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最显著的特征之一，它是在生物和非生物环境的选择压力下进化的。与此一致的是，许多专门的代谢物在有机体相互作用中起到化学防御或信号的作用。芥蓝属植物中存在的硫代葡萄糖苷-黑芥子酶系统是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依赖于一个激活步骤，该步骤通过黑芥子酶的水解和下游反应完成，具有巨大的额外结构多样性的潜力，例如通过指定蛋白。PEN2是一种非典型的黑芥子酶，可以在没有组织损伤的情况下引发硫代葡萄糖酸盐的分解，这拓宽了人们对这种在组织破裂时会引爆芥子油炸弹的经典观点。PEN2是16种β-葡萄糖苷酶(PEN2/PYK10)中的一员，其中一些还被证明能水解硫代葡萄糖苷，并有不同的表达模式。硫代葡萄糖苷、黑芥子酶和特定蛋白积累的时空调节表明，硫代葡萄糖苷的分解途径可以被组织成一个模块系统，根据器官、发育阶段和环境条件来控制特定分解产物的形成。以拟南芥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说明子蛋白(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’黑芥子酶一起参与种子发育和萌发过程中硫代葡萄糖苷的转运途径，(3)根据表达部位的硫代葡萄糖苷图谱，NSP是否对硫代葡萄糖苷无糖元侧链具有一定的特异性，以及(4)拟南芥Col-0-β葡萄糖苷酶PEN2/PYK10分支的成员是否确实是底物专一性黑芥子酶，其产物受NSP影响。为了验证这些假说，我们将结合对重组酶的生化研究和利用具有操纵分解产物谱的突变体进行的生物测试，以及对NSP缺乏反应的基因表达调控的研究。在对硫代葡萄糖苷-黑芥子酶系统进行了100多年的研究后，对硫代葡萄糖苷的生物学作用的了解仍然是零散的。该项目将深入了解不同硫代葡萄糖苷分解产物的形成和特定器官的功能，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.