Overexpression of a novel feedback-desensitized Δ1-pyrroline-5-carboxylate synthetase increases proline accumulation and confers salt tolerance in transgenic Nicotiana plumbaginifolia

Abstract

∆1-Pyrroline-5-carboxylate synthetase (P5CS) is a key enzyme of proline biosynthesis in plants. The Nicotiana plumbaginifolia mutant RNa was previously reported as salt tolerant, overproducing proline, and having reduced feedback inhibition of proline biosynthesis. A mutation in an RNa P5CS gene changing a conserved glutamate codon at position 155 into a glycine codon (E155G) is a possible explanation for the phenotypes in the RNa mutant. In this work, the E155G mutation was introduced in the Arabidopsis thaliana P5CS1 cDNA driven by the CaMV 35S promoter and expressed in wild type N. plumbaginifolia (P2) and in the RNa mutant. The E155G substitution decreased feedback inhibition of P5CS by proline in transgenic lines. Under normal conditions, proline content and growth were comparable in the transgenic lines and the P2 control. In contrast, when plants were exposed to salt stress, proline levels in all transgenic lines increased compared to P2. Growth rate was also less affected by salt treatment in the transgenic lines than in the controls. Semi-quantitative RT-PCR showed higher p35S-P5CS transgene derived mRNA levels in plants submitted to salt stress than in non-stressed plants. To evaluate the role of the CaMV 35S promoter during salt stress, N. plumbaginifolia lines harbouring a p35S-GUS cassette were investigated. Increased GUS activities were observed when submitting leaf discs of transformants to salt stress compared to normal conditions. This result can partly explain increased proline content in the p35S-P5CS transgenic plants when exposed to salt stress.