Comparative withanolide profiles, gene isolation, and differential gene expression in the leaves and roots of Withania somnifera

Abstract

SummaryWithania somnifera plants derive their therapeutic properties by synthesising biologically-active, steroidal lactones known as withanolides. However, little is known about the genes that encode the enzymes involved in this biosynthetic pathway. Significant differences in the spectra of withanolides detected in the aerial and underground organs of plants suggest differential expression of distinct organ-specific pathways and genes. Since expressed sequence tags (ESTs) provide a robust method for gene prospecting, and few ESTs are available for W. somnifera in the public databases, this study used an EST-based approach to analyse gene expression profiles in W. somnifera plants. Separate leaf and root cDNA libraries of W. somnifera were generated and yielded 495 leaf and 76 root ESTs. Two of the leaf ESTs were then converted into full-length gene sequences using RACE procedures. Genes for sterol methyl transferase (SMT1) and obtusifoliol-14 -demethylase (ODM), whose products are claimed to be involved in the biosynthetic pathway that leads to withanolides, were thus cloned. Withaferin-A contents, as well as the abundance of ODM and SMT1 gene transcripts, were found to be higher in leaves than in roots, and increased in both organs as the plants matured. Genes encoding 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMGR) were also isolated via W. somnifera leaf mRNA using RT-PCR. Subtractive hybridisation between leaf and root cDNA populations yielded 36 leaf-abundant and 47 root-abundant ESTs, some of which showed sequence (80%) homology to glucosyltransferases, which may be significant in view of the highly glucosylated nature of withanolides.