RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Abstract

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol-glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.

Third DWF1 paralog in Solanaceae, sterol Δ 24 -isomerase, branches withanolide biosynthesis from the general phytosterol pathway

“…Several studies have reported the effect of manipulation of the general phytosterol pathway on withanolides, but no withanolide-specific activities have, to our knowledge, been identified. One W. somnifera glucosyltransferase has been implied to glycosylate both withanolides and phytosterols ( 34 ). We have shown here that 24ISO is a key enzyme in the production of withanolides, catalyzing the committing isomerization of 24-methylenecholesterol to 24-methyldesmosterol.…”

Molecular Cloning and Functional Characterization of a Sterol 3-O-Glucosyltransferase Involved in Biosynthesis of Steroidal Saponins in Trigonella foenum-graecum

“…If the sugar moieties are added after the formation of diosgenin, following the route 1 as shown in Figure 1 , the reduction of the glycosides would in turn stimulate biosynthesis of the alycone backbone (i.e., diosgenin) through a feedback regulation. Through RNAi silencing of a UDP-glycosyltransferase, this type of the feedback regulation has also been observed for biosynthesis of withaferin A-derived saponins in W. somnifera ( Saema et al, 2015 ) and ginsenosides in Panax ginseng ( Lu et al, 2017 ). However, our data do not support this premise, as the aglycone diosgenin was not increased, and even was slightly decreased in the TfS3GT2 -silenced lines ( Figure 5 ).…”

Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application

“…Leaf explants ultrasonicated at 47 KHz ± 6% for 10 s showed higher gus expression as compared to directly infected explants. The protocol was used to analyse in vivo enzymatic action of one SGT ( WsSGTL1 ) of W. somnifera by Saema et al ( 2015 , 2016 ). RNAi silencing (Saema et al, 2015 ) as well as overexpression (Saema et al, 2016 ) of WsSGTL1 gene has been achieved in transgenic W. somnifera .…”

“…The protocol was used to analyse in vivo enzymatic action of one SGT ( WsSGTL1 ) of W. somnifera by Saema et al ( 2015 , 2016 ). RNAi silencing (Saema et al, 2015 ) as well as overexpression (Saema et al, 2016 ) of WsSGTL1 gene has been achieved in transgenic W. somnifera . As expected, reduction in the level of glycosylated products observed in transgenic with silenced WsSGTL1 transcript.…”