An Arabidopsis GCMS chemical ionization technique to quantify adaptive responses in central metabolism.

Abstract

We present a methodology to survey central metabolism in 13CO2-labeled Arabidopsis (Arabidopsis thaliana) rosettes by ammonia positive chemical ionization-gas chromatography-mass spectrometry. This technique preserves the molecular ion cluster of methyloxime/trimethylsilyl-derivatized analytes up to 1 kDa, providing unambiguous nominal mass assignment of >200 central metabolites and 13C incorporation rates into a subset of 111 from the tricarboxylic acid (TCA) cycle, photorespiratory pathway, amino acid metabolism, shikimate pathway, and lipid and sugar metabolism. In short-term labeling assays, we observed plateau labeling of ∼35% for intermediates of the photorespiratory cycle except for glyoxylate, which reached only ∼4% labeling and was also present at molar concentrations several fold lower than other photorespiratory intermediates. This suggests photorespiratory flux may involve alternate intermediate pools besides the generally accepted route through glyoxylate. Untargeted scans showed that in illuminated leaves, noncyclic TCA cycle flux and citrate export to the cytosol revert to a cyclic flux mode following methyl jasmonate (MJ) treatment. MJ also caused a block in the photorespiratory transamination of glyoxylate to glycine. Salicylic acid treatment induced the opposite effects in both cases, indicating the antagonistic relationship of these defense signaling hormones is preserved at the metabolome level. We provide complete chemical ionization spectra for 203 Arabidopsis metabolites from central metabolism, which uniformly feature the unfragmented pseudomolecular ion as the base peak. This unbiased, soft ionization technique is a powerful screening tool to identify adaptive metabolic trends in photosynthetic tissue and represents an important advance in methodology to measure plant metabolic flux.