Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis)

Abstract

Fruits of sweet orange (Citrus sinensis), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O-glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O-rutinosides or O-neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7-O-rhamnoside, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside, implying that the enzyme has flavonoid 7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities in vivo.

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

“…Flavonol 3‐ O ‐rhamnosides from Camellia sinensis can alter the tea infusion's silky, mouth‐drying and mouth‐coating sensation at very low threshold concentration (Scharbert and Hofmann, ; Dai et al ., ), 8‐prenylkaempferol 3‐ O ‐rhamnoside (baohuoside II) from Epimedium pseudowushanense is used in traditional Chinese medicine to invigorate the kidneys and strengthen muscles and bones (Feng et al ., ). Quercetin 7‐ O ‐rhamnoside is known to accumulate at high concentration in Citrus sinensis and is widely used as food additives (Liu et al ., ), and it also possesses strong inhibitory activity against porcine epidemic diarrhea virus (PEDV; Choi et al ., ). Kaempferol 3‐ O ‐rhamnoside can eliminate β‐amyloid toxicity by modulating monomers and remodeling oligomers and fibrils to non‐toxic aggregates, and may be a good candidate for incorporation into the therapeutic strategy for treatment of Alzheimer's disease (Holler et al ., ).…”

“…The rhamnosyltransferase is the key enzyme involved in the biosynthesis of rhamnosides. However, only four flavonol rhamnosyltransferases, that is, A. thaliana UGT78D1 (Jones et al ., ) and UGT89C1 (Yonekura‐Sakakibara et al ., ), E. pseudowushanense PF3RT (Feng et al ., ) and C. sinensis UGT76F1 (Liu et al ., ), have been identified to date.…”

Genome-wide characterization, evolution and expression profiling of UDP-glycosyltransferase family in pomelo (Citrus grandis) fruit

“…Several UGT genes have been functionally characterized in plants, such as Arabidopsis thaliana [5], Zea mays [18], Triticum aestivum [26], Brassica rapa [27] and Prunus persica [20]. In citrus, only three UGTs have been functionally identi ed, Cm1_2RhaT from pomelo, Cs1,6RhaT and CsUGT76F1 from sweet orange associated with avonoid glycosylation [13][14][15][16], and three putative terpenoid UGTs, CsUGT1, CsUGT2 and CsUGT3 [17]. However, no large-scale analysis was found in citrus.…”

“…All UGT members were divided into 16 phylogenetic groups, including 14 conservative groups (A-N) identi ed in Arabidopsis [2], and two newly identi ed groups O and P found in other plants, such as grapevine [5]. Cm1_2RhaT (100% amino acid sequence identity to Cg1g023820) from pomelo and Cs1,6RhaT from sweet orange that were identi ed as avonoid 7-O-UGTs [13,15,16], were clustered in group A. CsUGT76F1, located in group H, was identi ed as being involved in the biosynthesis of avonoid 7-O-glucosides and 7-O-rhamnosides in sweet orange [14]. Arabidopsis UGT73C6 ( avonol-3-O-rhamnoside-7-O-glucosyltransferase) [21] and strawberry FaGT7 ( avonol-3-O-glucosyltransferase) [8] were located in group D. Other UGTs responsible for avonol-3-O-glycosylation were located in group F, including UGT78D1 from Arabidopsis [22], and VvGT5 and VvGT6 from grapevine [7].…”

Evolutionary and ecological functional genomics, from lab to the wild

“…Three RNA‐seq papers (Lister et al ., ; Mortazavi et al ., ; Nagalakshmi et al ., ) on Arabidopsis, yeast and mice, respectively, mark the start of this tool's use for functional genomic studies. Contemporarily, RNA‐seq is used at ever‐larger scales for functional characterization of developmental, environmental response and economically important phenotypes (Becker et al ., ; Feng et al ., ; Giacomello et al ., ; Leydon et al ., ; Liu et al ., ), and is often used to validate epigenomic measurements (Wang et al ., ). There are numerous studies that employ whole‐genome gene expression analysis not just for model plant species and crops, but also for lesser‐known plant species including Japanese lawn grass (Xie et al ., ), Cunninghamia lanceolata (Cao et al ., ), mangrove fern (Zhang et al ., ), wild oil‐tea camelia (Chen et al ., ), curry tree (Meena et al ., ) and Banksia (He et al ., ).…”