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Despite substantial evidence suggesting that hydrogen gas (H2) can enhance osmotic tolerance in plants, the conventional supply method of hydrogen-rich water (HRW) poses challenges for large-scale agricultural applications. Recently, magnesium hydride (MgH2), a hydrogen storage material in industry, has been reported to yield beneficial effects in plants. This study aimed to investigate the effects and underlying mechanisms of MgH2 in plants under osmotic stress. Mung bean seedlings were cultured under control conditions or with 20% polyethylene glycol (PEG)-6000, with or without MgH2 addition (0.01 g L−1). Under our experimental conditions, the MgH2 solution maintained a higher H2 content and longer retention time than HRW. Importantly, PEG-stimulated endogenous H2 production was further triggered by MgH2 application. Further results revealed that MgH2 significantly alleviated the inhibition of seedling growth and reduced oxidative damage induced by osmotic stress. Pharmacological evidence suggests the MgH2-reestablished redox homeostasis was associated with activated antioxidant systems, particularly the ascorbate–glutathione cycle. The above observations were further supported by the enhanced activities and gene transcriptional levels of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. Overall, this study demonstrates the importance of MgH2 in mitigating osmotic stress in mung bean seedlings, providing novel insights into the potential agricultural applications of hydrogen storage materials.

Molecular hydrogen (H2) is crucial for agricultural microbial systems. However, the mechanisms underlying its influence on crop yields is yet to be fully elucidated. This study observed that H2-based irrigation significantly increased strawberry (Fragaria × ananassa Duch.) yield with/without nutrient fertilization. The reduction in soil available nitrogen (N), phosphorus (P), potassium (K), and organic matter was consistent with the increased expression levels of N/P/K-absorption-related genes in root tissues at the fruiting stage. Metagenomics profiling showed the alterations in rhizosphere microbial community composition achieved by H2, particularly under the conditions without fertilizers. These included the enrichment of plant-growth-promoting rhizobacteria, such as Burkholderia, Pseudomonas, and Cupriavidus genera. Rhizobacteria with the capability to oxidize H2 (group 2a [NiFe] hydrogenase) were also enriched. Consistently, genes related to soil carbon (C) fixation (i.e., rbcL, porD, frdAB, etc.), dissimilar nitrate reduction (i.e., napAB and nrfAH), and P solublization, mineralization, and transportation (i.e., ppx-gppA, appA, and ugpABCE) exhibited higher abundance. Contrary tendencies were observed in the soil C degradation and N denitrification genes. Together, these results clearly indicate that microbe-mediated soil C, N, and P cycles might be functionally altered by H2, thus increasing plant nutrient uptake capacity and horticultural crop yield.

Abstract 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.

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.

Abstract Background Plant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2. Results The inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2, 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2, which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture. Conclusions A dominant dwarfing gene, BnaC04.BIL1, encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.

Abstract Background Rice black-streaked dwarf virus (RBSDV) causes one of the most important rice virus diseases of plants in East Asia. However, molecular mechanism(s)controlling rice resistance to infection is largely unknown. Results In this paper, we showed that RBSDV infection in rice significantly induced nitric oxide (NO) production. This finding was further validated through a genetic approach using a RBSDV susceptible (Nipponbare) and a RBSDV resistant (15HPO187) cultivar. The production of endogenous NO was muchhigher in the 15HPO187 plants, leading to a much lower RBSDV disease incidence. Pharmacological studies showed that the applications of NO-releasingcompounds (i.e., sodium nitroprusside [SNP] and nitrosoglutathione [GSNO]) to rice plants reduced RBSDV disease incidence. After RBSDV infection, the levels of OsICS1, OsPR1b and OsWRKY 45 transcripts were significantly up-regulated by NO in Nipponbare. The increased salicylic acid contents were also observed. After the SNP treatment, protein S-nitrosylation in rice plants was also increased, suggesting that the NO-triggered resistance to RBSDV infection was partially mediated at the post-translational level. Although Osnia2 mutant rice produced less endogenous NO after RBSDV inoculation and showed a higher RBSDV disease incidence, its RBSDV susceptibility could be reduced by SNP treatment. Conclusions Collectively, our genetic and molecular evidence revealed that endogenous NO was a vital signal responsible for rice resistance to RBSDV infection.

