Abiotic Stress Resistance

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    Genetic engineering of metabolic pathways in

    abiotic stress resistance

    Abiotic stresses:

    Drought

    Salinity

    Temperature

    Oxidative stress

    Chilling

    Freezing

    High temperature

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    Drought Stress

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    Drought will be most important abiotic stress in the next 20 years

    Water will become scarce and probably wars will start

    on this between countries.

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    Findings of NCAR (National Centre for Atmospheric Research)

    (Published in J. of Hydrometeorology, Dec. 2004)

    Area hit by severe drought increased from nearly 12%

    in 1970 to 30% in early 2000.

    50% of this change is due to global warming rather than decrease

    in rainfall or snowfall.

    Drought increased in last 30 years : much of Europe, Asia, Western

    And South Africa, eastern Australia and Canada.

    Down to Earth, Feb 15, 2005

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    Water stress

    Signal perception

    Signal transduction

    Gene expression

    Gene Products

    Functional Proteins Regulatory Proteins

    Water channel proteins * Transcription factors

    Osmolyte biosynthetic enzymes * Protein KinasesChaperons * Phospholipase C

    LEA * 14-3-3protein

    Proteinases

    Detoxifying enzymes

    Stress Tolerance Stress response

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    Root to shoot signaling:

    a) Message synthesized by roots

    b) Role of this message in the devt. of the root itself under stress.

    With the help of the message (chemical or electrical) shoots sense

    soil drying through roots.

    This message instructs shoots to conserve water for useduring later stages of growth.

    Closing stomata

    Decreasing water loss as a

    First line of defence

    Decreases transpirational area

    by inhibiting leaf expansion

    by

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    What happens to the ABA when it reaches the stomata

    after traveling through the xylems?

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    During the last decade, intensive studies at the molecular level have unravelled

    the signal intermediates in ABA guard cells drought signaling pathway.

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    From the Agriculture perspective we need to develop

    desiccation tolerance plants.

    WHAT SHOULD BE THEAPPROACH?

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    TRANSGENIC APPROACHES TO ENHANCE DROUGHT

    TOLERANCE BY INCORPORATING MULTIPLE GENES.

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    What is the best source of

    these genes?

    Answer is

    RESURRECTION PLANTS

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    What is the Unique feature

    of these plants? Can withstand complete dryness

    (RWC 1%)

    Still viable after rehydration

    Full physiological activity resumewithin several hours

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    Unique features of these plants

    Many survive even at RWC of4% while the lethal RWC of

    most crop plants and mesophytes

    is 30-50%. [32% Pigeon pea,

    50% Soyabean; Ref.Sinclair (1980)]

    Can loose over 95% of their

    water content survive in their

    dried state for prolonged

    periods and revive rapidly

    when water is available.

    Myrothamnus fl abell if oli a

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    Craterostigma plantagineum

    Hydrated Dried Rehydrated

    This and many other studies shows that ABA responsive genes

    expressed in dehydrating plants of these species may be responsible

    for their amazing desiccation tolerance.

    ABA induces desiccation tolerance in Craterostigma calli.

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    reflectance

    transpiration

    Drought tolerance

    SUN

    cooling

    Pest tolerance

    PHWL

    Epicuticular wax

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    Epicuticular wax

    Protection from mechanical damage (Walker 1998,Eigenborde 1996)

    Primary defence against pathogens(Carver et al 1996)

    Site of interaction with insect and microbes

    Scatter and reflect light (less heat load,better mesophyll function)

    Restricts non-stomatal water loss

    Can contribute for drought tolerance and WUE (Samdur et al.,2003)

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    VLCFA

    Aldehydes

    Alkanes

    Secondary alcohol

    Ketone

    Aldehydes

    Primary alcohol

    Esters

    Acyl reductionDecarbonylation

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    C 18

    C 26

    C 28

    C 30

    C 32

    OH

    O

    Aldehydes

    OH

    1o Alcohols

    Esters

    OH

    AldehydesO

    AlkanesCO2

    OH 2 o Alcohols

    OKetones

    Acyl Reduction Decarbonylation

    Fatty acid

    Elongation

    Cer 1

    Cer 6

    O

    Figure2:The flow chart representing the Epicuticular waxBiosynthetic pathway (Kunst and Samuels,2003)

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    March 2005

    WXP1- Wax production 1

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    Medicago truncatula a novel model plant for legume biological studies.

