Characterization of a UGT84 Family Glycosyltransferase Provides New Insights into Substrate Binding and Reactivity of Galloylglucose Ester-Forming UGTs.

Galloylated plant specialized metabolites play important roles in plant-environment interactions and in the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs. By employing site-directed mutagenesis of candidate amino acids, enzyme assays with analogous substrates, and kinetic analysis, we elucidated key amino acid sites for PgUGT84A23 substrate binding and reactivity. The galloylglucose ester-forming UGT84s have not been shown to glycosylate genistein (an isoflavonoid) in vivo. Unexpectedly, amino acids highly conserved among UGT84s that affect specifically the binding of genistein but not gallic acid or other tested sugar acceptors were identified. This result suggests that genistein may resemble the substrate profile for the enzyme ancestor of the galloylglucose ester-forming UGTs and recruited during transition from a general to a more specialized defense function. Overall, a better understanding of the structure-function relationship of UGT84s will facilitate enzyme engineering for the production of pharmaceutically and industrially valuable glycosylated compounds.

Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway.

In contrast to the numerous reports on the human therapeutic applications of hydrolyzable tannins (HTs), genes involved in their biosynthesis have not been identified at the molecular level from any plant species. Although we have previously identified candidate HT biosynthetic genes in pomegranate using transcriptomic and bioinformatic analyses, characterization of in planta enzyme function remains a critical step in biochemical pathway elucidation. We here report the establishment of a pomegranate (Punica granatum) hairy root culture system that produces HTs. Agrobacterium rhizogenes strains transformed with a binary vector harboring a yellow fluorescent protein (YFP) gene were used for hairy root induction, allowing visual, non-destructive, detection of transgene incorporation. It also demonstrated that the pomegranate hairy root culture system is suitable for expressing heterologous genes (YFP in this case). Expression of 26 putative UDP-glycosyltransferase (UGT) genes, obtained from a pomegranate fruit peel (a tissue highly abundant in HTs) RNA-Seq library, were verified in wild type and hairy roots. In addition, two candidate UGTs for HT biosynthesis were identified based on HPLC and differential gene expression analyses of various pomegranate tissues. Together with in vitro enzyme activity assays, the hairy root culture system holds great promise for revealing the undivulged HT biosynthetic pathway using pomegranate as a model system.

Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum).

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-ß-D-glucopyranoside (ß-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 ß-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.

The multiplicity of hairy root cultures: prolific possibilities.

Hairy root cultures (HRCs), induced by Agrobacterium rhizogenes infection, have been established from a wide variety of plant species. HRCs accumulate phytochemicals to levels comparable to that of intact plants and are usually stable in their biosynthetic capacity. When optimized for liquid cultures, hairy roots can be grown in industrial-scale bioreactors providing a convenient, abundant and sustainable source of phytochemicals. Due to their ease of propagation and growth in confined environments, HRCs have also been used in recent years in the synthesis of recombinant therapeutic proteins, especially those that have been challenging to express in bacteria, yeast and mammalian expression systems. Although phytochemicals are recognized for their important roles in plant and human health, large gaps still exist in understanding how phytochemicals (in particular, secondary/specialized metabolites) are synthesized in plants. This review presents recent developments and findings in phytochemical and recombinant protein production, as well as new revelations in gene discovery and biochemical pathway elucidation, by the utilization of HRCs. Although many challenges still exist for industrial applications of HRCs, the immediate future of this diverse system, especially for the bench-side scientists, is still found to be promising and abounding in possibilities.