The delayed senescence in harvested blueberry by hydrogen-based irrigation is functionally linked to metabolic reprogramming and antioxidant machinery.

Molecular hydrogen is beneficial for fruits quality improvement. However, the mechanism involved, especially cellular metabolic responses, has not been well established. Here, the integrated widely targeted metabolomics analysis (UPLC-MS/MS) and biochemical evidence revealed that hydrogen-based irrigation could orchestrate, either directly or indirectly, an array of physiological responses in blueberry (Vaccinium spp.) during harvesting stage, especially for the delayed senescence in harvested stage (4 °C for 12 d). The hubs to these changes are wide-ranging metabolic reprogramming and antioxidant machinery. A total of 1208 distinct annotated metabolites were identified, and the characterization of differential accumulated metabolites (DAMs) revealed that the reprogramming, particularly, involves phenolic acids and flavonoids accumulation. These changes were positively matched with the transcriptional profiles of representative genes for their synthesis during the growth stage. Together, our findings open a new window for development of hydrogen-based agriculture that increases the shelf-life of fruits in a smart and sustainable manner.

Hydrogen Fertilization with Hydrogen Nanobubble Water Improves Yield and Quality of Cherry Tomatoes Compared to the Conventional Fertilizers.

Although hydrogen gas (H2)-treated soil improves crop biomass, this approach appears difficult for field application due to the flammability of H2 gas. In this report, we investigated whether and how H2 applied in hydrogen nanobubble water (HNW) improves the yield and quality of cherry tomato (Lycopersicon esculentum var. cerasiforme) with and without fertilizers. Two-year-long field trials showed that compared to corresponding controls, HNW without and with fertilizers improved the cherry tomato yield per plant by 39.7% and 26.5% in 2021 (Shanghai), respectively, and by 39.4% and 28.2% in 2023 (Nanjing), respectively. Compared to surface water (SW), HNW increased the soil available nitrogen (N), phosphorus (P), and potassium (K) consumption regardless of fertilizer application, which may be attributed to the increased NPK transport-related genes in roots (LeAMT2, LePT2, LePT5, and SlHKT1,1). Furthermore, HNW-irrigated cherry tomatoes displayed a higher sugar-acid ratio (8.6%) and lycopene content (22.3%) than SW-irrigated plants without fertilizers. Importantly, the beneficial effects of HNW without fertilizers on the yield per plant (9.1%), sugar-acid ratio (31.1%), and volatiles (20.0%) and lycopene contents (54.3%) were stronger than those achieved using fertilizers alone. In short, this study clearly indicated that HNW-supplied H2 not only exhibited a fertilization effect on enhancing the tomato yield, but also improved the fruit's quality with a lower carbon footprint.

Microencapsulation as a Protective Strategy for Sialylated Immunoglobulin G: Efficacy in Alleviating Symptoms of Dextran Sulfate Sodium-Induced Colitis in Mice and Potential Mechanisms.

Sialylated immunoglobulin G (IgG) is a vital glycoprotein in breast milk with the ability to promote the growth of Bifidobacterium in gut microbiota and relieve inflammatory bowel disease (IBD) symptoms in vitro. Here, it was found that the microcapsules with sialylated IgG could protect and release sialylated IgG with its structure and function in the intestine. Furthermore, the sialylated IgG microcapsules alleviated the clinical symptoms (body weight, feed quantity, and colon length loss), decreased disease activity index score, suppressed the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IFN-γ, and MCP-1) and endotoxin (lipopolysaccharide), and enhanced the intestinal mucosal barrier (Claudin1, Muc2, Occludin, and ZO-1) in dextran sulfate sodium (DSS)-induced colitis mice. Additionally, the sialylated IgG microcapsules improved the gut microbiota by increasing the relative abundance of critical microbe Bifidobacterium bifidum and promoted the production of short-chain fatty acids (SCFAs). Correlation analysis indicated that the key microbes were strongly correlated with pro-inflammatory factors, clinical symptoms, tight junction protein, and SCFAs. These findings suggest that the sialylated IgG microcapsules have the potential to be used as a novel therapeutic approach for treating IBD.

