Extraction, Purification, Sulfated Modification, and Biological Activities of Dandelion Root Polysaccharides.

In this study, polysaccharides were extracted at a rate of 87.5% ± 1.5% from native dandelion roots, and the dandelion root polysaccharides (DRPs) were then chemically modified to obtain sulfated polysaccharides (SDRPs) with a degree of substitution of 1.49 ± 0.07. The effects of modification conditions, physicochemical characterizations, structural characteristics, antioxidant properties, hypoglycemic activity, and proliferative effects on probiotics of DRP derivatives were further investigated. Results showed that the optimum conditions for sulfation of DRPs included esterification reagents (concentrated sulfuric acid: n-butanol) ratio of 3:1, a reaction temperature of 0 °C, a reaction time of 1.5 h, and the involvement of 0.154 g of ammonium sulfate. The DRPs and SDRPs were composed of six monosaccharides, including mannose, glucosamine, rhamnose, glucose, galactose, and arabinose. Based on infrared spectra, the peaks of the characteristic absorption bands of S=O and C-O-S appeared at 1263 cm-1 and 836 cm-1. Compared with DRPs, SDRPs had a significantly lower relative molecular mass and a three-stranded helical structure. NMR analysis showed that sulfated modification mainly occurred on the hydroxyl group at C6. SDRPs underwent a chemical shift to higher field strength, with their characteristic signal peaking in the region of 1.00-1.62 ppm. Scanning electron microscopy (SEM) analysis indicated that the surface morphology of SDRPs was significantly changed. The structure of SDRPs was finer and more fragmented than DRPs. Compared with DRPs, SDRPs showed better free radical scavenging ability, higher Fe2+chelating ability, and stronger inhibition of α-glucosidase and α-amylase. In addition, SDRPs had an excellent promotional effect on the growth of Lactobacillus plantarum 10665 and Lactobacillus acidophilus. Therefore, this study could provide a theoretical basis for the development and utilization of DRPs.

Correction: Novel aerosol treatment of airway hyper-reactivity and inflammation in a murine model of asthma with a soluble epoxide hydrolase inhibitor.

[This corrects the article DOI: 10.1371/journal.pone.0266608.].

Transcriptomic analysis provides insights into the molecular mechanism of melatonin-mediated cadmium tolerance in Medicago sativa L.

Cadmium (Cd), a toxic element, often makes a serious threat to plant growth and development. Previous studies found that melatonin (Mel) reduced Cd accumulation and reestablished the redox balance to alleviate Cd stress in Medicago sativa L., however, the complex molecular mechanisms are still elusive. Here, comparative transcriptome analysis and biochemical experiments were conducted to explore the molecular mechanisms of Mel in enhancing Cd tolerance. Results showed that 7237 differentially expressed genes (DEGs) were regulated by Mel pretreatment to Cd stress compared to the control condition in roots of Medicago sativa L. Besides, in comparison with Cd stress alone, Mel upregulated 1081 DEGs, and downregulated 1085 DEGs. These DEGs were mainly involved in the transcription and translation of genes and folding, sorting and degradation of proteins, carbohydrate metabolism, and hormone signal network. Application of Mel regulated the expression of several genes encoding ribosomal protein and E3 ubiquitin-protein ligase involved in folding, sorting and degradation of proteins. Moreover, transcriptomic analyse suggested that Mel might regulate the expression of genes encoding pectin lyase, UDP-glucose dehydrogenase, sucrose-phosphate synthase, hexokinase-1, and protein phosphorylation in the sugar metabolism. Therefore, these could promote sucrose accumulation and subsequently alleviate the Cd damage. In conclusion, above findings provided the mining of important genes and molecular basis of Mel in mitigating Cd tolerance and genetic cultivation of Medicago sativa L.

Temporal bright light at low frequency retards lens-induced myopia in guinea pigs.

