Responsive modes of Medicago sativa proline dehydrogenase genes during salt stress and recovery dictate free proline accumulation

Free proline accumulation is an innate response of many plants to osmotic stress. To characterize transcriptional regulation of the key proline cycle enzymes in alfalfa (Medicago sativa), two proline dehydrogenase (MsPDH) genes and a partial sequence of Delta(1)-pyrroline-5-carboxylate dehydrogenase (MsP5CDH) gene were identified and cloned. The two MsPDH genes share a high nucleotide sequence homology and a similar exon/intron structure. Estimation of transcript levels during salt stress and recovery revealed that proline accumulation during stress was linearly correlated with a strong decline in MsPDH transcript levels, while Delta(1)pyrroline-5-carboxylate synthetase (MsP5CS) and MsP5CDH steady-state transcript levels remained essentially unchanged. MsPDH transcript levels dramatically decreased in a fast, salt concentration-dependent manner. The extent of salt-induced proline accumulation also correlated with salt concentrations. Salt-induced repression of MsPDH1 promoter linked to the GUS reporter gene confirmed that the decline in MsPDH transcript levels was due to less transcription initiation. Contrary to the salt-dependent repression, a rapid induction of MsPDH transcription occurred at a very early stage of the recovery process, independently of earlier salt treatments. Hence our results suggest the existence of two different regulatory modes of MsPDH expression; the repressing mode that quantifies salt concentration in an as yet unknown mechanism and the "`rehydration''-enhancing mode that responds to stress relief in a maximal induction of MsPDH transcription. As yet the components of salt sensing as well as those that might interact with MsPDH promoter to reduce transcription are still unknown.