GENETIC ENGINEERING FOR DROUGHT RESISTENCE IN RICE

7/29/2019 GENETIC ENGINEERING FOR DROUGHT RESISTENCE IN RICE

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GENETIC ENGINEERING FOR DROUGHT

RESISTENCE IN RICE

SUBMITTED BY:

CHANDRAKANT SINGH

M.Sc. PLANT PHYSIOLOGY


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DROUGHT RESISTENCE

It is a water deficit stress, because the environmentalconditions either reduce the soil water potential orincrease the leaf water potential due to hot, dry, orwindy conditions.

High salt conditions caused water deficiency becausethe soil water potential is decreased, making it moredifficult for roots to extract water from environment.

High ambient temperatures caused water deficientcondition due increased water loss by evaporation .

Freezing temperature also caused osmotic stress dueto reduction in water potential.


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Drought situation can be classified as either

terminal or intermittent.

During terminal drought , the availability of

soil water decreases progressively and this

leads to premature plant death.

Intermittent drought is the result of finite

periods of inadequate irrigation occurring at

one or more intervals during the growingseason and is not necessarily lethal.


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Mechanism of drought resistance

Mechanism of drought resistance can be

grouped into three categories:

Drought escape

Drought avoidance

Drought tolerance


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Drought escape is the ability of plant to complete

its life cycle before serious soil and plant waterdeficit develop. Early rice (90-120 days)

Drought avoidance is the ability of plants to

maintain relatively high tissue water potentialdespite a shortage of soil moisture. e.g. leafrolling, thick cuticle, deep root

Drought tolerance is the ability to withstandwater-deficit with low tissue water potential. e.g.osmotic adjustment, green leaf retention


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Osmoprotactans

LEA protein:Hydrophilicity with which LEA protein is provided,

catch enough water from exterior space of cell inorder to protect plant cell against water stress.

LEA proteins wildly exist in various plants and LEAgenes have been well isolated and studied asdrought-inducible genes.

Dehydrin, a sort of the most important LEA

proteins, is hypothesized to stabilize membranesand macromolecules during cellular dehydrationand is considered to involve in the dormancy ofdrought-tolerant plants.


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Proline: Proline, one of the most common compatible

osmolytes, is one of the most particular on studyingrelated with plant osmoregulation in water-stressed

plants. In plants, L-proline is synthesized from L-glutamic acid

via delta(1)-pyrroline-5-carboxylate (P5C) by twoenzymes, P5C synthetase (P5CS) and P5C reductase(P5CR).

Under the drought stress or water stress, prolineaccumulation is preceded by a rapid increase of themRNA level of P5CS.


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Glycine betaine:

Glycine betaine (GB) is another importantosmolyte, existing in many organisms. Membersof the Chenopodiaceae, such as sugar beet andspinach, accumulate glycine betaine in response

to drought stress.Plants synthesize the osmoprotectant glycine

betaine via choline betaine aldehyde glycinebetaine.

Two enzymes are involved in the pathway,choline monooxygenase (CMO) and betainealdehyde dehydrogenase (BADH).


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Soluble sugars:The accumulation of the soluble sugars that play

a role in the osmoregulation for droughtresistance in plants, is often observed under

drought stress or dehydration.Soluble sugars include levan, trehalose, sucrose,

etc.

Levan is soluble in the cell of plants, and deducesthe water potential of the cell, and participatesosmoregulation of the cell when encounteringdrought stress.


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Trehalose affects sugar metabolism as well asosmoprotection, and trehalose also is able to restrainthe transforming of the cellular phosphatide fromcrystalline liquid state to solid state and stabilizes the

structure of the high molecular compound againstseveral environmental stresses, such as drought anddesiccation.

The accumulation of sucrose is helpful to promote the

ability to resist drought, and sucrose is important indrought resistance of plant as well as storage andmetabolism of the energy.


