Functional characterization of UDP‐rhamnose‐dependent rhamnosyltransferase involved in anthocyanin modification, a key enzyme determining blue coloration in Lobelia erinus

Abstract

SUMMARYBecause structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3 0 5 0 -O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.

Biosynthesis of the anti‐diabetic metabolite montbretin A: glucosylation of the central intermediate mini‐MbA

“…Similarly, in flavonoid biosynthesis in Arabidopsis thaliana , the sequence of xylosylation and glucosylation at C‐2″ or C‐6″, respectively, of cyanidin 3‐ O ‐glucoside, can vary (Morita et al ., ; Sawada et al ., ). In contrast, acylated anthocyanins in lobelia ( Lobelia erinus ) known as lobelinins appear to be synthesized through decorations of the anthocyanin core that follow a specific sequence starting with the formation of a coumaroyl rutinoside at the 3‐OH group of the anthocyanin core, followed by the glucosylation and malonylation on the 5‐OH before the anthocyanin B‐ring is modified (Hsu et al ., ).…”

“…Completion of the trisaccharide moiety does not precede the acylation that produces mini‐MbA. Formation of the trisaccharide moiety at the myricetin C‐ring (Figure ) is followed by the decoration of the myricetin B‐ring, which are the later steps similar to other flavonoid biosynthetic pathways (Yamazaki et al ., ; Hsu et al ., ). The montbretia UGTs responsible for the remaining formation of the rhamnosyl xyloside disaccharide moiety at the 4′‐hydroxy of the B‐ring of MbA remain to be identified.…”

“…CcUGT3 catalyzes the fourth step in MbA biosynthesis, the conversion of mini‐MbA into MbA‐XR 2 , which involves formation of a 1,2‐linked trisaccharide chain using an acylated flavonol disaccharide substrate. A variety of GGTs mediating the formation of acceptor‐disaccharides, mainly flavonoid disaccharides, have been described (Yonekura‐Sakakibara et al ., ; Di et al ., ; Li et al ., ; Rodas et al ., ; Hsu et al ., ). Less is known about GGTs catalyzing the formation of more complex glycosyl chains.…”

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A

“…Using metabolite profiling, enzyme assays, and characterization of cloned UGTs and ATs, we resolved the first three steps in the assembly of MbA: the rhamnosylation of myricetin to yield MR, followed by the glucosylation of MR to form the disaccharide MRG, and next the acylation of MRG to form mini-MbA (Figure 9). A similar pattern of glycosylation occurs in the biosynthesis of lobelinin in edging lobelia (Lobelia erinus) (Hsu et al, 2017), where stepwise assembly of a disaccharide at the 3-hydroxy group of the anthocyanin core precedes the glycosylation on other positions of the Tobacco leaves were infiltrated with Agrobacterium transformed with plasmids carrying the promoter-UGT constructs 35S pro :UGT77B2, 35S pro :UGT709G2, 35S pro :CcAT1, and 35S pro :CcAT2 or the gene for eGFP. Leaves were collected at day 4 after infiltration.…”

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

“…They have recently attracted extensive research interest due to their broad bioactivity. They play important roles in plant growth and development (Kuhn et al ., ), protect them from UV radiation and bacterial and fungal infections (Hectors et al ., ), and are also responsible for the plant color (Hsu et al ., ). They also possess various human health‐promoting activities (e.g.…”