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. H2induecd 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.