    Small diploid genome

    Self fertile

    Short life cycle

    A large number of Ests have been sequenced

    Whole genome sequencing is in progress

    Closely related to the worlds most important forage legume alfalfa

    It is the fourth most widely grown crop in U.S.

    G

    http://images.google.co.in/imgres?imgurl=http://www.ccrc.uga.edu/web/personnel/hahn/potted.gif&imgrefurl=http://www.ccrc.uga.edu/web/personnel/hahn/mtimages.htm&h=512&w=768&sz=313&tbnid=4n_Fe0ourr8J:&tbnh=94&tbnw=141&hl=en&start=16&prev=/images%3Fq%3DMedicago%2Btruncatula%2Bplant%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3DG
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    Growth and development

    Transgenics were obtained under 35S promoter by Agrobacterium

    Mediated Transformation.

    Relatively slow growth rate delayed the flowering time

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    Trifoliates had more glaucous appearance

    Difference in glaucous was more prominent in adaxial side

    I t f WXP1 i ti l d ti

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    wing coiled crystalline structure decreases

    Complemented by increase in density of tubular and plate like

    wax crystals

    Impact of WXP1 overexpression on cuticular wax production

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    Total wax load is significantly higher in transgenic leaves comparedto control.

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    PHWL

    Top second and third leaf showed decreased water loss

    D h l

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    T47 is performing better 3days of drought stress

    Drought tolerance

    WT T47

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    Recovery after 10days of drought stress and rewatering

    WT T47

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    Recovery after 3 cycles of drought- rewatering treatments in green house

    WT T47

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    Wax Transcription factor

    The SHINE Clade of AP2 Domain Transcription Factors

    Activates Wax Biosynthesis, Alters Cuticle Properties, and

    Confers Drought Tolerance when Overexpressed in

    Arabidopsis

    Asaph Aharoni,a Shital Dixit,a Reinhard Jetter,b Eveline Thoenes,a Gert van

    Arkel,a and Andy Pereiraa

    The plant cell, 2004

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    Wax composition

    Shn 6 fold increase in wax coverage

    WT-equal amounts of compounds from both pathway

    Shn-partial towards decarbonylation pathway

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    15d old seedlings of Arabidopsis

    Exposed to 9 to 11d dehydration

    Seedlings were watered

    Recovery for a week

    Checking the drought tolerance

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    Salinity StressSalt StressCaused by concentrations greater than that required for

    optimum growth of a typical crop plant (1500 ppm or25 mM Na+)

    Oceans are the principal sources of salt

    99.991% of water is in the oceans where typically Na+ is 460 mMand Cl- is 540 mM.

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    Biogeochemical cyclingwater droplets containing salt are carried by wind over landwhere these evaporate and deposit salt onto the soil and increase soil salt content.

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    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

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    Na+

    Na

    +external

    I A: Suaeda maritima, Atriplex nummularia

    I B:Atriplex hastata, Spartina townsendii and sugar beet

    II : Cotton, Barley, Tomato, Common bean and soyabean

    III : Fruit trees, Avacado, stone fruits etc.,

    How does salinity effect plant growth?

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    How does salinity effect plant growth?Salinity decreases plant growth, yield and market quality.

    1. Short-term effect (minutes to days)

    External osmotic effectsReduced leaf expansionLowered stomatal conductance

    2. Long-term effects (weeks to months)

    Internal ionic effect-specific ion toxicityInjurious conc.of ions:Na+, Cl-, SO4

    Chlorosis and Necrosis.

    Eventual death.

    Non-saline SalineCytosolic Concentration: Na (1-10 mM)

    K (100-200 mM)Ratio shifts.

    Degree of shift determines the

    degree of reduction in growth.

    Transgenics Expressing Different Salt Stress Genes

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    Osmoprotectants Transporters

    Transgenics Expressing Different Salt Stress Genes

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    Proline Biosynthesis

    Glutamic Acid.

    Glutamic gama

    semi aldehyde

    P5C -Synthase

    Pyroline- 5 -Carboxylase

    Proline

    P5C-Synthase

    Stress

    induciblepromoter

    ABRE

    Constitutive

    PromoterCamv-35

    Introduce to

    the plant

    Clone

    into a

    vector(35S-P5CS)(AB P5CS)

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    Free proline accumulation in finger millet plants over expressing P5CS

    Three weeks old finger millet seedlings were subjected to water deficit

    stress for 5 days. The extent of accumulation of free proline were

    determined at the end of stress and also one day after re-watering.

