Hydrogen-induced osmotic tolerance is associated with nitric oxide-mediated proline accumulation and reestablishment of redox balance in alfalfa seedlings

Abstract Although hydrogen (H2) and nitric oxide (NO) are respectively suggested to enhance plant tolerance against osmotic stress, the corresponding causal link is still elusive. In this report, the application of hydrogen-rich water (HRW) strengthened the production of NO in PEG-stressed alfalfa seedling roots, followed by the obvious alleviation of seedling growth inhibition. Comparatively, significant but weaker responses in phenotypes were observed in the plants supplemented with nitrogen-rich water, indicating that the role of HRW was H2-related. The application of tungstate, an inhibitor of the NO synthetic enzyme nitrate reductase (NR), showed the similar blocking response in the phenotype, suggesting that NR might be the major source of NO involved in above H2 actions. Proline synthesis was stimulated by H2 and NO, both of which were supported by the increased Δ1-pyrroline-5-carboxylate synthetase (P5CS) activities, the decreased proline dehydrogenase (ProDH) activities, and corresponding transcripts. The addition of H2 and NO could increase antioxidant defence in stressed plants, confirmed by the histochemical staining for reactive oxygen species (ROS) production and lipid peroxidation, representative antioxidant enzyme activities, and transcripts. Thus, redox balance was reestablished. When NO scavenger was applied, NO and proline syntheses, redox balance, and thereafter osmotic tolerance induced by H2, were severely impaired. Additionally, H2-triggered S-nitrosylation was obviously inhibited by the removal of endogenous NO level. Together, above results discovered the involvement of NO-induced proline and redox balance in H2-triggered osmotic tolerance.