilated carbon

might be used as the backbone for the synthesis of sugars and other osmolytes which can

protect the cells from osmolysis during low temperature stress GSTs are the enzymes

involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione

(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in

response to various stresses such as metal stress oxidative stress phytohormone treatment

inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards

et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and

Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice

seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were

differentially expressed significantly under at least one of the abiotic stress conditions

studied (Jain et al 2010) The expression of catalase remained constant during cold treatment

in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase

enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002

Wang et al 2005) in our case we found a stable expression level even in the WT plants

The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the

scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the

APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995

Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are

quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase

Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of

cold acclimation in wheat which might be due to the increased activity of the plastid bound


APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress

in Pancratium maritimum

The results presented here suggest that genes encoding antioxidant enzymes may

play a pivotal role in cold response in partial protection of the WT plants All the genes for

antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant

enzymes may be used to protect plant cells from oxidative stress resulting from exposure to

cold temperature However the constant expression of all the enzymes in both the transgenic

lines throughout the stress period may suggest that the plants are not experiencing any stress

(due to the presence of transgene) and hence the production of ROS might be less

Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines

Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time

intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as

template for PCR to determine the transcript accumulation of signaling molecules and enzymes

involved in the biosynthesis of Proline β-tubulin was used as the reference gene

PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT


MAP related kinases catalyze reversible phosphorylations reactions and are

important for relaying signals to various targets during stress They function via kinase-

phosphatase cascades which involve in sequential phosphorylation of a kinase by its

upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be

more in WT plants during stress which implies that the low temperature signals are

relayed to nucleus through MAPKK However in our study the expression of MAPKK

was found to be constant in both the transgenic lines at all time points We presume that

the transgenic lines are not experiencing the cold stress because of the presence of AFP

under constituitive promoter 35S and hence MAPKK would not have been activated

A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and

osmotic stress signal transduction A MAPK with specific involvement in drought and

salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing

et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased

tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive

regulator of drought and salt stress Some MAPK and MAPKK proteins have also been

shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)

Plant mitochondria possess a unique respiratory pathway in addition to the main

cytochrome pathway The alternative pathway is a non-phosphorylating electron transport

pathway and the electron flow through the pathway reduces the molecular oxygen to

water without the conservation of energy in the form of ATP A variety of biotic and abiotic

stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX

genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002


Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at

all time points in our studies whereas the there were no significant change in transcript

accumulation in transgenic tobacco lines During cold stress the respiration capacity of

the alternative pathway is increased significantly in WT plants and this enhancement

seems to be coupled with the AOX transcript accumulation High levels of the AOX

protein and the alternative pathway partly would have lead to freezing tolerance of the

WT plants in the present study As the transgenic plants possibly were not experiencing

the effect of cold stress the respiration might occur through normal cytochrome pathway

due to which the alternative pathway might not have been activated hence AOX

transcript levels remains same in transgenic plants

Three classes of phospholipases PLD PLC and PLA2 are identified and studied

widely for their roles in lipid derived signalling pathways PLC and PLA2 are the

well-researched signaling enzymes in plant and animal systems PLC produces the

second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes

the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was

reported to accumulate in response to cold treatment (Ruelland et al 2002) and water

deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of

diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in

remodelling of membrane phospholipids The activity of the plasma membrane

H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)

and changes in the properties of membrane transport proteins like plasma membrane

ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)


The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time

points whereas expression levels of both the genes in AFP 41 were found to be slightly

higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible

explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be

ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it

may be involved in transmitting the stress signals The change in expression level of the

genes in AFP 41 could also be due to the insertional effect of the transgene in the plant

genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis

(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could

contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to

produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second

messengers that can activate the protein kinase C and trigger Ca2+

release respectively

The role of PLA may be regulating the composition of plasma membrane lipid

composition during low temperature stress however the signaling pathway through

which AFP induction is still not clear It cannot be ruled out that the AFP signaling

occurs through the activation of PLC but further studies are needed to validate the data

Many plants respond to cold and other abiotic stress by accumulating high amount

of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-

betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT

plants as compared to the transgenic plants (both the lines) in our studies The free

proline was also quantified in the leaf samples of transgenic and WT plants before and

after cold stress This analysis revealed that proline level was increased in WT plants


during stress as compared to that of the transgenic plants which resembles as that of our

sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible

solute that protects cell membranes and proteins against the adverse effect of inorganic

ions and temperature extremes by scavenging reactive oxygen species at high

concentration It has been reported that cold acclimation studies in WT Arabidopsis

resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR

results showed that the P5CS from Jatropha curcas was induced by drought and salt

stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline

content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)

Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level

as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP

expression is not expected to increase the P5CS expression in transgenic tobacco plants

and it is clear that AFP might act independent of osmolyte accumulation route in order to

protect the plant cells

Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants

516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in

tomato plants

516 (a) 516 (b)


535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of

proline biosynthesis in plants during LT stress plants also accumulate soluble sugars

which are thought to involve in protecting plant cells from damage caused by cold stress

through different routes like serving as osmoprotectants interacting with the lipid

bilayer etc In our study the amount of soluble sugars increased in WT during stress but

there was no significant change in both tobacco and tomato transgenic lines tested

[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance

that is independent of sugar accumulation mechanism

Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP

517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines

536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are

disrupted at low temperatures that results in leakage of electrolytes which further leads to an

increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to

low temperature can be measured by the increments in tissue conductivity due to the leakage of

electrolytes and ions Membrane damage due to various stress have been evaluated through

measurements of the rates of solute and ionic leakage At controlled conditions the leakage of

WT and transgenic lines were more or less similar in the present study As the temperature was

reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is

517 (a) 517(b)


further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less

in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is

stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which

it shows more tolerance In WT plants the membrane damage is responsible for the higher

leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay

it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato

and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco

lines were healthy when compared to the WT plants

Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants

518 (a) in tobacco lines and 518 (b) in tomato lines

537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were

chosen for low temperature stress treatment to study the phenotype To assess the cold

stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold

chamber grown for 3 days The experiments revealed the drastic phenotypic changes

indicating superior chilling tolerance of transgenic plants in comparison to WT plants

The growth of WT plants was retarded during the prolonged low temperature treatment

however the growth of the tobacco transgenic lines was not affected (fig 519 520) The

length of root stem number of leaves area of leaves were also affected in WT plants

518(a) 518(b)

243


These results indicated clearly that transgenic lines showed better cold tolerance and

enhanced recovery from the cold treatment and the WT plants did not survive A very

similar observation was reported where an insect AFP (MpAFP 149) conferred cold

tolerance in tobacco transgenic lines and performed well whereas WT plants suffered

from chlorosis wilting after exposure (Wang et al 2008)

Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants

Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks

AFP 32 after two week

stress

AFP 32 before

stress

WT after two week

stress

WT before

stress

AFP 41 before stress


stress


Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants

Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the

effect of cold stress on morphology of plants Lower panel shows the plants which are grown at

normal conditions (which do not exhibit any phenotypic changes)

Plants after two weeks of stress

Plants after one week stress

Plants before stress

Plants after three weeks of stress

One month old plants grown at

controlled conditions

WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION

Improved tolerance against different stress by single gene manipulation was

reported earlier for genes involved in transcriptional activation membrane integrity and

other metabolic functions As tobacco is a model system most of the analyses were

performed only in transgenic tobacco lines In the present study molecular biochemical

physiological and phenotypic analysis did not show any induction andor change in

transgenic plants however most of the genes studied were upregulated in WT plants

The leakage of electrolytes was less in transgenic plants during cold treatment than the

WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold

stress could be through the enhanced cell membrane stability It can be concluded that

AFP integration in the genome of tobacco and tomato imparted a low temperature

tolerance in transgenic plants Field studies are required to further confirm the

effectiveness of the AFP transgenic plants in the real high altitude situations


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3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants

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10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by

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12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC

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Biologia Plantarum 546-12

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1213-15

14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione

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15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different

mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in

Arabidopsis Plant Physiology 127918-927

16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the

cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells

Physiologia Plantarum 9311-18

17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts

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79094-97

18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed

tropical legumes Phytochemistry 231007-1015

19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione

reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta

13321-25

20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and

intracellular glutathione transport systems in plants Trends in Plant Science 6

486-492

21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense

functions of tocopherol ascorbate and glutathione In Photoprotection

Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp

Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands


22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit

triggers phospholipase D activity in the resurrection plant Craterostigma

plantagineum Plant Cell 12111-123

23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative

Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society

of Plant Physiologists Rockville MD

24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)

