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RationaleMass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin‐related compounds using stable‐isotope‐labelled (SIL) indole‐3‐acetic acid (IAA) doped into agarose substrate. MethodsWild‐typeArabidopsis thalianaseedlings,sur2andwei8 tar2loss‐of‐function mutants, andYUC1gain‐of‐function line were grown for 3 days in the dark in standard growth medium. SIL‐IAA was doped into a 1% low‐melting‐point agarose gel and seedlings were gently laid on top for IR‐MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post‐acquisition by normalization of auxin‐related compounds to SIL‐IAA in the agarose. Amounts of auxin‐related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest. ResultsIAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR‐MALDESI analyses. Indole‐3‐acetaldoxime was increased insur2mutants compared with wild‐type and other mutants. Other auxin‐related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants. ConclusionsAgarose was shown to be an appropriate sampling surface for IR‐MALDESI mass spectrometry imaging ofArabidopsisseedlings. SIL‐IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations.