It is not the strongest of the species that survive, nor the most intelligent, but the one most responsive to change.

Charles R.Darwin

welcome

31-03-2015 1

TARA SINGH RAWATJr. MSc.

PALB-4248Submitted to Dr. D. DAYAL DOSS

ADVANCED CENTRE FOR PLANT BIOTECHNOLOGYGKVK,UAS, BANGALORE

31-03-2015 2

INTRODUCTION• As we all know that Agriculture is totally dependent on climate.• So a variety of Abiotic Stresses causing a serious crop loss of about

>50 % on an average thus limiting the agricultural productivity world wide.

• By 2025, 30% of crop production will be at risk due to the declining water availability.

• World Bank projects that the climate change will depress crop yields by 20% or more by the year 2050. (Narendra Tuteja, 2012)

• Efforts have been made by Plant breeder in developing abioticstress resistant crop plants but are not sufficient enough.

• Thus the role of Transgenic Approach in crop improvement has become of great importance in assuring worlds future food security.

31-03-2015 3

FRACTION OF WORLD’S ARABLE LAND AFFECTED WITH ABIOTIC STRESSES

31-03-2015 4

DROUGHT

26%

MINERAL TOXICITY

/DEFICIENCY

20%

FREEZING

15%

Drought accounts alone for 50 % of losses caused by biotic and abioticstresses

VERSATILITY OF ABIOTIC STRESSES

31-03-2015 5

STRESS

ABIOTIC

WATER DEFICIT

EXCESS

TENPERATURE HIGH

LOW

SALT/ION

TOXICITY

DIFFICIENCY

AIR POLLUTION

OTHERS

BIOTIC

COMPLEXITY OF PLANT RESPONSE TO ABIOTIC STRESSES

31-03-2015 6

Stress characteristics

Duration

Frequency

Severity

Stress combination

Plant characters

Genotypes

Tissue in question

Developmentstage

Result

Survival

Death

Response

Resistance

Susceptible

Many factor determine stress responseSt

ress

31-03-2015 7

Plant breeders and geneticists have utilized naturalvariability for stress tolerance within germplasm.

One special advantage of genetic engineering is theability to transform plants with genes from otherspecies rather than upregulating an already existingplant stress response.

RELEVANCE OF GENETIC ENGINEERING

31-03-2015 8

DEFINITIONSGENETIC ENGINEERING

The artificial manipulation, modification and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of an organism.

(Encyclopaedia Britanica 15 edn.)

STRESSStress can be defined as an influence that is outside the normal

range of homeostatic control.(Lerner, 1999)

RESISTANCEThe capacity of an organism or a tissue to withstand a effects of

a harmful environmental agent.

31-03-2015 9

DEFINITIONSTRANSGENIC

Off, relating to, or being an organism whose genome hasbeen altered by the transfer of a gene from sexually

incompatible species.

DROUGHT

An extended period of deficient rainfall < 75% as compared to normal rainfall of the region is called drought.

RESURRECTION PLANT

Plant species with special attribute to withstand against abiotic stresses include algae, bryophytes, lichenes, ferns and some angiosperm. Eg. Selaginella lepidophylla

31-03-2015 10

DEFINITIONSAcclimation

Increase in resistance as result of exposure to prior stress,adjustments in response to stress, changes In steady state physiology.

AdaptationGenetically determined level of resistance acquired by process of selection over many generation (evolutionary improvements).

Cross-resistanceResistance to one stress induced by acclimation to other.

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APPROACHES FOR RESISTANCE AGAINST ABIOTIC STRESS

• Improving protection from stress.

Eg. Oxidative stress is protected By SOD enzyme.

• Reducing sensitivity to stress.

Eg. Drought tolerance, salt tolerance and chilling tolerance.

31-03-2015 12

RESPONSIVE GENES FROM EXTREMOPHILES

Tolerance against abiotic stresses is genetically controlled.

Xerophyta viscosa.

• A modle African Extreamophile

• Can survive extremes of

dehydration and regain normal

life on rehydration

• Eleven gene have been

isolated

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Xerophyta

viscosa

XvPer1,

XvPrx2XvSAP1

XvVHA-C1

XvCAM

XvT8

XvG6

XvGols, Xvlno1,

XvALDR4

XvERD-15

1

2

3

4

5

67

8

OTHER SOURCES OF RESPONSIVE GENES

• Arabidopsis thaliana

• Nicotiana tabacum, N. Plumbaginifolia

• Spinach

• Holomonas elongata

• Saccharomyces sp.

• E. Coli

• Arthrobacter globiformis

( Grover et al.,2003,Current Science.)

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Winter Flounder Fish- Antifreeze protien

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Hordeum vulgare- Hb gene

Oriza sativa- Cu/Zn SOD

Vitroscilla stercoraria- VHb gene

Anasystis nidulans- Fatty acid desaturase gene

GENES INDUCED BY ABIOTIC STRESSESThe product of genes whose expression is induced by abiotic

stresses are classified in two groups.

Proteins that protect cell from dehydration.a- Enzyme involved in production of osmoprotectantsb- Late embryogenesis abundant proteinsc- Antifreeze proteinsd- Chaperones e- Detoxifying enzymes

Proteins involved in inducing transcription of stressresponsive genes.

a- TFsb- Protien kinasesc- Enzymes involved in phosphoinositide metabolism

31-03-2015 16

BADHCDH

GENES INVOLVED IN SYNTHESIS OF OSMOPROTECTANTS

Osmoprotectants helps plants in two ways by -a- acting as a cytoplasmic osmolytes.b- protecting and stabilizing macromolecule from

damage induced by abiotic stresses.

