Functional Characterization of a New Tea (Camellia sinensis) Flavonoid Glycosyltransferase

Abstract

Tea (Camellia sinensis) is an important commercial crop, in which the high content of flavonoids provides health benefits. A flavonoid glycosyltransferase (CsUGT73A20), belonging to cluster IIIa, was isolated from tea plant. The recombinant CsUGT73A20 in Escherichia coli exhibited a broad substrate tolerance toward multiple flavonoids. Among them, kaempferol was the optimal substrate compared to quercetin, myricetin, naringenin, apigenin, and kaempferide. However, no product was detected when UDP-galactose was used as the sugar donor. The reaction assay indicated that rCsUGT73A20 performed multisite glycosidation toward flavonol compounds, mainly forming 3-O-glucoside and 7-O-glucoside in vitro. The biochemical characterization analysis of CsUGT73A20 showed more K7G product accumulated at pH 8.0, but K3G was the main product at pH 9.0. Kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro. Besides, the content of five flavonol-glucosides was increased in CsUGT73A20-overexpressing tobaccos (Nicotiana tabacum).

Glucosyltransferase CsUGT78A14 Regulates Flavonols Accumulation and Reactive Oxygen Species Scavenging in Response to Cold Stress in Camellia sinensis

“…Interestingly, at least two product peaks were detected when using kaempferol and quercetin as acceptor substrates, indicated that CsUGT78A14 performed multiposition glycosylation on flavonols. The above-mentioned products were identified by retention time, parent ions (H adducts), and daughter ion spectra (MS/MS) in comparison with standards (Zhao et al, 2017). The main products of CsUGT78A14-1 was identified as a diglucoside by comparison of its parent and daughter ion spectra with a reference compound ( Figure 1D) (Zhao et al, 2017), in addition to two monoglucoside products, identified as kaempferol-3-glucoside, and -7-glucoside ( Figure 1C), when kaempferol was used as substrate.…”

“…Recently, 132 UGTs were identified in a transcriptome database of the tea plant. The function of most of them were unknown, except four UGTs (CsUGT78A14, CsUGT78A15, and CsUGT82A22, CsUGT73A20) which exhibited catalytic activity toward phenolic acids and flavonoid (Cui et al, 2016;Zhao et al, 2017), and three UGTs involved in the glycosylation of aroma (Ohgami et al, 2015;Jing et al, 2019;Song et al, 2018). Numerous studies have indicated that some UGTs are involved in the regulation of plant growth and development in responses to biotic and abiotic stresses (Tognetti et al, 2010;von Saint Paul et al, 2011;Liu et al, 2015;Palareti et al, 2016), and improved freezing tolerance in Arabidopsis (Schulz et al, 2016).…”

UGT74AF3 enzymes specifically catalyze the glucosylation of 4-hydroxy-2,5-dimethylfuran-3(2H)-one, an important volatile compound in Camellia sinensis

“…To study UGTs related to HDMF glycosylation, the candidate genes TEA012191, TEA016995, TEA031670, and TEA016084 were randomly selected from groups E, G, H, and L, respectively. Genes from group D were not selected because some genes in this group have been reported to encode flavonoid glucosyltransferases in tea plants 27 . TEA031670 could not be isolated as a full-length gene.…”

“…For a long time, because the transgenic system of tea plants has not been successfully established, the in vivo functional verification of tea plant genes has only been carried out in heterologous plants 2,27 . In this study, the in planta function of UGT74AF3 was first investigated in tobacco, where overexpression of UGT74AF3a resulted in an increased HDMF glucoside content compared to that of the control (Fig.…”

“…More than 300 UGTs were found in the tea plant genome 24,25 , but the function of most of them is unknown, except for that of four UGTs (UGT78A14, UGT78A15, UGT82A22, and UGT73A20), which exhibited catalytic activity toward phenolic acids and flavonoid 26,27 , and three UGTs involved in the glycosylation of geraniol 28 and (Z)-3-hexen-1-ol 2 . However, the genes coding for HDMF glycosyltransferases in tea plants are not yet known.…”