Two UGT84 family glycosyltransferases catalyze a critical reaction of hydrolyzable tannin biosynthesis in pomegranate (Punica granatum)

Abstract

Hydrolyzable tannins (HTs) play important roles in plant herbivore deterrence and promotion of human health. A critical step in HT production is the formation of 1-O-galloyl-β-D-glucopyranoside (β-glucogallin, ester-linked gallic acid and glucose) by a UDP-glucosyltransferase (UGT) activity. We cloned and biochemically characterized four candidate UGTs from pomegranate (Punica granatum), of which only UGT84A23 and UGT84A24 exhibited β-glucogallin forming activities in enzyme assays. Although overexpression and single RNAi knockdown pomegranate hairy root lines of UGT84A23 or UGT84A24 did not lead to obvious alterations in punicalagin (the prevalent HT in pomegranate) accumulation, double knockdown lines of the two UGTs resulted in largely reduced levels of punicalagins and bis-hexahydroxydiphenyl glucose isomers. An unexpected accumulation of galloyl glucosides (ether-linked gallic acid and glucose) was also detected in the double knockdown lines, suggesting that gallic acid was utilized by an unidentified UGT activity for glucoside formation. Transient expression in Nicotiana benthamiana leaves and immunogold labeling in roots of pomegranate seedlings collectively indicated cytosolic localization of UGT84A23 and UGT84A24. Overall, functional characterization and localization of UGT84A23 and UGT84A24 open up opportunities for further understanding the regulatory control of HT metabolism in plants and its coordination with other biochemical pathways in the metabolic network.