Molecular Mechanism Underlying Hydrogen Gas-alleviation Of Osmotic And Salt Stress And Extension Of Cut Flower Freshness

Originally,hydrogen gas(H2)was considered a physiologically inert gas.In 1975,it has been proved that high-pressure H2 therapy significantly improved skin cancer of mice in Science.However,H2 biological effects had not yet attracted enough attention.H2 could be used as a selective antioxidant to improve cerebral ischemic injury of rats in 2007,and above study was published in Nature Medicine.It is known to all that exogenous H2 could alleviate paraquat-induced oxidant damage in alfalfa reported by Plant Cell and Environment in 2013.As was reported in Plant Physiology in 2014,H2 administration could enhance drought tolerance of Arabidopsis.Since then,the study of H2 biological effects and the related molecular mechanism has gradually been payed attention.At present,according to different application fields of H2,H2 biology mainly includes hydrogen medicine and hydrogen agriculture.The practical application corresponding to hydrogen agronomy is hydrogen agriculture.Hydrogen agriculture is a new discipline that explored the regularity of hydrogen agriculture by using physiology,biochemistry,molecular biology,and genetic methods.Hydrogen agriculture is a green and ecological agriculture,and hydrogen agriculture mainly includes the plantation,animal husbandry,and aquaculture.It is mainly divided into facility-horticulture hydrogen agriculture,farmland hydrogen agriculture,and home hydrogen agriculture.Hydrogen agriculture was associated with research fields.This research started with the study of drought and salt tolerance in plant and the mechanism of flower freshness.Alfalfa,Arabidopsis,and lisianthus cut flower were used for experimental materials respectively,to explore the molecular mechanism of H2 to enhance plant drought and salt tolerance and extend the freshness of fresh cut flowers by using pharmacology,molecular biology and genetic methods.These findings aimed to provide a theoretical and practical basis for the widespread use of H2 in agriculture.Here,these results are as follows:Although H2 and NO are respectively suggested to enhance plant tolerance against osmotic stress(mimicking the drought stress),the crosstalk of them is still elusive.Here,the application of hydrogen-rich water(HRW)strengthened NO production in PEG-stressed alfalfa seedling roots,followed by obviously alleviating inhibition of seedling growth.Comparatively,significant but weaker responses in phenotype were observed in plants supplemented with nitrogen-rich water,indicating that the roles of HRW was H2-related.Above responses of H2 were inhibited by the removal of NO with the scavenger(s)of NO.The application of tungstate,an inhibitor of the NO synthetic enzyme nitrate reductase(NR),showed the similar blocking response in phenotype,suggesting that NR might be the major source of NO involved in above H2 actions.Proline synthesis was also stimulated by H2 and NO,both of which were supported by the increased ?1-pyrroline-5-carboxylate synthetase(P5CS)activities,the decreased proline dehydrogenase(ProDH)activities,and corresponding transcripts.The addition of H2 and NO could increase antioxidant defense in stressed plants,both of which were confirmed by the histochemical staining for ROS production and lipid peroxidation,representative antioxidant enzyme activities and transcripts.Thus,redox balance was reestablished.When NO scavenger was applied,proline synthesis,redox balance,and thereafter osmotic tolerance induced by H2,were severely impaired.Additionally,H2-triggered S-nitrosylation was obviously inhibited by the removal of endogenous NO level.Together,above results discovered the involvement of NO-induced proline and redox balance in H2-triggered osmotic tolerance.Ample studies confirmed that exogenous H2 or endogenous melatonin(MT)could effectively improve plant performance under salinity condition.However,whether and how endogenous H2 involved in salinity responses and the crosstalk between endogenous H2 and MT has yet to be elucidated.Here,exogenous H2 could enhance salinity tolerance of wild-type(WT).However,exogenous H2 failed to alter salt-sensitivity of atsnat mutants,which is compromised for either MT synthesis or MT signaling,implicating that