氢农学:研究进展与展望

Hydrogen agronomy: research progress and prospects # # 氢农学:研究进展与展望 1 first-author 1 1 2 3 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:wbshenh@njau.edu.cn †E-mail:wbshenh@njau.edu.cn wbshenh@njau.edu.cn College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Center of Hydrogen Science, Shanghai Jiao Tong University, Shanghai 200240, China Center of Hydrogen Science, Shanghai Jiao Tong University, Shanghai 200240, China School of Design, Shanghai Jiao Tong University, Shanghai 200240, China School of Design, Shanghai Jiao Tong University, Shanghai 200240, China Agriculture is the foundation of social development. Under the pressure of population growth, natural disasters, environmental pollution, climate change, and food safety, the interdisciplinary “new agriculture” is becoming an important trend of modern agriculture. In fact, new agriculture is not only the foundation of great health and new energy sources, but is also the cornerstone of national food security, energy security, and biosafety. Hydrogen agronomy focuses mainly on the mechanism of hydrogen gas (H 2 ) biology effects in agriculture, and provides a theoretical foundation for the practice of hydrogen agriculture, a component of the new agriculture. Previous research on the biological effects of H 2 focused chiefly on medicine. The mechanism of selective antioxidant is the main theoretical basis of hydrogen medicine. Subsequent experiments have demonstrated that H 2 can regulate the growth and development of plant crops, edible fungus, and livestock, and enhance the tolerance of these agriculturally important organisms against abiotic and biotic stresses. Even more importantly, H 2 can regulate the growth and development of crops by changing the soil microbial community composition and structure. Use of H 2 can also improve the nutritional value and postharvest quality of agricultural products. Researchers have also shown that the biological functions of molecular hydrogen are mediated by modulating reactive oxygen species (ROS), nitric oxide (NO), and carbon monoxide (CO) signaling cascades in plants and microbes. This review summarizes and clarifies the history of hydrogen agronomy and describes recent progress in the field. We also argue that emerging hydrogen agriculture will be an important direction in the new agriculture. Further, we discuss several scientific problems in hydrogen agronomy, and suggest that the future of hydrogen agronomy depends on contributions by multiple disciplines. Important future research directions of hydrogen agronomy include hydrogen agriculture in special environments, such as islands, reefs, aircraft, and outer space. 农业是社会发展的基础.在人口增长、自然灾害、环境污染、气候变化和食品安全等诸多压力下,跨学科的新农业正在成为现代农业的发展方向之一.新农业不仅是大健康和新能源的基础,同时也是国家粮食安全、能源安全以及生物安全的基石.氢农学主要研究农业领域中氢气(H 2 )生物学效应的相关机理,为从属于新农业的氢农业实践提供相关理论基础.过去,H 2 生物学效应的研究主要集中在医学方面,选择性抗氧化是氢医学的主要理论依据.随后的实验表明,H 2 也可以调节农作物、食用微生物和家畜的生长发育,增强它们对各种胁迫的耐受性.更重要的是,H 2 还可以改变土壤中微生物的群落结构,进而调节农作物生长.使用H 2 还可以提高农产品的营养价值和采后品质.研究人员还表明,H 2 的生物学功能是通过调控植物与微生物等体内活性氧(ROS)、一氧化氮(NO)和一氧化碳(CO)信号级联来介导的.本文总结了氢农学的发展历程,综述了相关研究的新进展.我们认为崭露头角的氢农业将是新农业发展的方向之一.我们还讨论了目前氢农学存在的一些科学问题,指出氢农学的未来需要结合多学科的优势.此外,例如岛礁、飞行器和外太空等特殊环境下的氢农业也将成为氢农学的重要研究方向. Hydrogen gas (H 2 ) Hydrogen agronomy New agriculture 氢气(H 2 ) 氢农学 新农业 Introduction 1 Many years ago, the significance of hydrogen gas (H 2 ) to human life became apparent. As early as in 1937, Paneth ( 1937 1937 ) reported that the tropospheric mixing ratio of H 2 in the atmosphere was about 0.5 ppmv (parts per million by volume), equivalent to 34.5 ppbw (parts per billion by weight). Although the content of hydrogen is very low, it plays an important role in maintaining oxidation state of earth atmosphere (Constant et al., 2009 2009 ). H 2 affects almost every aspect of our lives. For example, due to its high energy density and ability to store and retain energy after combustion, H 2 has been widely used, especially in the energy industry (Árnason and Sigfússon, 2000 2000 ) for applications such as hydrogen-powered fuel cell vehicles (Dresselhaus and Thomas, 2001 2001 ). The sources of atmospheric H 2 are diverse. According to previous work (Novelli et al., 1999 1999 ; Constant et al., 2009 2009 ), photochemical oxidation reactions of methane (CH 4 ) and non-methane hydrocarbons (NMHC) are the largest sources of H 2 in atmosphere (about 19 Tg/year (1 Tg=1×10 12 g)). In addition to photochemical oxidation reactions, biomass combustion (about 16 Tg/year) and the fossil fuel industry (about 15 Tg/year) are close secondary and tertiary sources of atmospheric H 2 (Novelli et al., 1999 1999 ). H 2 is also generated from nitrogen-fixing legumes, as a type of byproduct during nitrogen (N 2 ) fixation (Novelli et al., 1999 1999 ). The proportion of electrons utilized