Characterization of alternative oxidase (AOX) gene expression in response to

methyl salicylate and methyl jasmonate pre-treatment and low temperature in

tomatoes Journal of Plant Physiology 1631049-1060

25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein

produced endogenously in winter rye leaves Plant Physiology 100593-596

26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial

ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp

Environment 1211-215

27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a

phosphatidylinositol-specific phospholipase C is induced by dehydration and salt

stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of

United States of America 923903-3907

28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication

tolerance Trends in Plant Science 6431-438

29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically

expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress


30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-

induced RD29A and RD29B promoters by constitutively active Arabidopsis

MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770

31


Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can

inhibit a calmodulin-stimulated Ca2+

pump (ACA2) located in the endoplasmic

reticulum of Arabidopsis Proceedings of National Academy of Sciences of United

States of America 976224-6229

32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of

tandem- arranged alternative oxidase genes in rice are increased by low

temperature Gene 203121-129

33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal

transduction in guard cells is mediated by phospholipase D activity Proceedings of

National Academy of Sciences United States of America 9612192-12197

34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression

analysis suggests overlapping and specific roles of rice glutathione S-transferase

genes during development and stress responses BMC genomics doi1011861471-

2164-11-73

35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study

of frost tolerance and antioxidant activity in cereals Plant Science 164301-306

36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper

MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice

recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic

Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment

32336-348

37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress

signaling in plants A mitogen-activated protein kinase pathway is activated by cold

and drought Proceedings of National Academy of Sciences of United States of

America 9311274-11279

38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif

M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial

mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant

Cell Tissue and Organ Culture 103267-277


39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)

Proline induces the expression of salt stress-responsive proteins and may improve

the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental

Botany 542553-2562

40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S

Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase

(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK

Plant Cell 122247-2258

41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and

the recrystallization of ice Cryobiology 3223-34

42 Knight H (2000) Calcium signaling during abiotic stress in plants International

Review of Cytology 195269-325

43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic

characterization of three phosphoinositide-specific phospholipase C isoforms from

potato Plant Physiology 116239-250

44 Kung CC (1998) Characterization of a pea gene responsive to low temperature

Botanical Bulletin of Academia Sinica 399-15

45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound

phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature

Biotechnology 22427-433

46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in

sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93

47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat

catalase expressed in transgenic rice can improve tolerance against low temperature

stress Physiologia Plantarum 116317-327

48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards

understanding and application Trends in Biotechnology 8358-362


49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)

Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the

redox potential of the cellular glutathione redox buffer Plant Journal 52973-986

50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)

Extraction of high-quality genomic DNA from latex-containing plants Analytical

Biochemistry 31585-89

51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive

oxygen gene network of plants Trends in Plant Science 9490-498

52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular

components in plants Annual Review of Plant Biology 58459-481

53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants

Biochimica et Biophysica Acta 1389222-272

54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on

illumination Plant and Cell Physiology 211295-1307

55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species

generation and antioxidant systems in plant mitochondria Physiologia plantarum

129185-195

56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the

level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment


57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP

dependent proton accumulation in isolated oat root plasma membrane vesicles


58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents

on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane

vesicles Biochimica et Biophysica Acta 1021133-140

59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in

Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK

The EMBO Journal 156564-6574


60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)

Expression profile of oxidative and antioxidative stress enzymes based on ESTs

approach of citrus Genetics and Molecular Biology 30872-880

61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and

tolerance in developing maize seedlings changes in antioxidant system oxidation

of proteins and lipids and protease activities The Plant Journal 101017-1026

62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-

induced oxidative stress in maize seedlings and a regulatory role for hydrogen

peroxide Plant Cell 665-74

63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum

induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116

409-418

64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione

S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco

seedlings during stress Nature Biotechnology 15988-991

65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of

phospholipases C and D is an early response to a cold exposure in Arabidopsis

suspension cells Plant Physiology 130999-1007

66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a

single Ca2+

-dependent protein kinase confers both cold and saltdrought tolerance

on rice plants Plant Journal 23319-327

67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant

Cell 11691-706

68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated

oxygen species in wheat (Triticum aestivum L) seedlings responses to cold

acclimation Journal of Plant Physiology 155762-768

69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in

transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant

Physiology 1131177-1183


70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to

dehydration and low temperature differences and cross talk between two stress

signaling pathways Current Opinion in Plant Biology3217-223

71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J

McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze

protein from grass Nature 406256

72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant

defense and stress responses Current Opinion in Plant Biology 5430-436

73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd

J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel

antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391

74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux

through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana

modifies photosynthetic acclimation at low temperatures and the development of

freezing tolerance Plant Cell amp Environment 26523-535

75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of

wheat alternative oxidase gene WAOX1a alters respiration capacity and response to

reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp

Genetic Systems 81349-354

76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of

photosystem II from photodamage Biochimica et Biophysica Acta1708352-361

77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding

glutathione-S-transferase Proceedings of the National Academy of Sciences of


78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)

