The adverse effects of intake nitrite upon human health are well known. However, eating fruits and vegetables is one of the main pathways to absorb nitrite because of nitrogen assimilation in plants. This study demonstrated that during storage of tomato fruit, the production of endogenous hydrogen (H2) was decreased, in parallel with nitrite accumulation and the senescence rate. Furthermore, exogenously applied H2 could delay the decreased fruit H2 production and senescence, but importantly, nitrite accumulation was blocked. Consistently, the activities and transcripts of nitrate reductase (NR; catalyzing the synthesis of nitrite) and nitrite reductase (NiR; responsible for the reduction of nitrite to ammonium), were either inhibited or increased, respectively, by 0.585 mM H2. Decreased or increased nitrite synthesis was observed when tungstate (an inhibitor of NR) or 2,6-dichloroindophenol sodium salt (a putative inhibitor of H2 synthesis) were applied separately. Time-course analysis revealed that the decrease in vitamin C, a well-known nitrite scavenger, was blocked by H2. Overall, this study strongly revealed that nitrite accumulation during storage of tomato fruit was prevented by H2. This study describes potential applications for H2 in agriculture and food industry, especially in the preservation of fruit and vegetable products. Abbreviations: Ar, argon; DCPIP, 2,6-dichloroindophenol sodium salt; GC, gas chromatograph; H2, hydrogen gas; HPLC, high-performance liquid chromatography; HRW, hydrogen-rich water; N, Newton; N2, nitrogen; NO, nitric oxide; NiR, nitrite reductase; NO2−, sodium nitrite; NO3−, sodium nitrate; NR, nitrate reductase; ONOO−, peroxynitrite anion; qPCR, Real-time quantitative reverse transcription-PCR; RNS, reactive nitrogen species; ROS, reactive oxygen species.

Exogenous hydrogen-rich water (HRW) improves vase life and quality of cut flowers, but the effects of endogenous hydrogen gas (H-2) are not known. During the vase of cut lisianthus flowers, endogenous H-2 concentrations decreased and redox homeostasis was impaired. The supplementation with HRW containing 0.078 mmol L-1 H-2 blocked, but 2,6-dichlorophenolindophenol (DCPIP; a putative inhibitor of H-2 synthesis) increased, endogenous H-2 production. Senescence of cut flowers was delayed by H-2, but accelerated by DCPIP. Also, decreased vase life by DCPIP was blocked by H-2 administration. These beneficial roles of H-2 were associated with less lipid peroxidation, and the increased activities of superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. Compared with DCPIP alone, the soluble protein, total chlorophyll, and proline contents were elevated when H-2 and DCPIP were added together. Overall, endogenous H-2 prolongs vase life of lisianthus cut flowers in part by maintaining redox homeostasis.

Although hydrogen (H-2) 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 H-2-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 H-2 actions. Proline synthesis was stimulated by H-2 and NO, both of which were supported by the increased Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) activities, the decreased proline dehydrogenase (ProDH) activities, and corresponding transcripts. The addition of H-2 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 H-2, were severely impaired. Additionally, H-2-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 H-2-triggered osmotic tolerance.

RNAseq information of rice roots under various Cd stresses conditions. (XLSX 12Â kb)

Although previous results showed that exogenous hydrogen (H2) alleviated aluminum (Al) toxicity, the detailed mechanism remains unclear. Here, we reported that the exposure of germinating rice seeds to Al triggered H2 production, followed by a decrease of GA/ABA ratio and seed germination inhibition. Compared to inert gas (argon), H2 pretreatment not only strengthened H2 production and alleviated Al-induced germination inhibition, but also partially reestablished the balance between GA and ABA. By contrast, a GA biosynthesis inhibitor paclobutrazol (PAC) could block the H2-alleviated germination inhibition. The expression of GA biosynthesis genes (GA20ox1 and GA20ox2) and ABA catabolism genes (ABA8ox1 and ABA8ox2), was also induced by H2. Above results indicated that GA/ABA might be partially involved in H2 responses. Subsequent results revealed that compared with Al alone, transcripts of miR398a and miR159a were decreased by H2, and expression levels of their target genes OsSOD2 and OsGAMYB were up-regulated. Whereas, miR528 and miR160a transcripts were increased differentially, and contrasting tendencies were observed in the changes of their target genes (OsAO and OsARF10). The transcripts of Al-tolerant gene OsSTAR1/OsSTAR2 and OsFRDL4 were up-regulated. Above results were consistent with the anti-oxidant defense, decreased Al accumulation, and enhanced citrate efflux. Together, our results provided insight into the mechanism underlying H2-triggered Al tolerance in plants.

