Distinct metabolic flux modes through the tricarboxylic acid cycle in mesophyll and guard cells revealed by GC-MS-based 13C-positional isotopomer analysis

Summary 13 C-Metabolic flux analysis ( 13 C-MFA) have greatly contributed to revealing the regulation of plant metabolism. However, mass spectrometry (MS) approaches have hitherto been limited in their power to deduce flux information due to lack of positional information. Here we established an MS-based 13 C-positional isotopomer labelling approach and performed a multi-species/cell-types analysis based on previous 13 C-MFA to compare flux modes through the tricarboxylic acid (TCA) cycle and associated pathways in mesophyll (MCs) and guard cells (GCs). Both cell types showed high 13 C-enrichment in pyruvate. However, GCs and sink MCs, but not source MCs showed high 13 C-incorporation into Glu/Gln following provision of 13 C-sucrose. Only GCs showed higher 13 C-enrichment in the carbon 1 atom of Gln, which is derived from PEPc-mediated CO 2 fixation. Increased 13 C-enrichment in the carbon 1 of Glu was also observed in both trxo1 and ntra ntrb mutants, but not in wild type Arabidopsis plants, following provision of 13 C-glucose. Our results suggest that the mitochondrial thioredoxin system restricts the fluxes from PEPc and glycolysis to Glu in illuminated MCs and reveal that fluxes throughout the TCA cycle of GCs resemble those of sink MCs but operate different non-cyclic flux modes to support Gln synthesis in the light.