AUTHOR=Sharma Arti , Rather Gulzar A. , Misra Prashant , Dhar Manoj K. , Lattoo Surrinder K. 

TITLE=Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00842

DOI=10.3389/fpls.2019.00842

ISSN=1664-462X

ABSTRACT=<p><italic>Withania somnifera</italic> (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (<italic>CYP85A69</italic>) from <italic>W. somnifera</italic>. The full length <italic>WsCYP85A69</italic>, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, <italic>in vitro</italic> conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a <italic>m/z</italic> value of 506 [M+ACN]<sup>+</sup>. <italic>In planta</italic> transient over-expression of <italic>WsCYP85A69</italic> significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of <italic>WsCYP85A69</italic> resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of <italic>WsCYP85A69</italic> in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of <italic>WsCYP85A69</italic> resulted in the identification of several potential <italic>cis</italic>-regulatory elements. Elicitations, given on the basis of identified <italic>cis</italic>-regulatory elements, demonstrated methyl jasmonate as an effective inducer of <italic>WsCYP85A69</italic>. Overall, these empirical findings suggest that functional characterization of <italic>WsCYP85A69</italic> may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering.</p>