S-Nitrosylation of GSNOR and LCD integrates NO and H2S signaling to regulate cadmium-induced programmed cell death in tomato

Cadmium (Cd), a non-essential and toxic heavy metal, severely disrupts plant physiological and biochemical processes by inducing programmed cell death (PCD). Nitric oxide (NO) and hydrogen sulfide (H₂S) are key signaling molecules involved in plant stress responses, but the molecular mechanisms underlying their crosstalk in Cd-induced PCD remain elusive. Here, we first demonstrated that Cd-triggered PCD is accompanied by NO bursts, where NO dynamically modulates PCD progression—exacerbating cell death when depleted and alleviating it when present. Proteomic analysis of S-nitrosylated proteins revealed that differential S-nitrosylation targets in Cd-induced vs. NO-alleviated PCD are enriched in carbohydrate metabolism and amino acid metabolism, with unique targets in cofactor/vitamin metabolism and lipid metabolism. Additionally, S-nitrosylation of proteins involved in porphyrin/chlorophyll metabolism and starch/sucrose metabolism contributes to Cd-induced leaf chlorosis, while in vivo S-nitrosylation of SEC23 (protein transport), ubiquitinyl hydrolase 1, and pathogenesis-related protein 1 was confirmed, with their expressions upregulated in Cd-induced PCD but downregulated by NO treatment (consistently observed in tomato seedlings with elevated S-nitrosylation levels). Building on this foundation, further investigation using GSNOR (S-nitrosoglutathione reductase, a key regulator of NO homeostasis) and LCD (L-cysteine desulfhydrase, a core enzyme for H₂S biosynthesis) knockout and overexpressing transgenic tomato (Solanum lycopersicum L.) demonstrated that both GSNOR and LCD inhibit Cd²⁺-induced PCD. GSNOR and LCD knockout plants exhibited increased Cd sensitivity and enhanced cell death compared to wild-type controls. Mechanistically, S-nitrosylation of GSNOR at Cys47 and LCD at Cys225 altered their subcellular localization, reduced their enzymatic activities, promoted Cd²⁺ uptake, and thereby accelerated PCD. Notably, S-nitrosylation attenuated the interaction between GSNOR and LCD during PCD progression. Collectively, our findings establish that NO modulates Cd-induced PCD via protein S-nitrosylation, and GSNOR-LCD interactions, together with their post-translational S-nitrosylation, constitute a critical regulatory node integrating NO and H₂S signaling in plant responses to Cd stress. These results provide novel insights into the molecular network underlying heavy metal-induced PCD and the regulatory roles of S-nitrosylation in NO-H₂S crosstalk.