Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng

H B Shilpashree,S J Sudharshan,Dinesh A Nagegowda,Ajit K Shasany

doi:10.1038/s41598-022-05634-9

H B Shilpashree, S J Sudharshan + Show 2 more

Open Access

https://doi.org/10.1038/s41598-022-05634-9

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Abstract

The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress.

Highlights

Withania somnifera (Ashwagandha, Indian ginseng or winter cherry) is a medicinal plant of high repute belonging to the Solanaceae family
Identification of WsCYP450s involved in secondary metabolism
The results demonstrated that change in the expression of these genes by silencing or overexpression resulted in significant differential modulation of certain withanolides indicating their role in one or more steps of the withanolides biosynthetic pathway

Summary

Introduction

Withania somnifera (Ashwagandha, Indian ginseng or winter cherry) is a medicinal plant of high repute belonging to the Solanaceae family. Plant extracts of W. somnifera and purified withanolides have demonstrated diverse pharmacological activities such as anti-inflammatory, anti-tumor, cardioprotective, neuroprotective and anti-bacterial properties[1,2]. Despite their immense pharmacological and therapeutic potential, commercial exploitation of withanolides has been limited owing to low in planta accumulation (in the range of 0.001–0.5% of DW) resulting in limited availability in purified forms[3]. It was shown that WsCYP85A69 catalyzed in vitro conversion of 6-deoxocastasterone into c astasterone[13] Despite some of these above studies, the knowledge on the in planta role of CYP450s in withanolides biosynthesis and defense is limited. We present the molecular characterization of three genes encoding CYP450s and show the in planta involvement in withanolides biosynthesis and defense using silencing and overexpression studies

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