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.
Highlights
Tannins are phenolic compounds capable of binding to proteins and interacting with cellulose, lignin, pectin, starch and alkaloids [1]
Since transcriptional control has been suggested to be the primary mode of regulation for specialized metabolite accumulation [31], UGT85K15, UGT84A23 and UGT73AL1 were proposed to be involved in hydrolyzable tannins (HTs) biosynthesis as their transcript accumulation either correlated with punicalagin levels in different pomegranate tissues or was very high in fruit peel (Ono et al, 2012)
It is worth noting that UGT84A23 and UGT84A24 enzyme assay products were treated with NaOH to verify the galloyl glucose linkage as glucose esters are generally less stable than glucose ethers under alkaline conditions
Summary
Tannins are phenolic compounds capable of binding to proteins and interacting with cellulose, lignin, pectin, starch and alkaloids [1]. Previous studies in oak (Quercus robur) leaves demonstrated that β-glucogallin was produced from conjugation of gallic acid and UDP-glucose by a UDP-glucosyltransferase (UGT) activity [8, 9]. Genes encoding βglucogallin forming UGTs have been cloned and biochemically characterized from grape (Vitis vinifera; VvgGT1-3), tea (Camellia sinensis; UGT84A22) and oak (Q. robur; UGT84A13) and were proposed to be involved in the biosynthesis of galloylated proanthocyanidins and HTs, respectively [11,12,13]. The recombinant grape, tea and oak UGTs synthesized glucose esters from a variety of BA and cinnamic acid derivatives [11,12,13]. The grape, oak and tea UGT activities towards β-glucogallin formation have far not been investigated through manipulation of gene expression in tissue cultures or the native plant systems
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