Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana.

BACKGROUND: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. RESULTS: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. CONCLUSION: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

Mutations in the uridine diphosphate glucosyltransferase 76G1 gene result in different contents of the major steviol glycosides in Stevia rebaudiana.

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, 'N01-N05', with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants 'N01-N05' with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in 'N01'. By contrast, 'N02' exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In 'N03', 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in 'N04' and an insertion of a 68 base fragment in 'N05' were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.