Data on molecular cloning and characterization of three glycosyltransferases involved in the anthocyanin biosynthetic pathway in Clematis cultivar

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posted on 2025-03-03, 08:48 authored by Yoshikazu Tanaka

Fig. S1. HPLC profile of the anthocyanins of Clematis cultivar Violet sepals. (A) and deduced anthocyanin structures on the basis of their MS(+/-) , MS2(+/-) (B) and the reported anthocyanin structure (C) (Peak d, Sakakuchi et al., 2013). The peak d (retention time=16.7 min) corresponds to Compound I in Figure 1. Its MS(+/-) and MS2(+/-) are shown. Fig. S2. Clustering the family 1 glycosyltransferases and sequence alignment to show the critical residue to determine sugar donor. (A) Phylogenetic tree of the family 1 glycosyltransferases involved in flavonoid biosynthesis, plant name (Arabidopsis thaliana, At), function, and accession number are shown. Clematis family 1 glycosyltransferase sequences are marked with a star and the three enzymes studied here are shown in bold. The clades shown are: A3GT: anthocyanidin 3-O-glycosyltransferase, A3GGT: anthocyanidin 3-O-glycoside glycosyltransferase, F3′/7GT: flavonoid 3′ or 7-O-glycosyltransferase, A5GT: anthocyanin 5-O-glycosyltransferase, HBAGT: hydroxybenzoic acid glycosyltransferase. (B) A part of amino acid sequences (the accession numbers are shown) to compare anthocyanidin or flavonoid 3-O-galactosyltransferases and 3-O-glucosyltransferase. The upper five proteins are galactosyltransferase having the histidine residue (shown with the arrow), while the lower seven are glucosyltransferases having glutamine residue. The numbers are of Clematis anthocyanidin 3-O-galactosyltransferase sequence. Ac: Actinidia chinensis, Ar: Aralia cordata, Ph: Petunia hybrida, Vm: Vigna mungo, At: A. thaliana, Fa: Fragaria ananassa, Gt: Gentiana triflora, Pf: Perilla frutescens, Vv: Vitis vinifera Fig. S3. Examples of Clematis anthocyanidin 3-O-galactosyltransferase reactions. A. A reaction to delphinidin, B. Delphinidin 3-O-galactoside and cyanidin 3-O-galactoside, C. A reaction to cyanidin, D. A reaction to pelargonidin. The spectrum of each compound was shown. Galactosylation resulted in a shorter wavelength shift of absorption maximum. Fig. S4. Examples of Clematis anthocyanidin 3-O-galactoside-2''-O-glucosyltransferase reactions. A. A reaction to cyanidin 3-O-galactoside, B. A reaction to cyanidin 3-O-glucoside, C. Cyanidin 3-O-sophoroside, The spectrum of each compound is shown. No significant wavelength shifts were observed for glucosylation. Fig. S5. LC-MS analysis of the anthocyanidin 3-O-galactoside-2''-O-glucosyltransferase reaction from cyanidin 3-O-galactoside and UDP-glucose. A. Elution profile and MS(+). The product has matched m/z to cyanidin 3-O-glucosyl-(1→2)-galactoside (m/z 611.1599 [M]+, C27H31O16+=611.1607). B. MS/MS pattern of the product (m/z 287.0545 [M]+ corresponding cyanidin, C15H11O6+=287.0550) is consistent with that of cyanidin di-hexosides (absence of cyanidin+hex). C. MS/MS pattern of the substrate. Fig. S6. Examples of Clematis anthocyanin 3’-O-glucuronosyltransferase reaction A. A reaction to delphinidin 3-O-galactoside, B. A reaction to cyanidin 3-O-galactoside, C. A reaction to cyanidin 3-O-sophoroside Fig. S7. Color shift assay of 3’-glucuronosydation of anthocyanins with AlCl3. P1 (presumably delphinidin 3-O-glucoside-3′-O-glucuronide) and P2 (presumably delphinidin 3-O-galactoside-3′-O-glucuronide) changed their color toward blue by treatment with AlCl3 while product 3 (presumably cyanidin 3-O-galactoside-3′-O-glucuronide) did not show the blue color shift. The results indicated that 3′-hydroxy group was glucuronisydated. Fig. S8. LC-MS analysis of the anthocyanin 3′-O-glucuronosyltransferase reactions from cyanidin 3-O-galactoside (A) and cyanidin 3-O-sophoroside (C). The products (m/z 625.1394 and 787.1917) derived from cyanidin 3-O-galactoside and cyanidin 3-O-sophoroside were glucuronidated compounds, respectively. MS/MS detected a compound matching cyanidin glucuronide (m/z 463.0867 (B) and 463.0872 (D)). Table S1. List of biosynthetic genes isolated in this study (<a href="https://doi.org/10.50826/bnmnsbot.51.1_35" target="_blank">Related Materials 1</a>) . The search was carried out in April 28, 2024