Stress Responses in Alfalfa (Medicago sativa L.) XVIII: Molecular Cloning and Expression of the Elicitor-Inducible Cinnamic Acid 4-Hydroxylase Cytochrome P450

We have isolated cDNA clones encoding a novel class of plant cytochrome P450 by screening an alfalfa cDNA expression library with an antibody against an avocado P450 (CYP73A1) that is expressed during fruit ripening but that has not yet been ascribed an in vivo activity. The cDNAs encode a polypeptide of M r 58,000, p I 9.3, containing sequence similarity (25 to 31% overall at the amino acid level) to the avocado P450 and mammalian, yeast, and bacterial P450s, particularly in the presumed heme binding region. Expression studies in yeast confirmed that the P450 clones encode cinnamic acid 4-hydroxylase (CA4H), a key enzyme of the phenylpropanoid pathway in plants. CA4H is encoded by a small gene family in alfalfa, which does not appear to contain homologs of the avocado P450. CA4H transcripts are strongly induced by fungal elicitor at the onset of accumulation of isoflavonoid phytoalexins in alfalfa cell suspension cultures.