to generate H 2 in the root nodule nitrogenases is about 40%–60% (Schubert and Evans, 1976 1976 ). H 2 circulates all across the globe. The whereabouts of H 2 entering the earth atmosphere are also diverse. Previous research found that H 2 was consumed mainly by soil microbes, and oxidized by hydroxyl radicals (Ichimasa et al., 1989 1989 ; Derwent et al., 2001 2001 ; Constant et al., 2009 2009 ). About 80% of atmospheric H 2 (about 56 Tg/year) is absorbed by soil, through a microbial-mediated process (Constant et al., 2009 2009 ). Because H 2 is consumed by soil microorganisms adjacent to the nodules, relatively little soil H 2 gets into the atmosphere (about 3 Tg/year) (Novelli et al., 1999 1999 ; Constant et al., 2009 2009 ). The 56 Tg/year of soil H 2 represents significant energy, about 8 EJ/year. Because this energy source is taken up by soil microbial populations, it is not surprising that H 2 has biological effects on agricultural systems. Although the production and release of H 2 from algae, plants, microorganisms, and animals have been reported, the biological functions of H 2 were long unknown (Gaffron, 1939 1939 ; Gest and Kamen, 1949 1949 ; Renwick et al., 1964 1964 ; Czerkawski, 1972 1972 ). Dole et al. ( 1975 1975 ) reported positive effects of high-pressure H 2 on skin cancer in mice, which is the earliest report of medical hydrogen research recognized by the scientific community. Later, Ohsawa et al. ( 2007 2007 ) found that inhaling 2% to 4% (volume/volume ratio, v/v) of H 2 can protect against ischemic brain damage in rats by selectively reducing toxic reactive oxygen species (ROS), including hydroxyl radicals and peroxynitrite anions. These findings sparked a new wave of research on H 2 . By 2019, 42 scientific research institutions in China had participated in research on hydrogen biology, and a landmark event is the establishment of the Center of Hydrogen Science at Shanghai Jiao Tong University (Shanghai, China). The new center is headed by Prof. Wen-jiang DING, Academician of Chinese Academy of Engineering. In order to expand commercial applications of H 2 , some companies have also begun to focus their attention on hydrogen medicine and hydrogen agriculture. In the history of hydrogen biology, hydrogen medicine was ahead of hydrogen agronomy. The effects of H 2 as an anti-oxidant (Ohsawa et al., 2007 2007 ), anti-inflammatory (Zhao et al., 2013 2013 ), and anti-apoptotic agent (Liu et al., 2015 2015 ) have been elucidated by several animal models or small-scale clinical trials (Ohta, 2014 2014 ; Lu and Sun, 2018 2018 ). Moreover, H 2 can not only inhibit tumors (Dole et al., 1975 1975 ; Saitoh et al., 2008 2008 ), but it also has positive effects on oxidative stress-related diseases, such as ischemia reperfusion injury (Ohsawa et al., 2007 2007 ), acute ischemic disease (Du et al., 2014 2014 ), Parkinson’s disease (Fu et al., 2009 2009 ), Alzheimer’s disease (Hou et al., 2018 2018 ), and atherosclerosis (Song et al., 2012 2012 ). Although the hypothesis of H 2 as a selective antioxidant is widely accepted, in some cases, it also still questioned by academia, and the molecular mechanisms underlying anti-oxidation, anti-inflammatory, and anti-apoptosis effects of H 2 in medicine need to be further elucidated (Ge et al., 2017 2017 ; Shen and Sun, 2019 2019 ). More importantly, the potentially important development of hydrogen agronomy cannot be ignored. Several research groups have found that H 2 has important biological effects on crops. Dong et al. ( 2003 2003 ) put forward the concept of “H 2 fertilization,” which was a milestone in the advancement of hydrogen agronomy. H 2 can change the structure of microbial communities and ultimately promotes soil fertility by promoting community growth of beneficial microorganisms. Thus, the essence of H 2 effects in agronomy is as a type of special biofertilizer. In this role, H 2 produced from symbiotic Rhizobium leguminosarum had been shown to promote crop rotation (Golding and Dong, 2010 2010 ). Furthermore, some studies suggested that H 2 might enhance plant tolerance towards abiotic and biotic stresses (Xie et al., 2012 2012 , 2014 2014 ; Jin et al., 2013 2013 ; Zeng et al., 2013 2013 ; Su et al., 2018 2018 ), and improve the quality of vegetables and fruits (Hu et al., 2014 2014 , 2018 2018 ; Su et al., 2014 2014 ). With support from the Ministry of Science and Technology of the People’s Republic of China, hydrogen agronomy is developing quickly. By 2019, 85 projects on hydrogen biology have been supported by the National Natural Science Foundation of China (Shen and Sun, 2019 2019 ), of which nine were related to hydrogen agronomy. From the perspective of species, these hydrogen agronomy projects focused on alfalfa ( Medicago sativa ) (Cui et al., 2013 2013 , 2014 2014 ; Chen et al., 2014 2014 ; Su et al., 2018 2018 ) and several horticultural plants, such as tomatoes ( Solanum lycopersicum ) (Zhang et al., 2019 2019 ) and marigolds ( Tagetes erecta ) (Zhu and Liao, 2017 2017 ). Hydrogen agronomy is related to a diverse range of physiological functions, including plant responses related to abiotic stress (Xie et al., 2012 2012 , 2014 2014 , 2015 2015 ; Cui et al., 2013 2013 , 2014 2014 , 2020 2020 ; Jin et al., 2013 2013 , 2016 2016 ; Xu et al., 2013 2013 , 2017 2017 ; Zeng et al., 2013 2013 ; Chen et al., 2014 2014 , 2017 2017 ; Wu et al., 2015 2015 ; Dai et al., 2017 2017 ; Su et al., 2018 2018 ), root development (Lin et al., 2014 2014 ; Zhu et al., 2016 2016 ; Cao et al., 2017 2017 ; Zhu and Liao, 2017 2017 ), and quality of agricultural products (Hu et al., 2014 2014 , 2018 2018 ; Su et al., 2014 2014 ; Zhang XY et al., 2018 2018 ; Su et al., 2019 2019 ; Zhang YH et al., 2019 2019 ). A long list of obvious and significant questions in the field of hydrogen agronomy awaits deeper scientific investigation. Hydrogen agronomy: laboratory-based research 1 Agricultural status in China 2 Food is the fuel of life. As important sources of food, agriculture is one of the foundations of social development, and occupies an important position in people’s lives. According to data from the National Bureau of Statistics of the People’s Republic of China, the agricultural output and its gross output value keep sustainable increase (Table 1 1 ). However, pressures from increasing population, reduction in arable land, extreme weather, and increasing demand for cereals like maize ( Zea mays ), for both fodder and fuel, are important factors restricting the development of agriculture in China (Pimentel, 1996 1996 ; Lewandrowski et al., 1997 1997 ; Lesk et al., 2016 2016 ; Nicolopoulou-Stamati et al., 2016 2016 ). Data source: the National Bureau of Statistics of the People’s Republic of China (https://data.stats.gov.cn/easyquery.htm?cn=C01) Year Gross agricultural output value (billion CNY) Grain output (1×10 4 t) Fruit output (1×10 4 t) Tea output (1×10 4 t) Meat output (1×10 4 t) 2013 4894.39 63 048.20 22 748.10 188.72 8632.77 2014 5185.11 63 964.83 23 302.63 204.93 8817.90 2015 5420.53 66 060.27 24 524.62 227.66 8749.52 2016 5565.99 66 043.51 24 405.24 231.33 8628.33 2017 5805.98 66 160.72 25 241.90 246.04 8654.43 2018 6145.26 65 789.22 25 688.35 261.04 8624.63 Soil plays a key role in agricultural development and biodiversity of agricultural production systems (Maeder et al., 2002 2002 ; Young and Crawford, 2004 2004 ; Mijangos et al., 2006 2006 ; Melman et al., 2019 2019 ; Qaswar et al., 2020 2020 ). Soil degradation is a serious problem not only in China but also throughout the world. Crop acreage during last 6 years in China shows a volatile trend, and maize and rice are the first and second major crops (Fig. 1 1 ), based on data released by the Ministry of Natural Resources of the People’s Republic of China and the United States Department of Agriculture. Although the main reasons for reduction in crop acreage include ecological land transfer and agricultural structure adjustment, according to official findings, the potential contradiction between growing population and cropland cannot be easily ignored. As we all know, modern agriculture is closely integrated not only with traditional planting and breeding industries, but also with secondary industries, including manufacturing and food processing, and with tertiary industries, such as transportation, technology, and information services (Macrae et al., 1993 1993 ; Li et al., 2018 2018 ; Lytos et al., 2020 2020 ). Hydrogen agronomy has also no exception. Data sources: the National Bureau of Statistics of the People’s Republic of China (https://data.stats.gov.cn) and the United States Department of Agriculture (https://apps.fas.usda.gov/psdonline/app/index.html#/app/downloads) Hydrogen agronomy needs to be developed 2 Although the core of both hydrogen agronomy and hydrogen medicine is human health, the practices of these two programs are different, including targets, methods of H 2 supply, and degree of difficulty (Table 2 2 ). The ultimate practical goal of hydrogen medicine is human health (Ohta, 2011 2011 , 2014 2014 ). Unlike hydrogen medicine, hydrogen agronomy is not only a laboratory-based research study, but also has potential applications for agricultural production, dubbed hydrogen agriculture, with the goal of growing food in an affordable, healthy, and sustainable manner for human beings (Shen and Sun, 2019 2019 ). HRW: hydrogen-rich water Program Research model Method of H 2 supply Degree of difficulty Reference 6 Hydrogen medicine Rat Inhalation of H 2 Difficult Ohsawa et