Genes for Glycinebetaine Biosynthesis-- Effective osmolyte accumulated during water stress

by Bacteria, Cyanobacteria and members of Chenopodiacae.

- Several crop like potato, tomato, rice, tobacco do not accumulate it but can be made to accumulate by transgenesis.

- It is obtained in two step-Choline Betaine aldehyde Glycinebetaine31-03-2015 17

Contd.. Two enzyme are involved in glycinebetaine

biosynthetic pathway.

1. Choline Dehydrogenase (CDH) in E. coli and Cholinemonoxygenase in Spinach.

2. Betainealdehyde dehydrogenase (BADH).

- Bacterial CDH is most useful enzyme it not only catalyze the oxidation of choline into betainealdehyde but also convert BA into glycinebetaine.

- E. coli betA gene encoding CDH has been cloned and used in transgenesis.

(Jean A.P., et al. 1997)

31-03-2015 18

Genes for Trehalose Biosynthesis -

• Trehalose is a non-reducing sugar.

• Bacteria have five different biosynthetic pathway

but in fungi, plants and animals have only one such pathway.

UDP-Glucose-6-phosphate Trehalose-6-phoshate

(TPS-Trehalose-6-phosphate synthase )

(TPP- Trehalose Phosphatase) Trehalose

• TPS1 Gene from budding yeast have been cloned and used for engineering drought and salinity resistance in crop plants.

( Dan Tau et al.,2008)

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TPS

TPP

Myrothamnus flabellifolia

DriedRehydrated

Sugars as compatible solutes

Trehalose is the osmolyte of choice in the most dessication

tolerant plants

Glucose 6-phosphate TPS

otsAT6.Phosphate TrehaloseTPP

otsB

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SPONTANEOUS CYCLIZATION

Genes for Proline Biosynthesis -

• In plant it is produced from ornithine under normal condition but under stress it is made directly from glutamate.

• P5CS – PYRROLINE-5-CARBOXYLATE

SYNTHATASE

• P5CR- PYRROLINE-5-CARBOXYLATE

REDUCTASE

• Gene was obtained from

Soybean and Mothbean(Baocheng Zhu et al.1998. ,Moss J.P. Et al.1992.)

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P5CS

P5CS

P5CR

GLUTAMATE

ᵞ-GLUTAMYL PHOSPHATE

GLUTAMIC -ᵞ-SEMIALDEHYDE

∆-PYRROLINE-5-CARBOXYLATE

PROLINE

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Even the constitutive expression of functional genes

reduced plant growth

A

WT

B

IP

C

CP

D

WT IP CP

E

WT IP CP

Tobacco transgenics expressing P5CS

IP-inducible promoter (synthetic ABRE); CP-constitutive promoter

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GENE WITH DRE AND DREB TRANSCRIPTION FACTORS

• Dehydration Response Element regulates the gene expression in response to drought, salinity and freezing.

• There are four types of DREB protien. DREB1, DREB2, DREB3 and DREB4. (Peng Xianjun et al. 2011)

• DREB1A and DREB2B binds to DRE and activate transcription of genes with DRE sequence.

• cDNA of these two protein along with 35S promotor was used • Which gave strong constitutive expression of stress inducible gene like

rd29a, kinl, cor6.6/kin2, cor47/rd17, corlSa and erdlo, confered resistance to salt, drought and freeze stress in Arabidopsis.

• CRT/DRE binding protien CBF1 also confer resistance to freeze stress.

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Wheat transgenics over expressing DREB-1A on stress inducible promoter

This signifies the importance of transcription factors

DREB1A Control

31-03-2015 25Alessandro Pellegrineschi et. al,2003

CATALASE

GENE INVOLVED IN SYNTHESIS OF ANTIOXIDANT• Superoxide Dismutase Gene (SOD).

• Classes of SOD enzyme.a. Cu/Zn SOD found in cytoplasm and chloroplast

b. Mn -SOD found in mitochondria

c. Fe- SOD found in chloroplast

d. Ni-SOD found in prokaryote

• Peroxidases and Catalases operate with SOD for antioxidantdefense mechanism.

• Remove H2O2 produced by SOD

• O2 2 H2O2 H2O + O2

Over production of SODs gene in tobaco led to chilling and droughttolerance and gene was obtained from potato and N. plumbaginifolia

plant.

(M. Van Montagu and E Galun,2014)31-03-2015 26

SOD

EARLY RESPONSE TO DEHYDRATION GENES

• XvERD15, an early-responsive gene to stress from Xerophyta viscosa

• Genes that are upregulated in the early response to stress are not well understood.

• ERD15 in Arabidopsis and its homologues in various other plants have been shown to be upregulatedwithin 1 hr post-exposure to dehydration and high salinity stress treatments.

• A cDNA showing homology to ERD15 was isolated from a library generated by low temperature stress treatment of Xerophyta and was subsequently named XvERD15. ( Ming –Yi Lee ,2005)

31-03-2015 27

GENES MAINTAINING CELL MEMBRANE INTEGRITY

1. Late embryogenesis abundant protein

2. Heat Shock Protein

• One such protien XvSAP1 Incoded by XvSAP1 gene is multifunctional protein obtained from X. viscosaplant.