Purpose: Bright light conditions are supposed to curb eye growth in animals with experimental myopia. Here we investigated the effects of temporal bright light at very low frequencies exposures on lens-induced myopia (LIM) progression. Methods: Myopia was induced by application of -6.00 D lenses over the right eye of guinea pigs. They were randomly divided into four groups based on exposure to different lighting conditions: constant low illumination (CLI; 300 lux), constant high illumination (CHI; 8,000 lux), very low frequency light (vLFL; 300/8,000 lux, 10 min/c), and low frequency light (LFL; 300/8,000 lux, 20 s/c). Refraction and ocular dimensions were measured per week. Changes in ocular dimensions and refractions were analyzed by paired t-tests, and differences among the groups were analyzed by one-way ANOVA. Results: Significant myopic shifts in refractive error were induced in lens-treated eyes compared with contralateral eyes in all groups after 3 weeks (all P < 0.05). Both CHI and LFL conditions exhibited a significantly less refractive shift of LIM eyes than CLI and vLFL conditions (P < 0.05). However, only LFL conditions showed significantly less overall myopic shift and axial elongation than CLI and vLFL conditions (both P < 0.05). The decrease in refractive error of both eyes correlated significantly with axial elongation in all groups (P < 0.001), except contralateral eyes in the CHI group (P = 0.231). LFL condition significantly slacked lens thickening in the contralateral eyes. Conclusions: Temporal bright light at low temporal frequency (0.05 Hz) appears to effectively inhibit LIM progression. Further research is needed to determine the safety and the potential mechanism of temporal bright light in myopic progression.

A Review: Systemic Signaling in the Regulation of Plant Responses to Low N, P and Fe.

Plant signal transduction occurs in response to nutrient element deficiency in plant vascular tissue. Recent works have shown that the vascular tissue is a central regulator in plant growth and development by transporting both essential nutritional and long-distance signaling molecules between different parts of the plant's tissues. Split-root and grafting studies have deciphered the importance of plants' shoots in receiving root-derived nutrient starvation signals from the roots. This review assesses recent studies about vascular tissue, integrating local and systemic long-distance signal transduction and the physiological regulation center. A substantial number of studies have shown that the vascular tissue is a key component of root-derived signal transduction networks and is a regulative center involved in plant elementary nutritional deficiency, including nitrogen (N), phosphate (P), and iron (Fe).

Haem Oxygenase 1 is a potential target for creating etiolated/albino tea plants (Camellia sinensis) with high theanine accumulation.

Theanine content is highly correlated with sensory quality and health benefits of tea infusion. The tender shoots of etiolated and albino tea plants contain higher theanine than the normal green tea plants and are valuable materials for high quality green tea processing. However, why these etiolated or albino tea plants can highly accumulate theanine is largely unknown. In this study, we observed an Arabidopsis etiolated mutant hy1-100 (mutation in Haem Oxygenase 1, HO1) that accumulated higher levels of glutamine (an analog of theanine). We therefore identified CsHO1 in tea plants and found CsHO1 is conserved in amino acid sequences and subcellular localization with its homologs in other plants. Importantly, CsHO1 expression in the new shoots was much lower in an etiolated tea plants 'Huangkui' and an albino tea plant 'Huangshan Baicha' than that in normal green tea plants. The expression levels of CsHO1 were negatively correlated with theanine contents in these green, etiolated and albino shoots. Moreover, CsHO1 expression levels in various organs and different time points were also negatively correlated with theanine accumulation. The hy1-100 was hypersensitive to high levels of theanine and accumulated more theanine under theanine feeding, and these phenotypes were rescued by the expression of CsHO1 in this mutant. Transient knockdown CsHO1 expression in the new shoots of tea plant using antisense oligonucleotides (asODN) increased theanine accumulation. Collectively, these results demonstrated CsHO1 negatively regulates theanine accumulation in tea plants, and that low expression CsHO1 likely contributes to the theanine accumulation in etiolated/albino tea plants.

