Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance

Abstract

Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ∆1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.