    0

    10

    20

    30

    40

    50

    60

    70

    100 % FC 50 % FC 30 % FC Recovery

    Proline(mm

    ol/gFW)

    WT

    AB-P5CS

    35S-P5CS

    Eff t f S lt t S dli th i fi ill t

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    Effect of Salt stress on Seedling growth in finger millet

    Seeds of wild type and transgenic finger millet over expressing P5CSwere

    subjected to salt (NaCl) stress for 5 days. The growth of the P5CStransformed

    finger millet seedlings was far better than the untransformed control.

    WT AB-P5CS 35S-P5CS

    0 mM NaCl

    200 mM NaCl

    400 mM NaCl

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    Phenotype of 6-week old wild type and P5CS seedlings as affected by salinity (200mM NaCl)

    stress. Seeds were germinated and maintained on MS medium containing 200mM NaCl. The

    plates were kept in a controlled environment at 240C under constant light.

    1. Plants

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    Choline

    CH2OH

    CH2

    H3C-N+-CH3

    CH3

    O2 2H2O

    2Fd (red) 2Fd (ox)

    CMO

    CHO

    CH2

    H3C-N+-CH3

    CH3

    Betaine

    aldehyde

    NAD+ NADH

    BADH

    COO-

    CH2

    H3C-N+-CH3

    CH3

    Betaine

    2. Escher ichia coli

    NAD+ NADH

    CDH

    CH2OH

    CH2

    H3C-N+-CH3

    CH3

    Choline

    NAD+ NADH

    BADH

    CHO

    CH2

    H3C-N+-CH3

    CH3

    Betaine

    aldehyde

    COO-

    CH2

    H3C-N+-CH3

    CH3

    Betaine

    3. Arhrobacter globif ormisCH2OH

    CH2

    H3C-N+-CH3

    CH3

    Choline

    2O2 2H2O2

    COD

    COO-

    CH2

    H3C-N+-CH3

    CH3

    Betaine

    Biosynthetic pathway of Glycine betaine

    synthesis in some natural accumulators.

    BETAINES P th i Mi i

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    BETAINES

    Pathway in plants

    Chloine

    Betaine aldehyde

    Glycine betaine

    CMO

    BADH

    Pathway in Microorganism-

    Ar throbacter globiformis

    Chloine

    Glycine betaine

    CODA

    Introduce into

    plants

    Transgenic plants

    Cloned into

    a vector

    Glycine betaine production in transgenic plants:

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    Transgene Host plant Accumulation of glycine

    betaine

    Stress tolerance tested

    Barley badh Tobacco

    peroxisome

    Not tested Not tested

    Spinach badh Tobacco

    chloroplast

    20mmol g-1 FW Not tested

    Spinach cmo Tobacco

    chloroplast

    < 0.05 mmol g-1 FW Not tested

    E.colibetB Tobacco

    Chloroplast

    Not tested Not tested

    E.coli betA Tobcco

    Cytosol

    Not tested Salt

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

    betA Rice 5.0 mmol g-1 FW Drought, Salt

    A.globiformiscodA

    ArabidopsisChloroplast

    1.2 mmol g-1 FW Salt, chilling, Freezing,Heat

    CodA Rice 5.3 mmol g-1 FW Salt, chilling

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

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

    cox Tobacco 13 mmol g-1 DW Salt

    I Pl t

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    Fructose-6-phosphate

    mtlD

    Mannitol-1-phosphate

    Non-specific phosphatase

    Mannitol

    NAD

    HPr P-HPr

    In Plants

    In E.coli

    (pHPr-phosphorylated heat stable protein)

    NADH

    Pi

    Mannitol biosynthetic pathway in transgenic tobacco

    (Mannitol dehydrogenase)

    Phenotype of mtlD transgenic finger millet under water deficit stress

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    MTLD-1 MTLD-3

    WTMTLD-5

    yp g g

    100%FC 50%FC 30%FC 100%FC 50%FC 30%FC

    100%FC 50%FC 30%FC100%FC 50%FC 30%FC

    Cyclicitols: (Sugar Alcohols)

