Leaves and Roots of Micropropagated Plants Adopt Different Pathways for Withanolide Biosynthesis in Withania Coagulans: A Comparative Transcriptome Study

Abstract

Withania coagulans (Stocks) Dunal commonly known as paneerbandh due to its milk coagulating properties, is a widely used medicinal herb, and micropropagated plants of the plant to produce some novel withanolides. Trasncscriptome sequencing of leaves and roots of micropropagated plants of W. coagulans assembled total 8.08 and 6.35 GB of raw reads into 292,074 and 16,474 high quality reads, out of which 267,119 and 15,758 unigenes were identified in WcL and WcR, respectively. Further, 40.6% WcL and 55.05% WcR unigenes were annotated using more than one database. Metabolic process and cellular components were identified as dominant categories in gene ontology, while total 20,927 WcL and 2,474 WcR unigenes were mapped to different biological pathways. KEGG classification aided in identification of genes involved in biosynthesis of withanolide precursor, 24-methylenecholesterol. All the genes related to withanolide precursor biosynthesis were present only in WcL, indicating de novo biosynthesis of withanolides, while absence of some rate limiting enzymes in WcR indicated towards biosynthesis of withanolides through salvage pathways. GTs, MTs and CYP450s were identified as putative genes involved in conversion of 24-methylenecholesterol to different withanolides. Differential expression of these genes further revealed details of enzymes involved in biosynthesis of tissue-specific withanolides. Further, withanolide profiling through HPLC analysis ascertained the differential biosynthesis and accumulation of withanolides in both the tissues in relatively very less concentration confirmed their biosynthesis through salvage mechanism in roots. Therefore, the present study can be fruitful for future research and product development of W. coagulans through pathway engineering. Moreover, the SSRs identified in this study can be used in marker assisted breeding and selection of biochemically elite varieties of W. coagulans.