Biological roles of nitrile formation in the network of glucosinolate breakdown pathways in plants
Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
Plant Physiology
Final Report Abstract
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. Most of its effects in plant-insect and plantmicrobe interactions depend on an activation step accomplished through hydrolysis by myrosinases (thioglucoside glucosidases, TGG) and downstream reactions with a great potential for additional structural diversification, e.g. by specifier proteins such as nitrilespecifier proteins (NSPs). The classical view of this 'mustard oil bomb' that detonates upon tissue disruption has been broadened by the discovery of so-called 'atypical' myrosinases (βglucosidases of the BGLU18-BGLU33 clade) some of which initiate glucosinolate breakdown without prior tissue damage. In this project, we combined biochemical studies on recombinant NSPs and BGLU enzymes with biotests using mutant Arabidopsis thaliana lines to expand our understanding of the organization and biological roles of the glucosinolatemyrosinase system with focus on below-ground interactions. We characterized the newly established mutant nsp134, an A. thaliana line deficient in root NSPs, with respect to its glucosinolate and glucosinolate breakdown products in root and leaf homogenates. Using nsp134 plants as well as lines impaired in aliphatic or indole glucosinolate biosynthesis, we demonstrated that besides glucosinolate biosynthesis the ability to form nitriles is critical for the establishment of the rhizosphere microbial community of A. thaliana. Our results are in agreement with breakdown of glucosinolates in the intact root prior to metabolite excretion to the rhizosphere, but future studies are required to understand the responsible mechanisms. As A. thaliana roots are rich in indole glucosinolates, we studied the influence of NSP1-NSP5 (recombinantly expressed in Escherichia coli) on product formation upon in vitro indole glucosinolate hydrolysis catalyzed by root myrosinase TGG4 (recombinantly expressed in Pichia pastoris). This showed that all five NSPs are, in principle, able to generate considerable amounts of nitrile upon hydrolysis of indol-3-ylmethylglucosinolate (I3M), 1-methoxy-I3M and 4-methoxy-I3M. However, the NSPs appear to differ in their activity upon hydrolysis of 4-methoxy-I3M, a compound known to play a role in above-ground antipathogen defense. We also expressed two β-glucosidases of the BGLU18-BGLU33 clade functionally in P. pastoris and found that they have distinct substrate spectra. Our results lay the foundation for further evaluation of the complex machinery involved in glucosinolate breakdown in A. thaliana and its biological roles in future projects.
Publications
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Assessing the roles of glucosinolates and their different breakdown product types in below-ground interactions. International Conference of the German Society of Plant Sciences, Bonn, poster
Hielscher A., Ravindran B.M., Stauber E.J. & Wittstock U.
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Impaired glucosinolate biosynthesis and berakdown product formation affect microbial composition in the rhizosphere of Arabidopsis thaliana. International Conference of the German Society of Plant Sciences, Bonn, poster
Chroston E.C.M., Ravindran B.M., Stauber E.J., Meier K., Bziuk N., Smalla K. & Wittstock U.
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The Impact of Nitrile-Specifier Proteins on Indolic Carbinol and Nitrile Formation in Homogenates of Arabidopsis thaliana. Molecules, 27(22), 8042.
Chroston, Eleanor C. M.; Hielscher, Annika; Strieker, Matthias & Wittstock, Ute
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Assessing the effects of glucosinolates and their different breakdown product types on the rhizosphere bacterial community. International Conference of the German Society of Plant Sciences, Halle, poster
Hielscher A., Chroston E.C.M., Bziuk N., Stauber E.J., Ravindran B.M., Smalla K. & Wittstock U.
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Glucosinolate structural diversity shapes recruitment of a metabolic network of leaf-associated bacteria. Nature Communications, 15(1).
Unger, Kerstin; Raza, Syed Ali Komail; Mayer, Teresa; Reichelt, Michael; Stuttmann, Johannes; Hielscher, Annika; Wittstock, Ute; Gershenzon, Jonathan & Agler, Matthew T.
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Plant glucosinolate biosynthesis and breakdown pathways shape the rhizosphere bacterial/archaeal community. Plant, Cell & Environment, 47(6), 2127-2145.
Chroston, Eleanor C. M.; Bziuk, Nina; Stauber, Einar J.; Ravindran, Beena M.; Hielscher, Annika; Smalla, Kornelia & Wittstock, Ute
