Novel guard cell sink characteristics revealed by a multi-species/cell-types meta-analysis of 13 C-labelling experiments

The tricarboxylic acid (TCA) cycle is an important metabolic pathway to underpin stomatal movements, given that respiration is thought to be the main energy source for guard cell (GC) metabolism. However, it is still unclear how the metabolic fluxes throughout the TCA cycle and associated pathways are regulated in GCs. Here we used a 13 C-positional isotopomer approach and performed a multi-species/cell-types analysis based on previous 13 C-labelling studies carried out using Arabidopsis rosettes, maize leaves, Arabidopsis source and sink leaves, and isolated GCs from Arabidopsis and tobacco. We aimed to compare flux modes through the TCA cycle and associated pathways in GCs and leaves, which are mostly composed by mesophyll cells (MCs). Mesophyll cells showed high 13 C-enrichment into alanine and aspartate following provision of 13 CO 2 , whilst GCs and sink MCs showed high 13 C-incorporation into glutamate/glutamine following provision of 13 C-sucrose. Only GCs showed high 13 C-enrichment in the carbon 1 atom of glutamine, which is derived from phospho enol pyruvate carboxylase (PEPc)-mediated CO 2 assimilation. The PEPc-mediated 13 C-incorporation into malate was similar between GCs and MCs, but GCs had higher 13 C-enrichment and accumulation of fumarate than MCs. The metabolic fluxes throughout the TCA cycle of illuminated GCs resemble those of sink MCs, but with different contribution from PEPc, glycolysis and the TCA cycle to glutamate/glutamine synthesis. We further demonstrate that transamination reactions catalysed by alanine and aspartate amino transferases may support non-cyclic TCA flux modes in illuminated MCs.