Abstract Background Hydrogen gas (H2) is hypothesised to play a role in plants that are coping with stresses by regulating signal transduction and gene expression. Although the beneficial role of H2 in plant tolerance to cadmium (Cd) has been investigated previously, the corresponding mechanism has not been elucidated. In this report, the transcriptomes of alfalfa seedling roots under Cd and/or hydrogen-rich water (HRW) treatment were first analysed. Then, the sulfur metabolism pathways were focused on and further investigated by pharmacological and genetic approaches. Results A total of 1968 differentially expressed genes (DEGs) in alfalfa seedling roots under Cd and/or HRW treatment were identified by RNA-Seq. The DEGs were classified into many clusters, including glutathione (GSH) metabolism, oxidative stress, and ATP-binding cassette (ABC) transporters. The results validated by RT-qPCR showed that the levels of relevant genes involved in sulfur metabolism were enhanced by HRW under Cd treatment, especially the genes involved in (homo)glutathione metabolism. Additional experiments carried out with a glutathione synthesis inhibitor and Arabidopsis thaliana cad2–1 mutant plants suggested the prominent role of glutathione in HRW-induced Cd tolerance. These results were in accordance with the effects of HRW on the contents of (homo)glutathione and (homo)phytochelatins and in alleviating oxidative stress under Cd stress. In addition, the HRW-induced alleviation of Cd toxicity might also be caused by a decrease in available Cd in seedling roots, achieved through ABC transporter-mediated secretion. Conclusions Taken together, the results of our study indicate that H2 regulated the expression of genes relevant to sulfur and glutathione metabolism and enhanced glutathione metabolism which resulted in Cd tolerance by activating antioxidation and Cd chelation. These results may help to elucidate the mechanism governing H2-induced Cd tolerance in alfalfa.

Abstract Abundant studies revealed that multi-walled carbon nanotubes (MWCNTs) are toxic to plants. However, whether or how MWCNTs influence lateral root (LR) formation, which is an important component of the adaptability of the root system to various environmental cues, remains controversial. In this report, we found that MWCNTs could enter into tomato seedling roots. The administration with MWCNTs promoted tomato LR formation in an approximately dose-dependent fashion. Endogenous nitric oxide (NO) production was triggered by MWCNTs, confirmed by Greiss reagent method, electron paramagnetic resonance (EPR), and laser scanning confocal microscopy (LSCM), together with the scavenger of NO. A cause-effect relationship exists between MWCNTs and NO in the induction of LR development, since MWCNT-triggered NO synthesis and LR formation were obviously blocked by the removal of endogenous NO with its scavenger. The activity of NO generating enzyme nitrate reductase (NR) was increased in response to MWCNTs. Tungstate inhibition of NR not only impaired NO production, but also abolished LR formation triggered by MWCNTs. The addition of N G-nitro-l-arginine methyl ester (l-NAME), an inhibitor of mammalian nitric oxide synthase (NOS)-like enzyme, failed to influence LR formation. Collectively, we proposed that NO might act as a downstream signaling molecule in MWCNT control of LR development, at least partially via NR.

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.