• Codes for a membrane-associated signalling protein.

• Transgenic E. Coli, Arabidopsis and Tobaco plants showed resistant to salinity, drought, cold, high temperature and high light intensity.

(Dahlia Garwe et al.2003)

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LEA• Late Embryogenesis Abundant proteins (LEA proteins) are

proteins in animals and plants that protect other proteins from aggregation, desiccation or osmotic stresses.

• Most LEA proteins are part of a more widespread group of proteins called hydrophilins.

• They are considered to be intrinsically unstructured proteins, forming random coiled proteins in solution.

• LEA proteins were classified into at least seven groups (nine groups in Arabidopsis thaliana based on amino acid sequence homology and specific motifs).

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31-03-2015 30

DIFFERENT GROUP OF LEA PROTEIN

The possible functions of LEA proteins include

• Binding and replacement of water • Ion sequestration • Maintenance of protein and membrane structure • Molecular chaperones • Membrane stabilization and• Nuclear transport of specific molecules

One class of LEAs, is dehydrins, which have detergent and chaperone-like properties, stabilize membranes, proteins, and cellular compartments during stress.

31-03-2015 31

31-03-2015 32

CHAPERONES

• Chaperone are specific stress-associated proteins,

which are responsible for protein synthesis, targeting,

maturation and degradation, and function in protein

and membrane stabilization, and protein renaturation.

• HSPs, which can be divided into five conserved families, have been

shown to have particularly important stress-related chaperone functions

in plant.

• HSPs, which are induced by heat, have been implicated in plant cell

protection mechanisms under drought stress .

• HSPs maintain or repair companion protein structure and target

incorrectly aggregated and non-native proteins for degradation and

removal from cells.31-03-2015 33

HsP60

• One such protein, NtHSP70-1, was constitutively

overexpressed in tobacco .The drought

tolerance of transgenic seedlings was increased

and their optimum water content was maintained

after progressive drought stress.

• HSP24 from Trichederma harzianum was found

to confer significantly higher resistance to salt,

drought, and heat stress when constitutively expressed in Saccharomyces cerevisiae .

(Cho EK, Hong CB, 2006)

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31-03-2015 35

GENE INVOLVED IN ION HOMOEOSTSIS

• Osmotic stresses also disrupt ionic equilibrium of the cell due to cytotoxic build up of sodium and chlorine ions.

• Homoeostasis is maitained by Na+/H+ transporter in the vacuolar membrane.

• V-ATPase is involved in Na+ transport.

• XvVHA-c1 gene codes for this V-ATPase in Xerophyts viscosa .

31-03-2015 36

Recovery growth after 13 days of stress

Transgenics expressing AVP1 showed enhanced

drought recovery in tomato

WT AVP1

AVP1 enhances the root growth and hence better survival at the

end of stress and high recovery growth on stress alleviation

Park et al., 2005; PNAS 102: 5231-03-2015 37

GENE ENCODING CALCIUM BINDING PROTIEN

• In response to stresses like low temperature , drought and ABA Ca2+ concentration in cell increases.

• Calmodulin is highly conserved receptor in plants which is induced by a number of stresses provide protection against these stresses

• XvCaM gene encodes a classical calmodulin protein is being used

31-03-2015 38

DROUGHT and ENGINEERING DROUGHT RESISTANCE

TYPES OF DROUGHT

1. Meteorological Drought- rainfall < 25 % of the average of the region.( <50 %- severe drought)

2. Agricultural Drought- lack of rainfall result in insufficient moisture in the root zone.

3. Hydrological Drought- extended dry period leading to marked deplition of surface water leading to drying up of reservoir, lacks ,stresms, rivers and fall in ground water level.

31-03-2015 39

About 70% of cropped area is rain-fed

The rain-fed area contributes about 36% to total production

Water is the most overriding limitation

India – under low precipitation zone and high ET

31-03-2015 40

0 10 20 30 40 50 60 70 80 90

Jammu & Kashmir

Uttar pradesh

Orissa

Madhya Pradesh

Bihar

west bengal

Haryana

maharastra

Andhra Pradesh

Gujarath

Rajasthana

Tamil Nadu

Karnataka

Different states

Pe

rce

nt

Percent drought prone area in different states in india

percent

Differen

t state

Percent drought prone area in different states of India

31-03-2015 41

DROUGHT RESISTANCE MECHANISM

Postponement(avoidance)

Ability to maintain tissue hydration

ToleranceAbility to function while dehydrated

EscapeAbility to complete life cycle during wet period

(short life cycle) WINTER WHEAT

31-03-2015 42

DROUGHT RESPONSE

31-03-2015 43

Pyramiding the drought traits

Genotype withdrought traits

Root, wax, WUE

Genes codingfor droughtmechanism

Multiple gene construct

Increased

productivity under

drought

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45

Delay of onset of drought-induced senescence

Figure 19.34

itp gene: from Ti plasmid PSARK: senescence-associated protein

kinase promoter Require only 30% of total water

needed Produce 4~5X higher level of biomass

31-03-2015

NCED rate limiting step in ABA biosynthesisConversion of neoxanthine to xanthoxin

