Regulation of proline biosynthesis in plants subjected to osmotic stress

Drought and salinity stresses are the limiting factors for plant growth and productivity. Adaptation of plants to environmental stresses involves morphological, physiological as well as biochemical changes including accumulation of compatible solutes, such as proline, manitol, and betaine. More than 80% of plant species accumulate proline under stress conditions, and proline is thought to play an important role in plant cells for adaptation to osmotic stress. Proline accumulation has shown to increase osmotolerance in bacteria and plants.; In plants, proline is synthesized from both glutamate and ornithine, and the glutamate pathway for proline production is predominant in plants under osmotic stress and nitrogen starvation. Two enzymes, {dollar}{bsol}Delta{bsol}sp1{dollar}-pyrroline-5-carboxylate synthetase (P5CS) and {dollar}{bsol}Delta{bsol}sp1{dollar}-pyrroline-5-carboxylate reductase (P5CR), are involved in proline biosynthesis from glutamate. The first enzyme of the pathway, P5CS, is the rate-limiting enzyme in the biosynthesis of proline. A P5CS cDNA isolated from Vigna aconitifolia was expressed in E. coli, and the expressed enzyme was purified to homogeneity. Vigna P5CS exhibits two enzymatic activities, {dollar}{bsol}gamma{dollar}-glutamyl kinase ({dollar}{bsol}gamma{dollar}-GK) and glutamate-5-semialdehyde (GSA) dehydrogenase. The {dollar}{bsol}gamma{dollar}-GK activity of the purified P5CS was detected by the hydroxamate assay and by a ({dollar}{bsol}sp{lcub}14{rcub}{dollar}C) glutamate assay. The native molecular mass of the P5CS was found to be 450 kDa with six identical subunits. The Vigna P5CS showed a {dollar}K{bsol}sb{lcub}m{rcub}{dollar} of 3.6 mM for glutamate while the {dollar}K{bsol}sb{lcub}m{rcub}{dollar} for ATP was 2.7 mM. The {dollar}{bsol}gamma{dollar}-GK activity of the P5CS is competitively inhibited by proline and ADP while its GSA dehydrogenase activity is insensitive to proline inhibition. In addition, a protein inhibitor of the P5CS was detected in the plant cell. Western blot analysis, using polyclonal antibody raised against Vigna P5CS enzyme, showed that the level of the P5CS protein was enhanced in Vigna roots treated with NaCl. Two amino acid residues of the P5CS were identified to be involved in proline binding and a single substitution of an alanine for a phenylalanine at position 129 of the P5CS protein resulted in a significant reduction of proline feedback inhibition. The 50% inhibition values of {dollar}{bsol}gamma{dollar}-GK activity of the wild-type and the mutant P5CS were observed at 5 mM and 960 mM of proline, respectively. Other properties of the mutant P5CS remained unchanged. These results may allow genetic manipulation of proline biosynthesis and overproduction of proline in plants for conferring water stress tolerance.; The promoter of Arabidopsis P5CS gene was isolated and analyzed using a {dollar}{bsol}beta{dollar}-glucuronidase (GUS) reporter gene in transgenic plants. NaCl stress induced GUS activity to the maximum level within 3 hr while dehydration continued to enhance the GUS activity up to 24 hr. The AtP5CS gene promoter was found to contain two transcription start sites, and dehydration stimulated the transcription predominantly from the downstream site. Abscisic acid (ABA) failed to induce AtP5CS gene expression although ABA is known to increase during water stress. These observations suggest that induction of the AtP5CS gene is the response of plants to salt and water stresses. Although ABA is not directly involved in the AtP5CS gene expression, it is possible that ABA is involved in a post-transcription regulation of the P5CS gene expression and/or regulation of proline dehydrogenase (PDH) gene expression. A reciprocal increase and decrease in the levels of the P5CS and the PDH leads to proline accumulation in plants.