Strawberry Yield Improvement by Hydrogen-Based Irrigation Is Functionally Linked to Altered Rhizosphere Microbial Communities.

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.

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.

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.

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.

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.

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.

Preharvest application of hydrogen nanobubble water enhances strawberry flavor and consumer preferences.

The improvement of fruit flavor is a challenge for producers and breeders. This study investigated the effects and mechanisms of preharvest hydrogen nanobubble water (HNW) application on the flavor of cultivated strawberry (Fragaria × ananassa 'Benihoppe'). Compared with surface water, HNW enhanced the volatile profiles, sugar-acid ratio, and sensory attributes (e.g., aroma, flavor, and overall liking) with/without fertilizer application. Meanwhile, flavor components such as esters (e.g., ethyl hexanoate), acids (e.g., hexanoic acid), and soluble sugars (including glucose, fructose, and sucrose) significantly contributed to increased strawberry flavor achieved with HNW. Importantly, HNW may alleviate the negative effects of fertilizers on strawberry fruit aroma. Further study elucidated that the aroma-related genes (including FaLOX, FaADH, FaAAT, FaQR, FaOMT, and FaNES1) were involved in the accumulation of specific volatiles after HNW treatment. This study provided evidence that the practical application of H2 can improve horticultural product quality at a lower carbon cost.

Hydrogen Nanobubble Water Delays Petal Senescence and Prolongs the Vase Life of Cut Carnation (Dianthus caryophyllus L.) Flowers.

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.

Hydrogen Commonly Applicable from Medicine to Agriculture: From Molecular Mechanisms to the Field.

The emerging field of hydrogen biology has to date mainly been applied in medicine. However, hydrogen biology can also enable positive outcomes in agriculture. Agriculture faces significant challenges resulting from a growing population, climate change, natural disasters, environmental pollution, and food safety issues. In fact, hydrogen agriculture is a practical application of hydrogen biology, which may assist in addressing many of these challenges. It has been demonstrated that hydrogen gas (H2) may enhance plant tolerance towards abiotic and biotic stresses, regulate plant growth and development, increase nutritional values, prolong the shelf life, and decrease the nitrite accumulation during the storage of vegetables, as well as increase the resilience of livestock to pathogens. Our field trials show that H2 may have a promising potential to increase yield and improve the quality of agricultural products. This review aims to elucidate mechanisms for a novel agricultural application of H2 in China. Future development of hydrogen agriculture is proposed as well. Obviously, hydrogen agriculture belongs to a low carbon economy, and has great potential to provide "safe, tasty, healthy, and high-yield" agricultural products so that it may improve the sustainability of agriculture.

Magnesium Hydride-Mediated Sustainable Hydrogen Supply Prolongs the Vase Life of Cut Carnation Flowers via Hydrogen Sulfide.

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.

A Novel Mechanism Underlying Multi-walled Carbon Nanotube-Triggered Tomato Lateral Root Formation: the Involvement of Nitric Oxide.

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 NG-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.

Methane-induced lateral root formation requires the participation of nitric oxide signaling.

Although methane (CH4)-induced lateral root (LR) formation has been discovered, the identification of downstream signaling compounds has yet to be fully elucidated. Here, we report a unique mechanism for the involvement of nitric oxide (NO) in the above CH4-mediated pathway in tomato (Solanum lycopersicum L.) and Arabidopsis thaliana. NO was produced rapidly in the root tissues of tomato seedlings when CH4 was administrated exogenously. The scavenging of NO with its scavengers prevented lateral root primordia formation and thereafter lateral rooting triggered by CH4. Gene expression analysis revealed that similar to the responses of sodium nitroprusside (SNP; a NO-releasing compound), CH4-induced SlCYCA2;1, SlCYCA3;1, and SlCDKA1 transcripts, and -downregulated SlKRP2 mRNA, were differentially abolished when endogenous NO was removed by its scavengers. Changes in the lateral root-related miRNA genes (SlmiR160 and SlmiR390a) and their target genes (SlARF16 and SlARF4), exhibited similar tendencies. Similar to those results in tomato, the addition of CH4 and SNP could obviously induce NO production and LR formation in Arabidopsis seedlings, which were correlated with the transcriptional profiles of representative LR-related genes. Combine with these findings in tomato and Arabidopsis thaliana, our results showed that NO might act, at least partially, as the downstream signaling molecule for CH4 control of lateral rooting.

The role of methane in plant physiology: a review.

Methane (CH4), one of the most important greenhouse gases, has conventionally been considered as a physiologic inert gas. However, this perspective has been challenged by the observation that CH4 has diverse biological functions in animals, such as anti-inflammatory, antioxidant, and anti-apoptosis. Meanwhile, it has now been identified as a possible candidate of gaseous signaling molecule in plants, although its biosynthetic and metabolic pathways as well as the mechanism(s) of CH4 signaling have not fully understood yet. This paper aims to review the available evidence for the biological roles of CH4 in regulating plant physiology. Although currently available reports do not fully support the notion of CH4 as a gasotransmitter, they do show that CH4 might be produced by an aerobic, non-microbial pathway from plants, and plays important roles in enhancing plant tolerance against abiotic stresses, such as salinity, drought, heavy metal exposure, and promoting root development, as well as delaying senescence and browning. Further results showed that CH4 could interact with reactive oxygen species (ROS), other gaseous signaling molecules [e.g., nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S)], and glutathione (GSH). These reports thus support the idea that plant-produced CH4 might be a component of a survival strategy of plants. Finally, the possibility of CH4 application in agriculture is preliminarily discussed.

The complete chloroplast genome of Osmanthus serrulatus rehd., a natural sweet olive endemic to China.

Osmanthus serrulatus is an endemic tree in southwest China. Here, the complete chloroplast genome of this species was assembled and characterised from high-throughput data. The entire circular genome of O. serrulatus was 155,465 bp in size, which composed of large single-copy (LSC) and small single-copy (SSC) regions of 86,545 bp and 17,494 bp, respectively, and separated by a pair of inverted repeat (IR) regions of 25,713 bp each. The genome contained 133 genes, including 88 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall GC content of the genome is 37.8%. A phylogenetic tree reconstructed by 35 chloroplast genomes reveals that O. serrulatus is most related with O. yunnanensis.

Hydrogen sulfide acts downstream of methane to induce cucumber adventitious root development.

Previous results have shown that hydrogen sulfide (H2S), mainly catalyzed by l-cysteine desulfhydrase (DES) in plants, triggers adventitious rooting. The objective of this study was to test whether H2S is involved in methane (CH4)-induced adventitious root development in cucumber explants. First, we observed that the activities of DES, endogenous H2S production, and thereafter adventitious root development were induced by CH4 and NaHS (an H2S donor). Some responses were sensitive to hypotaurine (HT; a scavenger of H2S), showing that endogenous H2S production and adventitious rooting were obviously blocked. The development of adventitious root primordia was also impaired. Further molecular evidence revealed that CH4-induced gene expression of CsDNAJ-1, CsCDPK1, CsCDPK5, CsCDC6 (a cell-division-related gene), CsAux22D-like, and CsAux22B-like (two auxin-signaling genes), several molecular markers responsible for adventitious rooting, were blocked by the co-treatment with HT. The occurrence of CH4-elicited S-sulfhydration during the above responses was also sensitive to the removal of endogenous H2S, suggesting the requirement of H2S. Taken together, our results reveal a vital role of endogenous H2S in CH4-triggered cucumber adventitious root development, and thus provide a comprehensive window into the complex signaling transduction pathway in CH4-mediated root organogenesis.