Abstact

NADPH-dependent thioredoxin reductase C (NTRC) is ubiquitously localized across all plastid types, including both chloroplasts and nonphotosynthetic plastids, serving as a central regulator in redox homeostasis. It plays pivotal roles in peroxidation resistance, redox signaling, tetrapyrrole biosynthesis, starch metabolism and photoperiod regulation. The NTRC/2-Cys peroxiredoxin (2CP)-mediated antioxidant defense system is also critically involved in counteracting biotic stress from pathogens. Relative studies demonstrate that the interaction between NTRC and 2CP is essential for modulating and integrating redox functions in chloroplasts. However, the molecular mechanism underlying the NTRC-2CP interaction remains elusive. In this study, we characterized the three-dimensional structure of Nicotiana benthamiana NTRC (NbNTRC) and resolved the crystal structure of its NTR domain by X-ray crystallography. Furthermore, we investigated the molecular basis of NbNTRC-Nb2CP interaction and stability using in vitro assays, including pull-down, size-exclusion chromatography, microscale thermophoresis and thermal stability assays. In summary, our work identifies two cysteine residues crucial for the intrinsic stability of the complex between NbNTRC and Nb2CP and the intermolecular interaction between them, determines the structure of the NbNTRC NTR domain and provides novel mechanistic insights into the NbNTRC-Nb2CP regulatory complex.