H2 supplied via ammonia borane stimulates lateral root branching via phytomelatonin signaling.

A reliable and stable hydrogen gas (H2) supply will benefit agricultural laboratory and field trials. Here, we assessed ammonia borane (AB), an efficient hydrogen storage material used in the energy industry, and determined its effect on plant physiology and the corresponding mechanism. Through hydroponics and pot experiments, we discovered that AB increases tomato (Solanum lycopersicum) lateral root (LR) branching and this function depended on the increased endogenous H2 level caused by the sustainable H2 supply. In particular, AB might trigger LR primordia initiation. Transgenic tomato and Arabidopsis (Arabidopsis thaliana) expressing hydrogenase1 (CrHYD1) from Chlamydomonas reinhardtii not only accumulated higher endogenous H2 and phytomelatonin levels but also displayed pronounced LR branching. These endogenous H2 responses achieved by AB or genetic manipulation were sensitive to the pharmacological removal of phytomelatonin, indicating the downstream role of phytomelatonin in endogenous H2 control of LR formation. Consistently, extra H2 supply failed to influence the LR defective phenotypes in phytomelatonin synthetic mutants. Molecular evidence showed that the phytomelatonin-regulated auxin signaling network and cell-cycle regulation were associated with the AB/H2 control of LR branching. Also, AB and melatonin had little effect on LR branching in the presence of auxin synthetic inhibitors. Collectively, our integrated approaches show that supplying H2 via AB increases LR branching via phytomelatonin signaling. This finding might open the way for applying hydrogen storage materials to horticultural production.

TaRLK-6A promotes Fusarium crown rot resistance in wheat.

The plasma membrane-localized phytosulfokine receptor-like protein TaRLK-6A, interacting with TaSERK1, positively regulates the expression of defense-related genes in wheat, consequently promotes host resistance to Fusarium crown rot.

Molecular hydrogen positively regulates nitrate uptake and seed size by targeting nitrate reductase.

Although the sources of molecular hydrogen (H2) synthesis in plants remain to be fully elucidated, ample evidence shows that plant-based H2 can regulate development and stress responses. Here, we present genetic and molecular evidence indicating that nitrate reductase (NR) might be a target of H2 sensing that positively regulates nitrogen use efficiency (NUE) and seed size in Arabidopsis (Arabidopsis thaliana). The expression level of NR and changes of NUE under control and, in particular, low nitrogen supply were positively associated with H2 addition supplied exogenously or through genetic manipulation. The improvement in nitrate assimilation achieved by H2 was also mediated via NR dephosphorylation. H2 control of seed size was impaired by NR mutation. Further genetic evidence revealed that H2, NR, and nitric oxide can synergistically regulate nitrate assimilation in response to N starvation conditions. Collectively, our data indicate that NR might be a target for H2 sensing, ultimately positively regulating nitrate uptake and seed size. These results provide insights into H2 signaling and its functions in plant metabolism.

Argon-stimulated nitric oxide production and its function in alfalfa cadmium tolerance.

Recent results showed that argon may have great potential in both medicines (especially) and agriculture. However, how argon positively influences crop physiology remains elusive. Here, we observed that the stimulation of nitric oxide (NO) production upon cadmium (Cd) stress in hydroponic alfalfa root tissues was strengthened by argon-rich water and/or a NO-releasing compound. The pharmacological results further indicated that above potential source of NO stimulation achieved by argon might be attributed to NO synthase (NOS) and nitrate reductase (NR). Under hydroponic and pot conditions, the improvement of Cd tolerance elicited by argon, confirmed by the alleviation in the plant growth inhibition, oxidative damage, and Cd accumulation, was sensitive to the scavenger of NO. These results suggested a crucial role of argon-induced NO synthesis in response to Cd stress. Subsequent evidence showed that the improved iron homeostasis and increased S-nitrosylation were also dependent on argon-stimulated NO. Above results were matched with the transcriptional profiles of representative target genes involved in heavy metal detoxification, antioxidant defence, and iron homeostasis. Taken together, our results clearly indicated that argon-stimulated NO production contributes to Cd tolerance by favoring important defense strategies against heavy metal exposure.

