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In this article we will discuss about the production of osmo-protective compounds.
Plants exhibit certain degree of defences against various environmental stresses by producing various osmoprotectants (Table 15.1). Accumulation of osmoprotectants and osmolytes like quarternary amines, amino acids and sugar alcohols increases their stress tolerance. Stress tolerant plants accumulating such compounds are more effective in tackling stress problem than others.
Excess accumulation of osmoprotectants enhances osmotic potential of the cell. Thus, balancing the osmotic potential of an externally increased osmotic pressure and also stabilize the membrane and macromolecular structure especially in the protection of vital proteins.
Production of Glycine Betaine:
Glycine betaine is widely distributed in several families of the flowering plants. It is a quaternary amine extremely soluble in water. Glycine betaine, due to its methyl groups, has the potential to interact with hydrophilic as well as hydrophobic molecule and consequently stabilize proteins and membranes. Glycine betaine, in general, protects plants’ cells from salt stress by maintaining osmotic balance with the surrounding environment. Several plant crops such as potato, rice and tomato do not accumulate osmoprotectant i.e., glycine betaine.
Synthesis of glycine betaine in plants is a two-step conversion. In the species belonging to Chenopodiacease and Poaceae families, glycine betaine is synthesized by oxidation of choline, catalyzed by choline monoxygenase (CMO) and converted into betaine aldehyde. In the second step, betaine aldehyde is converted to glycine betaine in presence of betaine aldehyde dehydrogenase. These two enzymes involved in betaine synthesis are found in the stroma of chloroplast and are induced by stress. Choline monoxygenase activity is light dependent. Both these genes have been cloned in several plants.
Biosynthesis of glycine betaine in E. coli takes different pathway. Conversion of choline to glycine betaine is aided by choline dehydrogenase (CDH) via betaine aldehyde as intermediate. In the subsequent step, conversion by betaine aldehyde dehydrogenase exhibit strong similarity between plants and bacteria. Biosynthesis of glycine betaine is still simpler and it is one step conversion in certain bacteria. In Arthrobacter globuliformis, choline oxidase (COD) directly converts choline into glycine betaine.
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In view of enhancing stress tolerance using transgenics, bacterial betaA gene encoding choline dehydrogenase (CDH) was introduced into tobacco. Transgenic tobacco conferred 80 percent increase in salt tolerance on comparison with wild type, grown at 300 mM NaCl.
Another possibility of exploiting strategy for increasing stress tolerance is the introduction of gene for choline oxidase (COD gene) from Arthrobacter globuliformis. Transgenic plants expressing this enzyme catalyze both steps in coverting choline into glycine betaine.
Both salt and freezing tolerant plant was created by introducing COD A gene into Arabidopsis using transit peptide to be localized in the chloroplast. Accumulation of glycine betaine upto 50 mM was established in transgenic plants. In addition, transgenic Arabidopsis expressing bacterial choline oxidase has also exhibited enhanced tolerance to light stress in terms of photosynthetic activity.
Proline:
Amino acid proline resembles glycine betaine structurally. Proline acts as osmoprotective agent. The p5cS gene from Vigna aconitifolia (moth bean), encoding bifunctional enzyme r- glutamyl kinase and glutamic-r-semialdehyde dehydrogenase, has been used to transform tobacco. This enzyme catalyzes the conversion of glutamate to pyrroline-carboxylase which is then reduced to proline. Transgenic tobacco expressing the enzyme produced 10-15 fold more proline than control plant.
Sugar Alcohol:
Sugar alcohol like mannitol D-ononitol helps in the stabilization of subcellular membrane and macromolecular structure. The bacterial gene mltd for mannitol production was expressed in Arabidopsis. Over production of mannitol in transgenic plants survives high salinity and increased tolerance to stress. Similarly, gene for myoinositol-o-methyl transferase (IMTI) has been transferred to tobacco and provides excellent protection against tolerance.
Trehalose:
Trehalose, a non-reducing disaccharide has been overproduced in tobacco by the expression of TPS1 gene encoding trehalose-6-phosphate synthase taken from yeast. Transgenic tobacco exhibits improved water retention and desiccation tolerance. In another approach for increased stress tolerance fructon producing enzyme was introduced into tobacco. This was achieved by transferring bacterial SacB gene from Bacillus subtilis. The SacB encodes the enzyme fructosyl transferase.