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DREB(dehydration responsive element

binding factor) gene

Liu et al. (1998) isolated and analyzed twocDNA clones that encode DRE bindingproteins, DREB1A and DREB2A by using the

yeast one-hybrid screening technique.Both the DREB1A and DREB2A proteins

specifically bound to the DRE sequence invitro and activated the transcription of the b-glucuronidase reporter gene driven by theDRE sequence inArabidopsis leaf protoplasts.


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Biotechnological approach for drought

resistance Mainly two approaches namely targeted and shotgun

approach facilitate genetic engineering to obtain transgenic

plants conferring drought resistance.1. Targeted approach:

For synthesis of these polyamine, proline, glycine betaineand utilizes the related genes to transfer them fromdifferent sources to crop plants.

The gene TPS1 found in yeast encodes for trehalose-6-phosphate synthetase and is involved in biosynthesis of

trehalose. Another gene, P5CS, encodes for pyrroline-5- carboxylate

synthetase which is involved in proline synthesis, and theover-production of proline confers drought resistance.


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The genes betA encoding for choline dehydrogenase andbetB encoding for betaine aldehyde dehydrogenase areinvolved in the biosynthesis of glycine betaine andaccumulation of glycine betaine confers drought resistance.

2. Shotgun approach:

This approach to obtain the desired gene is indirect. Arandom analysis of stress-related alteration in cell processand gene expression is employed.

Transgenic rice carrying barley hva1 gene produced through

this approach has shown drought resistance. Gene hva1encodes for a group of three LEA (late embryogenesisabundant) proteins which gets accumulated in vegetativeorgans during drought condition.


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Marker-assisted selection

Molecular markers such as restriction fragment lengthpolymorphism (RFLP), random amplified polymorphicDNA (RAPD) and isozyme will facilitate to developdrought-resistant genotypes more effectively as their

expressions are independent of environmentaleffects.

The application of marker-assisted selection in evolvingdrought resistant genotypes is in an experimental

stage; more specifically just identification of RFLPmarkers associated with osmotic adjustment.


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CASE STUDIES


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Over-expression of OsDREB genes lead to enhanced

drought tolerance in rice

DREB transcription factors, which specifically interact with C-repeat/DRE (A/GCC

GAC), play an important role in plant abiotic stress tolerance by controlling the

expression of many cold or/and drought-inducible genes in an ABA-independent

Pathway.

Three novel rice DREB genes, OsDREB1E, OsDREB1G, and OsDREB2B, which are

homologous to Arabidopsis DREB genes and which specifically bound to C-

repeat/ DREB where isolated.

Transgenic rice plants analysis revealed that over-expression of OsDREB1G and

OsDREB2B in rice significantly improved their tolerance to water deficit stress,while over-expression of OsDREB1E could only slightly improved the tolerance to

water deficit stress, suggesting that the OsDREBs might participate in the stress

response pathway in different manners.

Qiang Chen et al.(2008).China


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The survival rate of the transgenic rice under water

deficit stress.


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Transgenic analysis of rice DREB genes.

Transgenic rice harboring OsDREB1G, OsDREB2B showed the enhanced tolerance

to water deficit, but transgenic rice harboring OsDREB1E was not significant;


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Over-expression of a LEA gene in rice improves

droughtresistance under the field conditions

LEA protein gene OsLEA3-1 wasidentifiedand over-expressed in rice to test

the drought resistance of transgenic lines under the field conditions OsLEA3-1

is induced by drought, salt and abscisic acid (ABA), but not by cold stress.

The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibitsstrong activity under drought- and salt-stress conditions

Three expression constructs consisting of the full-length cDNA driven by the

drought-inducible promoter ofOsLEA3-1 (OsLEA3-H), the CaMV 35S promoter

(OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed intothe drought sensitivejaponica rice.

