Cytochrome P450 CYP81 family: ISOFLAVONE HYDROXYLASES and disease resistance in soybean.

The phenylpropanoid pathway is a source of many specialized metabolites, such as flavonoids, isoflavonoids, anthocyanins, lignin, and phytoalexins. Among several protein families involved in the regulation and functionality of this pathway, cytochrome P450 monooxygenases are one of the major enzyme families. The P450 enzymes are categorized into different families, with each family performing a different type of reaction. Some examples include hydroxylation, epoxidation, decarboxylation, N- and O-dealkylation, nitration, and C–C bond cleavage. CYP81 family of P450 enzymes is known to participate in the hydroxylation of isoflavonoid aglycons in legumes. This enzyme class is known as isoflavone hydroxylase (IFH) that provides building blocks for the synthesis of phytoalexins in legume plants. IFH is the first rate-limiting P450 enzyme that initiates the biosynthesis of phytoalexin glyceollin in soybean catalyzing the conversion of daidzein to hydroxydaidzein. Production of glyceollin is an induced response in soybean in response to environmental stress. The goal of this research is to identify and characterize IFHs in soybean to uncover their role in stem and root rot disease caused by Phytophthora sojae. Here we report that the soybean genome contains 12 candidate IFH (GmIFH) genes that belong to the CYP81 family. Expression of the candidate IFHs using the heterologous yeast expression system and enzyme assay using microsomal proteins revealed that six GmIFH are functional, showing varied hydroxylation activity with five types of isoflavones aglycons, including daidzein, genistein, formononetin, glycitein, and biochanin A. Transcriptome analysis of soybean roots infected with P. sojae revealed that the six functional GmIFHs are induced upon pathogen infection and their transcripts accumulate at higher level in resistant soybean cultivar compared to susceptible cultivar. In conclusion, our study demonstrates that GmIFHs play an important role in the resistance toward P. sojae.