Hydrogen agriculture is recently recognized as an emerging and promising approach for low-carbon society. Since shorter retention time for hydrogen gas (H2) in conventional electrolytically produced hydrogen-rich water (HRW) limits its application, seeking a more suitable method to produce and maintain H2 level in HRW for longer time remain a challenge for scientific community. To solve above problems, we compared and concluded that the H2 in HRW prepared by ammonia borane (NH3·BH3) could meet above requirement. The biological effects of HRW prepared by NH3·BH3 were further evaluated in seedlings of rapeseed, the most important crop for producing vegetable oil worldwide. Under our experimental conditions, 2 mg/L NH3·BH3-prepared HRW could confer 3-day-old hydroponic seedlings tolerance against 150 mM sodium chloride (NaCl), 20% polyethylene glycol (PEG; w/v), or 100 μM CdCl2 stress, and intensify endogenous nitric oxide (NO) accumulation under above stresses. The alleviation of seedlings growth stunt was confirmed by reducing cell death and reestablishing redox homeostasis. Reconstructing ion homeostasis, increasing proline content, and reducing Cd accumulation were accordingly observed. Above responses were sensitive to the removal of endogenous NO with its scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl‐imidazoline‐1‐1‐oxyl‐3‐oxide (cPTIO; 100 μM), reflecting the requirement of NO functioning in the regulation of plant physiology achieved by NH3·BH3-prepared HRW. The application of 1 mM tungstate, an inhibitor of nitrate reductase (NR; an important NO synthetic enzyme), showed the similar blocking responses in the phenotype, suggesting that NR might be the major source of NO involved in above H2 actions. Together, these results revealed that HRW prepared by NH3·BH3 could enhance rapeseed seedlings tolerance against abiotic stress, thus opening a new window for the application of H2 in agricultural production.

Bispyribac-sodium (BS) is an herbicide often used to control weeds in rice fields. Hydrogen-rich water (HRW) has recently been recommended for alleviating adverse effects, but whether and how HRW alleviates the injury to rice from exposure to BS is still largely unknown. In this study, a greenhouse hydroponic experiment showed that BS alone could substantially inhibit the plant height and fresh weight of both indica and japonica rice seedlings. For indica rice, its pretreatment with HRW at 75% saturation could markedly alleviate the impact on its size but not so with either 50% or 100% HRW. For japonica rice, all the concentrations of HRW used in this study (50%, 75%, and 100% HRW) were capable of reversing the plant size reductions. Further results revealed that the HRW supplement could increase the activity of antioxidative enzymes, including that of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), which assist in the effective removal of ROS (reactive oxygen species). Subsequent results demonstrated a weaker inhibition of the acetolactate synthase (ALS) enzyme within five days by BS in rice seedlings pretreated with HRW than those receiving the BS treatment alone, and that the HRW pretreatment can hasten the rate at which BS is degraded in rice. Taken together, these findings strongly suggest that pretreatment with HRW may offer a promising and effective strategy to improve the ability of rice to tolerate BS.

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.

Faba bean (Vicia faba L.) is one of the most widely grown cool season legume crops in the world. Winter faba bean normally has a vernalization requirement, which promotes an earlier flowering and pod setting than unvernalized plants. However, the molecular mechanisms of vernalization in faba bean are largely unknown. Discovering vernalization-related candidate genes is of great importance for faba bean breeding. In this study, the whole transcriptome of faba bean buds was profiled by using next-generation sequencing (NGS) and single-molecule, real-time (SMRT) full-length transcriptome sequencing technology. A total of 29,203 high-quality non-redundant transcripts, 21,098 complete coding sequences (CDS), 1,045 long non-coding RNAs (lncRNAs), and 12,939 simple sequence repeats (SSRs) were identified. Furthermore, 4,044 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these differentially expressed transcripts were found to be enriched in binding and transcription factor activity, electron carrier activity, rhythmic process, and receptor activity. Finally, 50 putative vernalization-related genes that played important roles in the vernalization of faba bean were identified; we also found that the levels of vernalization-responsive transcripts showed significantly higher expression levels in cold-treated buds. The expression of VfSOC1, one of the candidate genes, was sensitive to vernalization. Ectopic expression of VfSOC1 in Arabidopsis brought earlier flowering. In conclusion, the abundant vernalization-related transcripts identified in this study will provide a basis for future researches on the vernalization and faba bean breeding and established a reference full-length transcriptome for future studies on faba bean.

