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Stevia rebaudiana  (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of  UGT76G1 . By comparing the SG profiles among independent transgenic events, we demonstrated that altering  UGT76G1  expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in  E .  coli , we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional  UGT76G1  transcripts, and possibly produce enzyme variants within the plant.

An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function