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

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  (Related Materials 1) .

The search was carried out in April 28, 2024