Hydrogen gas enhances Arabidopsis salt tolerance by modulating hydrogen peroxide-mediated redox and ion homeostasis

Hydrogen gas (H₂) plays a crucial role in mitigating salt stress in plants, but the underlying mechanisms is largely unknown. Herein, we employed the pharmacological, molecular, and genetic approaches to investigate the positive roles of hydrogen peroxide (H₂O₂) in endogenous H₂-induced salt tolerance of Arabidopsis thaliana. H₂-induecd salt tolerance of CrHYD1 (hydrogenase 1 gene from Chlamydomonas reinhardtii) transgenic Arabidopsis was blocked by H₂O₂ scavenger or NADPH oxidase inhibitor. When RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) genes (AtrbohD or AtrbohF) were mutated, salt sensitivity of CrHYD1/atrboh (especially CrHYD1/atrbohD) hybrids was increased, but diminished by exogenous H₂O₂ administration. Salt-stimulated endogenous H₂ enrichment consequently resulted in the rapid reactive oxygen species (ROS) accumulation under early salt stress, and the expression of AtrbohD (especially) and AtrbohF in CrHYD1 plants was higher than those in the wild-type (WT), suggesting that endogenous H₂ could induce Atrboh-dependent ROS burst to respond salt stress. Further, H₂-induced less 3,3'-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) stain in CrHYD1 plants was reversed under salt stress when either H₂O₂ was removed or Atrbohs were mutated, which could be explained by higher H₂O₂ and thiobarbituric acid reactive substances (TBARS) levels, as well as lower antioxidant enzyme activity. Additionally, H₂-induced Na⁺ discharge and K⁺ accumulation in CrHYD1 plants under salt stress were blocked by either H₂O₂ removal or Atrboh knockout, which was validated by higher Na⁺/K⁺ ratios and lower ion transport-related gene expression. Our findings not only elucidate that endogenous H₂ enhanced Arabidopsis salt tolerance by reestablishing H₂O₂-dependent ion and redox homeostasis, but provide new insights into the mechanisms of plant salinity responses.