Functional characterization of WsMYC2 via artificial microRNA mediated silencing and transient over-expression displayed significant regulatory role vis-à-vis withanolides and stigmasterol biosyntheses in Withania somnifera. Further, metabolic intensification corroborated well with higher expression levels of putative pathway genes. Additionally, copious expression of WsMYC2 in response to exogenous elicitors resulted in enhanced withanolides production. Withania somnifera, a high value multipurpose medicinal plant, is a rich reservoir of structurally diverse and biologically active triterpenoids known as withanolides. W. somnifera has been extensively pursued vis-à-vis pharmacological and chemical studies. Nonetheless, there exists fragmentary knowledge regarding the metabolic pathway and the regulatory aspects of withanolides biosynthesis. Against this backdrop, a jasmonate-responsive MYC2 transcription factor was identified and functionally characterized from W. somnifera. In planta transient over-expression of WsMYC2 showed significant enhancement of mRNA transcript levels which corroborated well with the enhanced content of withanolides and stigmasterol. Further, a comparative analysis of expression levels of some of the genes of triterpenoid pathway viz. WsCAS, WsCYP85A, WsCYP90B and WsCYP710A in corroboration with the over-expression and silencing of WsMYC2 suggested its positive influence on their regulation. These corroboratory approaches suggest that WsMYC2 has cascading effect on over-expression of multiple pathway genes leading to the increased triterpenoid biosynthesis in infiltered plants. Further, the functional validation of WsMYC2 was carried out by artificial micro-RNA mediated silencing. It resulted in significant reduction of withanolides and stigmasterol levels, indicative of crucial role of WsMYC2 in the regulation of their biosyntheses. Taken together, these non-complementary approaches provided unambiguous understanding of the regulatory role of WsMYC2 in context to withanolides and stigmasterol biosyntheses. Furthermore, the upstream promoter of WsMYC2 presented several cis-regulatory elements primarily related to phytohormone responsiveness. WsMYC2 displayed inducible nature in response to MeJA. It had substantial influence on the higher expression of WsMYC2 which was in consonance with enhanced accumulation of withanolides.
Keywords: Artificial micro RNA; Elicitations; MYC2; Over-expression; Phytohormones; Triterpenoids; Withania somnifera; Withanolides.