A Fast and Precise Method To Identify Indolic Glucosinolates and Camalexin in Plants by Combining Mass Spectrometric and Biological Information

摘要

In this manuscript, a fast and accurate identification and quantitation by mass spectrometry of indolicglucosinolates and camalexin involved in defense in Arabidopsis thaliana are described. Two elicitation systems, inoculation withBotrytis cinerea and treatment with AgNO3, were used in Col-0 wild-type and mutant genotypes impaired in the biosynthesis ofthe selected metabolites. Identification of analytes was carried out by nontargeted LC/ESI-QTOF-MS profiling. Confirmation ofindolic glucosinolates and camalexin was achieved by their absence in the cyp79B2/B3 and pad3 mutants as well as theirrespective fragmentation upon collision-induced dissociation. Camalexin accumulation was induced only after AgNO3 treatment,whereas all indolic glucosinolates were constitutively present. Inoculation with Botrytis did not influence camalexin concentrationbut caused most aliphatic and indolic glucosinolates contents to decrease. Only the pen 3.1 mutant showed increased indolicglucosinolate levels after Botrytis or AgNO3 treatments. In addition, profiles of secondary metabolite in nontreated Col-0 andmutant plants were analyzed by means of partial least squares coupled to discriminant analysis (PLS-DA), and differences in thebasal levels of indolic glucosinolates and tryptophan between cyp79B2/B3 plants and the rest of genotypes, including Col-0, werefound. This probably has to be taken into consideration when comparing stress responses of Col-0 and cyp79B2/B3. The use ofmutants carrying alterations in biosynthetic pathways is proposed as a useful strategy to identify secondary metabolites.