Hydrogen sulfide maintains nitric oxide homeostasis through PpWRKY33-PpNR and PpGSNOR to enhance cold tolerance of peach fruit

Hydrogen sulfide (H 2 S) regulates nitric oxide (NO) biosynthesis in peach ( Prunus persica ) fruit stress response, but the underlying mechanisms are unclear. In this study, electronic tongue (E-tongue) and electronic nose (E-nose) assays demonstrated that H 2 S treatment alleviated the deterioration of taste and aroma in peach fruit during chilling injury (CI) development. Exogenous H 2 S (20 μL · L −1 ) induced an early peak in intracellular NO levels, and correlation analysis further indicated that moderate NO concentrations (relative intensity of NO fluorescence: 1.63−3.88) were critical for maintaining fruit quality during long-term cold storage. Since overall NO levels reflect a balance between production and scavenging, we identified the H 2 S-responsive transcription factor PpWRKY33 as a transcriptional activator of peach nitrate reductase ( PpNR ), a key gene responsible for NO synthesis. The conserved β 2 and β 3 strands in the DNA-binding domain of PpWRKY33 specifically recognized the W-box in the PpNR promoter. Transient overexpression of PpWRKY33 in tobacco leaves and peach fruit increased NR expression, NR activity, and NO content. Furthermore, H 2 S (400−800 μmol · L −1 NaHS) enhanced S-nitrosoglutathione reductase (GSNOR) activity in vitro in a concentration-dependent manner. Flexible molecular docking indicated that H 2 S-derived persulfidation at the cysteine 271 (Cys271) of PpGSNOR altered the structure and electrostatic potential of the nicotinamide adenine dinucleotide (NADH)-ligand pocket, promoting NADH-PpGSNOR interactions for scavenging excess NO. Our findings uncover a fine-tuning mechanism for NO homeostasis in which H 2 S coordinately activates the PpWRKY33- PpNR pathway and NADH-dependent PpGSNOR activity, enhancing cold tolerance of peach fruit.