Dynamics of withanolide biosynthesis and accumulation in relation to temporal expression pattern of pathway genes in Withania somnifera a comparative study between in vitro and in vivo tissues

Abstract

Withania somnifera, commonly known as Ashwagandha is a constituent of over 200 newlineformulations in Ayruvedha, Siddha and Unani medicine. The major chemical constituents of this newlineplant are withanolides, which are steroidal lactone in nature. But, under natural conditions, W. newlinesomnifera possesses restricted levels of withanolides then, alternatives for obtaining withanolides newlinein better yields are imperative. The objective of the present study was to identify the growth newlinestage of in vitro and in vivo tissues at which maximum withanolide accumulation occurs and to newlinecompare the expression of selected pathway genes between in vitro and field grown tissues. The newlinestudy was also aimed to compare the total metabolites at different growth stages and different newlinetissues using GC-MS technique. Many of the metabolites detected were recorded to be present in newlinesignificantly in higher intensities among in vitro root cultures in comparison with field grown newlineroot tissues. Therapeutically important metabolite, namely vanillic acid was detected only in in newlinevitro root tissues. In vitro grown root and leaf tissues were then harvested at different growth newlineperiod and analyzed for major withanolide accumulation. Both in vitro leaf and root tissues newlineexhibited significantly higher accumulation of Withaferin A and Withanolide A at 45th day of newlineculture. Both the field grown tissues recorded significant accumulation of withanolides at the newlineyellow berry stage of growth. The expression analysis of selected pathway genes of withanolide newlinebiosynthesis was then performed using qRT-PCR. The expression of Squalene epoxidae (SE) newlineand Glucosyl transferase (GT) seemed to follow a similar pattern with relatively higher newlineexpression in in vitro roots compared to leaves at all three time periods analyzed. Among the newlinefield grown samples, high expression of Farnesyl pyrophospahte (FPPS) was seen among leaf tissue at all stages, whereas the SE showed robust upregulation in root tissue at yellow berry stage. We then investigated the expression pattern of selected pathway genes