RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides

Withanolide biosynthetic pathway.

<p>IPP, isopentenyl pyrophosphate; MVA, mevalonate; MEP, 2-C-methyl-D-erythritol 4-phosphate GPPS, geranyl pyrophosphate synthase; FPPS, farnasyl pyrophosphate synthase; SS, squalene synthase; SE squalene epoxidase; CAS, cycloartenol synthase; LAS, lanosterol synthase; LS, lupeol synthase; BAS, beta amyrin synthase; AAS, alpha amyrin synthase.</p

<i>WsCAS</i> transcript expression analysis and withanolide accumulation in Withania silencing and overexpressing lines.

<p>(A) RealTime PCR analysis of <i>WsCAS</i> silenced lines. (B) Withanolide content in silenced lines. (C) Real-Time PCR analysis of <i>WsCAS</i> overexpressing lines. (D) Withanolide content in <i>WsCAS</i> overexpressing lines.</p

Profiling of <i>WsCAS</i> expression and withanolide in Withania seedlings under different elicitor conditions.

<p>(A) Seedling before and after treatment of cold, wounding, SA 2mM, SA 5mM, MeJ 200μM, MeJ 500μM and heating (a-i) respectively. (B) Real time-PCR analysis of <i>WsCAS</i>. (C) Quantitative withanolide profile.</p

Molecular characterization of Withania silenced lines and overexpressing lines of <i>WsCAS gene</i>.

<p>(A) PCR amplification with <i>bar</i> primers showing amplicon of ≈400 bp. Lane M 100 bp ladder; lane–C untransformed shoot; lane +C plasmid DNA; lane 1–9, <i>in vitro</i> transformed shoots; (B) Southern blot analysis of transgenic silenced lines of <i>Withania</i> with <i>bar</i> PCR probe. (C) PCR amplification of pIGCAS transformants with <i>npt</i>II primers showing amplicon of 500bp. Lane- 1 to 6 transformed lines. (D) Southern blot analysis of overexpresing lines.</p

Full length <i>WsCAS</i> gene with three selected silencing sites for silencing constructs preparation in <i>Withania somnifera</i>.

<p>19 bp silencing sites were enclosed in red colored boxes. Arrows indicate the region of start of amplification of particular fragment incorporating silencing sites.</p