Sequencing analysis and enzyme activity assay of SrUGT76G1 revealed the mechanism toward on/off production of Rebaudioside-A in stevia plants

Stevia plants are well-known for their ability to synthesize steviol glycosides (SGs), a natural sweetener blend. The principal SGs include stevioside (STV) and Rebaudioside-A (Reb-A), with the latter exhibiting superior sweetness and organoleptic properties. UDP glucosyltransferase-76G1 (UGT76G1) is responsible for converting STV to Reb-A, determining the intensity of sweetness. A better understanding of the structure/activity of SrUGT76G1 could provide insights into Reb-A production in stevia plants. To this end, a combination of enzymatic assays and sequencing analysis was performed using two stevia genotypes (Brazilian and Spanish) with contrasting Reb-A production capabilities (off/on). Relative expression of SrUGT76G1 gene showed remarkably higher expression ( threefold) in Spanish samples compared to Brazilian ones. Foliar protein fractions (crude or partially purified extract) from Brazil plants were unable to convert STV into Reb-A under in vitro conditions, resulting in undetectable levels of Reb-A by HPLC. Molecular analyses revealed that the Brazilian SrUGT76G1 gene not only presents a premature stop codon, resulting in the absence of PSPG motif responsible for the binding of glycosyl groups, but also exhibits mutations affecting key amino acid residues in the acceptor-binding pocket. These alterations provide a plausible explanation for the Brazilian protein inability to catalyze the transformation of STV into Reb-A.