Novel Bioassay for the Discovery of Inhibitors of the 2-<i>C</i>-Methyl-<i>D</i>-erythritol 4-Phosphate (MEP) and Terpenoid Pathways Leading to Carotenoid Biosynthesis

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.2-C-甲基-D-赤藓醇-4-磷酸（2-C-methyl-d-erythritol 4-phosphate，简称MEP）途径可在质体（plastids）中催化合成异戊烯基二磷酸（isopentenyl diphosphate）。该途径是类胡萝卜素（carotenoids）、生育酚（tocopherols）、质体醌（plastoquinone）、叶绿素植醇链，以及脱落酸（abscisic acid）和赤霉素（gibberellins）等物质合成前体的核心分支节点。因此，该途径的功能紊乱会对植物产生有害影响。为此，我们开发了一种可定量测定类胡萝卜素途径碳流的体内生物测定法（in vivo bioassay）：将离体叶片切段置于八氢番茄红素去饱和酶抑制剂（phytoene desaturase inhibitor）中培养，以诱导八氢番茄红素积累。任何能够降低八氢番茄红素积累水平的化合物，均有可能干扰MEP途径中的某一步骤，或是抑制牻牛儿基牻牛儿基二磷酸（geranylgeranyl diphosphate）的合成。我们通过靶向MEP途径各步骤的已知抑制剂验证了这一研究思路，并通过测试已知靶向MEP途径与类胡萝卜素途径之外过程的代表性除草剂，证实了该体内生物测定法的特异性。尽管部分抑制剂对该测定法存在非特异性作用等局限性，但该方法可实现对八氢番茄红素合成上游相关酶类新型抑制剂的快速筛选。