Study On The Mechanism Of Tomato SlMPK2 In The Process Of High Temperature Stress

Mitogen activated protein kinase(MAPK)cascade is an important way for cells to convert various extracellular environmental stimuli into various intracellular physiological and biochemical reactions.It involves the physiological processes of plants in response to various environmental stresses.MAPK cascade system is a three-cascade model composed of MAPKKK,MAPKK and MAPK.MAPK phosphorylation and other signals activate specific downstream targets,such as transcription factors and cytoskeletal proteins,to transmit cascade signals.High temperature and extreme climate are one of the main environmental factors affecting tomato yield.Continuous high temperature environment usually leads to abnormal growth of tomato.Therefore,it is of great significance to study the molecular and physiological mechanisms of tomato in response to high temperature for improving the heat tolerance of tomato.Our previous studies have shown that tomato SlMPK1 plays a negative role in regulating thermotolerance.At the same time,we found a highly homologous gene SlMPK2 with SlMPK1 gene,but its function is not clear.In this study,on the one hand,we used gene recombination technology to obtain recombinant tomato plants with SlMPK2 overexpression and interference,and then heterologous overexpression of SlMPK2 in Arabidopsis thaliana,and then observed the phenotypic differences under high temperature stress and measured the relevant physiological indicators to verify the function.On the other hand,Y2H technology was used to screen tomato cDNA library induced by heat,and the proteins that may interact with SlMPK2 were obtained.Some of the proteins were verified by Y2H point-to-point and BiFC technology.Finally,the function of SlMPK2 upstream interacting protein SlMPKK9 was studied to explore the mechanism of SlMPK2 under high temperature stress.The main results are as follows:1.SlMPK2 negatively regulated the thermotolerance of tomato under high temperature stress.The SlMPK2 gene was cloned,and the overexpression and interference vector of the gene was constructed.The interference strain line of SlMPK2 was obtained by Agrobacterium mediated transformation of tomato cotyledons.The phenotype and physiological changes were observed.Under high temperature stress,the phenotype of SlMPK2 overexpression plants,such as growth state,plant height,fresh weight,and root length,was significantly weaker than wild type;Chlorophyll content degradation was faster;The activities of antioxidant enzymes such as SOD,POD,APX,CAT and GR were significantly lower than that of wild type;The results of DAB and NBT staining further showed that the overexpression plants of SlMPK2 accumulated too much ROS,and the oxidative damage was more serious than that of wild type.In contrast,SlMPK2 interference plants with the phenotype,chlorophyll,antioxidant enzyme activity,and ROS level of the plants showed that the thermotolerance of SlMPK2 interferes was stronger than that of wild type.2.SlMPK2 negatively regulates thermotolerance of Arabidopsis under high temperature stress.The plants with SlMPK2 overexpression were obtained by Agrobacterium tumefaciens mediated Arabidopsis flower dipping method,and the plants with SlMPK2 overexpression were treated with high temperature.The results showed that the seed germination of SlMPK2 overexpression Arabidopsis was significantly worse than that of WT,and the growth status of SlMPK2 overexpression Arabidopsis was also significantly worse than that of WT,the hypocotyl length of Arabidopsis was also significantly shorter than that of WT.The above results further demonstrated that SlMPK2 played a negative role in plant response to high temperature stress.3.The yeast two hybrid library was screened successfully.The bait expression vector pGBKT7-SlMPK2 was successfully constructed,and its self-activation and virulence were tested.The results showed that the bait protein SlMPK2 was non-toxic to yeast cells but had self-activating activity.Further screening for the optimal concentration of 3-AT inhibited its self-activation,it was found that 20 mM 3-AT inhibited its self-activation in SD/-Trp/-Leu/-his/-Ade/+AbA+X-α-gal.The self-activation of SlMPK2 could be effectively inhibited on medium without affecting the interaction of positive control yeast.The pGADT7 plasmid of heat-induced cDNA library was transferred into yeast cells containing pGBKT7-SlMPK2 plasmid.After two rounds of screening,30l positive colonies were obtained,and l29 proteins were identified by PCR,sequencing and sequence alignment.Some proteins appeared repeatedly in the screening library,including some proteins known to interact with plant MAPK,such as SlMKK9(Solyc03g097920),SlSPRH1(Solyc06g053700),which indicated that the results of Y2H screening library were reliable.4.Y2H and BiFC were used to verify the interacting proteins.The N-terminal of fluorescent protein YFP was fused with SlMKK9,8MFU and 9DGC respectively to construct the bimolecular fluorescent vectors pSPYNE-SlMKK9,pSPYNE-8MFU and pSPYNE-9DGC.After Co-infecting tobacco with Agrobacterium tumefaciens containing pSPYCE-SlMPK2,the fluorescence was observed by laser confocal microscope.The results showed that there was fluorescence in tobacco leaf cells,which proved that SlMPK2 interacted with SlMKK9,8MFU and 9DGC proteins.5.SlMKK9 negatively regulated the thermotolerance of tomato and Arabidopsis under high temperature stress.SlMKK9 overexpression and interference vectors were constructed to obtain tomato genetic transformation plants,and the high temperature tolerance phenotype and physiological indexes were determined.The results showed that the phenotypic and physiological changes of SlMKK9 overexpression transgenic plants were consistent with that of SlMPK2 overexpression plants under high temperature stress,the phenotypic trend of slmkk9 interference plants was consistent with that of slmpk2 interference plants,which indicated that SlMKK9 negatively regulate the thermotolerance of tomato under high temperature stress.At the same time,SlMKK9 overexpression in Arabidopsis plants and SlMKK9 homologous atmkk9 mutant(AtMKK9-TDNA insertion)were obtained.The results showed that the seed germination rate of SlMKK9 overexpression plant was significantly slower than that of wild type,and the germination rate was also significantly lower than that of wild type.Moreover,the germination rate of AtMKK9 Arabidopsis seeds was significantly higher than that of wild type after heat treatment,and SlMKK9 was supplemented to eliminate this difference.In addition,the growth status of SlMKK9 overexpressed Arabidopsis plants was significantly worse than that of WT plants after high temperature treatment;The hypocotyl length of Arabidopsis was also significantly shorter than that of WT.The results obtained here showed that SlMPK2 was involved in the response of tomato to high temperature stress,and there was a negative regulatory mechanism.The changes of antioxidant enzyme activity,ROS metabolism balance and oxidative stress degree mediated by SlMPK2 are one of the main regulatory mechanisms.At the same time,SlMKK9,as upstream interaction protein,may participate in the SlMKK9-SlMPK2 cascade in the response of tomato to high temperature stress mediated by SlMPK2.