The Role Of MAPK Involved In Ameliorating The Nitrate Stress By H₂S

Soil secondary salinazation is one of the major abstacles to the vegetable production in greenhouse,which seriously inhibited the growth and quality of vegetable.The main anion in soil of greenhouse is N03-.Mitogen-activated protein kinase(Mitogen-Activated ProteinKinase,MAPK)cascade signaling system,including MAPKKK,MAPKK and MAPK kinase,plays very important role regulate in cell growth and development and responding to external environmental stimuli.H2S is a third gasotransmitters following NO and CO.Researches show that certain concentrations of exogenous H2S can relieve salt,drought stress,ion toxicity,metal stress,etc..In this paper,we studied the effect of exogenous H2S on the growth and physiological characteristics of tomato under NO3-stress using H2S donor,scavengers,MAPK inhibitor PD98059.The main results were presented as follows:1.The effects of different concentration(0,50,100,300,500,1000μpmo1/L)NaHS NaHS on the growth of tomato seedlings were investigated.The results showed that:different NaHS concentration have different effect on tomato seedlings.There no significant difference between 50 μmol/LNaHS treatment with the control.However,100 μmol/L NaHS enhanced the growth of tomato seedlings.The height,fresh weight,dry weight,chorophyll contents net photosynthetic rate,gas conductance,cellular C02 contents,transpiration rate,antioxidance enzyme activites,antioxidants(AsA and GSH),nitrogen metabolism enzymes(NR,GOGAT,GS)activities,endogenous H2S content and AtLCD activity increased significantly.High concentration of NaHS(300-1000μmol/L)inhibited the tomato seedling growth.2.We investigated the effect of gas transmitter H2S(by adding H2S donor NaHS)on the physiological and biochemical characteristics of hydroponic growth tomato seedlings under nitrate stress.The results showed that the shoot height,root length,fresh weight and dry weight,chlorophyll a and b contents,net photosynthetic rate,gas conductance and transpiration rate increased significantly after NaHS added for 1,3,5 days,while the intracellular CO2 concentration decreased,compared with the nitrate stress treatment alone.The MDA and H2O2 contents increased significantly after nitrate stress treatment.However,when NaHS was added,the MDA and H2O2 contents decreased,while the antioxidant enzyme activities of superoxide dismutase,catalase,peroxidase,and ascorbate peroxidase increased,besides,the antioxidant substances of ascorbate and GSH contents increased.The activities of nitrate reductase,glutathione synthetase,and glutamate synthase increased after NaHS was added.NaHS treatment stimulated the increase of L-desulfhydrase activity,which in turn induced accumulation of H2S content.These results indicate that the alleviation of the nitrate stress damage on tomato by H2S might be fulfilled through increasing the antioxidant enzyme activities and the antioxidant substances contents.3.The expression SlMAPK family under nitrate stress with or without NaHS was analyzed by RT-PCR.The result showed that NO3-(N)induced gene expression SlMAPK,especially SIMAPK3,6,9,12,13.Exogenous NaHS to the nitrate solution exdecreased the expression of SlMAPK3,5,6,9,10,11,12,13,16 indicating SlMAPK gene may be involved in the process of H2S relieving NO3-stress.Further,SlMAPK3,6,9,12,13 gene full-length cDNA were obtained.The overexpression plant vector pRI101-SIMAPK12 and prokaryotic expression vector pET32a-SlMAPK3 were constructed.4.The role of MAPK involved in ameliorating oxidative damage induced by nitrate in tomato seedlings were studied using H2S donor NaHS and MAPK inhibitor PD98059.The results showed that in tomato,under NO3-stress,MDA,H2O2 was significantly increased and antioxidant enzymes(SOD,POD,CAT)activities decreased,while APX activity increased.After adding exogenous H2S donor NaHS to N03-,the MDA and H2O2 content decreased and antioxidant enzymes(CAT,POD,SOD,APX)activities,AsA,GSH contents increased.When MAPK inhibitor PD98059 was added,the effect of NaHS was inhibited,indicating that MAPK participated in the process of H2S ameliorating NO3-stress.5.To overexpress CsNMAPK transgenic tobacco as experimental material,the use H2S donor NaHS,scavengers HT,PAG and MAPK inhibitors inhibitor PD98059,research MAPK participation of NO3-H2S stress-induced oxidative damage mitigation.The results show that NO3-treatment of wild-type tobacco and overexpression CsNMAPK transgenic tobacco plants,a significant increase in MDA and H2O2 content,applied after H2S donor NaHS,antioxidant enzymes(CAT,APX,POD,SOD)activity significantly increased exogenous,AsA and GSH were significantly increased,a significant increase in GR,DHAR MDHAR activity and a significant increase in the endogenous H2S content and AtLCD activity,H2S scavengers HT,PAG and MAPK inhibitor PD98059 inhibited the NaHS inhibitors of this effect,but PD98059 suppressing effective as HT and PAG significant over-expression in transgenic tobacco plants CsNMAPK effect is more prominent.6.We obtained tomato full-lengthcDNA sequence of AtLCD gene and Arabidopsis SlDCD)gene.Then the overexpression vector of pRI101-AtLCZD and pRI101-SlDCZD were contructed and transformated to tobacco by leaf discs.The transgenic tobacco were analyzed by genomic PCR and RT-PCR and the SIDCD transgenic tobacco plants were obtained.The flower time of SIDCD transgenic tobacco plants was earlier,compared with the wild type plants.