A methane-cGMP module positively influences adventitious rooting.

KEY MESSAGE: Endogenous cGMP operates downstream of CH4 control of adventitious rooting, following by the regulation in the expression of cell cycle regulatory and auxin signaling-related genes. Methane (CH4) is a natural product from plants and microorganisms. Although exogenously applied CH4 and cyclic guanosine monophosphate (cGMP) are separately confirmed to be involved in the control of adventitious root (AR) formation, the possible interaction still remains elusive. Here, we observed that exogenous CH4 not only rapidly promoted cGMP synthesis through increasing the activity of guanosine cyclase (GC), but also induced cucumber AR development. These responses were obviously impaired by the removal of endogenous cGMP with two GC inhibitors. Anatomical evidence showed that the emerged stage (V) among AR primordia development might be the main target of CH4-cGMP module. Genetic evidence revealed that the transgenic Arabidopsis that overexpressed the methyl-coenzyme M reductase gene (MtMCR) from Methanobacterium thermoautotrophicum not only increased-cGMP production, but also resulted in a pronounced AR development compared to wild-type (WT), especially with the addition of CH4 or the cell-permeable cGMP derivative 8-Br-cGMP. qPCR analysis confirmed that some marker genes associated with cell cycle regulatory and auxin signaling were closely related to the brand-new CH4-cGMP module in AR development. Overall, our results clearly revealed an important function of cGMP in CH4 governing AR formation by modulating auxin-dependent pathway and cell cycle regulation.

A Novel Wall-Associated Kinase TaWAK-5D600 Positively Participates in Defense against Sharp Eyespot and Fusarium Crown Rot in Wheat.

Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat.

Molecular Hydrogen Confers Resistance to Rice Stripe Virus.

Although molecular hydrogen (H2) has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. Here, after rice stripe virus (RSV) infection, H2 production was pronouncedly stimulated in Zhendao 88, a resistant rice variety, compared to that in a susceptible variety (Wuyujing No.3). External H2 supply remarkably reduced the disease symptoms and RSV coat protein (CP) levels, especially in Wuyujing No.3. The above responses were abolished by the pharmacological inhibition of H2 production. The transgenic Arabidopsis plants overexpressing a hydrogenase gene from Chlamydomonas reinhardtii also improved plant resistance. In the presence of H2, the transcription levels of salicylic acid (SA) synthetic genes were stimulated, and the activity of SA glucosyltransferases was suppressed, thus facilitating SA accumulation. Genetic evidence revealed that two SA synthetic mutants of Arabidopsis (sid2-2 and pad4) were more susceptible to RSV than the wild type (WT). The treatments with H2 failed to improve the resistance to RSV in two SA synthetic mutants. The above results indicated that H2 enhances rice resistance to RSV infection possibly through the SA-dependent pathway. This study might open a new window for applying the H2-based approach to improve plant disease resistance. IMPORTANCE Although molecular hydrogen has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. RSV was considered the most devastating plant virus in rice, since it could cause severe losses in field production. This disease was thus selected as a classical model to explore the interrelationship between molecular hydrogen and plant pathogen resistance. In this study, we discovered that both exogenous and endogenous H2 could enhance plant resistance against Rice stripe virus infection by regulating salicylic acid signaling. Compared with some frequently used agrochemicals, H2 is almost nontoxic. We hope that the findings presented here will serve as an opportunity for the scientific community to push hydrogen-based agriculture forward.

The somatic embryogenesis receptor kinase TaSERK1 participates in the immune response to Rhizoctonia cerealis infection by interacting and phosphorylating the receptor-like cytoplasmic kinase TaRLCK1B in wheat.

