N-hydroxy pipecolic acid methyl ester is involved in Arabidopsis immunity

The biosynthesis of N-hydroxy pipecolic acid (NHP) has been intensively studied, though knowledge on its metabolic turnover is still scarce. To close this gap, we discovered three novel metabolites via metabolite fingerprinting in Arabidopsis thaliana leaves. Exact mass information and fragmentation by mass spectrometry (MSMS) suggest a methylated derivative of NHP (MeNHP), a NHP-OGlc-hexosyl conjugate (NHP-OGlc-Hex) and an additional NHP-OGlc-derivative. All three compounds were formed in wildtype leaves but not present in the NHP deficient mutant fmo1-1. The identification of these novel NHP-based molecules was possible by a dual-infiltration experiment using a mixture of authentic NHP- and D9-NHP-standards for leaf infiltration followed by an UV-C treatment. Interestingly, the signal intensity of MeNHP and other NHP-derived metabolites increased in ugt76b1-1 mutant plants. This suggests a detour, for the inability to synthesize NHP-O-glucoside. For MeNHP, we unequivocally determined the site of methylation at the carboxylic acid function. MeNHP application by leaf infiltration leads to the detection of a MeNHP-OGlc as well as NHP, suggesting MeNHP-hydrolysis to NHP. This is in line with the observation that MeNHP-infiltration is able to rescue the fmo1-1 susceptible phenotype against Hyaloperonospora arabidopsidis Noco 2. Together these data suggest MeNHP as additional storage or transport form of NHP. HighlightIn this work, we identify N-hydroxy pipecolic acid (NHP) metabolites including methyl ester and complex glycosides. The application of methyl ester is able to rescue the disease phenotype of the biosynthesis deficient mutant of NHP.