Comparative Transcriptome Analysis of Different Chemotypes of Withania somnifera to Elucidate Biosynthetic Pathway for Specific Withanolides

Withania somnifera (L.) Dunal (Family, Solanaceae), is one of the most valuable medicinal plants synthesizing large number of pharmacologically active secondary metabolites known as withanolides. The pharmacological activities of the Withania include anti-osteoporotic, anti-arthritic, anti-aging, anti-cancer and recovery from neurodegenerative disorders. Despite of having great pharmaceutical activities, there is very limited information about the genes responsible for biosynthesis of these molecules. Phytochemical profiling suggests that differential withanolide biosynthesis in leaf and root of different chemotypes with differences in the content and/or nature of major withanolide moieties. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis as well as to manipulate the pathways for enhanced biosynthesis, we established transcriptome sequences of leaf and root tissues of three distinct chemotypes. For digital differential expression analysis, reads from each library were tagged, pooled, and assembled into large contigs. Through annotation based approach, full-length sequence of all the genes involved in biosynthesis of precursor molecule (24-methylene cholesterol) for withanolides have been established. The involvement of a set of genes in biosynthesis of precursor molecules has been validated through Virus Induced Gene Silencing (VIGS) approach. This study has also identified a number of differentially expressing transcripts encoding members of cytochrome P450s, glycosyltransferases, methyltransferase and transcription factor gene families which might be involved in chemodiversity in Withania. On the basis of identified genes, their differential expression patterns and molecular docking studies, involvement of putative candidate genes have been proposed to elucidate biosynthetic pathway for specific withanolides.