Singh Shefali, Pal Shaifali, Shanker Karuna, Chanotiya Chandan Singh, Gupta Madan Mohan, Dwivedi Upendra Nath, Shasany Ajit Kumar
Biotechnology Division, CSIR - Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, UP, India.
Physiol Plant. 2014 Dec;152(4):617-33. doi: 10.1111/ppl.12213. Epub 2014 Jun 3.
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.
在植物睡茄（Withania somnifera (L.) Dunal）中，假睡茄内酯生物合成被认为是在24-亚甲基胆固醇水平上从甾醇途径分化而来。该植物中甾醇和假睡茄内酯中间体的转化和转运尚未得到表征。为了了解甲羟戊酸（MVA）和2-C-甲基-D-赤藓糖醇-4-磷酸（MEP）途径对植物中甾醇和假睡茄内酯生物合成的影响，我们在烟草中过表达了WsHMGR2和WsDXR2，通过RNAi分析了瞬时抑制的效果，抑制了MVA和MEP途径，并给叶片组织饲喂不同的甾醇。与WsDXR2转基因株系相比，WsHMGR2的过表达增加了环阿屯醇、谷甾醇、豆甾醇和菜油甾醇。然而，WsDXR2转基因株系中胆固醇的增加略高。这通过瞬时抑制分析和途径抑制得到了进一步验证，在叶片中发现由于WsDXR2抑制导致胆固醇降低更高，而所有其他甾醇主要受WsHMGR2抑制的影响。转录本丰度和酶分析数据也与甾醇积累相关。与环阿屯醇、谷甾醇、豆甾醇和菜油甾醇相比，饲喂胆固醇并没有增加假睡茄内酯含量。因此，发现来自MVA和MEP途径的碳优先转运，从而区分了甾醇类型。总体结果表明，在植物中，MVA途径在为假睡茄内酯合成贡献中间体方面占主导地位，主要通过菜油甾醇/豆甾醇途径。
