BASF SE, Crop Protection, Speyerer Str. 2, 67117 Limburgerhof, Germany.
Plant Sci. 2013 Nov;212:60-71. doi: 10.1016/j.plantsci.2013.08.005. Epub 2013 Aug 29.
Small molecules affecting plant processes have been widely used as probes to study basic physiology. In agricultural practices some of these molecules have served as herbicides or plant growth regulators. Historically, most of the compounds were identified in large screens by the agrochemical industry, but also as phytoactive natural products. More recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. In the present review different approaches were evaluated for the identification of the mode of action (MoA) of phytoactive compounds. Based on the methodologies used for MoA identification, three approaches are differentiated: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach. Target sites of compounds targeting primary or secondary metabolism were identified most successfully with a phenotyping approach. Target sites for compounds that influence cell structure, such as cell wall biosynthesis or the cytoskeleton, or compounds that interact with the hormone system, were in most cases discovered by using a genetic approach. Examples showing the strengths and weaknesses of the different approaches are discussed in detail. Additionally, new techniques that could contribute to future MoA identification projects are reviewed. In particular, next-generation sequencing techniques may be used for the fast-forward mapping of mutants identified in genetic screens. Finally, a revised three-tiered approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st tier, which addresses compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes such as transcription and translation. Advanced studies based on these findings initiate the 2nd tier MoA characterization, either with further phenotypic characterization, starting a genetic screen or establishing a biochemical screen. At the 3rd tier, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.
小分子影响植物过程已被广泛用作探针来研究基本的生理学。在农业实践中，这些小分子中的一些被用作除草剂或植物生长调节剂。历史上，大多数化合物是通过农业化学工业的大规模筛选，以及植物活性天然产物的筛选来识别的。最近，新型植物活性化合物来源于学术界通过化学筛选进行的研究，以诱导感兴趣的特定表型。在本综述中，评估了不同的方法来鉴定植物活性化合物的作用模式 (MoA)。基于用于 MoA 鉴定的方法，区分了三种方法：表型鉴定方法、基于遗传筛选的方法和生化筛选方法。使用表型鉴定方法最成功地鉴定了靶向初级或次级代谢的化合物的靶标位点。靶向影响细胞结构的化合物（如细胞壁生物合成或细胞骨架）或与激素系统相互作用的化合物的靶标位点，在大多数情况下是通过遗传方法发现的。详细讨论了显示不同方法的优缺点的示例。此外，还回顾了有助于未来 MoA 鉴定项目的新技术。特别是，下一代测序技术可用于快速映射遗传筛选中鉴定的突变体。最后，提出了一种修订的植物活性化合物作用模式鉴定的三级方法。该方法包括第一层，用于解决化合物的稳定性、不同物种中效果的均匀性、一般细胞毒性以及对转录和翻译等常见过程的影响。基于这些发现的进一步研究启动了第二层 MoA 特征描述，要么通过进一步的表型特征描述、开始遗传筛选或建立生化筛选来进行。在第三层，酶测定或蛋白质亲和研究应显示化合物在假定靶标上的活性，并将体外效应与化合物的体内特征相关联。
