Hydrogen-induced tolerance against osmotic stress in alfalfa seedlings involves ABA signaling

Background and aims This study investigated the detailed mechanism underlying the alleviation of osmotic stress by exogenous hydrogen (H 2 ) in Medicago sativa . Methods By using biochemical and molecular approaches, the experiments were performed with the analyses of biomass, relative water content (RWC), lipid peroxidation, abscisic acid (ABA) content, antioxidant activities, and related gene expression profiles. Results H 2 application stimulated ABA production, which was accompanied by the regulation of ABA biosynthesis and deactivation/activation genes. Elevated H 2 -induced ABA synthesis was sensitive to tungstate, an inhibitor of ABA synthesis. Meanwhile, H 2 -alleviated osmotic stress, which was supported by the increases in biomass and RWC, and the reduction of lipid peroxidation, was impaired by the inhibition of ABA synthesis. Consistently, tungstate blocked H 2 -induced antioxidant defense. Molecular evidence revealed that miR528 was down-regulated by H 2 , showing a negative correlation with its target gene POD2 . When tungstate was added together, the decreased miR528 and increased POD2 transcripts were respectively blocked. Transcriptional factor genes involved in ABA signaling, including MYB102 , MYC2 , and ABF/AREB2 , were differentially upregulated by H 2 , but further impaired by the co-incubation with tungstate. Conclusions Collectively, our results suggested the possible role of ABA signaling in exogenous H 2 -mediated tolerance against osmotic stress in alfalfa.