Glycine Betaine pathway

Plant growth and productivity are adversely affected by nature in the form of various abiotic stress condition such as drought, flooding, salt, low and high temperature, oxidative stress factors, their cells protect themselves from high concentrations of intracellular salts by accumulating a variety of small organic metabolites that are collectively referred to as compatible solutes/osmoprotectants
Compatible solutes are soluble in water and non-toxic even at higher concentration.
These metabolites allow cells to retain water and help in avoiding disturbances in their normal function when exposed to abiotic stresses.

Compatible solutes are classified into three major groups;
(1)Polyol(e.g.mannitol, etc),
(2)Amino acids (e.g. proline)
(3)Quaternary amines (e.g. glycine betaine)

1. By Tanvi chauhan WELCOME

2. “ ENGINEERING OF GLYCINE BETAINE PATHWAY ITS IMPORTANT EFFORTS AND ASSOCIATED PROBLEMS ”

3. HIGHLIGHTS Introduction Osmoprotectants Definition of GB Structure & Function of GB Availability of the precursors of glycine betaine pathways Pathway for Biosynthesis of Glycine Betaine Mechanism and protective role of glycine betaine pathway GB protection of photosynthesis machinery and ROS detoxification during abiotic stress Stratgeies for genetic engineering in case of drought Genetic Engineering of Glycine Betaine Pathway In Plants Transgenic plants with GB Application of glycine betaine pathway Future prospects Case study Conclusion References

4. Introduction Plant growth and productivity are adversely affected by nature in the form of various abiotic stress condition such as drought ,flooding ,salt , low and high temperature , oxidative stress factors ,their cells protect themselves from high concentrations of intracellular salts by accumulating a variety of small organic metabolites that are collectively referred to as compatible solutes/ osmoprotectants Compatible solutes are soluble in water and non toxic even at higher concentration . These metabolites allow cells to retain water and help in avoiding disturbances in their normal function when exposed to abiotic stresses .

5. Osmoprotectants : Osmoprotectants are small, nontoxic molecules that raise the osmotic potential of the cytoplasm without disrupting metabolism and also stabilize protein and membrane structures . Osmoprotectants are small molecules that can benefit osmotically stressed cells in two ways: B y acting as nontoxic cytoplasmic osmolytes to raise osmotic pressure B y protecting enzymes and membranes against damage by salt levels.. osmotic osmoprotectant

6. Compatible solutes are classified into three major groups; (1) Polyol ( e.g.mannitol , etc), (2)Amino acids (e.g. proline ) (3)Quaternary amines (e.g . glycine betaine )   Many higher plants do not accumulate glycine betaine or any their Osmoprotectants, and this has led to interest in the metabolic engineering of the glycine betaine biosynthesis pathway as an approach for enhancing stress resistance. Higher plants synthesize glycine betaine in chloroplasts via the pathway.

7. (1) Polyols Polyols is a chemical class of osmoprotectent . It occurs in small families of higher plants like Rice,Spinach etc. Accumulation of polyols in various plant species is related to high tolerance to salt and drought stress ( Bohnert and Jensen, 1996). Polyols protect membranes and enzyme complexes from reactive oxygen (ROS) species mainly by interacting with enzymes. For e.g. Mannitol , an important member of the polyols , studied in model plants such as Arabidopsis and tobacco .

8. (2)Amino acids (e.g. proline ) Proline accumulation has been reported in various plant species during a wide range of abiotic stresses ( Hayat et al, 2012). In plants, proline accumulation has been reported during osmotic stress induced by salt and drought stresses (Delaney and Verma , 1993 ). The primary function of proline in plants is to counteract the osmotic effects by stabilizing protein structures and scavenging free radicals. Apart from the above, proline also serves to store carbon and nitrogen. Application of proline play an important role in enhancing plant stress tolerance. This role can be in the form of either osmoprotection or cryoprotection . For example, in various plant species growing under saline conditions, exogenously supplied proline provided osmoprotection and facilitated growth .

