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

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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.