Abstract Rice stripe virus (RSV) causes one of the most important rice virus diseases of plants in East Asia. However, the molecular mechanisms controlling rice resistance to RSV infection are largely unknown. Recently, several studies presented a novel model that melatonin (MT) and nitric oxide (NO) participate in the plant-pathogen interaction in a synergetic manner. In this study, there was a difference in MT content between two rice varieties that correlated with one being susceptible and one being resistant to RSV, which suggested that MT is related to RSV resistance. In addition, a test with two NO biosynthesis inhibitors revealed that NO inhibitor were able to increase the disease incidence of RSV. A pharmacological experiment with exogenous MT and NO showed that increased MT and NO in the MT-pretreated plants led to lower disease incidences; however, only NO increased in a NO-releasing reagent [sodium nitroprusside (SNP)] pretreated plants. The expressions level of OsPR1b and OsWRKY 45 were significantly induced by MT and NO. These results suggest that rice resistance to RSV can be improved by increased MT through a NO-dependent pathway.

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.

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/β-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.

Heterosis is most frequently manifested as the superior performance of a hybrid than either of the parents, especially under stress conditions. Nitric oxide (NO) is a well-known gaseous signaling molecule that acts as a functional component during plant growth, development, and defense responses. In this study, the Brassica napus L. hybrid (F1, NJ4375 × MB1942) showed significant heterosis under salt stress, during both germination and post-germination periods. These phenotypes in the hybrid were in parallel with the better performance in redox homeostasis, including alleviation of reactive oxygen species accumulation and lipid peroxidation, and ion homeostasis, evaluated as a lower Na/K ratio in the leaves than parental lines. Meanwhile, stimulation of endogenous NO was more pronounced in hybrid plants, compared with parental lines, which might be mediated by nitrate reductase. Proteomic and biochemical analyses further revealed that protein abundance related to several metabolic processes, including chlorophyll biosynthesis, proline metabolism, and tricarboxylic acid cycle metabolism pathway, was greatly suppressed by salt stress in the two parental lines than in the hybrid. The above responses in hybrid plants were intensified by a NO-releasing compound, but abolished by a NO scavenger, both of which were matched with the changes in chlorophyll and proline contents. It was deduced that the above metabolic processes might play important roles in heterosis upon salt stress. Taken together, we proposed that heterosis derived from F1 hybridization in salt stress tolerance might be mediated by NO-dependent activation of defense responses and metabolic processes.

Magnesium hydride (MgH2) is a promising solid-state hydrogen source with high storage capacity (7.6 wt%). Although it is recently established that MgH2 has potential applications in medicine because it sustainably supplies hydrogen gas (H2), the biological functions of MgH2 in plants have not been observed yet. Also, the slow reaction kinetics restricts its practical applications. In this report, MgH2 (98% purity; 0.5–25 μm size) was firstly used as a hydrogen generation source for postharvest preservation of flowers. Compared with the direct hydrolysis of MgH2 in water, the efficiency of hydrogen production from MgH2 hydrolysis could be greatly improved when the citrate buffer solution is introduced. These results were further confirmed in the flower vase experiment by showing higher efficiency in increasing the production and the residence time of H2 in solution, compared with hydrogen-rich water. Mimicking the response of hydrogen-rich water and sodium hydrosulfide (a hydrogen sulfide donor), subsequent experiments discovered that MgH2-citrate buffer solution not only stimulated hydrogen sulfide (H2S) synthesis but also significantly prolonged the vase life of cut carnation flowers. Meanwhile, redox homeostasis was reestablished, and the increased transcripts of representative senescence-associated genes, including DcbGal and DcGST1, were partly abolished. By contrast, the discussed responses were obviously blocked by the inhibition of endogenous H2S with hypotaurine, an H2S scavenger. These results clearly revealed that MgH2-supplying H2 could prolong the vase life of cut carnation flowers via H2S signaling, and our results, therefore, open a new window for the possible application of hydrogen-releasing materials in agriculture.

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.