al., 2007 2007 Rat Injection of hydrogen-rich saline Difficult Zhao et al., 2013 2013 Rat Injection of hydrogen-rich saline Difficult Wang et al., 2011 2011 Rat Drinking HRW Easy Fu et al., 2009 2009 Mice Feeding magnesium hydride Easy Kamimura et al., 2016 2016 Human Drinking HRW Easy Kang et al., 2011 2011 5 Hydrogen agronomy Hypsizygus marmoreus Watering HRW Easy Zhang et al., 2017 2017 Rice Soak in HRW Easy Xu et al., 2013 2013 Kiwifruit Soak in HRW Easy Hu et al., 2014 2014 Tomato Soak in HRW Easy Lu et al., 2017 2017 Lisianthus Watering HRW Easy Su et al., 2019 2019 Traditional agriculture consumes a large volume of agrochemicals, especially fertilizers and pesticides (Lowry et al., 2019 2019 ). As a gaseous signaling molecule, H 2 has been studied extensively in recent years. Hydrogen agronomy focuses mainly on the molecular mechanisms underlying hydrogen-rich water (HRW)-or H 2 -increased yield and/or H 2 -improved quality of agricultural products (Wang and Wei, 2016 2016 ; Ren PJ et al., 2017 2017 ; Shen and Sun, 2019 2019 ). Additionally, we should be aware of the problems probably caused by the application of H 2 on human society. In particular, H 2 is used for clean energy in industry, and future development of hydrogen economy in agriculture could also result in more anthropogenic emissions of H 2 . In developing hydrogen agriculture, the influence of increasing global H 2 concentration in atmosphere cannot be easily ignored. Therefore, the impact of hydrogen economy on human beings and the planet should be carefully evaluated in the near future. Hydrogen agronomy: past and present 1 Generally speaking, both hydrogen medicine and hydrogen agriculture address the biological effects of H 2 and its corresponding applications. Discoveries related to the research targets and mechanisms in hydrogen agronomy are summarized below (Fig. 2 2 ). The targets of hydrogen agronomy mainly include plants, animals, and microbes. Hydrogen gas (H 2 ) can influence reactive oxygen species (ROS), nitric oxide (NO), and carbon monoxide (CO) signaling in organisms. In addition, the soil microbial community composition and structure can be changed by soil hydrogen treatment, and the hydrogen-treated soil can improve plant growth H 2 biosynthesis 2 H 2 biosynthesis occurs mainly in plants and microbes. Gas chromatography (GC), hydrogen-specific sensors, and spectrophotometric methods are normally used to determine H 2 in biological contexts (Shen and Sun, 2019 2019 ). H 2 production was first observed in bacteria (Stephenson and Stickland, 1931 1931 ). Subsequently, release of H 2 was also found in green algae (Gaffron, 1939 1939 ). In algae and microbes, H 2 production depends mainly on hydrogenase (typically [Ni–Fe] and [Fe–Fe] hydrogenases) and nitrogenase (Das et al., 2008 2008 ). [Ni–Fe] hydrogenase is mainly present in some sulfate-reducing bacteria (Yagi and Higuchi, 2013 2013 ), and [Fe–Fe] hydrogenase is found in anaerobic prokaryotes such as clostridia and sulfate reducers; among eukaryotes, [Fe–Fe] hydrogenase has been found in some anaerobic eukaryotes (including fungi, cilates, and trichomonads) and some green algae, including Chlamydomonas reinhardtii (Lubitz et al., 2014 2014 ). In addition, a third type of hydrogenase, [Fe] hydrogenase, has been found in methanogenic archaea (Vogt et al., 2008 2008 ). For nitrogenase, H 2 is a byproduct of nitrogen fixation in rhizobia (Golding and Dong, 2010 2010 ). Although metabolic synthesis of H 2 has been demonstrated in microbes, algae, and higher plants, the mechanisms of H 2 generation still need further investigation. Su et al. ( 2019 2019 ) discovered that the photosynthetic electron transport chain was related to H 2 production. The generation of H 2 was reduced by suppressing above process using pharmacological approach. Some researchers are currently focusing attention on the relationship among environmental stress, phytohormones, and hydrogen production. Many studies have found that the production of H 2 from plants can be induced by drought, osmotic stress, salt damage, and heavy metal stress (Cui et al., 2013 2013 ; Zeng et al., 2013 2013 ; Jin et al., 2016 2016 ; Chen et al., 2017 2017 ). Phytohormones are also associated with hydrogen production. H 2 production from plants can be induced or influenced by auxin, abscisic acid, and ethylene (Zeng et al., 2013 2013 ; Xie et al., 2014 2014 ; Cao et al., 2017 2017 ). Since H 2 production is quickly induced in response to abscisic acid signaling, it cannot be easily ruled out that hydrogen production may be a non-enzymatic process. We also speculate that H 2 production and emission might be associated with energy metabolism in chloroplasts and mitochondria. The involvement of H 2 in the signaling pathways of various phytohormones (Shen and Sun, 2019 2019 ) suggests the possibility that H 2 may be a “universal signaling molecule.” Beginning of hydrogen agronomy: hydrogen fertilizer? 