9. (3) Quaternary amines (e.g. glycine betaine ) Betaines are quaternary ammonium compounds in which the nitrogen atom is fully methylated . Trimethylglycine was the first betaine discovered; Originally it was simply called betaine because, in the 19 th century, it was discovered in sugar beets. The most common betaines in plants include glycine betaine (GB; the most widely studied betaine). e.g of betaines : proline betaine , β- alanine betaine , choline - O -sulfate and 3-dimethylsulfoniopropionate these are also termed as by specific name glycine betaine . A sweet tasting crystalline alkaloid, C 5 H 11 NO 2 , found in sugar beets and other plants, used to treat certain metabolic disorders, especially an enzyme defect that causes excessive levels of homocysteine in the blood and urine.

10. DEFINITION OF GB Its Structure Glycine betaine (GB) is a small, water-soluble organic molecule that is essential to protect plants, animals, and bacteria against abiotic stress . Glycine betaine is a quaternary ammonium compound that occurs naturally in a wide variety of plants, animals and microorganisms. It is a dipolar but electrically neutral molecule at physiological pH Glycine betaine

11. Function of GB: The major role of Glybet in plants exposed to saline soil is probably protecting plant cells from salt stress by osmotic adjustment & protein stabilization . A physiological role of betaine in alleviating osmotic stress was proposed i.e. based on enhanced accumulation of betaine in some plants subjected to osmotic stress. Betaine may also stabilize the photosystem II protein-pigment complex in the presence of high NaCl concentrations. Glycine betaine (GB) is zwitter ionic fully N-methyl substituted derivatives of glycine. It Plays important role in higher plants such as maize, barley, sugarbeet and spinach. It protects various component of photosynthetic machinery and oxygen evolving photosystem II and maintains highly order state of membranes at non physiological temperature and high salt concentration. The major role of GB in plants exposed to saline soil and reduction of oxygen radical scavengers.

12. Availability of the precursors of glycine betaine pathways Choline Choline [(CH 3 ) 3 N+CH 2 CH 2 OH] is a methylated nitrogen compound that is a common constituent of eukaryotic membranes in the form of phosphatidylcholine and therefore should be widespread in the marine environment . It is a precursor of glycine betaine [(CH 3 ) 3 N+CH 2 COOH] , one of the most potent Osmoprotectants known. Choline biosynthesis occurs in the cytosol and is then transported to chloroplast for GB production in higher plants. In transgenic plants, choline availability is one of the main factors that limit GB accumulation. Based on these findings, choline availability in the chloroplast is crucial to GB biosynthesis and subsequent stress tolerance.

13. Pathway for Biosynthesis of Glycine Betaine: site of glycine betaine accumulation: The tolerance of plant to abiotic stress is influenced by two factors that are concentration and localization of GB in cells. There are many reports engineered Accumulation of GB in plants in which GB biosynthetic enzyme have been targeted to chloroplast. However , there are few studies shows that the enzymes have been targeted to either cytosol or mitochondria , or to both cytosol and chloroplast simultaneously. GlyBet occurs in some but not all higher plants, as well as in bacteria and other organisms Rice plant do not accumulate GB naturally but the highest level of accumulated GB have found in leaves of cod­A transgenic rice plants 53umol -1 FW while in a natural GB Accumulator maize the highest reported level of GB in leaves of bet A transgenic maize plant is 5.753umol -1 FW

14. Biosynthesis of glycine betaine pathway It is synthesized by a two-step oxidation of choline via betaine aldehyde , but different types of enzymes are involved . In E.coli , a membrane-bound, electron transfer-linked choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde . The aldehyde is then oxidized to GlyBet by a soluble, NAD-linked betaine aldehyde dehydrogenase (BADH). Bacteria have a soluble choline oxidase (COX) that carries out both oxidation steps and generates H2O2 . In plants , the first oxidation is mediated by a ferredoxin -dependent choline monooxygenase (CMO) and the second by BADH. the enzyme choline monooxygenase (CMO) first converts choline into betaine aldehyde and then a NAD dependent enzyme, betaine aldehyde dehydrogenase (BADH) produces glycinebetaine . These enzymes are mainly found in chloroplast stroma and their activity is increased in response to salt stress. All these enzymes have been used to engineer tobacco and other plants that lack GlyBet , generally by placing the transgenes under the control of the CaMV 35S promoter.