ABA-aldehyde

ZEP

NCED

XDH

AAO

zeaxanthin

violaxanthin

neoxanthin

xanthoxin

ABA

Phaseic acid

Osmoticstress

Ca2+

Phosphorilation

Transcription

factors

NCED

31-03-2015 46(Xiaoqiong Qin1 and Jan A.D. Zeevaart, 2002)

Ectopic overexpression of the cell wall invertase gene CIN1 ( Chenopodium rubrum ) leads to dehydration

avoidance in tomato

31-03-2015 47Alfonso Albacete et al. 7 0ct ,2014

(Asaph et al., 2004, Plant Cell)

Increased wax synthesis improved droughttolerance

Evidences

Transcriptional factors regulating wax biosynthesis

SHINE/WIN1-AP2 ERF Transcription factorsWT WXP1 transgenics

3d after drought stress

Zhang et al., 2005, Plant journal

31-03-2015 48

22 23WT 1 4

22 23WT 1 4

3 days after stress alleviation

Control

Performance of codA rice transgenics under moisture stress

31-03-2015 49

Hitesh Kathuria et. al, 2009

BIP-Sense WT BIP- Antisense

Alvim, et. al., 2002, Pl. Physiol. 126, 1042

Antisense expression of BIP gene

disrupts water stress tolerance

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BIP is a HSP70 molecular chaperone

(Zhang et al., 2004)

Drought and freezing tolerance in transgenic Brassica napus through constitutive expression of CBF1

DREB1A over expression in groundnut imparts dehydration tolerance. Pooja Bhatnagar, 2007

Transgenic Wild type

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Trehalose accumulation in rice plants confers

high tolerance levels to drought

NTC – non transformedR80, A05 – transgenic; A-Control B-drought

Garg et al., 2000; PNAS 99(25):15898-903

Regulated overexpression of E coli trehalose biosynthetic genes (otsA and otsB)

as a fusion gene increased drought tolerance in rice

31-03-2015 52

Expression of ethylene response factor JERF1 in rice improves tolerance to drought

31-03-2015 53

Zhang Z et al. 2010

31-03-2015 54

Monsanto’s Transgenic Drought Tolerant Maize

Agricultural biotechnology giant Monsanto has received the green light from the USDepartment of Agriculture to sell its transgenic drought-tolerant maize (corn)MON 87460.

31-03-2015 55

Hybrid seed sold under this trademark combine a novel transgenic trait (based on the bacterial cspB gene) with the best of Monsanto's conventional breeding programme

Drought Gard™ maizewas the first commercially available

transgenic (GM) drought tolerant crop

released in 2013

GENES IN COMMON for DROUGHT and SALT TOLERANCE

DROUGHT

1072 genes

SALT

2879 genes

31-03-2015 56

96

Salt Stress –

Caused by concentrations greater than that required for optimum

growth of a typical crop plant (1500 ppm or 25 mM Na+)

Oceans are the principal sources of salt –

99.991% of water is in the oceans where typically Na+ is 460 mM

and Cl- is 540 mM.

31-03-2015 57

SALT STRESS AND ENGINEERING SALT STRESS

31-03-2015 58

Salinity Impact on Crop Production Worldwide

World Land Surface Area 150 x 106 km2

Salt affected 9 x 106 km2 (6%)

Cultivated Land 15 x 106 km2

*Salt affected 2 x 106 km2 (13%)

Irrigated Land 2.4 x 106 km2

*Salt affected 1.2 x 106 km2 (50%)

*Problem is increasing

Negative Impacts of Salinity on Agriculture

Reduced yields on land that is presently cultivated

Limited expansion into new areas

31-03-2015 59

GENETIC ENGINEERING OF OSMOREGULATION

31-03-2015 60

TARGETED OSMOLYTES

CARBOHYDRATES

CYCLIC POLYOLS

SUGAR ALCOHOLSQUATERNARY

AMONIUM COUMPOUNDS

PROLINE

STORAGE POLYSACCHARIDESEg. Fructans

NON REDUCINGsugar

Glycine betaine production in transgenic plants:Transgene Host plant Accumulation of glycine

betaine

Stress tolerance tested

Barley badh Tobacco

peroxisome

Not tested Not tested

Spinach badh Tobacco

chloroplast

20mol g-1 FW Not tested

Spinach cmo Tobacco

chloroplast

< 0.05 mol g-1 FW Not tested

E.coli betB Tobacco

Chloroplast

Not tested Not tested

E.coli betA Tobcco

Cytosol

Not tested Salt

betA/betB Tobacco 0.035 mol g-1 FW Chilling, Salt

betA Rice 5.0 mol g-1 FW Drought, Salt

A.globiformis

codA

Arabidopsis

Chloroplast

1.2 mol g-1 FW Salt, chilling, Freezing,

Heat

CodA Rice 5.3 mol g-1 FW Salt, chilling

A.pascens cox Arabidopsis 19 mol g-1 DW Freezing, Salt

cox Brassica napus 13 mol g-1 DW Drought, Salt

cox Tobacco 13 mol g-1 DW Salt31-03-2015 61

Transgenic plants engineered to synthesize osmoprotectants other than glycine betaine:

