Tiorredoxinas regulam os fluxos metabólicos através do ciclo do ácido tricarboxílico e vias associadas de forma independente da luz

Abstract

The tricarboxylic acid (TCA) cycle is an important metabolic hub that provides substrates for mitochondrial respiration and the synthesis of several amino acids. In plants, the metabolic fluxes throughout the TCA cycle are inhibited in the light by transcriptional and posttranslational mechanisms. For example, genes associated with the TCA cycle have their expression reduced in light conditions. Furthermore, it has been shown that the mitochondrial thioredoxin (TRX) system is a negative regulator of the activity of TCA cycle enzymes in illuminated leaves. However, it is unknown how this system contributes to the regulation of mitochondrial metabolism in the absence of light. This knowledge is of fundamental importance since the TCA cycle is an important hub for regulating the trade-off between growth and stress tolerance. Here, we aimed to analyze the influence of the mitochondrial TRX system on the control of metabolic fluxes throughout the TCA cycle and associated pathways under light and dark conditions. For this, we performed an isotopiclabeling experiment with 13C-HCO3 in detached leaves of Arabidopsis thaliana L. (WT) and in knockout plants for the genes TRX o1 (trxo1.1), TRX h2 (trxh2.2), or both NADPH-dependent TRX reductase A and B (ntra ntrb). The leaves were subjected to 13C-HCO3 feeding and frozen after 0, 30 and 60 min under light or dark conditions. The data generated by gas chromatography coupled to mass spectrometer was used to analyze the metabolite profiling and the distribution of 13C throughout the metabolism derived from 13C-HCO3. In addition, relative and positional 13C incorporation into glucose and malate was measured using an approach recently established in our group. No 13C enrichment was observed in TCA cycle metabolites in illuminated WT leaves. However, succinate content increased while alanine decreased in the light, suggesting an alternative carbon source for succinate synthesis in WT. In the dark, there was a significant increase in the content of malate, citrate and succinate in WT, evidencing a greater activity of the TCA cycle under this condition, when compared to light. In contrast to WT, all mutants showed substantial changes in both metabolite content and 13C enrichment, regardless of the imposed condition (light or dark). Multivariate analyzes showed that the double mutant ntra ntrb showed more drastic metabolic changes, while the trxo1 and trxh2 mutants showed an intermediate metabolite profile, compared to WT under both light and dark conditions. Interestingly, we observed 13C enrichment glutamine in trxo1.1 in the light and in glutamate in trxh2 and ntrb ntrb in the dark. Furthermore, there was an increase in the 13C-enrichment at the carbon 4 of malate in trxh2.2 in the light, as result of the activity of the enzyme phosphoenolpyruvate carboxylase (PEPc). These results suggest that TRX o1 restricts carbon flux for glutamine synthesis, while TRX h2 restricts PEPc activity, both in the light. Taken together, our results provide evidence that the TRX system is an important regulator of metabolic pathways associated with the TCA cycle in both dark and light conditions.