15. Recently , a novel pathway for GB synthesis from glycine was found in two extremely halophilic microorganisms, Actinopolyspora halophilia and Ectothiorhodospira halochloris . In these microorganisms a three-step successive methylation of the amino residue is catalyzed by two enzymes, glycine sarcosine methyltransferase (GSMT) and sarcosine dimethylglycine methyltransferase (SDMT), with S - adenosylmethionine as the methyl group donor. Both GSMT and SDMT are capable of catalyzing the three steps of methylation because of their partially overlapping specificity for the substrates. Genes that encode the enzymes involved in the biosynthesis of GB have been cloned. They include genes for choline monooxygenase (CMO) and BADH from higher plants; CDH and BADH from Escherichia coli ; choline oxidase (i.e. codA from Arthrobacter globiformis and cox from Arthrobacter pascens ; and glycine sarcosine methyltransferase (GSMT) and sarcosine dimethylglycine methyltransferase (SDMT) from both Actinopolyspora halophilia and Ectothiorhodospira halochloris .

16. Mechanism and protective role of glycine betaine pathway

17. Main mechanisms have been proposed for GB's responsibility for enhanced stress tolerance : Osmotic adjustment controlling the absorption of water from the surroundings, Reactive oxygen species (ROS) scavenging Stabilization by GB of the highly ordered structures of certain complex proteins to prevent denaturation when plants or plant cells are exposed to stress conditions . Induction by GB of the expression of specific genes that encode reactive oxygen species (ROS) ­scavenging enzymes and subsequent depression of levels of ROS in plant cells and Prevention by GB of the accumulation of excess ROS, resulting in protection of the photosynthetic machinery NADPH from the combined effects of light stress and other kinds of stress as well as of ion­channel proteins and the integrity of cell membranes . The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavourable conditions.

18. GB protection of photosynthesis machinery and ROS detoxification during abiotic stress GB has also been implicated in protection of quaternary structure of proteins from damaging effects of environmental stresses. Many proteins are prone to aggregation under heat and salt stress there by , losing their native structure and activity. GB has been shown to protect the photosynthesis machinery by stabilizing the activity of repair proteins under high concentrations of NaCl . The role of GB in ROS detoxification is also evident by reduced accumulation of ROS in transgenic plants under water deficit stress

19. Strategies For Genetic Engineering e.g . In case of Drought Tolerance :

20. Genetic Engineering Of Glycine betaine p athway in p lants : GB confers osmoproctection in bacteria, plants and animals and protects cell components against harsh condition in vitro . Major cereals like wheat, maize and barley do not accumulate significant amount of GB naturally. This could be due to the production of transmuted transcripts for GB biosynthesizing enzyme (BADH) among these, rice is the only cereals that does not accumulate GB naturally as well as under stress condition . Like rice, many crop plants such as Arabidopsis, Mustard, Tobacco and Tomato do not accumulate GB and are therefore potential target for engineering the GB biosynthesis.

21. Use of GB biosynthetic genes in Rice transgenic plants   In Rice has two BADH and one CMO encoding genes, however, no GB accumulation occurs in rice under stress. The BADH transcripts are processed in an unusual manner in rice resulting in removal of translational initiation codon , loss of functional domains and premature stop codons . However, some correctly spliced BADH transcripts have also been reported from rice. Exactly similar observations were made for CMO transcripts in rice by same group.

22. The BADH transcripts are processed in an unusual manner in rice resulting in removal of translational initiation codon , loss of functional domains and premature stop codons . However, some correctly spliced BADH transcripts have also been reported from rice. Exactly similar observations were made for CMO transcripts in rice by same group. However, transgenic rice plants expressing functional BADH gene from barley could convert exogenously applied betaine aldehyde to GB at a level better than WT plants. Like rice many crop plants lack the ability to accumulate GB naturally during abiotic stress . Identification of genes of GB biosynthetic pathways has made it easy to engineer GB biosynthesis into non accumulators by transgenic approach for improved stress tolerance. This approach has been successfuly used in diverse plant species, e.g., Arabidopsis, tobacco, Brassica , Persimmon, tomato, maize, rice, potato and wheat to improve their abiotic stress tolerance. Availability of endogenous choline , therfore , could limit the GB biosynthesis in transgenic plants. However, levels of endogenous choline were not changed significantly in transgenic Arabidopsis and rice plants expressing codA gene. Therefore , availability of choline does not affect the GB synthesis in these transgenic plants probably due to synergism in demand and supply of choline metabolism.

