Integrative in Silico and in Vitro Evidence for Thioredoxin-Mediated Regulation of Nitrogen Assimilation Enzymes in Plants

Abstract The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle is the main pathway for assimilating inorganic ammonium into organic molecules, being a central hub connecting carbon and nitrogen metabolisms. Recent findings suggest that the mitochondrial thioredoxin (mTRX) system can coordinate metabolic fluxes from the tricarboxylic acid (TCA) cycle toward glutamate and glutamine in a mechanism apparently independent of the redox status of GS. It remains unclear whether this mechanism involves GOGAT and glutamate dehydrogenase (GDH), other enzymes involved in glutamate metabolism. Here, we carried out bioinformatics and biochemical analyses to investigate how the absence of TRXs affects the enzyme activity of GOGAT and GDH in leaves harvested at the end of the day (ED), end of the night (EN), and after a short high-light (HL) period. We used plants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntrab), alongside wild-type (WT) plants. Molecular docking revealed multiple stable interaction modes between TRX o1 and GOGAT, GDH1, and GDH2. The lowest-energy clusters indicate high-affinity binding, with distinct hotspots identified for each enzyme. These results suggest that TRX o1 may engage in several energetically favorable conformations relevant to functional or regulatory interactions. The activity of GOGAT and GDH was lower in both trxo1 and ntrab mutant lines at ED. In addition, trxo1 mutants exhibited a reduction in the potential quantum yield of PSII under HL stress, accompanied by higher activities of GOGAT and GDH compared with WT plants under HL conditions. Collectively, our results suggest that the mTRX system is important to modulate GOGAT and GDH activities, including under HL stress.