genetic engineering for drought resistence in rice
TRANSCRIPT
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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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