| Recently, ample evidence demonstrated that Arabidopsis nitric oxide-associated 1 (NOA1)-associated nitric oxide (NO) production and HY1 play important roles in salt tolerance signalling. Our previous results revealed that HO1/CO might confer an increased tolerance to salinity stress in wheat seedling roots, which is partially mediated by the NO signal. However, the genetic evidence for the crosstalk between NO and HY1 in plant responses against salinity stress remains elusive. Furthermore, H2 is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials, whereas the physiological significance and molecular mechanisms of H2 in drought and salinity tolerance are still largely unexplored. To fill this knowledge gap, the interaction network among nitrate reductase (NIA/NR)- and NIA1-dependent NO production and HY1 expression was studied at the genetic and molecular levels, the mechanisms of H2 in enhancing arabidopsis salt and drought tolerance were also characterized.Upon salinity stress, we found that HY1 mutant hy1-100, nia1/2/noa1, and nia1/2/noa1/hy1-100 mutants exhibited progressive salt hypersensitivity, all of which were significantly rescued by three NO-releasing compounds. By contrast, a subsequent experiment showed that compared with wild-type plants, the HY1 overexpression line exhibited salinity tolerance, whereas the application with tungstate and cPTIO resulted in significant reduced primary root growth of 35S:HY1-4 plants. Although NO- or HY1-deficient mutants showed a compensatory mode of upregulation of HY1 or slightly increased NO production, respectively, during 2 d of salt treatment, downregulation of ZAT10/12-mediated antioxidant gene expression (cAPX1/2 and FSD1) was observed after 7 d of treatment. Further evidence confirmed that pretreatment with the CO-releasing compound, CORM-2, as well as SNP (in particular; as a positive control), marginally alleviated the decreases of fresh weight, primary root growth, chlorophyll contents and stress-related genes expression profiles in nial/2/noal mutants caused by the following salinity stress. Interestingly, similar reciprocal responses were observed in the nia1/2/noa1/hy1-100 quadruple mutant, with the NO-releasing compounds exhibit the maximal rescuing responses. It is worth mentioning that, compared with the wild-type, the basal or NaCl-induced DAF-FM-associated fluorescence levels were gradually reduced in the roots of noal, nial/2, and even nial/2/noal plants. Together, these findings suggested that NO might occupy the dominant position in the compensatory and synergistic modes of NR/NOA1-dependent NO production and HY1 expression in mediating salt-tolerance signalling.Besides the characterization of the significant roles of NO and CO in plant, the signaling role of hydrogen gas (H2) has attracted increasing attention from plant and animal researchers. We further studied its physiological significance and molecular mechanism in the enhancement of plant salt tolerance. It was observed that in comparison with controls, the pretreatment with 50% H2-saturated MS liquid medium followed by NaCl stress for 6 and 48 hr significantly increased H2 production in seedlings, which also approximately mimicked a physiological response elicited by NaCl stress. As expected, the ameliorating responses of H2 against the NaCl-induced growth inhibition were observed, with more significant performance in the development of leaf tissues compared to roots. Further results showed that H2 pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS) overproduction and lipid peroxidation, which were measured by histochemical, real-time RT-PCR and western-blot analysis. Additionally, H2 pretreatment maintained ion homeostasis by regulating the antiporters and H+ pump responsible for Na+ exclusion (in particular) and compartmentation. Strikingly, genetic evidence suggested that SOS1 and cAPXl might be the target genes of H2 signalling. Overall, our findings indicate that H2 acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.Considering the fact that the signaling cascades for salt, osmotic and drought stresses share a common cascade in an ABA-dependent pathway, it would be noteworthy to identify whether and how H2 regulates the bioactivity of ABA-induced downstream components and thereafter biological responses, including stomatal closure and drought tolerance. In this study, we reported that ABA elicited a rapid and sustained H2 releasing and production in Arabidopsis, exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H2 production, a reduction in the stomatal aperture and enhanced drought tolerance. Subsequent results revealed that HRW stimulated a significant induction of NO and ROS syntheses associated with stomatal closure in the wild-type, which were individually abolished in nitric reductase (NR) mutant nial/2 or NADPH oxidase deficient mutant rbohF. Furthermore, we demonstrated that the rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. These data confirmed that the HRW-promoted NO generation is dependent on ROS production. In addition, both HRW and H2O2 failed to induce NO production or stomatal closure in nial/2 mutant, while HRW-promoted ROS accumulation was not impaired. In GORK-null mutant, stomatal closure induced by ABA, HRW, NO or H2O2 was partially suppressed. Together, above study defines a main branch of H2-regulated stomatal movement involved in the ABA signaling cascade, in which RbohF-dependent ROS and NR-associated NO production, and subsequent GORK activation, were causally involved. |
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