Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea

Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins. To investigate the regulation of these P450s in the phenylpropanoid biosynthetic pathway of pea (Pisum sativum), partial cDNAs representing four distinct P450s expressed in pea seedlings were cloned using a reverse transcription-polymerase chain reaction strategy. One of the corresponding full-length cDNA clones, designated CYP73A9, encodes pea trans-cinnamic acid 4-hydroxylase, which catalyzes the second core reaction in the phenylpropanoid pathway. As expected from its central role in the production of lignin precursors and defense compounds, northern analysis of poly(A)+ mRNA demonstrates that transcripts encoding CYP73A9 are induced appreciably within 3 h after wounding. A second cDNA clone, designated CYP82, encodes a novel P450, whose transcripts are also induced in response to wounding at approximately the same time as CYP73A9 transcripts. Despite the multitude of environmental stimuli known to induce expression of phenylpropanoid pathway enzymes, genomic DNA Southern analysis indicates that each of these P450s is encoded by a low copy number (possibly a single copy) gene family.