MT might be required for H2-induced salinity tolerance.Additionally,the WT,CrHYD1 transgenic lines(mimicking endogenous H2 production by overexpressing hydrogenasel gene(CrHYD1)from Chlamydomonas reinhardtii),and atsnat/CrHYD1-4 hybrids were challenged with NaCl stress to confirm above hypothesis.As expected,endogenous H2 levels in CrHYD1 transgenic lines(CrHYD1-3 and CrHYD1-4)and atsnat/CrHYD1-4 hybrids were higher than the WT,and CrHYD1 plants were tolerant to salinity stress compared with atsnat/CrHYD1-4 hybrids.Further results showed that endogenous H2 increased mRNA levels of serotonin N-acetyltransferase gene(SNAT)and MT levels in CrHYD1 plants,suggesting that MT might be downstream signaling of endogenous H2-induced salinity tolerance.Next,our results showed that compared with the WT,endogenous H2-induced lower Na+/K+ ratio in CrHYD1 plants.Gene expression analysis showed that the expression levels of ion transporter genes(SOS1,SOS2,SOS3,AKT1,and NHX1)in CrHYD1 plants were higher than those in the WT.However,the expression levels of above genes in atsnat/CrHYD1-4 hybrids were similar to those in the WT.Analysis of non-invasive micro-test technology showed that compared with the WT,higher Na+ effluxes,higher H+influxes,and lower K+effluxes were observed in CrHYD1 plants,and the above effects were reversed in atsnat/CrHYD1-4 hybrids.Additionally,endogenous H2 increased the activity and the gene expression of antioxidant enzyme in CrHYD1 plants,compared to the WT.However,the functions of endogenous H2 were repressed in atsnat/CrHYD1-4 hybrids.Collectively,these results confirmed that under salinity stress,endogenous H2 induced SNAT expression to promote MT accumulation,which activated antioxidant defense and regulated ion fluxes,thus maintaining redox and ion homeostasis.This work suggested SNAT-dependent MT,at least in enhancing salinity tolerance of Arabidopsis,might act downstream signaling of endogenous H2.Exogenous H2 administration could extend the freshness of postharvest lily and rose cut flowers and prolong the vase life of cut flowers.However,whether and how endogenous H2 extends the freshness of cut flowers remains unclear.In this study,our results confirmed that both endogenous H2 homeostasis and redox balance of cut flowers were impaired during the senescence.Analysis of endogenous H2 concentration by gas chromatography showed that exogenous H2(in the form of hydrogen-rich water)could increase endogenous H2 levels of cut flowers,but 2,6-dichlorophenol indophenol(DCPIP,an endogenous H2 synthesis inhibitor)inhibited endogenous H2 production.Analysis of vase life,flower diameter,and fresh weight of cut flowers showed that compared with control,exogenous H2 extended the freshness of cut flowers,but DCPIP application accelerated the senescence of cut flowers.However,exogenous H2 inhibited DCPIP-induced the reduction of vase life and the acceleration of aging.Further results showed that exogenous H2 could extend the freshness of cut flowers by delaying lipid peroxidation accumulation and enhancing antioxidant enzymes activities.In addition,compared with control,exogenous H2 significantly inhibited the degradation of soluble proteins and chlorophyll,and increased the proline accumulation.However,DCPIP application inhibited above effects of H2.Collectively,endogenous H2 extends the freshness of cut flowers by increasing antioxidant capacity and proline content and inhibiting the degradation of soluble protein and chlorophyll,following by prolonging vase life of lisianthus cut flowers.This study started from three important areas of hydrogen agriculture respectively.Our results revealed that exogenous H2 application could increase the osmotic tolerance of alfalfa by mobilizing NO signals,and melatonin was the downstream signalling of endogenous H2 in enhancing the salinity tolerance of Arabidopsis,respectively.Additionally,endogenous H2 could extend the freshness of lisianthus cut flowers.Above findings could promote the research of hydrogen agronomy,and provided the theoretical guidance for H2 application in agricultural production,horticultural cultivation,and households.Meanwhile,this study could lay the foundation for the development of farmland hydrogen agriculture,facility-horticulture hydrogen agriculture,and household hydrogen agriculture.