Characterization of two non-homologous nuclear genes encoding mitochondrial

alternative oxidase in common wheat Genes amp Genetic Systems 7781-88


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regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular

Biology 50571-599

80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp

Shinozaki K (1994) Two genes that encode Ca2+

-dependent protein kinases are

induced by drought and high salt stresses in Arabidopsis thaliana Molecular and

General Genetics 224331-340

81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases

alternative pathway capacity and alternative oxidase protein in tobacco Plant


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glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur

Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)

pp 261ndash265 SPB Academic Publishers The Hague The Netherlands

83 Wang CY (1995) Temperature preconditioning affects glutathione content and

glutathione reductase activity in chilled zucchini squash Journal of Plant


84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of

insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers

the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358

85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over

expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling

and salt stress Journal of American Society of Horticultural Science 130167-173

86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van

Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is

indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816

87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene

expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal

of Experimental Botany 582969-2981


88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways

and network integration In Abiotic stress tolerance in plants toward the

improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29

Springer Dordercht The Netherlands

89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze

protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies

in China 77-15

90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with

water stress- Evolution of osmolyte systems Science 2171214-1217

91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein

kinase pathway is involved in disease resistance in tobacco Proceedings of

National Academy of Sciences of United States of America 98741-746

92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold

acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp

Moller IM) pp 304ndash319 Springer-Verlag Berlin

93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants

responses and chilling resistance in maize Plant Physiology and Biochemistry 33

567-575

94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)

An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits

both antifreeze and antifungal activities Biologia Plantarum 55141-148

95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends

in Science 6520-527

96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and

characterization of P5CS gene from Jatropha curcas L African Journal of


256


51 INTRODUCTION

Plants have a multi-facetted response to low temperature stress and they combat

the stress by bringing a myriad of molecular biochemical cellular and physiological

changes (Singh et al 2002 Thomashow 1999) These changes are acquired by the

change in photosynthetic rates calcium influxes activation of kinasephosphatise

cascades (Singh et al 2002) alteration in the membrane lipid composition (Kung 1998)

accumulation of osmolytes like proline glycine betaine and soluble sugars (Chen amp

Murata 2002) and increasing the levels of various antioxidants (Prasad et al 1994)

A notable upregulation or down regulation in the specific gene transcripts of the above

mentioned process occurs when a low temperature stress is thrusted on the plants

With the onset of low temperature stress putative thermo sensors like Calcium

messengers at the cell membrane generates a series of stress signals that are further

transmitted and amplified through a cascade that include Ca2++ signaling and a stepwise

kinasephosphatase chain This cascade ultimately reaches the nucleus and transcription

factors which act as ―master switch to activate the gene expression Low temperature

activates transcription of many genes which are either up or downregulated (Shinozaki amp

Yamaguchi-Shinozaki 2000) The upregulated genes encodes mainly 2 groups of

proteins the first group includes proteins playing active role in stress tolerance LEA

proteins antifreeze proteins water channel proteins enzymes involved in sugar and

proline synthesis detoxification enzymes etc The second group includes those involved

in regulation of signal transduction and gene expression which are activated during the

stress that includes protein kinases transcription factors and the enzymes in phospholipid

metabolism like phospholipases This alteration in gene expression and accumulation of

202





understood




































SOD O2 -

+ O2 - + 2H

+ harr 2H2O2 + O2










204

























205

















































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256





understood




































SOD O2 -

+ O2 - + 2H

+ harr 2H2O2 + O2










204

























205

















































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
















SOD O2 -

+ O2 - + 2H

+ harr 2H2O2 + O2










204

























205

















































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

























205

















































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

















































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


































release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256










release












208

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

























209
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
















Tokyo Japan)