2 The essence of hydrogen agronomy in the narrow sense has been previously regarded as “hydrogen fertilizer.” Unlike traditional fertilizers that supply nutrients (N, P, and trace nutrients), “hydrogen fertilizer” exerts the roles of fertilizer in the soil through supplying H 2 (Dong et al., 2003 2003 ; Golding and Dong, 2010 2010 ). This “hydrogen fertilizer” is not a nutrient but exerts its beneficial effects through other mechanisms. In the forms of HRW and H 2 gas, H 2 is the protagonist of hydrogen agronomy, and its potentials in the production, transportation, storage, and sales of agricultural products have been repeatedly demonstrated (Hu et al., 2014 2014 , 2018 2018 ; Xu et al., 2017 2017 ; Ji et al., 2019 2019 ; Zhang et al., 2019 2019 ). Previous research has found that the rates of oxygen and carbon dioxide uptake in soils are influenced by H 2 (Dong and Layzell, 2001 2001 ). Further work demonstrated that soil uptake of H 2 , especially near nodulated legume roots, dramatically modifies microbial populations (McLearn and Dong, 2002 2002 ; Dong et al., 2003 2003 ; Stein et al., 2005 2005 ), by selectively enhancing the populations of plant growth-promoting rhizobacteria (PGPRs). This phenomenon is used to explain the benefits of using legumes in crop rotation with wheat and other non-legumes (Kirkegaard et al., 2008 2008 ). The weight of fixed nitrogen in one hectare of legume crop is about 200 kg per season; meanwhile, the volume of H 2 produced during above process is about 240 000 L (Dong et al., 2003 2003 ). The evolution of H 2 from roots of nodulated grain legumes fluctuates between 0.06 and 0.51 mmol/(h·g nodule dry weight) (Angus et al., 2015 2015 ). Therefore, it is not difficult to understand that H 2 can exert tremendous biological effects in agriculture. The growth performance of barley, soybean, canola, and spring wheat was improved in H 2 -treated soils compared with growth in untreated soils, and the tiller numbers of barley and spring wheat were increased by 36% and 40%, respectively (Dong et al., 2003 2003 ). H 2 is an obligate byproduct of nitrogen fixation in legume rhizobia. Dong and his colleagues proposed that H 2 , whether produced by nitrogenase or exogenously applied, could have an important “fertilization” effect in rotations with legume crops (Dong et al., 2003 2003 ; Golding and Dong, 2010 2010 ). H 2 may then have some functions to partly minimize the use of chemical fertilizer. The relationships between microbes in soils and plants are very close (Yang and Crowley, 2000 2000 ). H 2 can change the microbial community structure and thus influence plant growth and development (Dong et al., 2003 2003 ). Research methods of hydrogen agronomy 2 Hydrogen agronomy is an interdisciplinary science that combines physiological, biochemical, molecular biology, genetics, and multi-omics to study the related scientific principles of hydrogen agriculture. Each method has its own unique characteristics. Integration of these various disciplines and use of multiple methods are needed to study hydrogen agronomy from multiple angles and at multiple organizational levels. 3.3.1 Physiological and biochemical approaches Physiology and biochemistry form the basis of hydrogen agronomy research. By measuring indicators related to physiology and biochemistry, we can intuitively understand hydrogen agronomy. Early research on hydrogen agronomy mainly focused on easily measurable parameters, such as yield, quality, and organ development. Golding and Dong ( 2010 2010 ) found that H 2 , as a “fertilizer,” promoted rotation in non-leguminous crops, including influencing yield. The role of H 2 in improving drought tolerance of alfalfa ( M . sativa ) was confirmed via pharmacological experiments (Jin et al., 2013 2013 ). In addition, lateral root and adventitious root formation, as easily observed phenotypes, have been widely used to study the influence of H 2 on plant development (Zhu et al., 2016 2016 ; Cao et al., 2017 2017 ). Moreover, the use of phytohormones and their synthetic inhibitors, as well as other biologically active substances, are effective experimental tools for studying hydrogen agronomy; these experiments provide strong evidence that H 2 may be an essential component of the complex signaling network (Zeng et al., 2013 2013 ; Xie et al., 2014 2014 ; Su et al., 2018 2018 ). These physiological and biochemical approaches are effective and useful methods for studying hydrogen agronomy. 