Crosstalk between melatonin and reactive oxygen species in fruits and vegetables post-harvest preservation: An update.

Fruits and vegetables contain numerous nutrients, such as vitamins, minerals, phenolic compounds, and dietary fibers. They reduce the incidence of cardiovascular diseases and the risk of certain chronic diseases, and improve the antioxidant and anti-inflammatory capacity. Moreover, melatonin was found in various fruits and vegetables species. Melatonin acts as a multifunctional compound to participate in various physiological processes. In recent years, many advances have been found that melatonin is also appraised as a key modulator on the fruits and vegetables post-harvest preservation. Fruits and vegetables post-harvest usually elicit reactive oxygen species (ROS) generation and accumulation. Excess ROS stimulate cell damage, protein structure destruction, and tissue aging, and thereby reducing their quality. Numerous studies find that exogenous application of melatonin modulates ROS homeostasis by regulating the antioxidant enzymes and non-enzymatic antioxidants systems. Further evidences reveal that melatonin often interacts with hormones and other signaling molecules, such as ROS, nitric oxide (NO), hydrogen sulfide (H2S), and etc. Among these 'new' molecules, crosstalks of melatonin and ROS, especially the H2O2 produced by RBOHs, are provided in fruits and vegetables post-harvest preservation in this review. It will provide reference for complicated integration of both melatonin and ROS as signal molecules in future study.

Theanine, a tea-plant-specific non-proteinogenic amino acid, is involved in the regulation of lateral root development in response to nitrogen status.

Glutamine synthetase type I (GSI)-like proteins are proposed to mediate nitrogen signaling and developmental fate by synthesizing yet unidentified metabolites. Theanine, the most abundant non-proteinogenic amino acid in tea plants, is the first identified metabolite synthesized by a GSI-like protein (CsTSI) in a living system. However, the roles of theanine in nitrogen signaling and development are little understood. In this study we found that nitrogen deficiency significantly reduced theanine accumulation and increased lateral root development in tea plant seedlings. Exogenous theanine feeding significantly repressed lateral root development of seedlings of tea plants and the model plant Arabidopsis. The transcriptomic analysis revealed that the differentially expressed genes in the roots under theanine feeding were enriched in the apoplastic pathway and H2O2 metabolism. Consistently, theanine feeding reduced H2O2 levels in the roots. Importantly, when co-treated with H2O2, theanine abolished the promoting effect of H2O2 on lateral root development in both tea plant and Arabidopsis seedlings. The results of histochemical assays confirmed that theanine inhibited reactive oxygen species accumulation in the roots. Further transcriptomic analyses suggested the expression of genes encoding enzymes involved in H2O2 generation and scavenging was down- and upregulated by theanine, respectively. Moreover, the expression of genes involved in auxin metabolism and signaling, cell division, and cell expansion was also regulated by theanine. Collectively, these results suggested that CsTSI-synthesized theanine is likely involved in the regulation of lateral root development, via modulating H2O2 accumulation, in response to nitrogen levels in tea plants. This study also implied that the module consisting of GSI-like protein and theanine-like metabolite is probably conserved in regulating development in response to nitrogen status in plant species.

Theanine metabolism and transport in tea plants (Camellia sinensis L.): advances and perspectives.

Theanine, a tea plant-specific non-proteinogenic amino acid, is the most abundant free amino acid in tea leaves. It is also one of the most important quality components of tea because it endows the "umami" taste, relaxation-promoting, and many other health benefits of tea infusion. Its content in tea leaves is directly correlated with the quality and price of green tea. Theanine biosynthesis primarily occurs in roots and is transported to new shoots in tea plants. Recently, great advances have been made in theanine metabolism and transport in tea plants. Along with the deciphering of the genomic sequences of tea plants, new genes in theanine metabolic pathway were discovered and functionally characterized. Theanine transporters were identified and were characterized on the affinity for: theanine, substrate specificity, spatiotemporal expression, and the role in theanine root-to-shoot transport. The mechanisms underlying the regulation of theanine accumulation by: cultivars, seasons, nutrients, and environmental factors are also being rapidly uncovered. Transcription factors were identified to be critical regulators of theanine biosynthesis. In this review, we summarize the progresses in theanine: biosynthesis, catabolism, and transport processes. We also discuss the future studies on theanine in tea plants, and application of the knowledge to crops to synthesize theanine to improve the health-promoting quality of non-tea crops.