23. Heat tolerance Figure High temperatures also limit the growth and productivity of plants. in vitro experiment indicated that GB protects some enzymes and protein complexes from heat induced destabilization. Therefore, it has been postulated that GB increases resistance to high temperature stress. More recent experiments showed that transformed Arabidopsis that accumulated GB exhibited enhanced tolerance to high temperatures during the imbibition and germination of seeds, as well as during the growth of young seedlings. It also seems likely that GB might alleviate the effects of heat shock because the extent of the induction of Heat shock proteins was significantly reduced in these transgenic plants.

24. Salt tolerance: Figure It has been demonstrated, through studies of both plant physiology and genetics, that the level of accumulated GB is correlated with the degree of salt tolerance. Transgenic Arabidopsis plants that produced COD in their chloroplasts not only acquired resistance to high concentrations of NaCl during germination but also were able to tolerate high levels of salt during the subsequent growth of seedlings and mature plants. Transformation of tobacco with a gene for CDH also enhanced plant growth under salt stress, although the level of GB was much lower than that in ‘COD engineered’ Arabidopsis.  

25. Transgenic plants with GB The GB biosynthetic genes in transgenic plants proved very effective in conferring stress tolerance compared to that of other osmoprotectant genes. Several studies have reviewed the important roles of GB in transgenic plants under various abiotic stresses. Transgenic plants such as Arabidopsis, eucalyptus, tobacco, rice, tomato, potato and wheat with GB biosynthetic genes have showed increased GB accumulation and Metabolic engineering of plants for GB biosynthesis . A number of transgenic plants with GB biosynthetic genes have been tested for GB accumulation and the resultant salt, drought and temperature tolerance . The GB accumulation was targeted in the chloroplast, in most of the transgenic plants, where its increased concentration conferred protection against various abiotic stresses, particularly salt, and drought stresses. Overall, in transgenic plants the accumulated GB content and the resultant stress tolerance is believed to be influenced by three factors: choline (precursor for GB) availability, type of transgene of the GB biosynthetic pathway and the type of promoter (constitutive and stress-inducible). In some GB-transgenic plants, it was reported that the accumulated GB not only conferred stress tolerance but also improved reproductive and yield components such as flowers and fruits.

26. Transformation of tobacco with a gene for CDH also enhanced plant growth under salt stress, although the level of GB was much lower than that in ‘COD-engineered’ Arabidopsis Arabidopsis plants that produced COD in their chloroplasts not only acquired resistance to high concentrations of NaCl during germination but also were able to tolerate high levels of salt during the subsequent growth of seedlings and mature plants. In addition, Brassica juncea and Japanese persimmon ( Diospyros kaki ) have been successfully transformed to tolerate salt stress through the introduction and overexpression of a gene for COD

27. APPLICATION OF GLYCINE BETAINE PATHWAY Drought in case of glycine betaine pathway- Drought trigger a wide variety of plant responses, Ranging from cellular metabolism to changes in growth rate and crop yields. The naturally occurring quartenary ammonium compound GB has received attention as a compatible solute that may aid in drought tolerance by allowing mentainance of turgor pressure . Salinity and glycine betaine- GB give adverse effect of salt stress by changing photosynthetic activity in many crop like Tomato, Maize Wheat and Sunflower which mainly occur due to stomatal limitation . Net photosynthetic rate and stomatal conductance showed a significant positive relationship and positively correlated with substomatal carbon dioxide .

28. Low temperature and glycine betain - Chilling injury that cause physical and physiological changes induced by exposure to low temperature is another primary factor which limit crop production worldwide. Exogenous GB is effective in inducing cold tolerance in unhardened and cold hardening plant of Strawberry. Oxidative stress and Glycine betaine Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. R O S inactivate enzymes and damage important cellular components This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation .

