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

Abstract

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.

Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis

“…5 ). Previous reports show that CAS overexpression and silencing resulted in an increase and decrease of withanolide content respectively 24 . In endophyte-inoculated plants, increased expression of CAS , SMT and ODM were correlated with enhanced withanolides production.…”

“…Overexpression of squalene synthase ( SQS ), a key regulatory gene of withanolide biosynthesis in W. somnifera could increase the content of withaferin A and withanolide A in the leaves up to 4–4.5 fold 23 . Overexpression of cycloartenol synthase ( CAS ) in W. somnifera increased the withanolide content to the extent of 1.06 to 1.66 fold 24 . Use of cell suspension 25 , adventitious 26 , 27 , and hairy root culture 28 for improvement of withanolide production have also been tried.…”

The Integration of Metabolomics and Next-Generation Sequencing Data to Elucidate the Pathways of Natural Product Metabolism in Medicinal Plants

“…According to the difference in the substituted groups of C-17 side chain, withanolides can be divided into two types; type A with a δ-lactone or δ-lactol and type B with γ-lactone or γ-lactol side chain [217]. Some recent investigations have identified putative regulatory and structural genes involved in withanolide biosynthesis [218][219][220][221].…”

Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd

“…This result corresponded to the DEUGs of the PtCAS genes after MeJA treatment. Similarly, Mishra et al [ 19 ] reported that transcript levels of the WsCAS ( Withania somnifera cycloartenol synthase) gene of the W. somnifera seedling, which contains a large amount of steroidal triterpene saponins, increased up to 1.65-fold after MeJA treatment. Taken together, these results suggest that the PtCAS genes may contribute to secondary steroidal triterpenoid biosynthesis as well as to phytosterols in P. tenuifolia .…”

“…It is likely that the PtCAS1 gene is constantly expressed to produce phytosterols and the PtCAS2 gene is responsible for the biosynthesis of steroidal compounds to protect themselves against changing or harsh environments such as pathogens coming into contact with flowers. In addition, CAS is activated to produce steroidal triterpenoids as secondary metabolites, like withanolide [ 19 ]. After 12 h of MeJA treatment on the seedlings of P. tenuifolia , the transcripts of the two CAS genes were higher than that of the control ( Figure 6 B).…”