Osmoprotect

ant

Transgenes Crop

plants

Accumulation Stress

tolerance

Proline

Mothbean

P5CS

Tobacco

Rice

soyabean

-

-

4 mg g-1 FW

Salt,

Drought, Salt

Osmotic, Heat

Anti-proDH Arabidopsis 0.6 mg g-1 FW Salt

Mannitol E.coli mtlD Arabidopsis

Tobacco

10 g g-1 FW

mol g-1 FW

Salt

Salt

Sorbitol Apple s6pdh Tobacco

Persimmon

61.5 mol g-1

FW

Oxidativestress

Salt

Trehalose Yeast tps1 Tobacco

Potato

3.2 g g-1 FW Drought

Drought

D-Ononitol Ice plant imt1 Tobacco 35 mol g-1 FW Drought, Salt

Fructans B.subtilis

sacB

Tobacco

Sugarbeet

0.35 mg g-1 FW

5 mg g-1 FW

Drought

Drought

Glutamine GS2 Rice - Salt, Chilling

Osmotin Osm1-Osm4 Tobacco - Drought, Salt31-03-2015 62

Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1 confers salt tolerance

31-03-2015 63

Zhou L et al.2014

• CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation

• Two transgenic AhCMO cotton lines used to study their salinity tolerance in both greenhouse and field under salinity stress

Increased Glycine betaine synthesis and salinity tolerance

Zhang et al., 2009, Mol Breeding, 23:289–298

GMO with high glycine betain

AhCMO

AhCMO

Glycine betain

31-03-2015 64

• Greenhouse study showed that on average, seedlings of thetransgenic lines accumulated 26 and 131% more glycinebetaine than those of non-transgenic plants under normal andsalt-stress (150 mmol l-1 NaCl) conditions respectively

Increased glycine betaine synthesis and salinity tolerance

Zhang et al., 2009, Mol Breeding, 23:289–29831-03-2015 65

WT L1 L2

31-03-2015 66

Examples of transgenic cotton with improved salt

tolerance.Gene Source Function Performance Reference

AtNHXl Arabidopsis vacuolar Na+/H+ antiporter salt tolerance ,increased biomass

He et al. 2005, 2007

TsVP Thellungiellahalophila

A H+-gene that causes accumulationof Na+ and Cl- in vacuoles.

same Lv et al. 2008

AVP1 Arabidopsis codes vacuolarpyrophosphatase

drought and salt toleranceincreased fibre yield

Pasapula et al. 2011

AhCMO Atriplexhortensis

synthesis of glycine betaine Improved salt tolerance increased plant biomass

Zhang et al. 2007, 2009

AnnBj1 Mustard Ca2+ dependent, phospholipidandcytoskeleton binding protein

Improved salt tolerance, relative water content and dry weight

Divya et al. 2010

31-03-2015 67

ENGINEERING PLANTS FOR LOW TEMPERATURE STRESS

Cold tolerance and cold Acclimation• Plants from temperate regions are chilling tolerant, although

most are not very tolerant to freezing but can increase their freezing tolerance by being exposed to chilling, non freezing temperatures, a process known as cold acclimation, which is associated with biochemical and physiological changes of genes with roles in freezing tolerance

• Chilling tolerance that is exhibited by temperate plants is not

entirely constitutive, and that at least part of it is developed

during exposure to chilling temperatures

31-03-2015 68

RESISTANCE AGAINST CHILLING

DESATURATION OF FATTY ACIDSIn higher plant only three enzyme are known to be involved in desaturation of saturated fatty acid

1. Stearoyl-ACT desaturase convert 18:O-ACP to 18:1c9-ACP

2. Phosphatidylglycerol convert 16:O to 16:1c33. Monogalactosyl diacylglycerol convert 16:O to

16:1c7 4. A Des9 gene from Anasystis nidulance is a broad

specificity desaturse gene.

31-03-2015 69

Proteins that probably function in cold

stress tolerance

• Chaperones,

• LEA proteins

• Osmotin

• Antifreeze proteins

• mRNA-binding proteins

• Key enzymes for osmolyte biosynthesis such as proline

• Water channel proteins, sugar and proline transporters

• Detoxification enzymes

• Proteinase inhibitors, ferritin, and lipid-transfer proteins.

31-03-2015 70

Antifreeze proteins

• Antifreeze proteins (AFPs) or ice structuring proteins (ISPs) refer to a class of polypeptides produced by certain vertebrates, plants, fungi and bacteria that permit their survival at low temperature.

• AFPs bind to small ice crystals to inhibit growth and crystallization of ice that would otherwise be fatal.

• Freeze avoidant: by preventing fluids from freezing (AFPs act as antifreezing agent).

• Freeze tolerant: by preventing freezing injury. AFPs act as cryoprotectant).

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31-03-2015 72

TEMPERATURE STRESS SIGNAL TRANSDUCTION

STRESS RESPONSESTRESS

Mutant ACYL-LIPID DESATURASE2 (ADS2) in Arabidopsis confer resistance to cold stress

• ADS2 mutant plants appear similar to the wild type under standard growth conditions but display a dwarf and sterile phenotype when grown at 6°C and also show increased sensitivity to freezing temperature.

• Fatty acid composition analysis demonstrated that ads2 mutant plants at 6°C have reduced levels of 16:1, 16:2, 16:3, and 18:3 and higher levels of 16:0 and 18:0 fatty acids compared with the wild type

Chen et al. , 201331-03-2015 73

Cold tolerance analysis of transgenic rice over expressing OsRAN1

(A) Two-week-old transgenic and WT plants were cold stressed at 4 °C for 84h and then transferred back to the normal condition for recovery.