Identification of key gene networks controlling polysaccharide accumulation in different tissues of Polygonatum cyrtonema Hua by integrating metabolic phenotypes and gene expression profiles.

Plant polysaccharides, a type of important bioactive compound, are involved in multiple plant defense mechanisms, and in particular polysaccharide-alleviated abiotic stress has been well studied. Polygonatum cyrtonema Hua (P. cyrtonema Hua) is a medicinal and edible perennial plant that is used in traditional Chinese medicine and is rich in polysaccharides. Previous studies suggested that sucrose might act as a precursor for polysaccharide biosynthesis. However, the role of sucrose metabolism and transport in mediating polysaccharide biosynthesis remains largely unknown in P. cyrtonema Hua. In this study, we investigated the contents of polysaccharides, sucrose, glucose, and fructose in the rhizome, stem, leaf, and flower tissues of P. cyrtonema Hua, and systemically identified the genes associated with the sucrose metabolism and transport and polysaccharide biosynthesis pathways. Our results showed that polysaccharides were mainly accumulated in rhizomes, leaves, and flowers. Besides, there was a positive correlation between sucrose and polysaccharide content, and a negative correlation between glucose and polysaccharide content in rhizome, stem, leaf, and flower tissues. Then, the transcriptomic analyses of different tissues were performed, and differentially expressed genes related to sucrose metabolism and transport, polysaccharide biosynthesis, and transcription factors were identified. The analyses of the gene expression patterns provided novel regulatory networks for the molecular basis of high accumulation of polysaccharides, especially in the rhizome tissue. Furthermore, our findings explored that polysaccharide accumulation was highly correlated with the expression levels of SUS, INV, SWEET, and PLST, which are mediated by bHLH, bZIP, ERF, ARF, C2H2, and other genes in different tissues of P. cyrtonema Hua. Herein, this study contributes to a comprehensive understanding of the transcriptional regulation of polysaccharide accumulation and provides information regarding valuable genes involved in the tolerance to abiotic stresses in P. cyrtonema Hua.

Compact multicolor two-photon fluorescence microscopy enabled by tailorable continuum generation from self-phase modulation and dispersive wave generation.

By precisely managing fiber-optic nonlinearity with anomalous dispersion, we have demonstrated the control of generating plural few-optical-cycle pulses based on a 24-MHz Chromium:forsterite laser, allowing multicolor two-photon tissue imaging by wavelength mixing. The formation of high-order soliton and its efficient coupling to dispersive wave generation leads to phase-matched spectral broadening, and we have obtained a broadband continuum ranging from 830 nm to 1200 nm, delivering 5-nJ pulses with a pulse width of 10.5 fs using a piece of large-mode-area fiber. We locate the spectral enhancement at around 920 nm for the two-photon excitation of green fluorophores, and we can easily compress the resulting pulse close to its limited duration without the need for active pulse shaping. To optimize the wavelength mixing for sum-frequency excitation, we have realized the management of the power ratio and group delay between the soliton and dispersive wave by varying the initial pulse energy without additional delay control. We have thus demonstrated simultaneous three-color two-photon tissue imaging with contrast management between different signals. Our source optimization leads to efficient two-photon excitation reaching a 500-µm imaging depth under a low 14-mW illumination power. We believe our source development leads to an efficient and compact approach for driving multicolor two-photon fluorescence microscopy and other ultrafast investigations, such as strong-field-driven applications.

