Genotype, age, tissue, and environment regulate the structural outcome of glucosinolate activation

Glucosinolates are the inert storage form of a two-part phytochemical defense system in which the enzyme myrosinase generates an unstable intermediate that rapidly rearranges into the biologically active product. This rearrangement step generates simple nitriles, epithionitriles, or isothiocyanates, depending on the structure of the parent glucosinolate and the presence of proteins that promote specific structural outcomes. Glucosinolate accumulation and myrosinase activity differ by plant age and tissue type and respond to environmental stimuli such as planting density and herbivory; however, the influence of these factors on the structural outcome of the rearrangement step remains unknown. We show that the structural outcome of glucosinolate activation is controlled by interactions among plant age, planting density, and natural genetic variation in Arabidopsis (Arabidopsis thaliana) rosette leaves using six well-studied accessions. We identified a similarly complex interaction between tissue type and the natural genetic variation present within these accessions. This raises questions about the relative importance of these novel levels of regulation in the evolution of plant defense. Using mutants in the structural specifier and glucosinolate activation genes identified previously in Arabidopsis rosette leaves, we demonstrate the requirement for additional myrosinases and structural specifiers controlling these processes in the roots and seedlings. Finally, we present evidence for a novel EPITHIOSPECIFIER PROTEIN-independent, simple nitrile-specifying activity that promotes the formation of simple nitriles but not epithionitriles from all glucosinolates tested.