Abstract Background Osmotic stress is a major abiotic stress limiting crop production by affecting plant growth and development. Although previous reports discovered that methane (CH4) has a beneficial effect on osmotic stress, the corresponding downstream signal(s) is still elusive. Results Polyethylene glycol (PEG) treatment progressively stimulated the production of CH4 in germinating mung bean seeds. Exogenous CH4 and sodium nitroprusside (SNP) not only triggered nitric oxide (NO) production in PEG-stressed plants, but also alleviated the inhibition of seed germination. Meanwhile, amylase activity was activated, thus accelerating the formation of reducing sugar and total soluble sugar. Above responses could be impaired by NO scavenger(s), suggesting that CH4-induced stress tolerance was dependent on NO. Subsequent tests showed that CH4 could reestablish redox balance in a NO-dependent fashion. The addition of inhibitors of the nitrate reductase (NR) and NO synthase in mammalian (NOS), suggested that NR and NOS-like protein might be partially involved in CH4-alleviated seed germination inhibition. In vitro and scavenger tests showed that NO-mediated S-nitrosylation might be associated with above CH4 responses. Conclusions Together, these results indicated an important role of endogenous NO in CH4-enhanced plant tolerance against osmotic stress, and NO-regulated redox homeostasis and S-nitrosylation might be involved in above CH4 action.

Whether or not hydrogen gas (H2) can reduce cadmium (Cd) toxicity in Ganoderma lucidum has remained largely unknown. Here, we report that Cd-induced growth inhibition in G. lucidum was significantly alleviated by H2 fumigation or hydrogen-rich water (HRW), evaluated by lower oxidative damage and Cd accumulation. Moreover, the amelioration effects of H2 fumigation were better than of HRW in an optimum concentration of H2 under our experimental conditions. Further results showed that H2-alleviated growth inhibition in G. lucidum was accompanied by increased nitric oxide (NO) level and nitrate reductase (NR) activity under Cd stress. On the other hand, the mitigation effects were reversed after removing endogenous NO with its scavenger cPTIO or inhibiting H2-induced NR activity with sodium tungstate. The role of NO in H2-alleviated growth inhibition under Cd stress was proved to be achieved through a restoration of redox balance, an increase in cysteine and proline contents, and a reduction in Cd accumulation. In summary, these results clearly revealed that NR-dependent NO might be involved in the H2-alleviated Cd toxicity in G. lucidum through rebuilding redox homeostasis, increasing cysteine and proline levels, and reducing Cd accumulation. These findings may open a new window for H2 application in Cd-stressed economically important fungi.

Improvements in the growth, yield, and quality of horticultural crops require the development of simply integrated, cost-efficient, and eco-friendly solutions. Hydrogen gas (H2) has been observed to have fertilization effects on soils by influencing rhizospheric microorganisms, resulting in improvements in crop yield and quality. Ample studies have shown that H2 has positive effects on horticultural crops, such as promoting root development, enhancing tolerance against abiotic and biotic stress, prolonging storage life, and improving postharvest quality of fruits, vegetables and cut flowers. In this review, we aim to evaluate the feasibility of molecular hydrogen application in horticulture and the strategies for its application, including H2 delivery methods, treatment timing, and the concentration of H2 applied. The discussion will be accompanied by outlining the effects of H2 and the likely mechanisms of its efficacy. In short, the application of H2 may provide novel opportunities for simple and cost efficient improvements of horticultural production in terms of increased yield and product quality but with low carbon dioxide emissions.