Background: The migration of cadmium (Cd) from contaminated soil to rice is a cause for concern. However, the molecular mechanism underlying the response of rice roots to various Cd stresses remains to be clarified from the viewpoint of the co-expression network at a system-wide scale. Results: We employed a comparative RNAseq-based approach to identify early Cd-responsive differentially expressed genes (DEGs) in rice 'Nipponbare' seedling roots after 1 h of high-Cd treatment. A multiplicity of the identified 1772 DEGs were implicated in hormone signaling and transcriptional regulation, particularly NACs and WRKYs were all upregulated under Cd stress. All of the 6 Cd-upregulated ABC transporters were pleiotropic drug resistance proteins (PDRs), whereas all of the 6 ZRT/IRT-like proteins (ZIPs) were consistently downregulated by Cd treatment. To further confirm our results of this early transcriptomic response to Cd exposure, we then conducted weighted gene co-expression network analysis (WGCNA) to re-analyze our RNAseq data in combination with other 11 previously published RNAseq datasets for rice roots exposed to diverse concentrations of Cd for extended treatment periods. This integrative approach identified 271 transcripts as universal Cd-regulated DEGs that are key components of the Cd treatment coupled co-expression module. A global view of the 164 transcripts with annotated functions in pathway networks revealed several Cd-upregulated key functional genes, including transporter ABCG36/OsPDR9, 12-oxo-phytodienoic acid reductases (OPRs) for JA synthesis, and ZIM domain proteins JAZs in JA signaling, as well as OsWRKY10, NAC, and ZFP transcription factors. More importantly, 104 of these, including ABCG36/OsPDR9, OsNAC3, as well as several orthologs in group metalloendoproteinase, plastocyanin-like domain containing proteins and pectin methylesterase inhibitor, may respond specifically to various Cd pressures, after subtracting the 60 general stress-responsive genes reported to be commonly upregulated following multiple stresses. Conclusion: An integrative approach was implemented to identify DEGs and co-expression network modules in response to various Cd pressures, and 104 of the 164 annotatable universal Cd-responsive DEGs may specifically respond to various Cd pressures. These results provide insight into the universal molecular mechanisms beneath the Cd response in rice roots, and suggest many promising targets for improving the rice acclimation process against Cd toxicity.

Metabolism of molecular hydrogen (H2) in bacteria and algae has been widely studied, and it has attracted increasing attention in the context of animals and plants. However, the role of endogenous H2 in lateral root (LR) formation is still unclear. Here, our results showed that H2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H2 production in tomato seedlings, and a contrasting response was observed in the presence of N-1-naphthyphthalamic acid (NPA), an auxin transport inhibitor. NPA-triggered the inhibition of H2 production and thereafter lateral root development was rescued by exogenously applied H2. Detection of endogenous nitric oxide (NO) by the specific probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in both NAA- and H2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H2 were prevented by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO) and the inhibitor of nitrate reductase (NR; an important NO synthetic enzyme). Molecular evidence confirmed that some representative NO-targeted cell cycle regulatory genes were also induced by H2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H2, Arabidopsis mutants nia2 (in particular) and nia1 (two nitrate reductases (NR)-defective mutants) exhibited defects in lateral root length. Together, these results demonstrated that auxin-induced H2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis.