Identification of elicitor-induced cytochrome P450s of soybean (Glycine max L.) using differential display of mRNA

Elicitor-inducible glyceollin biosynthesis in soybean depends on five presumably transcriptionally regulated cytochrome P450-dependent enzymes (P450s). In order to isolate corresponding cDNA clones, we devised a novel polymerase chain reaction (PCR)-based approach targeting P450s that are transcriptionally activated under glyceollin-inducing conditions. The differential display of mRNA (DD-RT-PCR) technique was performed with upstream primers based on the conserved heme-binding region of P450s, and ten different 3'-terminal partial P450 sequences were isolated. They were subsequently used to isolate nine different full-length cDNA clones from a cDNA library. As shown by Northern blot analysis, eight of the clones represented P450s, which were activated under glyceollin-inducing conditions similar to two enzymes of the glyceollin biosynthesis pathway, CHS and IFR. Therefore, these eight clones are candidate cDNAs for the glyceollin-related P450s. Functional expression in yeast identified one cDNA clone coding for cinnamate 4-hydroxylase. Thus, at least one of the isolated clones definitively encodes a P450 of the glyceollin pathway. Consequently, this approach offers a straightforward alternative to classical P450 isolation strategies via protein purification and should prove especially useful for isolating P450s that are expressed at a low level.