How to use environmentally friendly technology to enhance rice field and grain quality is a challenge for the scientific community. Here, we showed that the application of molecular hydrogen in the form of hydrogen nanobubble water could increase the length, width, and thickness of brown/rough rice and white rice, as well as 1000-grain weight, compared to the irrigation with ditch water. The above results were well matched with the transcriptional profiles of representative genes related to high yield, including up-regulation of heterotrimeric G protein β-subunit gene (RGB1) for cellular proliferation, Grain size 5 (GS5) for grain width, Small grain 1 (SMG1) for grain length and width, Grain weight 8 (GW8) for grain width and weight, and down-regulation of negatively correlated gene Grain size 3 (GS3) for grain length. Meanwhile, although total starch content in white rice is not altered by HNW, the content of amylose was decreased by 31.6%, which was parallel to the changes in the transcripts of the amylose metabolism genes. In particular, cadmium accumulation in white rice was significantly reduced, reaching 52% of the control group. This phenomenon was correlated well with the differential expression of transporter genes responsible for Cd entering plants, including down-regulated Natural resistance-associated macrophage protein (Nramp5), Heavy metal transporting ATPase (HMA2 and HMA3), and Iron-regulated transporters (IRT1), and for decreasing Cd accumulation in grain, including down-regulated Low cadmium (LCD). This study clearly showed that the application of molecular hydrogen might be used as an effective approach to increase field and grain quality of rice.

The short vase life of cut flowers limits their commercial value. To ameliorate this practical problem, this study investigated the effect of hydrogen nanobubble water (HNW) on delaying senescence of cut carnation flowers (Dianthuscaryophyllus L.). It was observed that HNW had properties of higher concentration and residence time for the dissolved hydrogen gas in comparison with conventional hydrogen-rich water (HRW). Meanwhile, application of 5% HNW significantly prolonged the vase life of cut carnation flowers compared with distilled water, other doses of HNW (including 1%, 10%, and 50%), and 10% HRW, which corresponded with the alleviation of fresh weight and water content loss, increased electrolyte leakage, oxidative damage, and cell death in petals. Further study showed that the increasing trend with respect to the activities of nucleases (including DNase and RNase) and protease during vase life period was inhibited by 5% HNW. The results indicated that HNW delayed petal senescence of cut carnation flowers through reducing reactive oxygen species accumulation and initial activities of senescence-associated enzymes. These findings may provide a basic framework for the application of HNW for postharvest preservation of agricultural products.

Abstract Background Both hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) are separately regarded as a highly reactive molecule involved in root morphogenesis. In this report, corresponding causal link governing lateral root formation was investigated. Methods By using pharmacological, anatomic, and molecular approaches, evidence presented here revealed the molecular mechanism underlying tomato lateral root development triggered by H2S. Results A H2S donor sodium hydrosulfide (NaHS) triggered the accumulation of H2O2, the up-regulation of RBOH1 transcript, and thereafter tomato lateral root formation. Above responses were sensitive to the H2O2 scavenger (dimethylthiourea; DMTU) and the inhibitor of NADPH oxidase (diphenylene idonium; DPI), showing that the accumulations of H2O2 and increased RBOH1 transcript were respectively prevented. Lateral root primordial and lateral root formation were also impaired. Further molecular evidence revealed that H2S-modulated gene expression of cell cycle regulatory genes, including up-regulation of SlCYCA2;1, SlCYCA3;1, and SlCDKA1, and the down-regulation of SlKRP2, were prevented by the co-treatment with DMTU or DPI. Above mentioned inducing phenotypes were consistent with the changes of lateral root formation-related microRNA transcripts: up-regulation of miR390a and miR160, and with the opposite tendencies of their target genes (encoding auxin response factors). Contrasting tendencies were observed when DMTU or DPI was added together. The occurrence of H2S-mediated S-sulfhydration during above responses was preliminarily discovered. Conclusions Overall, these results suggested an important role of RBOH1-mediated H2O2 in H2S-elicited tomato lateral root development, and corresponding H2S-target proteins regulated at transcriptional and post-translational levels.

