Metabolic fingerprinting reveals roles of Arabidopsis thaliana BGLU1, BGLU3 and BGLU4 in glycosylation of various flavonoids

Flavonoids are specialized metabolites that play important roles in plants, including interactions with the environment. The high structural diversity of this metabolite group is largely due to enzyme-mediated modifications of flavonoid core skeletons. In particular, glycosylation with different sugars is very common. In this study, the functions of the Arabidopsis thaliana glycoside hydrolase family 1-type glycosyltransferase proteins BGLU1, BGLU3 and BGLU4 were investigated, using a reverse genetics approach and untargeted metabolic fingerprinting. We screened for metabolic differences between A. thaliana wild type, loss-of-function mutants and overexpression lines and partially identified differentially accumulating metabolites, which are putative products and/or substrates of the BGLU enzymes. Our study revealed that the investigated BGLU proteins are glycosyltransferases involved in the glycosylation of already glycosylated flavonoids using different substrates. While BGLU1 appears to be involved in the rhamnosylation of a kaempferol diglycoside in leaves, BGLU3 and BGLU4 are likely involved in the glycosylation of quercetin glycosides in A. thaliana seeds. In addition, we present evidence that BGLU3 is a multifunctional enzyme that catalyzes other metabolic reactions with more complex substrates. This study deepens our understanding of the metabolic pathways and enzymes that contribute to the high structural diversity of flavonoids. Highlight The proteins BGLU1, BGLU3 and BGLU4 are involved in glycosylations of different, already glycosylated flavonoids in Arabidopsis thaliana . BGLU3 appears to be multifunctional, acting on several complex substrates. ### Competing Interest Statement The authors have declared no competing interest. * 2x35S : double enhancer cauliflower mosaic virus 35S AGT : acyl-glucose-dependent glycosyltransferase BGLU : β -glucosidase CDS : coding sequence CE : collision energy Col-0 : Columbia-0 ESI : electrospray ionization FC : fold change GH1 : glycoside hydrolase family 1 GT : glycosyltransferase Hex : hexosyl MS/MS : spectrum, fragment spectrum m / z : mass-to-charge ratio NL : neutral loss RT-qPCR : reverse transcriptase quantitative real-time PCR rH : relative humidity Rha : rhamnosyl RT : retention time RT-PCR : reverse transcriptase semi-quantitative PCR T-DNA : transfer DNA TT13 : TRANSPARENT TESTA13 UGT : UDP-glycosyltransferase; wt, wild type