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

Abstract

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.

In silico evaluation of the Eucalyptus transcriptome

“…The highest value was observed for cluster EGEQ FB1201B08.g, with a normalized expression of 43. This gene encodes a protein with high similarity to two cytochrome P450-dependent enzymes that were induced by wounding in peas (Frank et al, 1996) and by elicitors in soybeans (Schopfer and Ebel, 1998), making the gene an attractive target for further studies. Moreover, the expression value based in EST counting can be used to infer the strength of the corresponding promoters.…”

The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics

“…4) are planar aromatic molecules larger than simple phenylpropanoids. The most closely related P450 enzymes are CYP75 and CYP93, which are known or implied to metabolize flavonoids or isoflavonoids (Holton et al, 1993;Schopfer and Ebel, 1998). Molecules with a flavonoid-like structure are thus good potential substrates of CYP76.…”

Comparative Genomics of Rice and Arabidopsis. Analysis of 727 Cytochrome P450 Genes and Pseudogenes from a Monocot and a Dicot

“…Similarly, the CYP82 family sorts evolutionarily very close to the CYP81 family (bootstrap value of 985/1,000 iterations), and it is envisioned that the ancestor of the CYP81 and the CYP82 families split into a CYP82 family that resides only in dicots and a CYP81 family that resides in both monocots and dicots. Interestingly, members of the CYP82 family are reported to be highly stress responsive in pea (Pisum sativum), tobacco (Nicotiana tabacum), or soybean (Glycine max; Frank et al, 1996;Schopfer and Ebel, 1998;Ralston et al, 2001), but the function of these enzymes remains unknown.…”