Xiao et al.(2007)China


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RNA gel blot analysis ofOsLEA3-1 expression in 3-week old IRAT109 seedlings

treated with drought, 200 mM NaCl, 100 M ABA, and cold

GUS activity assay ofOsLEA3-1 promoter:: GUS in 3-week old transgenic plants treated bydrought stress (with water deprived from the hydroponic cultured plants), salt (200 mM

NaCl), ABA (100 M) and cold (4C) stress


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Molecular identification of transgenic plants.

Schematic diagram of the constructs for rice transformation. P represents CaMV 35S

(S), Actin1 (A), or HVA1-like promoter (H). LB and RB represent T-DNA left and right

border, respectively.

OsLEA3-S


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OsLEA3-H

OsLEA3-A


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Improvement of water use efficiency in rice by

expression of HARDY, an Arabidopsis drought

and salt tolerance geneThe expression of the Arabidopsis HARDY(HRD) gene in rice improves

water use efficiency, the ratio ofbiomass produced to the water used, by

enhancing photosynthetic assimilation and reducing transpiration.

The HRD gene, an AP2/ERF-like transcription factor, identified by again-of-function Arabidopsis mutant hrd-D having roots with enhanced

strength, branching, and cortical cells, exhibits drought resistance and

salt tolerance, accompanied by an enhancement in the expression of

abiotic stress associated genes.

HRD over expression in Arabidopsis produces thicker leaves with more

chloroplast-bearing mesophyll cells, and in rice, there is an increase in

leaf biomass and bundle sheath cells that probably contributes to the

enhanced photosynthesis assimilation and efficiency.

Karaba et al.(2007)India


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Fig. The hrd-D mutant phenotype in

Arabidopsis. (A) Rosette leaf phenotype of WT

and hrd-D mutant with smaller, slightly curled,

thicker deepgreen leaves.

(B) Cryo-fracture scanning electron microscopy

section of leaves of WT and hrd-D mutant,showing more mesophyll cell layers.

(C) Rootstructure of WT and hrd-D mutant,

showing more profuse secondary and

tertiary roots at the root base.

(D) Cross-section of WT and hrd-D roots,

showing increased cortical cell layers (lighterstained) and compact stele in the

mutant.


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Fig. Phenotype ofHRD over

expression in rice.

(A) Rice HRD over expression

line compared with WT

Nipponbare under well watered

(control) and water stress

(70% field capacity) conditions.

(B) Leaf cross-section ofWTand

HRD over expression lines,observed under fluorescence

microscope, revealing red

chlorophyll fluorescence and blue

vascular bundles surrounded by

the bundle sheath cells marked

with an arrow.(C) Number of bundle sheath cells

in WTcompared with HRD over

expressors, which show significant

increase


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Problems

There are many problems in application ofgenetic engineering to improve stress toleranceas follows:

Mechanisms for drought stress in trees havebeen little understood. So there are someproblems existing in application of geneticengineering for improving its drought resistance.

Sorts of drought-resistant genes are few andcannot satisfy the need of genetic engineeringfor improving drought tolerance of trees.


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Drought-tolerance are controlled by many genes.

So transgenic strategy of one gene is not perfect

and conduces some negative effects. For

example, the improvement of drought toleranceis often associated with abnormal growth of

trees.

Shortage of genes possessed independent patentalso restricts application of genetic techniques to

improve drought-resistance of trees.


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Future strategies

A single trait cannot confer drought resistance satisfactorily.

Therefore, breeding programme for drought resistance should aimat pyramiding a number of relevant traits in a crop.

Many different genes responsible for biosynthesis of differentsolutes and osmolytes conferring drought resistance should beconsidered for transfer in a crop plant at a time.

Attention should be concentrated on better understanding ofgenetic basis of drought resistance through antisense RNAtechnique, observing the effect of expression level of differentenzymes/ proteins in different biochemical pathways on droughtresistance.

A comparative assessment of various polypeptides produced in

response to drought, between sensitive and tolerant genotypesmay be used in identification of protein marker, which could help inproducing transgenic drought resistant plants.


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GENETIC ENGINEERING FOR DROUGHT RESISTENCE IN RICE