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    y ( g )

    Glucose 6 Phosphate Myoinositol D-Ononitol

    D-Pinnitol

    IMT1

    Trehalose:

    Non-reducing disaccharides

    TPS1 from yeast---transformed to tobacco.(Trehalose 6 PO4 synthase)

    Fructan:Soluble storage polysaccharides

    Sucrose FructanLevan sucrase

    Transgenic plants engineered to synthesize osmoprotectants other than glycine betaine:

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    Osmoprotectant Transgenes Crop plants Accumulation Stress tolerance

    Proline

    MothbeanP5CS 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 mg g-1 FW

    m mol g-1 FW

    Salt

    Salt

    Sorbitol Apples6pdh Tobacco

    Persimmon 61.5 m mol g-1 FW

    Oxidative stress

    Salt

    Trehalose Yeast tps1 Tobacco

    Potato

    3.2 m g g-1 FW Drought

    Drought

    D-Ononitol Ice plant imt1 Tobacco 35 m 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, Salt

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    Salt stress tolerance of

    transgenic plants

    http://localhost/var/www/apps/conversion/tmp/scratch_10/salt%20stress%20tolerance%20of%20transgenic%20plants.gifhttp://localhost/var/www/apps/conversion/tmp/scratch_10/salt%20stress%20tolerance%20of%20transgenic%20plants.gifhttp://localhost/var/www/apps/conversion/tmp/scratch_10/salt%20stress%20tolerance%20of%20transgenic%20plants.gifhttp://localhost/var/www/apps/conversion/tmp/scratch_10/salt%20stress%20tolerance%20of%20transgenic%20plants.gif
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    Transgenics Expressing Different Salt Stress Genes

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    Osmoprotectants Transporters

    g p g

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    Intracellular Ca pools

    disturbed

    Cell elongationCell production

    SALT STRESS

    Displacement of Ca2+ by Na+ from membranes

    Membrane permeabilityNa+ influx

    Membrane potential depolarized

    H+-ATPase activityH+ efflux

    Membrane potential restored

    Ca

    CaCa

    CaCa2+ uptakeK+ efflux

    CaCa

    Ca

    Initial and short-term effects of salt stress on the

    plasmalemma of root cells(Cramer et al., 1985)

    Ca imparts saline tolerance by increasing K/Na ratio

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    Ca imparts saline tolerance by increasing K/Na ratio.

    -Both by restricting Na entry as well as by decreasing

    K+ efflux.

    Control NaCl NaCl + CaCl2

    Na (mol.m-3) 3.0 74 44 (40%)K (mol.m-3) 180 132 151 (20%)K/Na ratio 60 1.8 3.4 (50% )

    (Ref: Munns, R., 1999)

    Maize

    Treatment K+ efflux

    % of Control

    225mM NaCl 68620

    225mM NaCl + 10mM

    CaCl2

    300133

    Cotton

    (Ref:Cramer, G.R., 1985)

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    1 2 3 4 5 6

    1: Control 2: 10mM CaCl2 3: 100mM NaCl

    4: 100mM NaCl +10mM CaCl2 5: 200mM NaCl 6: 200mM NaCl +10mM CaCl2

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    Cellular Homeostasis is the most important aspect and involves osmoregulation,

    compartmentation of Na, Calcium singatures and Calcium homeostasis.

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    Two good approaches has paid rich dividends for salt tolerance

    a) Compartmentalization of the excess Na into the vacuole

    b) Restrict the entry of Na by inactivating the

    gene HKT1, regulating Na entry.

    High Na+ H+

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    SOS1

    SOS2SOS3 Na

    +

    Ca2+

    ?

    H2O

    Transcriptional &

    Post transcriptional

    gene regulationH+

    V-ATPase

    PPase

    Vacuole

    H+

    Na+

    SOS2

    SOS3

    Ca2+

    ? HKT1

    Na+

    K+

    ACA4

    LCA1

    Regulation of ion (e.g., Na+ and K+) Homeostasis by the SOS pathway

    (Zhu, 2000)

