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.
We investigated the protective effect of a natural polyphenol, magnolol, on Saccharomyces cerevisiae cells under oxidative stress, and during aging. Our results showed the sensitivity of S. cerevisiae antioxidant gene deficient mutants (sod1∆, sod2∆, cta1∆, ctt1∆, gtt2∆ and tsa1∆) against hydrogen peroxide (H2O2) and menadione stress was rescued by magnolol as demonstrated in spot and colony forming unit counts. Yeast cells pretreated with magnolol showed decreased intracellular oxidation, lipid peroxidation and an increased level of reduced glutathione. Further, SOD1, CTA1 and GTT2 gene expression was examined by reverse transcription-polymerase chain reaction, and was found that magnolol significantly attenuated the upregulation of SOD1 and CTA1 genes under oxidative stress. Finally, longevity of the wild type and sod1 mutant cells were extended by magnolol, and also enhance stress resistance against oxidant stress during chronological aging.
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