RESUMEN
Abscisic acid (ABA) is crucial for plant water deficit (WD) acclimation, but how the interplay between ABA and guard cell (GC) metabolism aids plant WD acclimation remains unclear. Here, we investigated how ABA regulates GC metabolism and how this contributes to plant WD acclimation using tomato wild type (WT) and the ABA-deficient sitiens mutant. These genotypes were characterized at physiological, metabolic, and transcriptional levels under recurring WD periods and were used to perform a13C-glucose labelling experiment using isolated guard cells following exogenously applied ABA. ABA deficiency altered the level of sugars and organic acids in GCs in both irrigated and WD plants and the dynamic of accumulation/degradation of these compounds in GCs during the dark-to-light transition. WD-induced metabolic changes were more pronounced in sitiens than WT GCs. Results from the 13C-labelling experiment indicate that ABA is required for the glycolytic fluxes toward malate and acts as a negative regulator of a putative sucrose substrate cycle. The expression of key ABA-biosynthetic genes was higher in WT than in sitiens GCs after two cycles of WD. Additionally, the intrinsic leaf water use efficiency increased only in WT after the second WD cycle, compared to sitiens. Our results highlight that ABA deficiency disrupts the homeostasis of GC primary metabolism and the WD memory, negatively affecting plant WD acclimation. Our study demonstrates which metabolic pathways are activated by WD and/or regulated by ABA in GCs, which improves our understanding of plant WD acclimation, with clear consequences for plant metabolic engineering in the future.
RESUMEN
The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a13C-HCO3 labelling experiment in Arabidopsis leaves from wild type (WT) and mutants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntra ntrb) exposed to 0, 30 and 60 min of dark or light conditions. No 13C-enrichment in TCAC metabolites in illuminated WT leaves was observed. However, increased succinate content was found in parallel to reductions in Ala in the light, suggesting the latter operates as an alternative carbon source for succinate synthesis. By contrast to WT, all mutants showed substantial changes in the content and 13C-enrichment in TCAC metabolites under both dark and light conditions. Increased 13C-enrichment in glutamine in illuminated trxo1 leaves was also observed, strengthening the idea that TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCAC to glutamine. We further demonstrated that both photosynthetic and gluconeogenic fluxes toward glucose are increased in trxo1 and that the phosphoenolpyruvate carboxylase (PEPc)-mediated 13C-incorporation into malate is higher in trxh2 mutants, as compared to WT. Our results collectively provide evidence that TRX h2 and the mitochondrial NTR/TRX system regulate the metabolic fluxes throughout the TCAC and associated pathways, including glycolysis, gluconeogenesis and the synthesis of glutamine in a light-independent manner.