    Transport

    t i

    Mode of

    t t

    Membrane

    l ti

    Tools and systems

    d f

    K:Na

    S l ti it

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    protein transport location used for

    characterization

    Selectivity

    KAT/AKT

    Inward K+

    channels

    Passive

    diffusion

    Plasma

    membrane

    Molecular and

    electrophysilology in

    planta and inheterologous systems

    Highly selective

    for K+

    HKT1

    High affinity K+

    transporters

    Na+

    energizedNot known Molecular and

    electrophysilogy in

    heterologous systems

    Transports both

    Na+ and K+

    KUP or HAK

    High affinity K+

    transporter

    Not known Not known Molecular and

    radioisotopes inheterologous systems

    Some Na+

    permeability

    NSC

    Non-Selective

    cation channels

    Passive

    diffusion

    Plasma

    membrane

    Electro physiology in

    planta

    High Na+

    permeability

    AtNHX1Na+-H+ exchanger

    H+

    energized

    Vacuole andplasma

    membrane

    Molecular andradioisotopes in planta

    and in heterologous

    systems

    Not known

    LCT1

    Low-affinity

    cation transporter

    Not known Not known Molecular and

    radioisotopes in

    heterologous systems

    Transports both

    Na+ and K+

    Salient features of Calcium transporters

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    ACA4 LCA1 CAX1 CAX2Type of Transporter Ca2+-ATPases Ca2+-ATPases Ca2+/H+

    exchangersCa2+/H+

    exchangers

    Salt induced Yes Yes Not known Not known

    mRNA transcripts

    under stress

    Phenotype changes

    when over expressed

    Increases

    Nil

    Normaltransgenic

    plants

    Increases

    Nil

    Normaltransgenic plants

    Increases

    Not known

    Transcriptsunder cold stress

    Not known

    Over expression

    studies

    Done Done Not done Not done

    Effects on over

    expression

    Salt toleranceincreased

    Salt stressincreased

    Not known Not known

    Other functions Only calciumtransport

    Only calciumtransport

    Also involved inlow temperature

    acclimation

    Also involved inmetal transport

    Efficiency of

    sequestration

    Highlyefficient, highaffinity to Ca

    Highly efficient,high affinity to

    Ca

    Highly efficient,High affinity to

    Ca

    Low efficient,Low affinity to Ca

    Two lines of evidence suggest that plant Ca2+-ATPases

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    Expression of LCA1 in Tomato Expression of ACA4 in Arabidopsis

    gg p

    are involved in salt stress adaptation.

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    AtNHX1 over-expressionin Brassica

    (Salinity tolerance)

    X10E1 X10E2 X10E3 WT

    WT Transgenic

    Expression of Na/H+antiporter

    in tomato for salini ty tolerance

    SCIENCE VOL. 280 19 JUNE 1998

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    Salt

    stress

    Ionic

    stressSOS3SOS2 Ion transporters

    e.g. SOS1Ion homeostasis

    Homeostasis

    Osmotic

    stressMAPK

    cascade?Osmolytes

    Osmotic

    homeostasis

    Secondary

    stresses

    e.g. oxidation

    Detoxification

    Cell division

    and expansion

    ColdDrought

    ABA

    CBF/DREBStress proteins

    e.g. RD29A

    The three aspects of salt tolerance in plants- Homeostasis,

    detoxification and growth control (Zhu, 2001).

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    Species and genes

    Used in transformation

    Temperature stress:

    http://localhost/var/www/apps/conversion/tmp/scratch_10/species%20and%20genes%20used%20in%20transformation.pdfhttp://localhost/var/www/apps/conversion/tmp/scratch_10/species%20and%20genes%20used%20in%20transformation.pdfhttp://localhost/var/www/apps/conversion/tmp/scratch_10/species%20and%20genes%20used%20in%20transformation.pdf
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    physiological and/or structural damage (including plant death) inducedby non-freezing temperatures (0 - 120 C)

    physiological and/or structural damage induced by ice formation within

    tissues or organs

    Chilling stress:

    Freezing stress:

    metabolic and physiological damage induced by short or long term exposure

    to elevated temperature (>400 C)

    Heat stress:

    Temperature stress:

    Effect of temperature on plant growth:

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    Cool season crops complete most of their life cycle under cool

    conditions, 4 - 120C (broccoli, wheat, bluegrass)

    Warm season crops require higher temperatures for optimal

    growth , 22 - 300C (tomato, corn, melons)

    Perennial crops have adapted mechanisms for coping with

    temperature stress.

    Optimal growth temperature is a species characteristic.