3.3.2 Molecular and genetic approaches Molecular and genetic approaches are two important methods of biological research. Although the presence of hydrogenase in algae and certain bacteria has been confirmed, no homologous gene has been found in higher plants (Gaffron, 1939 1939 ; Golding and Dong, 2010 2010 ; Shen et al., 2018 2018 ). Therefore, finding candidate genes encoding a hydrogenase-like enzyme is of great biological and historical significance. At present, most molecular and genetic experiments in hydrogen agronomy have been carried out by using specific, signaling-impaired mutants including mutants deficient in ROS (Xie et al., 2014 2014 ) and nitric oxide (NO) (Cao et al., 2017 2017 ) signaling. In addition, the study of gene expression at transcriptional and translation levels is widely used in hydrogen agronomy. For example, Wu et al. ( 2015 2015 ) found that HRW could regulate expression of ion transporter genes, thus alleviating cadmium toxicity in Chinese cabbage ( Brassica campestris spp. chinensis ). The expression of microRNA528 ( miR528 ), miR160a , miR398a , and miR159a is also regulated by H 2 , which explains the phenotypes that alleviate aluminum stress in rice ( Oryza sativa ) seed germination (Xu et al., 2017 2017 ). The expression levels of jasmonic acid and salicylic acid receptors are also regulated by H 2 , indicating H 2 controls of plant resistance against disease (Zeng et al., 2013 2013 ). 3.3.3 “Multi-omics” approaches With the development of science and technology, the use of genomics, proteomics, or newly developed metabolomics technology is becoming more and more common. These approaches offer the potential of high-throughput experiment technology for hydrogen agronomy. At present, multi-omics approaches have been used to study the mechanisms of H 2 production in algae, and to characterize of some important genes, including HydA1 , HydA2 , Sulp , Tla1 , Sta7 , and PFL1 (Xu et al., 2019 2019 ). Similarly, some genes related to H 2 production have been discovered (Melis et al., 2000 2000 ; Vignais et al., 2001 2001 ; Volgusheva et al., 2013 2013 ). Using RNA-sequencing, Cui et al. ( 2020 2020 ) identified many differentially expressed genes in cadmium-stressed alfalfa ( M . sativa ) seedlings in the presence of HRW, including genes involved in glutathione and sulfur metabolism. More recently, Huang et al. ( 2020 2020 ) found that metabolism-related proteins, photosynthesis-related proteins, and stress response-related proteins might play positive roles in H 2 -promoted cucumber adventitious rooting by proteomic analysis. Use of multi-omics technology allows researchers to analyze the process of H 2 metabolism and understand the changes of the entire metabolic network. Since metabolomics technology has a lot of scientific potential in higher plants, metabolomics technology is bound to advance hydrogen agronomy in the near future. Mechanisms 2 Generally, the concentration of dissolved H 2 in HRW utilized in agriculture is about 78 μmol/L (10% HRW) (Cui et al., 2014 2014 ; Zhu and Liao, 2017 2017 ), and the H 2 content during fumigation tests is greater than 0.2 μmol/L (Hu et al., 2018 2018 ). Since H 2 concentration in the troposphere is about 0.5 ppmv (equivalent to 34.5 ppbw, about 22 nmol/L H 2 ) (Paneth, 1937 1937 ), and tropospheric H 2 can escape to upper atmosphere (Liu and Donahue, 1974 1974 ), it is reasonably deduced that the lower concentration of H 2 in the atmosphere could not directly influence the performance of plants on earth. However, there is ample evidence, showing that local H 2 in the soils and plants, when endogenously produced (especially in roots of legume plants, among other sources) and/or exogenously applied, could significantly alter plant responses (Shen and Sun, 2019 2019 ). Here, mechanism of hydrogen agronomy is accordingly summarized. 3.4.1 H 2 and ROS signaling Studies using various animal models have confirmed the effects of H 2 or hydrogen-rich saline on Alzheimer’s disease (Nishimaki et al., 2018 2018 ), cell apoptosis (Guo et al., 2015 2015 ), retinal light damage (Tian et al., 2013 2013 ; Qi et al., 2015 2015 ), and branch retinal vein occlusion (Long et al., 2019 2019 ). In animals, ROS are byproducts of oxidative phosphorylation. Excess ROS can be produced by smoking (Tanriverdi et al., 2006 2006 ; Grassi et al., 2010 2010 ), immobilization stress (Liu et al., 1996 1996 ), and ischemia/reperfusion injury (Zhao et al., 2013 2013 ). Besides their toxic effects, ROS also play an important role in cell signaling pathways, as has been shown in the previous work (Sauer et al., 2001 2001 ; Kim and Byzova, 2014 2014 ). In addition, the functions of H 2 are related to anti-apoptosis, metabolic diseases, and neurodegenerative diseases (Table 3 3 ). The main goal of hydrogen medicine is reduction of toxic ROS, especially selective scavenging of ROS (Ohsawa et al., 2007 2007 ). Unlike hydrogen medicine, H 2 may induce ROS signaling in plants. Xie et al. ( 2014 2014 ) found that H 2 first rapidly induced ROS signaling (as early as 10 min after treatment) and then mobilized NO signaling to promote stomatal closure in Arabidopsis. Further, by using a mutant deficient in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, we further confirmed that NADPH oxidase-dependent ROS were downstream signaling molecules of H 2 (Xie et al., 2014 2014 ). However, there is also strong evidence that H 2 might improve the antioxidant capacity of plants and reduce ROS accumulation (24 h after stress), thereby alleviating the inhibitory effects of metal pollution on seedling growth (Cui et al., 2014 2014 ; Dai et al., 2017 2017 ). Moreover, H 2 can increase superoxide dismutase (SOD) activity in kiwifruit ( Actinidia chinesis ), therefore maintaining ROS at a low level (Hu et al., 2014 2014 ). Additionally, H 2 enhances pyruvate kinase activity by reducing lipid peroxidation and intracellular ROS levels, thereby alleviating the damage of abiotic stress to Hypsizygus marmoreus (Zhang et al., 2017 2017 ). Ren A et al. ( 2017 2017 ) found that the homeostasis of ROS in Ganoderma lucidum can be regulated by H 2 . Overall, ROS play important roles in H 2 function in both hydrogen medicine and hydrogen agronomy. In agronomy, we propose that H 2 may quickly induce ROS signaling to trigger the gene expression of antioxidant genes. Afterwards, the redox homeostasis was reestablished. 3.4.2 Crosstalk between H 2 and NO In plants, NO generation is induced by phytohormones and environmental stimuli to trigger a wide range of cellular responses. Although NO has been widely studied, the causal relationship between NO and H 2 is a matter of strong research interest in the study of hydrogen agronomy. In Arabidopsis, NO was found to be involved in H 2 -induced stomatal closure and drought tolerance (Xie et al., 2014 2014 ). In addition, interaction between H 2 and NO was shown to be a factor in plant biotic stress and other abiotic stresses. For example, NO can contribute to H 2 -improved osmotic tolerance in alfalfa (Su et al., 2018 2018 ). Pharmacological experiments showed that H 2 can alleviate the inhibition of root growth caused by aluminum stress by inhibiting synthesis of NO in alfalfa (Chen et al., 2014 2014 ). H 2 also enhances the resistance of tomato ( S . lycopersicum ) to the fungal pathogen Botrytis cinerea by increasing polyphenol oxidase activity and NO content (Lu et al., 2017 2017 ). Furthermore, interactions between H 2 and NO have been found in plant organ development, especially in root development. For instance, the development of lateral roots in tomato and Arabidopsis is regulated by H 2 (Cao et al., 2017 2017 ). As a downstream signaling molecule, NO might participate in H 2 -induced cucumber adventitious roots (Zhu et al., 2016 2016 ). These research efforts have shown several apparent interactions between H 2 and NO. As a downstream signaling molecule, NO participates in H 2 -induced stress resistance, and in plant growth and development. These data strongly suggest that the interaction between H 2 and NO may be as tight as that of antigens and antibodies. 3.4.3 Crosstalk between H 2 and carbon monoxide For plant stress tolerance, carbon monoxide (CO) is also a potential downstream signaling molecule. Previous research reported that CO might be a downstream signaling molecule for H 2 control of drought tolerance in alfalfa (Jin et al., 2013 2013 ). Moreover, CO is involved in plant organogenesis induced by H 2 , especially in root development (Lin et al., 2014 2014 ). The expression of target genes related to adventitious root formation and auxin signaling is mediated by H 2 in CO-dependent fashion, thereby promoting the development of cucumber adventitious rooting. Additionally, CO may participate in the adventitious root development process induced by H 2 under drought stress, and reduce the oxidative damage as well (Chen et al., 2017 2017 ). According to current research, both H 2 and CO are thought to be involved in plant stress tolerance and adventitious root development, both of which closely interact with phytohormone activity (Shen and Sun, 2019 2019 ). Thus, the combination of genetic and multi-omics approaches should be adopted to reveal corresponding mechanisms in the near future. Nanotechnology and hydrogen agronomy 2 Although application of HRW is an effective and safe method to exert the biological effects of H 2 , the high diffusivity and the low solubility of H 2 in water normally results in several difficulties when used in hydrogen agronomy and thereafter in hydrogen agriculture. Yang et al. ( 2018 2018 ) packed the hydrogen-producing