Identification\nof a Flavanone 2‑Hydroxylase\nInvolved in Flavone <i>C</i>‑Glycoside Biosynthesis\nfrom Camellia sinensis

Tea contains a variety of flavone <i>C</i>-glycosides,\nwhich are important compounds that distinguish tea cultivars and tea\ncategories. However, the biosynthesis pathway of flavone <i>C</i>-glycosides in tea plant remains unknown, and the key enzymes involved\nhave not been characterized. In this study, a liquid chromatography–mass\nspectrometry method to determine 9 flavone <i>C</i>-glycosides\nwas developed, and the accumulation patterns of 9 flavone <i>C</i>-glycosides in tea plants were examined first. Then, an\nentry enzyme <i>Cs</i>F2H for flavone <i>C</i>-glycoside biosynthesis was identified, which had four cytochrome\nP450-specific conserved motifs and was targeted to the endoplasmic\nreticulum. Correlation analysis indicated that the expression level\nof <i>CsF2H</i> was positively correlated with all contents\nof 9 flavone <i>C</i>-glycosides. The recombinant <i>Cs</i>F2H could convert flavanone (naringenin) into the corresponding\n2-hydroxyflavonone (2-hydroxynaringenin), rather than into flavone\n(apigenin). Heterologous coexpression of <i>Cs</i>F2H and <i>Cs</i>CGT1 in yeast revealed that the substrate naringenin could\nbe enzymatically converted to flavone mono-<i>C</i>-glycosides\nvitexin and isovitexin under the catalytic control of <i>Cs</i>F2H and <i>Cs</i>CGT1 following dehydration. Gene-specific\nantisense oligonucleotide analysis suggested that suppressing <i>CsF2H</i> significantly reduced the levels of 9 flavone <i>C</i>-glycosides. Together, <i>Cs</i>F2H is the first\nkey enzyme that generates flavone <i>C</i>-glycosides through\nthe 2-hydroxyflavanone biosynthesis pathway in tea plants.