Is Ocular Accommodation Influenced by Dynamic Ambient Illumination and Pupil Size?

PURPOSE: We investigated ocular accommodative responses and pupil diameters under different light intensities in order to explore whether changes in light intensity aid effective accommodation function training. METHODS: A total of 29 emmetropic and myopic subjects (age range: 12-18 years) viewed a target in dynamic ambient light (luminance: 5, 100, 200, 500, 1000, 2000 and 3000 lux) and static ambient light (luminance: 1000 lux) at a 40 cm distance with refractive correction. Accommodation and pupil diameter were recorded using an open-field infrared autorefractor and an ultrasound biological microscope, respectively. RESULTS: The changes in the amplitude of accommodative response and pupil diameter under dynamic lighting were 1.01 ± 0.53 D and 2.80 ± 0.75 mm, respectively, whereas in static lighting, those values were 0.43 ± 0.24 D and 0.77 ± 0.27 mm, respectively. The amplitude of accommodation and pupil diameter change in dynamic lighting (t = 6.097, p < 0.001) was significantly larger than that under static lighting (t = 16.115, p < 0.001).The effects of light level on both accommodation and pupil diameter were significant (p < 0.001). CONCLUSION: Accommodation was positively correlated with light intensity. The difference was about 1.0 D in the range of 0-3000 lux, which may lay the foundation for accommodative training through light intervention.

Crosstalk between Melatonin and Reactive Oxygen Species in Plant Abiotic Stress Responses: An Update.

Melatonin acts as a multifunctional molecule that takes part in various physiological processes, especially in the protection against abiotic stresses, such as salinity, drought, heat, cold, heavy metals, etc. These stresses typically elicit reactive oxygen species (ROS) accumulation. Excessive ROS induce oxidative stress and decrease crop growth and productivity. Significant advances in melatonin initiate a complex antioxidant system that modulates ROS homeostasis in plants. Numerous evidences further reveal that melatonin often cooperates with other signaling molecules, such as ROS, nitric oxide (NO), and hydrogen sulfide (H2S). The interaction among melatonin, NO, H2S, and ROS orchestrates the responses to abiotic stresses via signaling networks, thus conferring the plant tolerance. In this review, we summarize the roles of melatonin in establishing redox homeostasis through the antioxidant system and the current progress of complex interactions among melatonin, NO, H2S, and ROS in higher plant responses to abiotic stresses. We further highlight the vital role of respiratory burst oxidase homologs (RBOHs) during these processes. The complicated integration that occurs between ROS and melatonin in plants is also discussed.

Novel aerosol treatment of airway hyper-reactivity and inflammation in a murine model of asthma with a soluble epoxide hydrolase inhibitor.

Asthma currently affects more than 339 million people worldwide. In the present preliminary study, we examined the efficacy of a new, inhalable soluble epoxide hydrolase inhibitor (sEHI), 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), to attenuate airway inflammation, mucin secretion, and hyper-responsiveness (AHR) in an ovalbumin (OVA)-sensitized murine model. Male BALB/c mice were divided into phosphate-buffered saline (PBS), OVA, and OVA+TPPU (2- or 6-h) exposure groups. On days 0 and 14, the mice were administered PBS or sensitized to OVA in PBS. From days 26-38, seven challenge exposures were performed with 30 min inhalation of filtered air or OVA alone. In the OVA+TPPU groups, a 2- or 6-h TPPU inhalation preceded each 30-min OVA exposure. On day 39, pulmonary function tests (PFTs) were performed, and biological samples were collected. Lung tissues were used to semi-quantitatively evaluate the severity of inflammation and airway constriction and the volume of stored intracellular mucosubstances. Bronchoalveolar lavage (BAL) and blood samples were used to analyze regulatory lipid mediator profiles. Significantly (p < 0.05) attenuated alveolar, bronchiolar, and pleural inflammation; airway resistance and constriction; mucosubstance volume; and inflammatory lipid mediator levels were observed with OVA+TPPU relative to OVA alone. Cumulative findings indicated TPPU inhalation effectively inhibited inflammation, suppressed AHR, and prevented mucosubstance accumulation in the murine asthmatic model. Future studies should determine the pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (i.e., concentration/dose responses) of inhaled TPPU to explore its potential as an asthma-preventative or -rescue treatment.