prodrug into a spherical mesoporous structure to increase hydrogen delivery time and to achieve higher concentrations. In addition, nanocarriers can also be modified to release H 2 in some specific areas or contexts, such as cancer cells (Zhao et al., 2018 2018 ; Kou et al., 2019 2019 ). Combining nanotechnology and H 2 agronomy should have great future potential. Hydrogen agronomy in future: a challenging 2 Unlike the processes used in hydrogen medicine, hydrogen agronomy has its own unique mechanisms (Table 3 3 ). In addition to ROS, the functions of H 2 are closely related to those of many other gaseous signaling molecules, including NO and CO. Hydrogen agronomy is a broad field that includes beneficial roles of H 2 in plants, microbes, and animals (Fig. 2 2 ). ROS, reactive oxygen species; NO, nitric oxide; CO, carbon monoxide; AKI, acute kidney injury; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; Nrf2, nuclear factor erythroid 2-related factor 2; ApoE4, apolipoprotein E4; HRW, hydrogen-rich water; Bcl-2, B cell lymphoma-2; ASK-1, apoptosis signal-regulating kinase 1; JNK, c-Jun N-terminal kinase; p-AMPK, phospho-adenosine monophosphate (AMP)-activated protein kinase; AIF, apoptosis-inducing factor Process Mechanism of action Reference Hydrogen medicine 2  Hydrogen-mediated oxidative stress H 2 can attenuate severe burn-induced early AKI by regulating the MAPKs, Akt, and NF-κB signaling pathway Guo et al., 2015 2015 H 2 can increase the antioxidant effect by activating the Nrf2 transcription system Kawamura et al., 2013 2013 2  Hydrogen-regulated metabolic diseases and neurodegenerative diseases H 2 can improve Alzheimer’s disease caused by apolipoprotein ApoE4 mutation Nishimaki et al., 2018 2018 HRW can reduce the damage of type 2 diabetes mellitus patients to glucose tolerance to a certain extent Kajiyama et al., 2008 2008 2  Hydrogen-regulated autophagy Hydrogen-rich saline can enhance anti-apoptosis of cells by regulating Bax/Bcl-2 ratio and ASK-1/JNK pathway Liu et al., 2015 2015 H 2 can increase the expression of phosphorylated p-AMPK, AIF, Caspase 3, etc., thereby enhancing the 5-fluorouracil-induced apoptosis of cancer cells Runtuwene et al., 2015 2015 Hydrogen agronomy 2  Hydrogen-mediated ROS signaling H 2 can reduce the accumulation of cadmium and mercury, improve the antioxidant capacity of plant seedlings, and reduce the accumulation of ROS Cui et al., 2014 2014 ; Dai et al., 2017 2017 H 2 can maintain a low-ROS level, thereby extending the shelf life of kiwifruit Hu et al., 2014 2014 2  Interaction of hydrogen and NO H 2 mobilizes NO signaling to promote stomata closure, thereby improving its drought tolerance Xie et al., 2014 2014 H 2 enhances the resistance of tomato fruits to Botrytis cinerea by increasing the activity of polyphenol oxidase and NO content Lu et al., 2017 2017 2  Interaction of hydrogen and CO H 2 regulates CO, a downstream signaling molecule that depends on heme oxygenase-1, thereby improving alfalfa’s drought tolerance Jin et al., 2013 2013 H 2 modulates target gene expression related to adventitious root development and auxin signaling pathway through the CO pathway, thereby promoting the development of cucumber adventitious roots Lin et al., 2014 2014 With the development of hydrogen science, some important papers and patents of hydrogen agronomy have been pointed out (Fig. 3 3 ). We searched hydrogen agronomy-related papers in the database of Web of Science, and found that the number of the papers shows an increasing tendency since 2012 (Fig. 4 4 ; Table S1 S1 ). Among these papers, the plant studies ranked first, at least in terms of the number of papers. More importantly, the development of hydrogen agronomy is just beginning. Although it was recently found that H 2 can alter enzyme activity (Ma et al., 2020 2020 ), there is still an urgent need for basic research to fully understand the process of endogenous H 2 metabolism and its consumption in agriculturally relevant plants and microbes, and also to identify the cellular targets or receptors of H 2 and their functions. Overall, hydrogen agronomy has developed gradually as a multidisciplinary and integrative field, and its interconnections among agronomy, medicine, chemistry, and other disciplines are apparent. Research on aquaculture and space agriculture may be the next direction in hydrogen agronomy. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B2000386 Project supported by the National Natural Science Foundation of China (No. 31972396), the Foshan Agriculture Science and Technology Project (Foshan City Budget, No. 140, 2019), and the Funding from Center of Hydrogen Science, Shanghai Jiao Tong University, China 2020-07-17 2020-09-03 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 21