Cloning and characterization of eight cytochrome P450 cDNAs from chickpea (Cicer arietinum L.) cell suspension cultures

Eight different P450 sequences were isolated from a cDNA library derived from cultured chickpea cells (cultivar ILC3279) elicited with a Phytophthora sojae (formerly megasperma) elicitor (Pmg-elicitor) by screening with heterologous and homologous probes. Screening with CYP73A1 from Helianthus tuberosus yielded several clones with one identical sequence. A full-length clone could be isolated and this sequence was assigned CYP73A19. Heterologous expression in yeast confirmed that CYP73A19 is the trans-cinnamic acid 4-hydroxylase of chickpea. Screening with a CYP81E2 polymerase chain reaction Fragment from chickpea yielded a CYP81E2 full-length sequence and two almost identical CYP81E3 sequences, differing in only 16 out of 498 amino acids; both share more than 85% homology with the isoflavone 2'-hydroxylase from licorice (Glycyrrhiza echinata L.). Using CYP93A1 as a probe, it was possible to isolate a full-length member of the CYP93 family, CYP93C3, that shares more than 80% homology with isoflavone synthase from soybean. In addition, partial sequences CYP81E3, CYP81E4 and CYP81E5 were also found in this screening. The use of a CYP82A2 probe derived from BAC F10N7 from Arabidopsis thaliana yielded only one sequence, CYP76F1. Rescreening with CYP81E4 and CYP81E5 did not result in the isolation of any new P450 sequences. Northern blot experiments revealed that all but the CYP76F1 are induced rapidly and transiently in cell cultures upon elicitor treatment. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.