Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 muM) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid B-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 8-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by MMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the B-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid B-hydroxylase cDNA from any plant species.

Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoid-specific CYP to be characterized at the molecular level. In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.

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.

The best characterized elicitors of the oomycete Phytophthora sojae for activating a multicomponent defense response, including the production of phytoalexins, in soybean (Glycine max L.) are the branched 1,3- and 1,6-linked beta-glucans that are structural polysaccharides of the hyphal walls of the pathogen. The soybean microsymbiont Bradyrhizobium japonicum synthesizes cyclic 1,3-1,6-beta-glucans that suppressed the fungal beta-glucan-induced phytoalexin response in soybean, indicating a novel mechanism by which the symbiotic bacteria might avoid a defense response that is normally activated in pathogenic interactions. A putative receptor (M-r = 75 kDa) for the P. sojae beta-glucans was isolated from soybean membranes and partially characterized. Partial amino acid sequences were used to raise an anti-peptide antiserum and to generate two oligonucleotides which served as primers for PCR using soybean cDNA as template. Northern blot experiments with a PCR product indicated that the glucan-binding protein mRNA has a size of about 2.4 kb. Since several species of the plant family Fabaceae were shown to possess related high-affinity beta-glucan-binding sites, glucan-based perception mechanisms may be a more common feature of this plant family. Following plasma membrane binding of the beta-glucan elicitor, the subsequent signal transduction might involve a rapid, transient increase in the cytosolic Ca2+ level and in permeability changes of the plasma membrane to Ca2+, H+, and Cl-. Production of the isoflavonoid phytoalexins, glyceollins, in soybean involves a multi-step biosynthetic pathway. Among the pathway enzymes are several cytochrome P450s. A gene family-specific differential display approach was used in the identification of several elicitor-inducible P450 cDNAs.