29. Future prospects Glycine betaine appears to play an important role in the responses of plant cells to a variety of stresses, and transgenic approaches have shed some light on the ways in which GB protects plants from stress. In present scenario, Current research efforts are focused on the elucidation of the mechanisms by which GB protects the cellular machinery in vivo and how, as a result, it enhances the tolerance of whole plants to environmental stress. Transgenic plants accumulate GB at levels of 50–100 m M at most, with substantial effects on stress tolerance. Further studies of transgenic plants are necessary, particularly at the cellular level, to address this issue and resolve the discrepancy. Finally, the application of new technologies such as DNA arrays and proteome analysis should help to reveal additional possible functions of GB in vivo , for instance the maintenance of transcription and translation under stress conditions

30. CASE STUDIES

31. Effect of water stress on proline metabolism and leaf relative water content in two high yeilding genotypes of groudnut ( Arachis hypogaea L. ) with contrasting drought tolrence .( Ranganayakulu , G.S., et al ., 2015. Jour. Of Exp. Bio. And Agri. Sci. Vol.3 no.1 pp) Objective: Comparative study was done out for two groundnut cultivars ( cv K-134 and cv JL-24, drought tolerant and drought sensitive, respectively) during water stress at different soil moisture levels [100 (control), 75, 50 and 25%] The, activities of pyrroline-5-carboxylate reductase (P-5-CR), proline oxidase , proline dehydrogenase (PDH) along with levels of quaternary ammonium compounds, leaf relative water content and chlorophyll stability were investigated . Case study 1

32. free proline content quantification Water stress resulted in a significant accumulation of free proline content in leaves of both groundnut cultivars cv K-134 accumulated relatively higher amounts of proline than cv JL-24 Water stress resulted 2.5 fold higher accumulation of proline in cv K-134 and 2.0 fold in cv JL-24 on day-5 at 25% SMLs. Procedure:

33. Furthermore, the tolerant cv K-134 shows a greater activity of pyrroline-5-carboxylate reductase and lesser inhibition of proline oxidase and proline dehydrogenase than susceptible cv JL-24. The greater proline levels were due to both the higher rates of proline synthesis and lower rate of proline oxidation in cv K-134 compared to cv JL-24 This study showed that water stress altered the proline metabolism and this alternation was significantly varied between the cultivars. Further, drought tolerance of cv K-134 can be justified by the higher accumulation of quaternary ammonium compounds ( glycine betaine ) which helps in the maintenance of leaf relative water content and higher chlorophyll stability during water stress as compared to cv JL-24. Findings

34. Case study 2 Accumulation of Glycinebetaine in Rice Plants that Overexpress Choline Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic Stress( Shirasawa .K., et al ., 2006. Annals of Botany 98: 565–571) Objective: Glycinebetaine (GB),is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice ( Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice.

35. Transgenic rice plants that over expressed a gene for CMO from spinach ( Spinacia oleracea ) were produced by Agrobacterium -mediated transformation After Southern blotting western blotting analysis Quantitation of glycine betaine and choline in rice leaves was quantified by NMR spectroscopy The tolerance of GB-accumulating plants to abiotic stress was investigated.

36. Findings Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 029–043 umol /g d . wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADH might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO expressing rice plants were not effective for accumulation of GB and improvement of productivity. The transformants also exhibited tolerance to salt stress and temperature stress in the seedling stage, not enough GB is accumulated in the plants to improve their productivity. The CMO from a higher plant, spinach, has proved to be less effective for the accumulation of GB in non-GB-accumulating rice plants than bacterial COD and CDH.