Chinese chive usually becomes decayed after a short storage time, which was closely observed with the redox imbalance. To cope with this practical problem, in this report, molecular hydrogen (H2) was used to evaluate its influence in maintaining storage quality of Chinese chive, and the changes in antioxidant capacity were also analyzed. Chives were treated with 1%, 2%, or 3% H2, and with air as the control, and then were stored at 4 ± 1 °C. We observed that, compared with other treatment groups, the application of 3% H2 could significantly prolong the shelf life of Chinese chive, which was also confirmed by the obvious mitigation of decreased decay index, the loss ratio of weight, and the reduction in soluble protein content. Meanwhile, the decreasing tendency in total phenolic, flavonoid, and vitamin C contents was obviously impaired or slowed down by H2. Results of antioxidant capacity revealed that the accumulation of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) was differentially alleviated, which positively matched with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and the improved activities of antioxidant enzymes, including superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). Above results clearly suggest that postharvest molecular hydrogen application might be a potential useful approach to improve the storage quality of Chinese chive, which is partially achieved through the alleviation of oxidative damage happening during the storage periods. These findings also provide potential theoretical and practical significance for transportation and consumption of perishable vegetables.

Our previous studies showed that hydrogen-rich water (HRW) promoted the biosynthesis of anthocyanin under UV-A in radish. However, molecular mechanism involved in the regulation of the anthocyanin biosynthesis is still unclear. In this study, the role of calcium (Ca2+) in HRW-promoted anthocyanin biosynthesis in radish sprouts hypocotyls under UV-A was investigated. The results showed that a positive effect of HRW on the content of cytosolic calcium and anthocyanin accumulation, mimicking the effects of induced CaCl2. Exogenous addition of Ca2+ chelator bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and inositol 1,4,5-trisphosphate (IP3) synthesis inhibitor neomycin partially reversed the facilitated effect of HRW. The positive effects of HRW on activity of anthocyanin biosynthetic-enzymes (L-phenylalanine ammonia-lyase, PAL; chalcone isomerase, CHI; dihydroflavonol 4-reductase, DFR and UDP glc-flavonoid 3-O-glucosyl transferase, UFGT) were reversed by EGTA and neomycin. Further tests confirmed that the upregulation of anthocyanin biosynthetic related genes induced by HRW was substantially inhibited by calcium antagonists. The possible involvement of CaM in HRW-regulated anthocyanin biosynthesis was also preliminarily investigated in this study. Taken together, our results indicate that IP3-dependent calcium signaling pathway might be involved in HRW-regulated anthocyanin biosynthesis under UV-A irradiation.

Although previous results showed that β-cyclodextrin-hemin complex (β-CDH) could induce tomato lateral root (LR) formation, the corresponding downstream messengers are still not fully understood. In this report, similar to the inducing effects of exogenously applied hydrogen peroxide (H2O2), we discovered that β-CDH elicited RBOH1 transcript upregulation, endogenous H2O2 accumulation, and thereafter tomato LR development. Above responses were sensitive to dimethylthiourea (DMTU) and ascorbic acid (AsA), two membrane-permeable scavengers of H2O2, showing that accumulation of H2O2 and LR formation were significantly blocked. The test with diphenyleneiodonium (DPI; the inhibitor of NADPH oxidase) revealed that H2O2 mainly produced by NADPH oxidase, might be involved in LR formation triggered by β-CDH. qPCR combined with pharmacological and anatomical analyses showed that β-CDH-modulated several marker genes responsible for LR formation, such as CYCA3;1, CYCA2;1, CYCD3;1, and CDKA1 (four cell cycle regulatory genes), ARF7 and RSI-1 (two auxin signaling genes), LAX3 (an auxin influx carrier), IAA14 (encoding a member of the Aux/IAA protein family), PIN3 and PIN7 (two auxin efflux carriers), isocitrate dehydrogenase [NADP], NADH-cytochrome b5 reductase 1, and L-ascorbate oxidase homolog genes (two reactive oxygen species-associated genes and one LR formation-related gene), were causally related to above H2O2 signaling. Particularly, representative proteins related to H2O2 metabolism and lateral rooting, were specifically induced in β-CDH-treated tomato seedlings. Overall, the results clearly suggested a vital role of H2O2 in the β-CDH-induced tomato LR formation, and β-CDH-elicited H2O2-related target proteins responsible for LR formation might be, at least partially, regulated at transcriptional and translational levels.

