Radial distribution of withanolides in tuberous roots of <i>Withania somnifera</i> is linked to ethylene-mediated regulation of <i>Ws24ISO2</i>

The medicinal benefits of Withania somnifera tuberous roots have been attributed mainly to the residing withanolides. However, their yield from the root is poor, and knowledge about withanolide biosynthesis is limited. A specific sterol Δ24-isomerase (24ISO) gene (a third paralog of DWF1, hereby named Ws24ISO1) produces the precursor for various withanolides. In the present study, we observed that 75% to 80% of withanolides accumulate in the region of the root outside the vascular cambium, which forms only 25% to 30% of the total biomass. Through de novo transcriptome analysis, we identified higher expression of Ws24ISO2 (a homolog of Ws24ISO1) in regions outside the cambium and showed its involvement in withanolide biosynthesis similar to Ws24ISO1. Also, stronger expression of Ws24ISO2 than Ws24ISO1 was observed in mature roots of W. somnifera. RNA in situ hybridization using probes for these transcripts further established the radial biosynthesis pattern of withanolides in the mature roots. Transcriptome analysis revealed the involvement of ethylene in withanolide biosynthesis by regulating Ws24ISO2 but not Ws24ISO1 expression, unlike jasmonate/oxylipin signaling, which regulated both these genes. In addition, W. somnifera transgenics overexpressing Ws24ISO2 showed increased withanolide accumulation in the regions inside the vascular cambium of the roots. Thus, the present work uncovers an ethylene-mediated radial distribution pattern of withanolides in W. somnifera roots and advances the knowledge on the regulation of their biosynthesis. This will be effective in designing further strategies to breed better varieties with improved root withanolide content.