Thioredoxin h1-targeted proteins in European beech (Fagus sylvatica L.): Insights into metabolic control, stress...

Seed viability and longevity underpin forest regeneration under changing environmental conditions. European beech (Fagus sylvatica L.) produces intermediate seeds that gradually lose viability during storage, largely through oxidative stress, which makes redox homeostasis central to their longevity. Here we investigated the role of thioredoxin h1 (TRX h1), a cytosolic thiol-disulfide oxidoreductase, in the embryonic axes of beech seeds. Using a His-tagged monocysteinic (Cys to Ser) trapping mutant of TRX h1 combined with data-dependent acquisition (DDA) proteomics, we identified 161 putative TRX h1 target proteins, which fall into functional categories related to genetic information processing, metabolism, environmental and cellular signaling, and protein homeostasis. Western blotting confirmed that target capture proceeded through DTT-reducible mixed-disulfide complexes, supporting a redox-based interaction. The targets include heat shock proteins (HSP70 and HSP90) and other chaperones, calcium-binding proteins (calmodulin and calreticulin), a methyl-CpG-binding protein, glycolytic and tricarboxylic acid (TCA) cycle enzymes, and antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and glutaredoxin. To link these targets to seed physiology, we quantified the thiol redox status of fresh and long-term stored seeds: total thiols were approximately 2.2-fold lower in stored than in fresh axes (21.0 ± 0.5 vs 45.9 ± 1.3 nmol GSH equivalents per axis), indicating that the reduced thiol pool, the substrate of the TRX h1 system, becomes depleted and more oxidized during storage. Together, these results suggest that TRX h1-dependent redox maintenance may contribute to redox homeostasis, energy metabolism and protein stability in beech embryonic axes, and provide a basis for improving seed storage and conservation strategies.