(B) Photographs of representative seedlings of WT and three transgenic lines were taken after 14 d of recovery

• Ran (RAs-related Nuclear protein) also known as GTP-binding nuclear protein. Ran is a small 25 kDa protein that is involved in transport into and out of the cell nucleus during interphase and also involved in mitosis

(Xu P et al. 2014)

31-03-2015 74

31-03-2015 75

31-03-2015 76

Different adaptation mechanisms of plants to high temperature

31-03-2015 77

Tolerance to heat stress

Heat shock proteins (HSPs)

HSP100 HSP90 HSP70 HSP60 SmHSP

Members appear to function as molecular chaperones.

Individual heat shock proteins have been transformed into plants in order to

enhance heat tolerance.

The rapid heat shock response is co-ordinated by a heat-shock transcription factor (HSF)

31-03-2015 78

Transgenes used to manipulate heat tolerance

Gene Protein Transgenic plant

AtHSF1 Heat shock transcription factor Arabidopsis

Hsp101 HSP100 class heat-shock protein Arabidopsis

Hsp70 HSP70 class heat-shock protein Arabidopsis

Hsp17.7 SmHSP (small heat-shock protein

family)

Carrot

TLHS1 Class I smHSP Tobacco

31-03-2015 79

• Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusabasmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium mediated transformation

• Diagrammatic representation of pUH-Athsp101 construct employed for rice transformation.

Heat-tolerant basmati rice by over-expression of hsp101

31-03-2015 80

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

• Comparison of survival of transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants

• 45 ◦C for 3 h and then were placed at 28 ◦C

• The optimum temperature for rice growth throughout its life cycle is 25–31 ◦C

Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

untransformed (C2) and transgenic lines (15and 43)

Heat-tolerant basmati rice by over-expression of hsp101

31-03-2015 81

OXYDATIVE STRESS

31-03-2015 82

Drought High Light Heat & Cold

Wounding

Ozone

Heavy metals

Pathogens

Senescence

Reactive Oxygen

Species

Oxidative stress

Scavenging mechanismAntioxidantsAntioxidants

Enzymes31-03-2015 83

How do they cause damage?

O -2 H2O2 OH*

Protein Membrane Lipids Other Cellularcomponents

Amino acid residues Lipid peroxide Nucleic Acids

CarbonyI DerivativesBreaking Intra-molecularCross linking lipid peroxidation

Protein degradation

31-03-2015 84

( malondialdehyde(MDA) and4-hydroxynonenal )

Oxidative stressROS detoxification

Any of the above mechanisms can reduce oxidative stress

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Several scavanging enzymes

cloned, characterized and validated

Coordinated expression of a few rate limiting enzymes maybring in oxidative stress response

Tobacco plants expressing catalase showed enhanced tolerance to oxidative stress

Kwon et. al., PCE, 25, 873; 200231-03-2015 86

Gene Host Stress tolerance

Mitochondrial Mn-SOD

Tobacco

Alfalfa chloroplast 2 X increase in SOD

Increased field drought

tolerance

Increased freezing tolerance

Chloroplast Cu/Zn-

SOD

Tobacco

chloroplast

3-15 X increase in SOD

Increased tolerance to high

light and chilling

Cytosolic Cu/Zn-SOD Tobacco cytosol 1.5-6 X increase in SOD

Reduced damage from acute

ozone exposure

Fe-SOD Arabidopsis Tobacco Protected plants from ozone

damage

Apx3 Tobacco Increased protection against

oxidative stress

Apx1 Arabidopsis Heat tolerance

GST/GPX Tobacco Increase stress tolerance

Nt107 (GST) Tobacco Sustained growth under cold

and salinity stress

NtPox (GPX) Arabidopsis Protects against oxidative

stress31-03-2015 87

Role of Ferritin

Regulates Haber-Weiss- reactions

(Fenton reaction)

Fe 2+ + H2O2 OH -

+ OH +

In presence of free Fe 2+ the most harmful

ROS ,OH – is formed from H2O2.

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Transgenic Control

Transgenics with ferritin show increased growth rate under anoxia

Goto et. al., 200031-03-2015

89

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Flooding stress conditions are distinguished based on the level of O2 in

the root environment

1 Hypoxia: conditions under which the reduction in available O2 starts to

become a limiting factor for ATP production through oxidative

phosphorylation

2 Anoxia: conditions under which ATP is only produced through

glycolysis, as no more O2 is available

Flooding stress tolerance

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• The two internal gaseous signals, oxygen and ethylene, are frequently associated with the responses of plants or plant parts surrounded by water

• Ethylene, accumulates to physiologically active levels in submerged tissues, due to production in almost every organ and hampered diffusion to the atmosphere .

• Elevated ethylene levels are important for the induction of morphological and anatomical traits upon flooding, such as formation of aerenchyma and adventitious roots, elongation etc

• Aerenchyma formation helps in diffusion of gas between roots, submerged parts etc.

• Plant with Arenchyma are able to maintain high amounts of ATP and reduces hypoxia.

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Ethylene Biosynthesis

Cold stressOxidative stressOsmotic stressMechanical stressUV stressPathogen attack

Flooding

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The tall etiolated

seedling has a

mutation in the

ethylene receptor

ETR1. The seedling

cannot detect ethylene.