Hydrogen gas (H2) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H2 in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H2 in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl2 and 50%-saturated HRW induced an increase of endogenous H2 in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H2 might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.

The inheritance of resistance to white tip disease (WTDR) in rice (Oryza sativa L.) was analyzed with an artificial inoculation test in a segregating population derived from the cross between Tetep, a highly resistant variety that was identified in a previous study, and a susceptible cultivar. Three resistance-associated traits, including the number of Aphelenchoides besseyi (A. besseyi) individuals in 100 grains (NA), the loss rate of panicle weight (LRPW) and the loss rate of the total grains per panicle (LRGPP) were analyzed for the detection of the quantitative trait locus (QTL) in the population after construction of a genetic map. Six QTLs distributed on chromosomes 3, 5 and 9 were mapped. qNA3 and qNA9, conferring reproduction number of A. besseyi in the panicle, accounted for 16.91% and 12.54% of the total phenotypic variance, respectively. qDRPW5a and qDRPW5b, associated with yield loss, were located at two adjacent marker intervals on chromosome 5 and explained 14.15% and 14.59% of the total phenotypic variation and possessed LOD values of 3.40 and 3.39, respectively. qDRPW9 was considered as a minor QTL and only explained 1.02% of the phenotypic variation. qLRGPP5 contributed to the loss in the number of grains and explained 10.91% of the phenotypic variation. This study provides useful information for the breeding of resistant cultivars against white tip disease in rice.

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H2S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H2S production as well as the activation of two H2S-synthetic enzymes activities, including L-cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H2S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H2S production triggered by Cd, but also alleviated Cd toxicity in a H2S-dependent fashion. By contrast, the inhibition of H2S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H2S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.

Greening of etiolated plants is predominantly stimulated by light but the complete molecular mechanism is still unknown. Multiple studies currently focus on the important physiological effects of heme oxygenase (HO)/carbon monoxide (CO) in plants. In this report, firstly, the role of HO/CO in light-induced de-etiolation process was investigated. We discovered that light could significantly increase HO activities, HO-1 gene expression, CO release, and chlorophyll accumulation, all of which were sensitive to zinc protoporphyrin (ZnPPIX), the potent inhibitor of HO-1, respectively. Both HO-1 inducer hematin (H) and CO aqueous solution were able to relieve etiolation in wheat seedling leaves under completely darkness by up-regulating endogenous HO/CO system, so as nitric oxide (NO) donor sodium nitroprusside (SNP) did. Similarly, endogenous NO production was also boost in response to light, SNP, hematin and CO aqueous solution in wheat seedling leaves. Additionally, the restoration of chlorophyll contents was blocked, when the inhibitor of mammalian nitric oxide synthase N(G)-nitro-L-arginine methylester hydrochloride (L-NAME) or the specific scavenger of NO 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) was added, respectively. Furthermore, the inducible effects of light were different from those of SNP, hematin, and CO on Pfr accumulation and PHYA transcripts. However, all of sodium nitroprusside (SNP), hematin, and CO could accelerate NO emission, which suggested that HO/CO in wheat seedlings de-etiolation under dark-light transition may have a cross talk with NO.

BACKGROUND: The metabolism of hydrogen gas (H(2)) in bacteria and algae has been extensively studied for the interesting of developing H(2)-based fuel. Recently, H(2) is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H(2) in plants as well as its signalling cascade remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H(2) in enhancing Arabidopsis salt tolerance. An increase of endogenous H(2) release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H(2)-saturated liquid medium, mimicking the induction of endogenous H(2) release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H(2) 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. Additionally, H(2) pretreatment maintained ion homeostasis by regulating the antiporters and H(+) pump responsible for Na(+) exclusion (in particular) and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H(2) signalling. CONCLUSIONS: Overall, our findings indicate that H(2) 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.