37. Transgenic potato plants ( Solanum tuberosum L. cv. Superior) with the ability to synthesize glycinebetaine (GB) in chloroplasts (referred to as SC plants) developed via the introduction of the bacterial choline oxidase ( codA ) gene under the control of an oxidative stress-inducible SWPA2 promoter. CASE STUDY 3 Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses(R. Ahmad. etal ., Plant Cell Rep (2008) 27:687–698) Increasing salinity can also significantly reduce the total and average yields of different potato cultivars, and the presence of 50 mM NaCl can result in a 50% reduction in the growth of potato plants Potato ( Solanum tuberosum L.) is one of the major food crops in many world regions, and ranks fourth in production after wheat, maize, and rice. Objective

38. Plant transformation and regeneration Plantlets, which were capable of developing good root systems on selection medium, were screened further for the presence of the codA gene via PCR. All subsequent experiments were conducted on the T0-generation of transgenic plants. Total RNA was extracted RT-PCR analysis Glycinebetaine analysis* Salt-stress treatment Drought-stress treatment PROCEDURE

39. Glycinebetaine analysis These extracts were treated with the strong anion exchange resin, AG 1-X8 (Bio-Rad, Hercules, CA, USA), liquid nitrogen frozen leaves were powdered with a ceramic mortar and pestle This powder (2 g) was then suspended in 2 mL of ice-cold methanol: chloroform: water (60:25:15) and thoroughly vortexed . An equal volume of distilled water was added to the tubes. The resultant homogenate was shaken gently for 10 min, and then centrifuged for 10 minutes at 5000rpm at room temperature. The upper methanol-water phase was transferred to clean tubes. The extracts were freeze-dried and dissolved in distilled water (2 mL ).

40. Micro Bio-spin chromatography columns (Bio-Rad, USA) were packed with AG 1-X8 resin via the addition of 1 mL of resin slurry and centrifuged at 5000rpm at room temperature Afterward, 1 ml of crude GB extract was loaded into the resin bed and centrifuged for 3 minutes at 5,000rpm. The resin was washed with 0.5 mL of distilled water and mixed with the previous flow-through fraction. GB was measured via high performance liquid chromatography, Purified GB was detected .

41. The GB peak and quantification was monitored using a UV detector . FINDING The transgenic lines harboring the codA gene evidenced enhanced tolerance to a variety of stresses. As the codA gene was driven by a stress-inducible promoter, the induction pattern of the codA gene under different stress conditions, including Methyl viogent salt, and drought verifies the stable integration and transcription of foreign genes in SC Plants tolerance of transgenic plants expressing stress induced and constitutive GB-synthesizing genes, indicating that the accumulation of biomass tends to be greater in the case of stress-induced GB producers than in constitutively GB-producing plants during salt stress.

42. CASE STUDY 4 Exogenous Proline and Glycine Betaine Mediated Up regulation of Antioxidant Defense and Glyoxalase Systems Provides Better Protection against Salt-Induced Oxidative Stress in Two Rice ( Oryza sativa L.) Varieties( H. Mirza ., etal., BioMed Research Internlt . Vol. 2014, 17 pg ) Objective: The roles of exogenous proline (Pro, 5mM) and glycine betaine (GB, 5mM) in improving salt stress tolerance in salt sensitive (BRRI dhan49 ) and salt tolerant (BRRI dhan54) rice ( Oryza sativa L.) varieties. Salt stresses (150 and 300mM NaCl for 48 h) endogenous Pro and increased lipid peroxidation and H2O2 levels. reduced significantly leaf relative water (RWC) chlorophyll ( chl ) content increased

43. Plant Materials and Stress Treatments. Measurement of Relative Water Content. Determination of Proline Content. Measurement of Lipid Peroxidation . Measurement of H 2 O 2 . Extraction and Measurement of Ascorbate and Glutathione. Determination of Protein. Enzyme Extraction and Assays. Statistical Analysis.

44. They suggests that exogenous application of Pro and GB increased rice seedlings’ tolerance to salt-induced oxidative damage by upregulating their antioxidant defense system where these protectants rendered better performance to taken rice variety and Pro can be considered as better protectant than GB. FINDINGS

45. Conclusion According Engineering transgenic plants with abiotic stress tolerance that utilized genes encoding osmoprotectants , and other stress-related functional proteins. In comparison to other genes, biosynthetic accumulation of glycine betaine, proline and other osmoprotectant genes in several transgenic crop plants have shown some improvement in abiotic stress tolerance. However, the success of these genes has been limited in the sense that most of the developed transgenic plants have been tested under controlled laboratory conditions response pathway

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