title: Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis.
abstract: Withanolides biosynthesis in the plant Withania somnifera (L.) Dunal is hypothesized to be diverged from sterol pathway at the level of 24-methylene cholesterol. The conversion and translocation of intermediates for sterols and withanolides are yet to be characterized in this plant. To understand the influence of mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways on sterols and withanolides biosynthesis in planta, we overexpressed the WsHMGR2 and WsDXR2 in tobacco, analyzed the effect of transient suppression through RNAi, inhibited MVA and MEP pathways and fed the leaf tissue with different sterols. Overexpression of WsHMGR2 increased cycloartenol, sitosterol, stigmasterol and campesterol compared to WsDXR2 transgene lines. Increase in cholesterol was, however, marginally higher in WsDXR2 transgenic lines. This was further validated through transient suppression analysis, and pathway inhibition where cholesterol reduction was found higher due to WsDXR2 suppression and all other sterols were affected predominantly by WsHMGR2 suppression in leaf. The transcript abundance and enzyme analysis data also correlate with sterol accumulation. Cholesterol feeding did not increase the withanolide content compared to cycloartenol, sitosterol, stigmasterol and campesterol. Hence, a preferential translocation of carbon from MVA and MEP pathways was found differentiating the sterols types. Overall results suggested that MVA pathway was predominant in contributing intermediates for withanolides synthesis mainly through the campesterol/stigmasterol route in planta.
authors.affiliation: Biotechnology Division, CSIR - Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, UP, India.
authors.forename: Shefali
authors.initials: S
authors.lastname: Singh
authors.forename: Shaifali
authors.initials: S
authors.lastname: Pal
authors.forename: Karuna
authors.initials: K
authors.lastname: Shanker
authors.forename: Chandan Singh
authors.initials: CS
authors.lastname: Chanotiya
authors.forename: Madan Mohan
authors.initials: MM
authors.lastname: Gupta
authors.forename: Upendra Nath
authors.initials: UN
authors.lastname: Dwivedi
authors.forename: Ajit Kumar
authors.initials: AK
authors.lastname: Shasany
mesh_terms: D001483:Base Sequence; D053898:Biosynthetic Pathways; D002244:Carbon / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D008798:Mevalonic Acid / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010840:Phytosterols / Q000737:chemistry / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D017422:Sequence Analysis, DNA; D012855:Sitosterols / Q000737:chemistry / Q000378:metabolism; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013265:Stigmasterol / Q000737:chemistry / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism; D032341:Withania / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D054358:Withanolides / Q000737:chemistry / Q000378:metabolism*
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D010840:Phytosterols; D010940:Plant Proteins; D012855:Sitosterols; D013261:Sterols; D013403:Sugar Phosphates; D014315:Triterpenes; D054358:Withanolides; C021273:campesterol; C025473:gamma-sitosterol; D002244:Carbon; D002784:Cholesterol; D013265:Stigmasterol; D004896:Erythritol; D008798:Mevalonic Acid; C100089:cycloartenol