Isolation and characterization of two different cDNAs of Delta(1)-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding Delta(1)-pyrroli ne-5-carboxylate synthase (P5CS), the first enzyme of the proline biosynthe tic pathway, were isolated from a lambda Zap-cDNA library constructed from salt stressed il Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open readi ng frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and pr oB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% ident ity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thali ana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS -1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT -PCR approach. A rapid increase in the steady-state transcript level of bot h genes in roots was observed by RT-PCR upon exposure of hydroponically gro wn 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducib le genes, yet a higher response was observed for MsP5CS-2.