    20C

    During slow freezing, ice forms outside the plasmalemma. Thislowers the vapor pressure, and pulls water from the cell until equilibrium

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    -20C

    -100C

    p p , p qis met. This phenomenon is known as freeze dehydration.

    Because water leaves the cell until the osmotic pressure insideequilibrates with outside, the organelles and membranes are exposedto high concentrations of salts and other cellular metabolites.

    Cells with higher concentrations of protective solutes such as sugarsshrink less to reach equilibrium, and lose less water. The protectivesolutes probably stabilize the membrane and proteins by H-bonding andby lowering the relative concentrations of salts and other damagingmetabolites to which cell organelles are exposed.

    During rapid freezing, ice mayform intracellularly because watercannot exit the cell fast enough toequilibrate. Intracellular freezing (inthe absence of cryoprotectants likeDMSO and glycerol) is usually lethalto plant cells, resulting in totalmembrane disruption and collapse of

    the cellular organization. Freezing Stress Approach: Mechanisms of freezing resistance.

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    During cold acclimation- plant produce a number of cold induced proteins that are

    assumed to play a role in the subsequent cold resistance.

    About 50 cold induced proteins have been identified in diff. Plant species.

    LEA proteins COR genes

    (cold responsive)

    According to their patterns of expression

    There are some examples of the Expression of Cold induced proteins in transgenic plants.

    Constitutive expression of Chloroplast targeted COR protein COR15a inArabidopsis

    improved freezing tolerance.

    Tolerance to heat stress:

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    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)

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    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

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    Secondary effect of abiotic stress:

    Production of reactive oxygen species

    Drought High Light Heat & Cold

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    Wounding

    Ozone

    Heavymetals

    Pathogens

    Senescence

    Reactive Oxygen

    Species

    Oxidative stress

    Scavenging mechanismAntioxidantsAntioxidants

    Enzymes

    O2.-Ascorbate-Glutathione cycle (Hlti-Well Asada cycle)

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    SOD

    Hydrogen peroxide is removed by ascorbate peroxidase and ascorbate is

    regenerated by this cycle

    APX: Ascorbate Peroxidase

    MDHA: Mono DeHydro Ascorbate

    DHAR: DeHydro Ascorbate reductase

    GR: Glutathione reductase

    DHA: DeHydro Ascorbate

    Oxidized

    Reduced

    SOD enzyme family:

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    SOD catalyzes the dismutation of superoxide to H2O2 and O2.

    SOD is present in most subcelluar compartments of the plant cell and is assumedto play a central role in the defence against oxidative stress.

    3 distinct types of SOD: based on metal cofactor.

    Cu/Zn SOD

    Mn-SOD

    Fe-SOD

    Cytosol/chloroplast

    Mitochondria

    Chloroplast

    However, they are not regulated coordinately, but independently according to the

    degree of oxidative stress experienced in the respective subcelluar compartments.

    Transgenes used to engineer tolerance to oxidative stress:Gene Host Stress tolerance

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    Ge e os S ess o e ce

    Mitochondrial Mn-SOD

    Tobacco

    Alfalfa chloroplast 2 X increase in SOD

    Increased field drought tolerance

    Increased freezing toleranceChloroplast 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 acuteozone exposure

    Fe-SODArabidopsis Tobacco Protected plants from ozone

    damage

    Apx3 Tobacco Increased protection against

    oxidative stressApx1 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 stress

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    NATURE BIOTECHNOLOGY VOL 23, MARCH 2004

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    (Plants exposed to a temp of60C for 2 days and returned to 220C for 5 days)

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    ( p p y y )

    (water withheld for 2 weeks)

    (Plants soaked in 600mM NaCl solution for 2 hrs and transferred to pots)

    Th b ti t th t b th th DREB1 DNA d th d29A

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    These observations suggest that both the DREB1 cDNA and the rd29A

    promoter used to improve the dehydration, salt and freezing tolerance of

    agriculturally important crops by gene transfer.

    The future transgenic approaches is uncertain:

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    Reluctance of most consumers to accept transgenically modified food.

    Single gene change may not make any difference.

    In the future, pyramiding regulatory genes controlling various aspects of

    tolerance (i.e., ionic, osmotic homeostasis and damage control) in a single

    transgenic plant is expected to yield very high levels of tolerance to salt and

    other related stresses.

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