Phylogenomic analysis of UDP-dependent glycosyltransferases provides insights into the evolutionary landscape of glycosylation in plant metabolism.

Glycosylated metabolites generated by UDP-dependent glycosyltransferases (UGTs) play critical roles in plant interactions with the environment as well as human and animal nutrition. The evolution of plant UGTs has previously been explored, but with a limited taxon sampling. In this study, 65 fully sequenced plant genomes were analyzed, and stringent criteria for selection of candidate UGTs were applied to ensure a more comprehensive taxon sampling and reliable sequence inclusion. In addition to revealing the overall evolutionary landscape of plant UGTs, the phylogenomic analysis also resolved the phylogenetic association of UGTs from free-sporing plants and gymnosperms, and identified an additional UGT group (group R) in seed plants. Furthermore, lineage-specific expansions and contractions of UGT groups were detected in angiosperms, with the total number of UGTs per genome remaining constant generally. The loss of group Q UGTs in Poales and Brassicales, rather than functional convergence in the group Q containing species, was supported by a gene tree of group Q UGTs sampled from many species, and further corroborated by the absence of group Q homologs on the syntenic chromosomal regions in Arabidopsis thaliana (Brassicales). Branch-site analyses of the group Q UGT gene tree allowed for identification of branches and amino acid sites that experienced episodic positive selection. The positively selected sites are located on the surface of a representative group Q UGT (PgUGT95B2), away from the active site, suggesting their role in protein folding/stability or protein-protein interactions.

Exploring the Phytochemical Landscape of the Early-Diverging Flowering Plant Amborella trichopoda Baill.

Although the evolutionary significance of the early-diverging flowering plant Amborella (Amborella trichopoda Baill.) is widely recognized, its metabolic landscape, particularly specialized metabolites, is currently underexplored. In this work, we analyzed the metabolomes of Amborella tissues using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). By matching the mass spectra of Amborella metabolites with those of authentic phytochemical standards in the publicly accessible libraries, 63, 39, and 21 compounds were tentatively identified in leaves, stems, and roots, respectively. Free amino acids, organic acids, simple sugars, cofactors, as well as abundant glycosylated and/or methylated phenolic specialized metabolites were observed in Amborella leaves. Diverse metabolites were also detected in stems and roots, including those that were not identified in leaves. To understand the biosynthesis of specialized metabolites with glycosyl and methyl modifications, families of small molecule UDP-dependent glycosyltransferases (UGTs) and O-methyltransferases (OMTs) were identified in the Amborella genome and the InterPro database based on conserved functional domains. Of the 17 phylogenetic groups of plant UGTs (A-Q) defined to date, Amborella UGTs are absent from groups B, N, and P, but they are highly abundant in group L. Among the 25 Amborella OMTs, 7 cluster with caffeoyl-coenzyme A (CCoA) OMTs involved in lignin and phenolic metabolism, whereas 18 form a clade with plant OMTs that methylate hydroxycinnamic acids, flavonoids, or alkaloids. Overall, this first report of metabolomes and candidate metabolic genes in Amborella provides a starting point to a better understanding of specialized metabolites and biosynthetic enzymes in this basal lineage of flowering plants.

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.

PgUGT95B2 preferentially metabolizes flavones/flavonols and has evolved independently from flavone/flavonol UGTs identified in Arabidopsis thaliana.

UDP-dependent glycosyltransferases (UGTs) convert aglycones into more stable, bioactive, and structurally diverse glycosylated derivatives. Pomegranate (Punica granatum L.) produces various glycosylated phenolic metabolites, e.g. hydrolyzable tannins (HTs), anthocyanins, and flavonoids, and constitutes an excellent system for investigating the corresponding UGT activities. Here we report the cloning and functional characterization of a pomegranate UGT, PgUGT95B2, which is highly active towards flavones and flavonols and can glycosylate at more than one position in the substrate molecule. Particularly, PgUGT95B2 has the strongest activity towards tricetin (flavone with a tri-hydroxylated B-ring) and can act at the 4'-O position of its B-ring. In addition, PgUGT95B2 was able to glycosylate flavones present in pomegranate metabolite extracts. Conversely, PgUGT95B2 did not produce a galloylglucose ester (precursor for HT biosynthesis) or anthocyanins in enzyme assays. Our phylogenetic analysis suggested an independent evolution of PgUGT95B2 and flavone/flavonol UGTs identified in the model plant Arabidopsis thaliana through convergent evolution or gene loss.

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 horizon line, linear perspective, interposition, and background brightness as determinants of the magnitude of the pictorial moon illusion.

A total of 110 undergraduate students participated in a series of three experiments that explored the magnitude of the moon illusion in pictures. Experiment 1 examined the role of the number and salience of depth cues and background brightness. Experiment 2 examined the role of the horizon line, linear perspective, interposition, and background brightness. In Experiment 3, comparative distance judgments of the moon as a function of linear perspective, interposition, and the size of the standard moon were obtained. The magnitude of the moon illusion increased as a function of the number and salience of depth cues and changes in background brightness. Experiment 2 failed to support the role of the horizon line in affecting the illusion. Experiment 3 provided additional support for the illusory distance component of the moon illusion.