Loss-of-function mutations in the ERF96 gene enhance iron-deficient tolerance in Arabidopsis.

Iron is an essential micronutrient for plant growth and development. Here we provide evidence for a role of ERF96 in iron-deficiency response in Arabidopsis thaliana. The ERF96-loss-of-function mutants were found to be more tolerant to iron-deficiency stress than wild type (WT) and to have higher iron and chlorophyll content. Further studies showed that the transcriptional levels of iron-uptake related genes IRT1, FRO2, AHA2, FIT and bHLH38 in mutants were significantly higher than in WT under iron deficiency. Comparative transcriptome analysis suggested that the differentially expressed genes (DEGs) between ERF96-loss-of-function mutant and WT under iron deficiency were mainly enriched in iron uptake and chlorophyll degradation. According to the specific analysis of these two kinds of DEGs, the expression of iron uptake and transport related genes in ERF96-loss-of-function mutant was higher and the expression of chlorophyll degradation related genes was lower under iron deficiency. Furthermore, loss-of-function of ERF96 influenced the plant hormone, especially auxin and ethylene signal transduction. Altogether, our results demonstrate that loss-of-function of ERF96 increased Fe uptake and chlorophyll level through ethylene and auxin signal pathway in the regulation of iron-deficiency response in Arabidopsis.

Theanine Improves Salt Stress Tolerance via Modulating Redox Homeostasis in Tea Plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is one of the most abundant secondary metabolites in tea. Its content largely determines green tea quality and price. However, its physiological roles in tea plants remain largely unknown. Here, we showed that salt stress significantly increased the accumulation of glutamate, glutamine, alanine, proline, and γ-aminobutyric acid, as well as theanine, in the new shoots of tea plants. We further found that salt stress induced the expression of theanine biosynthetic genes, including CsGOGATs, CsAlaDC, and CsTSI, suggested that salt stress induced theanine biosynthesis. Importantly, applying theanine to the new shoots significantly enhanced the salt stress tolerance. Similar effects were also found in a model plant Arabidopsis. Notably, exogenous theanine application increased the antioxidant activity of the shoots under salt stress, suggested by reduced the reactive oxygen species accumulation and lipid peroxidation, as well as by the increased SOD, CAT, and APX activities and expression of the corresponding genes. Finally, genetic evidence supported that catalase-mediated antioxidant scavenging pathway is required for theanine-induced salt stress tolerance. Taken together, this study suggested that salt stress induces theanine biosynthesize in tea plants to enhance the salt stress tolerance through a CAT-dependent redox homeostasis pathway.

Melatonin Confers Plant Cadmium Tolerance: An Update.

Cadmium (Cd) is one of the most injurious heavy metals, affecting plant growth and development. Melatonin (N-acetyl-5-methoxytryptamine) was discovered in plants in 1995, and it is since known to act as a multifunctional molecule to alleviate abiotic and biotic stresses, especially Cd stress. Endogenously triggered or exogenously applied melatonin re-establishes the redox homeostasis by the improvement of the antioxidant defense system. It can also affect the Cd transportation and sequestration by regulating the transcripts of genes related to the major metal transport system, as well as the increase in glutathione (GSH) and phytochelatins (PCs). Melatonin activates several downstream signals, such as nitric oxide (NO), hydrogen peroxide (H2O2), and salicylic acid (SA), which are required for plant Cd tolerance. Similar to the physiological functions of NO, hydrogen sulfide (H2S) is also involved in the abiotic stress-related processes in plants. Moreover, exogenous melatonin induces H2S generation in plants under salinity or heat stress. However, the involvement of H2S action in melatonin-induced Cd tolerance is still largely unknown. In this review, we summarize the progresses in various physiological and molecular mechanisms regulated by melatonin in plants under Cd stress. The complex interactions between melatonin and H2S in acquisition of Cd stress tolerance are also discussed.

