Identification of a Flavanone 2-Hydroxylase Involved in Flavone C-Glycoside Biosynthesis from Camellia sinensis

Tea contains a variety of flavone C-glycosides, which are important compounds that distinguish tea cultivars and tea categories. However, the biosynthesis pathway of flavone C-glycosides in tea plant remains unknown, and the key enzymes involved have not been characterized. In this study, a liquid chromatography-mass spectrometry method to determine 9 flavone C-glycosides was developed, and the accumulation patterns of 9 flavone C-glycosides in tea plants were examined first. Then, an entry enzyme CsF2H for flavone C-glycoside biosynthesis was identified, which had four cytochrome P450-specific conserved motifs and was targeted to the endoplasmic reticulum. Correlation analysis indicated that the expression level of CsF2H was positively correlated with all contents of 9 flavone C-glycosides. The recombinant CsF2H could convert flavanone (naringenin) into the corresponding 2-hydroxyflavonone (2-hydroxynaringenin), rather than into flavone (apigenin). Heterologous coexpression of CsF2H and CsCGT1 in yeast revealed that the substrate naringenin could be enzymatically converted to flavone mono-C-glycosides vitexin and isovitexin under the catalytic control of CsF2H and CsCGT1 following dehydration. Gene-specific antisense oligonucleotide analysis suggested that suppressing CsF2H significantly reduced the levels of 9 flavone C-glycosides. Together, CsF2H is the first key enzyme that generates flavone C-glycosides through the 2-hydroxyflavanone biosynthesis pathway in tea plants.