The sharp eyespot, caused by necrotrophic pathogen Rhizoctonia cerealis, often causes serious yield loss in wheat (Triticum aestivum). However, the mechanisms underlying wheat resistant responses to the pathogen are still limited. In this study, we performed a genome-wide analysis of somatic embryogenesis receptor kinase (SERK) family in wheat. As a result, a total of 26 TaSERK candidate genes were identified from the wheat genome. Only 6 TaSERK genes on the chromosomes 2A, 2B, 2D, 3A, 3B, and 3D showed obvious heightening expression patterns in resistant wheat infected with R. cerealis compared than those un-infected wheat. Of them, the transcripts of 3 TaSERK1 homoeologs on the chromosomes 2A, 2B, and 2D were significantly up-regulated in the highest level compared to other TaSERKs. Importantly, silencing of TaSERK1 significantly impaired wheat resistance to sharp eyespot. Further bio-molecular assays showed that TaSERK1 could interact with the defence-associated receptor-like cytoplasmic kinase TaRLCK1B, and phosphorylated TaRLCK1B. Together, the results suggest that TaSERK1 mediated resistance responses to R. cerealis infection by interacting and phosphorylating TaRLCK1B in wheat. This study sheds light on the understanding of the wheat SERKs in the innate immunity against R. cerealis, and provided a theoretical fulcrum to identify candidate resistant genes for improving wheat resistance against sharp eyespot in wheat.

Ammonia borane positively regulates cold tolerance in Brassica napus via hydrogen sulfide signaling.

BACKGROUND: Cold stress adversely influences rapeseeds (Brassica napus L.) growth and yield during winter and spring seasons. Hydrogen (H2) is a potential gasotransmitter that is used to enhance tolerance against abiotic stress, including cold stress. However, convenience and stability are two crucial limiting factors upon the application of H2 in field agriculture. To explore the application of H2 in field, here we evaluated the role of ammonia borane (AB), a new candidate for a H2 donor produced by industrial chemical production, in plant cold tolerance. RESULTS: The application with AB could obviously alleviate the inhibition of rapeseed seedling growth and reduce the oxidative damage caused by cold stress. The above physiological process was closely related to the increased antioxidant enzyme system and reestablished redox homeostasis. Importantly, cold stress-triggered endogenous H2S biosynthesis was further stimulated by AB addition. The removal or inhibition of H2S synthesis significantly abolished plant tolerance against cold stress elicited by AB. Further field experiments demonstrated that the phenotypic and physiological performances of rapeseed plants after challenged with cold stress in the winter and early spring seasons were significantly improved by administration with AB. Particularly, the most studied cold-stress response pathway, the ICE1-CBF-COR transcriptional cascade, was significantly up-regulated either. CONCLUSION: Overall, this study clearly observed the evidence that AB-increased tolerance against cold stress could be suitable for using in field agriculture by stimulation of H2S signaling.

Molecular hydrogen positively influences lateral root formation by regulating hydrogen peroxide signaling.

Although a previous study discovered that exogenous molecular hydrogen (H2) supplied with hydrogen-rich water (HRW) can mediate lateral root (LR) development, whether or how endogenous H2 influences LR formation is still elusive. In this report, mimicking the induction responses in tomato seedlings achieved by HRW or exogenous hydrogen peroxide (H2O2; a positive control), transgenic Arabidopsis that overexpressed the hydrogenase1 gene (CrHYD1) from Chlamydomonas reinhardtii not only stimulated endogenous hydrogen peroxide (H2O2) production, but also markedly promoted LR formation. Above H2 and H2O2 responses were abolished by the removal of endogenous H2O2. Moreover, the changes in transcriptional patterns of representative cell cycle genes and auxin signaling-related genes during LR development in both tomato and transgenic Arabidopsis thaliana matched with above phenotypes. The alternations in the levels of GUS transcripts driven by the CYCB1 promoter and expression of PIN1 protein further indicated that H2O2 synthesis was tightly linked to LR formation achieved by endogenous H2, and cell cycle regulation and auxin-dependent pathway might be their targets. There results might provide a reference for molecular mechanism underlying the regulation of root morphogenesis by H2.