01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256



01 WT tobacco WT

02 Tobacco AFP 32

03 Tobacco AFP 41

04 WT tomato WT

05 Tomato AFP 1

06 Tomato AFP 3








Total 50 microL
















































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256








































































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

















































period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


























period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




period)




process















further process




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




























table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




table 53




















217



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256



measured




























































Signaling Molecules

X 100


Final conductivity







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256







conditions

219


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


















250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA

220






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256






10kb

3kb

200bp

100bp

250bp

500bp

M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24

221












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256












SacI XbaI

11kb

54 (a)

54 (b)


54 (c)

11kb

100b

p

1kb

10kb

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256















Tobacco

DNA e a

quote from

the

document

or the

summary of

an

interesting

point You

can

position the

text box

anywhere

in the

document

Use the

Text Box

Tools tab to

change the

formatting

W

T

10kb

100bp

Genomic DNA

M 1 2 3 4 5 6

7

200bp

223








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256








600bp

100bp

750bp

1kb

M 1 2 3 4 5 6 7

11kb

M 1 2 3 4 5 6 7 8 9 10

10kb

200bp

100bp

1kb

224







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256







10 kb

1kb

100bp

11kb

M 1 2 3 4 5 6

WT Tomato DNA

10kb

M 1 2 3 4 5 6

1kb

100bp

Genomic DNA

225








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256








01 WT (Control) 3004 212

02 AFP 5 (Control) 5541 216

03 AFP 13 (Control) 3815 214

04 AFP 32 (Control) 5173 213

05 AFP 37 (Control) 8052 21

06 AFP 41(Control) 8165 211

07 WT (24 hour stress) 4581 216

08 AFP 5 (24 hour stress) 5479 211

09 AFP 13 (24 hour stress) 9334 214

10 AFP 32 (24 hour stress) 6330 209

11 AFP 37 (24 hour stress) 7102 204

12 AFP 41(24 hour stress) 6921 21

13 WT (48 hour stress) 4872 212

14 AFP 5 (48 hour stress) 5227 216

15 AFP 13 (48 hour stress) 7432 209

16 AFP 32 (48 hour stress) 5643 212

17 AFP 37 (48 hour stress) 2985 208

18 AFP 41(48 hour stress) 8639 212








Cold Stress




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




















AFP

β- Tubulin

RNA











24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256










24 hour recovery

48 hour stress

24 hour stress

Control

β- Tubulin

β- Tubulin

β- Tubulin

β- Tubulin

RNA

AFP

AFP

AFP

RNA

RNA

RNA


AFP



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256



















































reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256



























reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256







reference gene




β- tubulin

β- tubulin

β- tubulin

AFP

RNA

AFP

RNA

AFP

RNA

48 hour stress

24 hour stress

Control


WT AFP 32


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


































01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256










01 WT (Control) 10393 211



04 AFP 32 (Control) 4602 214

05 AFP 32 (3 days stress) 9007 211

06 AFP 32 (7 days stress) 6084 209

07 AFP 41 (Control) 9009 212

08 AFP 41 (3 days stress) 3911 214



during Cold Stress

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

















AFP 32 AFP 41 WT


SOD

APX

GPX

GST

Catalase

β- Tubulin

RNA
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
































































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256







































PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
















PLC

PLA

AOX

MAPKK

RNA

P5CS

β- Tubulin


AFP 32 AFP 41 WT
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
























































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256

































































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256










































tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


















tomato plants

516 (a) 516 (b)




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




















517 (a) 517(b)




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




















518(a) 518(b)

243










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256










stress

AFP 32 before

stress

WT after two week

stress

WT before

stress



stress












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256












WT AFP 32 AFP 41

WT AFP 32 AFP 41


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


54 CONCLUSION














55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


55 REFERENCES






Network India













83-116



144235-240












1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256







1213-15











79094-97





13321-25



486-492

































31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256





























31
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
















2164-11-73







32336-348




America 9311274-11279









Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256





Botany 542553-2562









































129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256


















129185-195

























409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256













409-418








single Ca2+




Cell 11691-706






































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256
































Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256




Biology 50571-599



































in China 77-15











567-575





in Science 6520-527




256








in China 77-15











567-575





in Science 6520-527




256


Documents

MOLECULAR, BIOCHEMICAL & PHYSIOLOGICAL ANALYSIS OF …shodhganga.inflibnet.ac.in/bitstream/10603/33751/5/... · 2018-07-02 · CHAPTER 5 MOLECULAR, BIOCHEMICAL & PHYSIOLOGICAL ANALYSIS