Arabidopsis

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•Deepwater rice responds to partial submergence by enhancing cell

division and elongation in the internodal regions of underwater stems, via

a mechanism triggered by entrapment of ethylene, which promotes

abscisic acid (ABA) degradation and increases gibberellic acids (GA) and

their downstream effects.

•Remarkably, stem elongation rates in deepwater varieties can reach 25

cm/day. This unusually robust underwater growth is controlled by three

quantitative trait loci (QTLs). Of these, the SNORKEL QTL on chromosome

12 encodes two ethylene responsive factor (ERF) DNA binding proteins,

SNORKEL1 (SK1) and SNORKEL2 (SK2), that are absent from the non-

deepwater rice accessions evaluated to date.

•A pronounced strong elongation growth response of these wild species

maintains sufficient aerial tissue above the air–water interface for efficient

photosynthesis and oxygen exchange with submerged organs.

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• SNORKEL genes belong to the ERF (Ethylene Response Factor) family of transcription factors, which are induced by ethylene.When plants are under water, ethylene accumulates in the plant.

• The ethylene then induces expression of these ERF genes.

SNORKEL1 and SNORKEL2 trigger remarkable internode elongation

via the hormone gibberellin.

• In contrast, SUB1A inhibits internode elongation.

Deepwater rice

Non-deepwater rice

Transcriptional response

No transcriptional response

SNORKEL1 & 2

Flooding

Flooding Non-deepwater rice does not have these genes!31-03-2015 96

Long-term flooding vs. flash flooding

• A few rice cultivars have adapted to areas where flash flooding is

common by learning how to “hold their breath”. These cultivars can

survive under water for up to 2 weeks.

• These cultivars do NOT use elongation as an escape strategy. They

become quiescent and stay submerged, avoiding the energy consumption

that is involved in elongation. For example, they increase anaerobic

respiration.

• The gene controlling this response, named SUB1, was identified and

cloned in 2006. Like the SNORKEL genes, it is also a member of the ERF

gene family.

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The SNORKEL ERFs SK1 and SK2, present in deepwater and floating rice, contribute to the GA-

mediated elongation growth that enables this remarkable extension of submerged shoots. The

phytohormone ethylene triggers the expression of SUB1A and the SNORKEL ERFs, although they

drive antithetical growth responses. SUB1 and SKs are members of the group VII ERF subfamily of

transcription factors.31-03-2015 98

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Constitutive and submergence-induced expression of Sub1A confers growth restriction and survival of

prolonged submergence.

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CASE STUDY 1International Journal of Molecular Sciences

Over expression of Arachis hypogaea AREB1 Gene Enhances Drought Tolerance by Modulating ROS Scavenging and Maintaining Endogenous ABA Content

Xiao-Yun Li 1, Xu Liu 2, Yao Yao 1, Yi-Hao Li 1, Shuai Liu 1, Chao-Yong He 1, Jian-Mei Li 1, Ying-Ying Lin 1 and Ling Li 1.

1- Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life

Science,South China Normal University, Guangzhou 510631, China.

2 - Molecular Analysis and Genetic Improvement Center, South China Botanical Garden, Chinese Academy of Science, Guangzhou 510650, China.

Received: 24 April 2013

Accepted: 31 May 2013

Published: 19 June 2013.

(Int. J. Mol. Sci. 2013, 14, 12827-12842)

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MATERIALS AND METHODPlant Materials• Seeds of Arabidopsis wild-type (WT) were surface sterilized in 70% ethanol for 2 min and in

1% sodium hypochlorite for 10 min. • The seeds were sown on MS (Murashige and Skoog) medium supplemented with 2%

sucrose and 0.8% agar. • Seeds were germinated and grown in a growth chamber under a daily cycle of 16 h light

and 8 h dark at 20 ± 2 °C.• Seven days after sowing, the seedlings were planted in plastic pots in a medium of

vermiculite, peat moss and perlite (1:2:1).•

Plasmid Construction and Arabidopsis TransformationThe full-length cDNA of AhAREB1 coding region, was generated by RT-PCR with the

following primers:5'-CTG AGATCT ATG AAC TTC AGG GGC TAT GGT GAT-3' and5'-CTGGGTGACC CTA CCA GGG ACC TGT AAC TGT CCTT-3'

Vector used - pCAMBIA1301 with 35S promoter The overexpression construct was introduced into Agrobacterium tumefaciens strain

• GV3101Assays of Seed Germination and Green Cotyledons; Growth of Roots• Drought Stress Tolerance Assays • Microarray Analysis• Quantitative PCR Assay

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In Situ NBT Staining and Measurement of SOD Activity• In situ accumulation of superoxide (O2−) was examined based on histochemical staining by

nitro blue tetrazolium (NBT)

• One unit SOD activity is defined as the amount of enzyme that will inhibit the rate of cytochrome c reduction by half under specific conditions.

In Situ DAB Staining and Measurement of CAT Activity• In situ accumulation of hydrogen peroxide (H2O2) was examined based on histochemical

staining by 3,3-diaminobenzidin (DAB).

• One unit of catalase will decompose 1.0 µmole of H2O2 per minute at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM.

Quantification of ABA Levels• To determine the ABA levels in WT and transgenic plants, 28 day-old seedlings were

dehydrated for 10 days.

