Functional characterization of Cinnamate 4-hydroxylase gene family in soybean (Glycine max)

Abstract

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion oftrans-cinnamic acid top-coumaric acid. Sincep-coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybeanC4Hgenes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified fourC4Hgenes in the soybean genome that are distributed into both class I and class II CYP73 family.GmC4H2,GmC4H14 and GmC4H20displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues.GmC4H10appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylatetrans-cinnamic acid with varying efficiencies.