Molecular Cloning and Evidence for Osmoregulation of the Δ 1-Pyrroline-5-Carboxylate Reductase (proC) Gene in Pea (Pisum sativum L.)

Several cDNA clones encoding Δ 1-pyrroline-5-carboxylate reductase (P5CR, L-proline:NAD$[\text{P}]^{+}$ 5-oxidoreductase, EC 1.5.1.2), which catalyzes the terminal step in proline biosynthesis, were isolated from a pea leaf library screened with a 32P-labeled Aval fragment of a soybean nodule P5CR cDNA (A. J. Delauney, D. P. S. Verma [1990] Mol Gen Genet 221: 299-305). DNA sequence analysis of one full-length 1.3-kb clone (pPPS3) indicated that the pea P5CR gene contains a single major open reading frame encoding a polypeptide of 28,242 Da. Genomic analysis suggested that two to three copies of the P5CR gene are present per haploid genome in pea. The primary structure of pea P5CR is 85% identical with that of soybean and exhibits significant homology to human, yeast, and Escherichia coli P5CR. The sequence of one of four highly conserved domains found in all prokaryotic and eukaryotic P5CRs is similar to the consensus sequence for the NAD(P)H-binding site of other enzymes. The pea P5CR cDNA hybridized to two transcripts, 1.3 and 1.1 kb in size, in polyadenylated RNA purified from leaf tissues of mature, light-grown plants (4 weeks old). Only the 1.3-kb transcript was detected in younger (1 week old) greened seedlings or in etiolated seedlings. In greened seedlings, steady-state levels of this 1.3-kb mRNA increased approximately 5-fold in root tissues within 6 h after plants were irrigated with 0.4 M NaCl, suggesting that expression of the P5CR gene is osmoregulated.