Nitric Oxide Emission From Mangrove Wetland And Regulation Of NO On Salt Tolerance For Mangrove Plants

The aim of the presented studies was to investigate the rate of NO release of the most frequently applied NO donors sodium nitroprusside(SNP).The influence of SNP concentration,light,temperature,pH and surge frequency on the rate of NO release was investigated.The rate of NO release from leaves of mangrove,including Bruguiera gymnorrhiza,Kandelia candel and Avicennia marina under the influence of different nitrogen sources,light intensity,oxygen was observed.Using static chamber-gas bag techniques and a chemiluminescence NO_x analyzer manufactured by Teledyne Inc.USA(model 200A),NO emission from mangrove wetland and diurnal variation pattern under the influence of environmental factor were carefully monitored. Based on above results,effect of NO on the leaf stomatal movement of Aegiceras corniculatum and the order and mechanism of NO on salt resistance in A.marina,A. corniculatum,K.candel and B.gymnorrhiza were investigated.1.The rate of NO release was affected by SNP concentration,light intensity, temperature and pH of solution and shake frequency.Dose-dependent correlation between the rate of NO release and SNP concentration,temperature were fitted to Logistic equation while dose-dependent correlation between the rate of NO release and pH of solution were fitted to exponential decrease equation.However,light intensity,shake frequency were fitted to exponential increase equation.Based on above results,it is likely to provide clue to interpret dose-dependent correlation between NO donor and physiological effect.2.Using chemiluminescence NO_x analyzer,the rate of NO release from leaves of mangrove,including B.gymnorrhiza,K.candel and A.marina under the influence of different nitrogen source,light,oxygen was investigated.Leaves of B.gymnorrhiza grown on nitrogen absence or ammonia,arginine as the N source did not emit NO, either in air or under anoxia,which was interpreted to indicate that nitrate reducase(NR)was the only NO source.However,when supplied with nitrite,NO emission in dark were higher than in light.Furthermore,NO emission increased strongly in anoxia.Under air condition,NO emission rate in leaves of B.gymnorrhiza grown on nitrite as N source were more than 7 times higher than those grown on nitrate as N source.Detached leaves of K.candel grown on nitrate as N source, emitted only small amount of NO in air whereas NO emission increased strongly under anoxia.NO emission rate in the light-dark was morn than 10 times higher than in the light under air condition.NO emission occurred in different treatment in K. candel leaves from nitrite-fed plants.Furthermore,NO emission rate increased in the light under air condition.However,leaves of A.marina from nitrite-fed plants emitted a little NO in the dark under anoxia condition.Our results suggested that different species of mangrove showed diverse NO emission model in response to nitrogen supply,light intensity and oxygen.Whether these variations were correlated to different regulate mechanism in response to environmental stress among different species were remained to be studied.3.Using static chamber-gas bag techniques and a chemiluminescence NO_x analyzer,NO emission from mangrove wetland in Mangrove Nature Reserves Jiulongjiang estuarine,Fugong Town,Longhai County,Fujian Province(24°29'N, 17°55'E)and diurnal variation pattern under the influence of environmental factor were carefully monitored.Our results suggest that during the period of non-waterlogging,the diurnal variation pattern in nude beach,with the maximum emission at 9:00-14:00,was determined by temperature.There were no significant correlations between light intensity and NO emission in nude beach.The diurnal variation pattern in mid tidal zones of B.gymnorrhiza region,with the maximum emission at 7:00-9:00,was not correlated with temperature variation.However,the diurnal variation pattern in high tidal zones of B.gymnorrhiza region,with the maximum emission at 15:00-23:00,was different from mid tidal zones and nude beach.Furthermore,there were no significant correlations between soil temperature, conductance,salinity,pH,Eh and NO emission from soil.4.The experiment is mainly to find out a best way to observe the stomatal movement of A.corniculatum,based on it to study the effects of NO on the leaf stomatal movement under different concentrations and different treated time,to study the effects of Ca²⁺in the regulation of stomatal movement induced by exogenous nitric oxide,to study the interactional