Molecular Hydrogen Improves Rice Storage Quality via Alleviating Lipid Deterioration and Maintaining Nutritional Values.

Improvement of the storage quality of rice is a critical challenge for the scientific community. This study assesses the effects of the irrigation with hydrogen nanobubble water (HNW) on the storage quality of rice (Oryza sativa 'Huruan1212'). Compared with ditch water control, after one year of storage at 25 °C and 70% RH, the HNW-irrigated rice had higher contents of essential amino acids, especially lysine. Importantly, the generation of off-flavors in the stored rice was significantly decreased, which was confirmed by the lower levels of volatile substances, including pentanal, hexanal, heptanal, octanal, 1-octen-3-ol, and 2-heptanone. The subsequent results showed that the HNW-irrigated rice not only retained lower levels of free fatty acid values, but also had increased antioxidant capacity and decreased lipoxygenase activity and transcripts, thus resulting in decreased lipid peroxidation. This study opens a new window for the practical application of HNW irrigation in the production and subsequent storage of crops.

An unexpected discovery toward argon-rich water amelioration of cadmium toxicity in Medicago sativa L.

Argon has organ-protective effects on animals. However, whether or how argon influences plant responses remains elusive. In this study, we discovered that the growth inhibition of hydroponically cultured alfalfa seedlings under 100 µM CdCl2 condition was significantly ameliorated by 100 % saturated argon-rich water (ARW). Less Cd uptake and accumulation were also observed in both root and shoot parts, which could be explained by the modified root cell walls, including the increased cell wall thickness, lignin content, and demethylation degree of covalently bound and ion-bound pectin, as well as the down-regulated expression of natural-resistance-associated-macrophage protein1 (Nramp1) encoding a heavy metal ion transporter in root tissue. The hindered Cd translocation from root to shoot achieved by ARW addition was validated by the decreased expression of heavy metal ATPase 2/4 (HMA2/4) in roots and decreased Cd content in xylem saps. The reestablished glutathione (GSH) homeostasis and redox balance, two important indicators of plant defense against Cd poisoning, were also observed. Further greenhouse experiments demonstrated that the phenotypic and physiological performances of alfalfa plants cultured in Cd-contaminated soil were significantly improved by irrigating with ARW. Above results implied that ARW confers plants tolerance against cadmium toxicity by impairing Cd uptake and accumulation and restoring GSH and redox homeostasis. These findings might open a new window for understanding argon biology in higher plants.

Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants.

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1-7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future.

A New Discovery of Argon Functioning in Plants: Regulation of Salinity Tolerance.

Argon, a non-polar molecule, easily diffuses into deeper tissue and interacts with larger proteins, protein cavities, or even receptors. Some of the biological effects of argon, notably its activity as an antioxidant, have been revealed in animals. However, whether and how argon influences plant physiology remains elusive. Here, we provide the first report that argon can enable plants to cope with salinity toxicity. Considering the convenience of the application, argon gas was dissolved into water (argon-rich water (ARW)) to investigate the argon's functioning in phenotypes of alfalfa seed germination and seedling growth upon salinity stress. The biochemical evidence showed that NaCl-decreased α/ß-amylase activities were abolished by the application of ARW. The qPCR experiments confirmed that ARW increased NHX1 (Na+/H+ antiporter) transcript and decreased SKOR (responsible for root-to-shoot translocation of K+) mRNA abundance, the latter of which could be used to explain the lower net K+ efflux and higher K accumulation. Subsequent results using non-invasive micro-test technology showed that the argon-intensified net Na+ efflux and its reduced Na accumulation resulted in a lower Na+/K+ ratio. NaCl-triggered redox imbalance and oxidative stress were impaired by ARW, as confirmed by histochemical and confocal analyses, and increased antioxidant defense was also detected. Combined with the pot experiments in a greenhouse, the above results clearly demonstrated that argon can enable plants to cope with salinity toxicity via reestablishing ion and redox homeostasis. To our knowledge, this is the first report to address the function of argon in plant physiology, and together these findings might open a new window for the study of argon biology in plant kingdoms.

