H 2 S‐Induced PAD3 Persulfidation Balances H 2 S‐HCN Homoeostasis for Osmotic Stress Tolerance in Arabidopsis

ABSTRACT Hydrogen sulphide (H 2 S), a novel gasotransmitter, regulates physiological processes in both plants and animals. Recently, hydrogen cyanide (HCN) has been proposed as a potential signalling molecule with functions similar to H 2 S, though their interplay remains unclear. Here, we elucidated their relationship in Arabidopsis thaliana . Fluorometric analysis confirmed phytoalexin deficient3 (PAD3) as an HCN synthase, with significantly elevated HCN levels in OE‐ PAD3 and reduced levels in the pad3 mutant. Subsequent experiments revealed that under salt stress, H 2 S enhanced ROS scavenging while HCN promoted accumulation, with the pad3 + H 2 S treatment showing significantly improved salt tolerance. Under drought conditions, H 2 S induced stomatal closure to reduce water loss, whereas HCN promoted stomatal opening, resulting in the pad3 + H 2 S treatment demonstrating optimal drought resistance. Oxygen consumption rate (OCR) measurements showed that HCN suppressed, while H 2 S enhanced cellular oxygen consumption. Notably, H 2 S and HCN cooperate to maintain respiratory homoeostasis under osmotic stress and enhance plant tolerance. Mechanistically, the biotin switch assay confirmed H 2 S‐mediated persulfidation of PAD3 at Cys440, thereby enhancing its enzymatic activity. Conversely, HCN suppresses the expression of H 2 S synthase‐encoding genes, thereby inhibiting H 2 S synthesis. This study demonstrates that H 2 S maintains H 2 S‐HCN homoeostasis through PAD3 persulfidation, thereby optimizing cellular OCR and consequently enhancing plant tolerance to osmotic stress.