Biological roles of nitrile formation in the network of glucosinolate breakdown pathways in plants

化学多样性是植物特化代谢的最显著特征之一，在生物和非生物环境的选择压力下进化。与此一致，许多专门的代谢物作为化学防御或作为信号在器官相互作用。芥子油苷-黑芥子酶系统是目前研究最多的植物化学防御系统之一。它在植物-昆虫和植物-微生物相互作用中的作用取决于通过黑芥子酶水解完成的活化步骤和具有额外结构多样化的巨大潜力的下游反应，例如通过特异性蛋白。这种“芥子油炸弹”在组织破裂时引爆的经典观点已经被PEN 2的发现所拓宽，PEN 2是一种“非典型”黑芥子酶，它在没有预先组织损伤的情况下启动芥子油苷分解。PEN 2是16种β-葡糖苷酶进化枝（PEN 2/PYK 10进化枝）的成员，其中一些还显示水解芥子油苷并具有不同的表达模式。芥子油苷，黑芥子酶和特异蛋白积累的空间和时间调节表明芥子油苷分解途径被组织为一个模块化系统，控制特定的分解产物的形成取决于器官，发育阶段和环境条件。以拟南芥（Arabidopsis thaliana）Col-0为模型，我们将测试（1）地下生物相互作用是否依赖于根腈特异性蛋白（NSP）以及“经典”和/或“非典型”黑芥子酶，（2）NSP是否与"经典“和”非典型“黑芥子酶一起参与发育和萌发种子中的硫代葡萄糖苷周转途径，（3）根据在其表达位点的硫代葡萄糖甙谱，NSP是否对硫代葡萄糖甙糖苷配基侧链具有某些特异性，以及（4）如果A.事实上，拟南芥Col-0 β-葡糖苷酶是底物特异性黑芥子酶，其产物谱受NSP影响。为了验证这些假设，我们将对重组酶的联合收割机生化研究与使用突变体A的生物试验相结合。拟南芥株系与操纵的分解产物谱和研究响应于NSP缺乏的基因表达调控。经过100多年的研究，芥子油苷-黑芥子酶系统的生物学作用的知识仍然是零碎的。该项目将深入了解不同硫代葡萄糖苷分解产物的形成和器官特异性功能，从而有助于更好地了解植物结构多样性的多方面作用。

Chemical diversity, one of the most distinguished features of plant specialized metabolism, evolves under selection pressures imposed by the biotic and abiotic environment. In agreement with this, many specialized metabolites act as chemical defenses or as signals in organismic interactions. The glucosinolate-myrosinase system present in the Brassicales is one of the best studied plant chemical defenses. Its effects in plant-insect and plant-microbe interactions depend on an activation step accomplished through hydrolysis by myrosinases and downstream reactions with a great potential for additional structural diversification, e.g. by specifier proteins. The classical view of this 'mustard oil bomb' that detonates upon tissue disruption has been broadened by the discovery of PEN2, an 'atypical' myrosinase which initiates glucosinolate breakdown without prior tissue damage. PEN2 is member of a clade of 16 beta-glucosidases (PEN2/PYK10 clade) some of which have also been shown to hydrolyze glucosinolates and have distinct expression patterns. The spatial and temporal regulation of glucosinolate, myrosinase and specifier protein accumulation suggests glucosinolate breakdown pathways to be organized as a modular system that controls formation of specific breakdown products depending on organ, developmental stage and environmental conditions. Using Arabidopsis thaliana Col-0 as a model, we are going to test (1) if below-ground biotic interactions depend on root nitrile-specifier proteins (NSPs) together with 'classical' and/or 'atypical' myrosinases, (2) if NSPs are involved in pathways of glucosinolate turnover in developing and germinating seeds together with 'classical' and 'atypical' myrosinases, (3) if NSPs possess some specificity with respect to the glucosinolate aglucone side chain according to the glucosinolate profile at their site of expression, and (4) if members of the PEN2/PYK10 clade of A. thaliana Col-0 beta-glucosidases are, in fact, substrate-specific myrosinases whose product profile is affected by NSPs. In order to test these hypotheses, we are going to combine biochemical studies on recombinant enzymes with biotests using mutant A. thaliana lines with manipulated breakdown product profiles and studies on regulation of gene expression in response to NSP deficiency. After more than 100 years of research on the glucosinolate-myrosinase system, knowledge on the biological roles of glucosinolates is still fragmentary. This project will provide insights into formation and organ-specific functions of different glucosinolate breakdown products and thereby contribute to a better understanding of the multifacetted roles of structural diversity in plants.