Phytomelatonin and gasotransmitters: a crucial combination for plant physiological functions.

Melatonin, a molecule that was first identified in animal tissues, has been confirmed to be involved as a potential phytohormone in a variety of plant physiological responses. It is considered primarily as an antioxidant with important actions in controlling reactive oxygen and reactive nitrogen species. In addition to its role in regulating plant growth and development, phytomelatonin is involved in protection against abiotic and biotic stresses. The 'gasotransmitter'-that is, a gaseous signaling molecule-is a new concept that has been advanced in the past two decades, with functions in animal and plant physiological regulation. Gasotransmitters including nitric oxide, carbon monoxide, hydrogen sulfide, methane, and, more recently identified, hydrogen gas are critical and indispensable in a wide range of biological processes. This review investigates the interrelationship between phytomelatonin and the above-mentioned gasotransmitters from the perspective of biosynthetic origin and functions. Moreover, the potential future research directions for phytomelatonin and gasotransmitters interactions are discussed.

Degradation of Carbendazim by Molecular Hydrogen on Leaf Models.

Although molecular hydrogen can alleviate herbicide paraquat and Fusarium mycotoxins toxicity in plants and animals, whether or how molecular hydrogen influences pesticide residues in plants is not clear. Here, pot experiments in greenhouse revealed that degradation of carbendazim (a benzimidazole pesticide) in leaves could be positively stimulated by molecular hydrogen, either exogenously applied or with genetic manipulation. Pharmacological and genetic increased hydrogen gas could increase glutathione metabolism and thereafter carbendazim degradation, both of which were abolished by the removal of endogenous glutathione with its synthetic inhibitor, in both tomato and in transgenic Arabidopsis when overexpressing the hydrogenase 1 gene from Chlamydomonas reinhardtii. Importantly, the antifungal effect of carbendazim in tomato plants was not obviously altered regardless of molecular hydrogen addition. The contribution of glutathione-related detoxification mechanism achieved by molecular hydrogen was confirmed. Our results might not only illustrate a previously undescribed function of molecular hydrogen in plants, but also provide an environmental-friendly approach for the effective elimination or reduction of pesticides residues in crops when grown in pesticides-overused environmental conditions.

Magnesium hydride confers copper tolerance in alfalfa via regulating nitric oxide signaling.

Magnesium hydride (MgH2) as a solid-state hydrogen source might be potentially applied in industry and medicine. However, its biological function in plants has not yet been fully discovered. In this report, it was observed that MgH2 administration could relieve copper (Cu) toxicity in alfalfa that was confirmed by a reduction in root growth inhibition. By using old MgH2 as a negative control, it was concluded that above MgH2 function was primarily derived from the releasing of molecular hydrogen (H2), but not caused by either magnesium metabolites or pH alteration. Further results revealed that Cu-triggered nitric oxide (NO) production was intensified by MgH2. Subsequent pharmacological and biochemical experiments suggested that nitrate reductase might be mainly responsible for NO production during above processes. Cu accumulation in the root tissues was also obviously reduced in the presence of MgH2. Meanwhile, increased non-protein thiols (NPTs) content and the deposition of Cu in cell wall of seedling roots could be used to explain the mechanism underlying MgH2-alleviated Cu toxicity via NO signaling. Further, the plant redox balance was reestablished since the Cu stress-modulated antioxidant enzymes activities, reactive oxygen species (ROS) accumulation, and oxidative injury detected by in vivo histochemical and biochemical analyses, were differentially abolished by MgH2. The above responses could be blocked by the removal of endogenous NO after the addition of its scavenger. Taken together, these results clearly suggested that MgH2 control of plant tolerance against Cu toxicity might be mediated by NO signaling, which might open a new window for the application of solid-state hydrogen materials in agriculture.