ResultsThe AhAREB1 Overexpression Greatly Improves Drought Tolerance and ABA Sensitivity in

Transgenic Arabidopsis Plants

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Drought tolerance and abscisic acid (ABA) sensitivity ofAhAREB1-overexpressed plants

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ConclusionsIn summary, they demonstrated that AhAREB1 acts as a transcriptional activator of stress-relative, ROS-modulated genes and ABA-induced genes under drought or dehydration stress, and it play an important role in drought stress tolerance via ABA homeostasis and control of ROS accumulation.

CASE STUDY 2

EsDREB2B, a novel truncated DREB2-type transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and transgenic tobacco

Xiaoshuang Li123, Daoyuan Zhang1*, Haiyan Li12, Yucheng, Wang1, Yuanming

Zhang1 andAndrew J Wood.

Corresponding author: Daoyuan Zhang zhangdy@ms.xjb.ac.cn1Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and

Geography, Chinese Academy of Sciences, Xinjiang Urumqi 830011, China2University of Chinese Academy of Sciences, Beijing 100049, China3Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901-6899, USA

Received:13 September 2013

Accepted:5 February 2014

Published:10 February 2014

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Growth of S. cerevisiae yeast cells transformed with the empty vector PYES2 and with the PYES2-EsDREB2B under different stress conditions

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Fresh weight and root architecture comparison of non-transformed (WT) plants and two EsDREB2B transgenic

tobacco lines under osmotic, salt, cold and heat stresses

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Comparison of the levels of proline, MDA and chlorophyll between WT and EsDREB2B-transformed tobacco after

osmotic, salt, cold and heat stresses

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Phenotype and leaf number comparison of non-transformed (WT) plants and two EsDREB2B transgenic tobacco lines

under osmotic, salt, cold and heat stresses

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ConclusionEsDREB2B is a promising candidate gene for the development of crops with multiple stress

tolerance .

TERMINATOR TECHNOLOGY• Terminator technology refers to plants that have

been genetically modified to render sterile seeds at harvest – it is also called Genetic Use Restriction Technology or GURTS.

• Genetic Use Restriction Technology (GURTs) is the “official” name for Terminator technology.

• Developed by Monsanto in association with Delta and Pine Land company and US department of agriculture in march 1998.

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TYPES

V-GURTs: This type of GURT produces sterile seeds, so the seed from this crop could not be used as seeds, but only for sale as food or fodder.

T-GURT: modifies a crop in such a way that the genetic enhancement engineered into the crop does not function until the crop plant is treated with a chemical that is sold by the biotechnology company.

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GENES SYSTEM INVOLVED

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• Gene System -1

contain a repressor gene from E coli under the control of constitutive chemical sensitive promotor.

• Gene System-2

contain a cre recombinase gene from Tn10 along with repressor binding site.

• Gene System -3

contain a RIP gene from Saponaria oficinalis under the control of LEA promotor interrupted by a spacer sequence.

MECHANISM

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A

B

• http://cls.casa.colostate.edu/transgeniccrops/flash/termin ator.swf

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Abiotic stress’ are major cause of concern for the global food security

Conventional knowledge has almost saturated in finding the solutions for the sprawling abiotic stress’ resulting due to climatic change and other causes.

GE has proved its worth in tweaking the plants’ ability to cope with the various abiotic stresses.

The main advantage of GE is that it can transcend across the species barrier.

Although much progress has been made through GE in taming stress’

Much is need to be done to realise the fulll potentiality of this technology.

CONCLUSION

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REFERENCESXiaoshuang , Daoyuan Zhang, Haiyan , Yucheng, Wang, Yuanming Zhang and Andrew J Wood, EsDREB2B, a novel truncated DREB2-type

transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and transgenic tobacco, BMC Plant Biology 2014, 14:44 .

.R. Munns, M. Tester, Mechanisms of salinity tolerance, Annu. Rev. Plant Biol. 59.(2008) 651–681.

J.M. Pardo, Biotechnology of water and salinity stress tolerance, Curr. Opin. Plant Biol. 21 (2010) 185–196

B-M Pooja, M. Jyostna Devi, D. Srinivas Reddy, M. Lavanya , Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions, Plant Cell Rep (2007) 26:2071–2082

T. M.Reguera, Z. Peleg, E. Blumwald, Targeting metabolic pathways for genetic engineering abiotic stress-tolerance in crops, Biochimica et BiophysicaActa 1819 (2012) 186–194

E. Blumwald, Sodium transport and salt tolerance in plants, Current Opinion in Cell Biology 2000, 12:431–434 Copyright © 2002 American Society of Plant Biologists

Xue-Chu Zhao, Xiang Qu Dennis, E. Mathews and G. Eric Schaller ,Effect of Ethylene Pathway Mutations upon Expression of the Ethylene Receptor ETR1 from Arabidopsis Plant Physiology December 2002 vol. 130 no. 4 1983-1991

Chawla H.S., INTRODUCTION TO PLANT BIOTECHNOLOGY, 2012, Oxford & IBH Publishing Co. Pvt Ltd, New Delhi.GUPTA PK, PLANT BIOTECHNOLOGY, 2010, Rastogi Publication, Meerut. P : 285-297.

• www.sciencedirect.com• www.brupt.com• www.pubmed.com • WWW.biomedcentral.com• www.mdpi/journal/ijms.com• www.econexus.info

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