Low noise, self-phase-modulation-enabled femtosecond fiber sources tunable in 740-1236†nm for wide two-photon fluorescence microscopy applications.

We have demonstrated widely tunable Yb:fiber-based laser sources, aiming to replace Ti:sapphire lasers for the nJ-level ultrafast applications, especially for the uses of nonlinear light microscopy. We investigated the influence of different input parameters to obtain an expansive spectral broadening, enabled by self-phase modulation and further reshaped by self-steepening, in the normal dispersion regime before the fiber damage. We also discussed the compressibility and intensity fluctuations of the demonstrated pulses, to reach the transform-limited duration with a very low intensity noise. Most importantly, we have demonstrated clear two-photon fluorescence images from UV-absorbing fluorophores to deep red dye stains.

Molecular hydrogen-induced salinity tolerance requires melatonin signalling in Arabidopsis thaliana.

Melatonin (MT) plays positive roles in salinity stress tolerance. However, the upstream signalling components that regulate MT are poorly understood. Here, we report that endogenous MT acts downstream of molecular hydrogen (H2 ) in the salinity response in Arabidopsis. The addition of hydrogen-rich water and expression of the hydrogenase1 gene (CrHYD1) from Chlamydomonas reinhardtii increased endogenous H2 and MT levels and enhanced salinity tolerance. These results were not observed in the absence of serotonin N-acetyltransferase gene (SNAT). H2 increased the levels of SNAT transcripts in the wild-type and CrHYD1 lines, which had lower Na+ /K+ ratios and higher levels of ion transport-related gene transcripts. These changes were not observed in atsnat/CrHYD1-4 hybrids. The increased MT-dependent Na+ extrusion observed in the CrHYD1 plants resulted, at least in part, from enhanced Na+ /H+ antiport across the plasma membrane. The endogenous H2 -induced MT-dependent regulation of ion and redox homeostasis was impaired in the atsnat/CrHYD1-4 hybrids. Taken together, these results demonstrate that MT-induced salinity tolerance is induced by a H2 signalling cascade that regulates ion and redox homeostasis in response to salinity.

Genetic elucidation of hydrogen signaling in plant osmotic tolerance and stomatal closure via hydrogen sulfide.

Although ample evidence showed that exogenous hydrogen gas (H2) controls a diverse range of physiological functions in both animals and plants, the selective antioxidant mechanism, in some cases, is questioned. Importantly, most of the experiments on the function of H2 in plants were based on pharmacological approaches due to the synthesis pathway(s) in plants are still unclear. Here, we observed that the seedling growth inhibition of Arabidopsis caused by low doses of mannitol could progressively recover by recuperation, accompanied with the increased hydrogenase activity and H2 synthesis. To investigate the functions of endogenous H2, a hydrogenase gene (CrHYD1) for H2 biosynthesis from Chlamydomonas reinhardtii was expressed in Arabidopsis. Transgenic plants could intensify higher H2 synthesis compared with wild type and Arabidopsis transformed with the empty vector, and exhibited enhanced osmotic tolerance in both germination and post-germination stages. In response to mannitol, transgenic plants enhanced L-Cys desulfhydrase (DES)-dependent hydrogen sulfide (H2S) synthesis in guard cells and thereafter stomatal closure. The application of des mutant further highlights H2S acting as a downstream molecule of endogenous H2 control of stomatal closure. These results thus open a new window for increasing plant tolerance to osmotic stress.