UGT76G1 polymorphism in Stevia rebaudiana: New variants for steviol glycosides conjugation

Steviol glycosides (SVglys) are secondary metabolites derived from terpenoids exhibiting high-sweetening properties produced in Stevia rebaudiana leaves. Their great diversity is due to the number, the position and the nature of glycosylations on the steviol aglycone. Steviol conjugation is mediated by uridine-diphosphate glycosyltransferases (UGTs). Four UGTs have been clearly identified as involved in SVglys metabolism: UGT74G1, UGT85C2, UGT76G1 and UGT73E1. Natural non-functional mutants with nonsense codon have yet been observed for UGT76G1. To investigate the variability of UGT76G1 functionality, natural mutants with low or no content of rebaudioside A and C were identified in a germplasm collection of Stevia rebaudiana. These compounds are known to be the direct products of UGT76G1 and their biosynthesis is governed by a single gene at the locus Rae (Rebaudioside A enablement). Crosses were done with remarkable accessions including phenotypes with low (0-3%) and high proportions (70%) of rebaudioside A and C, to investigate the functionality of the Rae locus in the parents. Seven variants of UGT76G1 were found, among them 4 lead to a functional protein and 3 lead to non-functional isoforms. Five of these variants are new. We found that non-functionality of UGT76G1 towards SVglys is not due to a premature nonsense codon, which appears to be an extreme case to explain the loss of functionality of an UGT. Variations in steviol glycoside profile in stevia leaves is partly due to UGT76G1 polymorphism: amino acid substitutions in parts of the protein involved in the substrate specificity can be found by sequence comparison.