Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Abstract

SummaryStevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2¢ and C-3¢ of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and 14 C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.

Gene transcription and steviol glycoside accumulation in Stevia rebaudiana under polyethylene glycol‐induced drought stress in greenhouse cultivation

“…Thus, the significant correlation between total SVgly accumulation and transcription of UGT85C2 suggests the UGT85C2 enzyme as a rate‐limiting step of SVgly biosynthesis. UGT76G1 modulates the synthesis of stevioside and rebaudioside A 9, 10. In the present study, the transcription of UGT74G1 was significantly reduced in polyethylene glycol‐treated plants, which is supported by the results of Yang et al .…”

“…In Stevia , geranylgeranyl‐diphosphate (GGDP) is converted into steviol by the consecutive action of four enzymes: copalyl diphosphate synthase (CPPS), ent‐kaurene synthase (KS), ent‐kaurene oxidase (KO), and ent‐kaurenoic acid hydroxylase (KAH). Different SVglys are formed by glycosylation of steviol by specific glycosyltransferases (UGTs) 8, 9, 10, 11 (Fig. 2).…”

Metabolism of acetaminophen (paracetamol) in plants—two independent pathways result in the formation of a glutathione and a glucose conjugate

“…2.4) is a key mechanism in the metabolic homoeostasis of plant cells. Potential acceptor molecules can be plant hormones (Hou et al 2004), endogenous synthesized secondary metabolites (Richman et al 2005) as well as xenobiotics (Messner et al 2003). With their multiple functions and the variety of possible substrates, glycosyltransferases play a major role in buffering the impacts of either biotic or abiotic challenges on the plant (Bowles et al 2005).…”

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

“…In Gentiana triflora, the blue flower pigmentation results from a delphinidin-3,5,3Ј-tri-Oglucoside (10). Skin color in grape varieties (11), bitterness in citrus (12,13), and sweetness in Stevia leaves (14) are due to the glycosylation of anthocyanin, flavonol (naringenin), or terpene (stevioside), respectively. Furthermore, glycosylation of secondary metabolites impacts disease resistance and plant development (15,16).…”