## 2 Results and Discussion
### Identification of UGT Candidates Involved in Dioscin Biosynthesis  
The biosynthesis of dioscin requires the addition of glucose and rhamnosgroups onto the C-3 hydroxyl group, which is catalyzed by UGTs (Figure 1). In the present study, 195 unigenes encoding UGTs with a sequence length above 1000 bps were obtained (Table S4). Inspection of these UGT unigenes revealed six unigenes (c73651_g1, c70118_g1, c61522_g1, c66977_g1, c60958_g1, and c59129_g1) annotated as a sterol 3-O-UGT and two unigenes (c60525_g2 and c69762_g2) as a UDP-rhamnose: rhamnsfarase (Table S7). These UGT unigenes would be possible candidates for dioscin biosynthesis. However, the expression trend of all the identified UGTs (Table S7) did not match the distribution pattern of dioscin in the tissue samples (Figure 2C). A similar case also has been reported for _Panax japonicus_ UGTs whose expression pattern across various tissues is different from the tissue-specificity for triterpen saponin biosynthesis in _Panax japonicus_[27]. These data indicate that glycosylation reaction governed by UGTs might not influence the tissue-specificity of saponin synthesis in some of the plant species. Figure 3: Phylogenetic analysis of potential CYP450 candidates from _D. zingiberensis_ transcriptome with previously published CYP450s from various metabolic pathways. For biochemical characterizations of the identified UGTs, it is necessary to know their full open reading frames (ORFs). Unfortunately, only partial transcripts of the two rhamnosyltransferase candidates were present in the transcriptome. Among the identified sterol 3-O-UGTs, the unigenes of c60958_g1 and c59129_g1 were expressed at very low levels in all the tissue samples, moreover, they were not at a full-length and thus were not subjected to further analysis. Within the rest of the 3-O-UGTs, only the c61522_g1 and c66977_g1 contain complete ORFS. The gene products of c61522_g1 and c66977_g1 were designated as Dz3 GT1 (NCBI accession no. MG488289) and Dz3 GT2 (NCBI accession no. MG488290), respectively, in this study. Dz3 GT2 shows only 56% amino acid identity to Dz3 GT1 but displays 99.8 % identity to the previously identified Dz3 GT from _D. zingiberensis_, the enzyme catalyzing a C3-glucosidase activity on diosgenin [28]. To further characterize the biochemical properties of Dz3 GT1 and Dz3 GT2, their ORFs were transferred into _E. coli_ cells, and the recombinant UGTs were purified by the use of their N-terminal fused tags. The recombinant protein was assayed with diosgenin and cholesterol followed by HPLC analysis. Both Dz3 GT1 and Dz3 GT2 were able to convert diosgenin to a new product (peak 1) which corresponds to trillin (diosgenin 3-O-glucoside) authentic standard (Figure 4A), suggesting their 3-O-glucosidase activities on diosgenin. When cholesterol was used as a substrate, a new product (peak 2) was observed from the assays with both _D. zingiberensis_ UGTs (Figure 4B). Based on the C3-glucosidase activities of Dz3 GTs described above, we assumed the peak 2 to be cholesterol 3-O-glucose, although the cholesterol 3-O-glucoside standard is not commercially available at present. Both Dz3 GT1 and Dz3 GT2 function redundantly in C3-O-glucosylation of diosgenin, the importance of Dz3 GT1 relative to Dz3 GT2 in dioscin formation is not clear. Further characterization of Dz3 GT1 and Dz3 GT2 is required to confirm their roles in dioscin formation by silencing their gene expression in _D. zingiberensis_. Figure 4: In vitro C3-glycosylation of diosgenin and cholesterol by the purified recombinant Dz3 GT1 and Dz3 GT2. HPLC profiles were shown for the assays with diosgenin (**A**) and cholesterol (**B**). CK means the control reaction. For the controls, the purified Dz3 GTs were excluded from the reaction mixtures. Both Dz3 GT1 and Dz3 GT2 were able to convert diosgenin to one new product (peak 1), corresponding to trillin (diosgenin 3-O-glucoside), which was not produced in the controls.