effects of NO and H₂O₂ in the regulation of stomatal movement.Our results suggest that the oxhide pastern is the best way to observe stomatal movement of A.corniculatum compared with the puddle method and the nail poish pastern.The inductions of stomatal closure by NO were enhanced by higher concerntration of Ca²⁺,and the role of NO appears to demonstrate to be obvious time and concentration effects.We also prove that H₂O₂ could probably act as upstream component of NO signaling and NO regulate H₂O₂ generation in stomatal closure in guard cells.5.The contents of photosynthetic pigment and proline,producing rate of superoxide radicals,contents of H₂O₂,MDA,AsA,GSH,polyphenol and Car, activities of the antioxidant enzymes involved in ROS scavenging invoked by SNP, were surveyed in A.marina seedling leaves under 600 mmol·L^-1NaCl stress.The results showed that the decrease in content of total chlorophyll in NaCl-treated seedlings was alleviated when 100μmol·L^-1SNP was supplied.NO promoted decrease in proline content.The activities of SOD,GPX and APX were promoted by NO,thus reduced the producing rate of superoxide radicals and content of H₂O₂ in A. marina leaves under NaCl stress.Meanwhile,Lipid peroxidation was alleviated by inhibited LOX activity.In addition,the oxidation of AsA was enhanced as well as contents of GSH,polyphenol and Car was increased by SNP treatment.It was also shown that APX and CAT are likely to be the major NO targets,which activities were regulated by NO,thus play important role in A.marina salt-tolerance.6.Three-month-old healthy seedlings of A.corniculatum a true mangrove,raised from propagules in normal nursery conditions were subjected to 350 mmol·L^-1NaCl under hydroponic culture.SNP was applied to investigate the effect of NO on activation potentials for antioxidative enzymes against salt stress.Changes in the contents of photosynthetic pigments,ASA,GSH and polyphenol in leaves,activities of the antioxidative enzymes including CAT,GPX and SOD in leaves were assayed to evaluate the effects of NO.Also,expression of proteins in leaves of A.corniculatum induced by NO were analyzed by two-dimensional gel electrophoresis(2-DE).The results showed that the content of total chlorophyll and carotenoid increased in NaCl-treated seedlings when 100μmol·L^-1SNP was supplied.NO treatment increased amount of thylakoid staking in the plastids.NO relieved the oxidative damage in leaves of A.corniculatum by changing the content of small molecule antioxidant and the activity of antioxidant enzyme.Firstly,NO treatment increased ASA and polyphenol content in NaCl-treated leaves,secondly,activities of SOD and CAT did not show change under NO treatment whereas GPX activity increased significantly when 100μmol·L^-1SNP was supplied.In our present study,NO enhanced salt tolerance in A.corniculatum mainly due to its regulating ability on the content of small molecule antioxidant and GPX activity.Furthermore,five up-regulated protein spots was observed in NO treated plants which suggested these protein may involved in salt resistance regulated by NO.7.Changes in the contents of photosynthetic pigments,soluble sugar,starch,total free amino acid,proline and polyphenol,levels of MDA and H₂O₂ in K.candel leaves, activities of the antioxidative enzymes including CAT,GPX and SOD in K.candel leaves were assayed to evaluate the effects of NO under 15‰and 30‰NaCl treatment.The results showed that the content of total chlorophyll and proline increased in NaCl-treated seedlings when 100μmol·L^-1SNP was supplied.NO relieved the oxidative damage in leaves of K.candel by enhancing activities of GPX and CAT,thus reduced H₂O₂ content,alleviate lipid peroxidation.GPX and CAT are likely to be the major NO targets,which play important role in K.candel under salt stress.8.Three-month-old healthy seedlings of B.gymnorrhiza a true mangrove,raised from propagules in normal nursery conditions were subjected to 500 mmol·L^-1NaCl under sand culture during 45 days.SNP was applied to investigate the effect of NO on activation potentials for antioxidative enzymes against salt stress.Changes in the contents of photosynthetic pigments,proline,ASA and Car,level of MDA and H₂O₂ in leaves,activities of CAT,GPX,APX and SOD in leaves were assayed to evaluate the effects of NO.The results showed that the content of proline increased.NO reduced H₂O₂ content by enhancing activities of GPX,APX and CAT.Furthermore, NO regulate antioxidative enzymes mainly by regulating antioxdative enzymes activites instead of altering the express of specific isoform bands.