| Aluminum toxicity is a major obstacle factor in acidic soils limiting crop growth.There are approximately 4 billion hm2 acid soils in the word,mainly in tropical,subtropical and temperate regions,especially in developing countries.There is a total area of 20.3 million hm2 acidic soils in china,which distribute in the southern 15 provinces.At the same time,due to the effects of applying the large number of chemical fertilizers and falling acid rain on the soil acidification,there is an increasing trend in A1 hazard area.Rice is a main food crop in the world.Rice A1 toxicity occurs mainly in the newly cultivated upland acid soil,limiting the absorption of mineral elements and rice growth.Nitrate and ammonium nitrogen are mainly two kinds of mineral nitrogen absorbed and used by rice,which plays an important role for rice growth.The apical plasma membrane(PM)H+-ATPase activity of plant is related to nitrogen absorption and also regulated by 14-3-3 protein,in which rice nitrogen absorption is changed,but the relationship of Al tolerance and N uptake in rice is studied less.Therefore,there is a theoretical and practical significance for studying the effect of A1 stress on nitrogen absorption of rice.In this study,using Al-sensitive cultivar rice(sterile line,Indica.Peak 1A),Al-resistant cultivar(Indica.Dian superior 35),screened from mainly grown 10 rice varieties,as the materials,the expression of root tip 14-3-3 protein and PM H+-ATPase and its interaction of two rice cultivar under Al stress response to the mechanism of Nt4+-N and NO3-N absorption is analyzed and studied at the physiological and molecular level.The main results were as follows:1.A relative elongation of the root,chromium azure dye and antioxidant enzymes activity(superoxide dismutase SOD,peroxidase POD,catalase CAT)as well as H2O2 and MDA content in rice root under different aluminum concentrations stress(0,20,50,100,200,400 ?mol·L-1 Al)for 10 rice varieties with hydroponic condition were analyzed for screening out the optimum A1 stress concentration and identifying aluminum tolerance in rice.The results showed that the relative elongation of peak 1A was only 55%,and those of the dian superior 35 was 75%under 50?mol·L-1 Al stress for 24 h,at the same time,chromazurine staining experiment showed that root tip coloring degree of peak 1A and dian superior 35 were deepening with increasing Al concent,but the peak 1A's coloring were more than dian superior 35.The content of aluminum in the peak 1A were higher than dian superior 35 under A1 stress,the MDA and H2O2 content in the peak 1A roots increased more quickly than that of the tian superior 35,the membrane lipid peroxidation in the root of peak 1A were more than dian superior 35.The activity of SOD,POD,CAT in the roots of the dian superior 35 were significantly higher than that of peak 1A.These indicated that the dian superior 35 had more anti oxidation ability than peak 1A under A1 stress.Thereby,the increases in antioxidant enzyme acitvity play important roles in rice resistance to Al stress.We concluded that the dian superior 35 is Al-resistant varieties and the peak 1A is Al-sensitive varieties.2.Nitrate(NO3+-N)and ammonium(NH4+-N)are the main two kinds of nitrogen forms absorbed directly and used by the plant roots.The mechanism of nitrogen uptake by rice was clarified by analyzing the Al induction degree and N uptake of rice cultivated with different nitrogen sources(NO3--N or NH4+-N)and the effect of PM H+-ATPase phosphorylation and its interaction with 14-3-3 protein on nitrogen absorption under Al stress.The results showed that more serious membrane damage in the peak 1A caused root plasma membrane peroxidation with different N as N source under Al stress and PM H+-ATPase activity and its interaction with 14-3-3 protein were small,compared with the dian superior 35.At the same treatment conditions,the PM H+-ATPase phosphorylation and the interaction level between 14-3-3 protein and phosphorylation of PM H+-ATPase in different nitrogen cultivated rice were NH4+-N/NO3--N(1:1)>NH4+-N>NO3--N.The nitrogen uptake in rice also showed the same trend as PM H+-ATPase phosphorylation.At the same nitrogen source,PM H+-ATPase phosphorylation and its interaction with14-3-3 protein in the peak 1A were weaker than that of the dian superior 35,as well as nitrogen absorption.These results indicated that the expression and interaction of 14-3-3 proteins and PM H+-ATPase was involved in regulation of N uptake under Al stress.3.In order to further investigate the role of expression and interaction of 14-3-3 proteins and PM H+-ATPase in the regulation of nitrogen uptake and Al-resistant under Al stress,It was investigated that the effects of exogenous AsA on physiological response of rice growth by using the dian superior 35 and Peak 1A as the materials,which clarify that exogenous AsA alleviate physiological and biochemical mechanism of rice growth under Al stress.These results showed that exogenous AsA could further improve the activity of SOD,POD,CAT and APX in the two kinds of rice roots,reduce the aluminum content in rice root,effectively remove H2O2 accumulation and decrease the degree of membrane lipid peroxidation induced by A1 stress in rice roots.At the same time,As A could enhance PM H+-ATPase activity and the ability of pump H+ by improving PM H+-ATPase activity and its interaction with 14-3-3 protein for providing the energy of nitrate uptake,thereby improving the absorption ability of nitrate in rice under Al stress.4 It was investigated that whether the change of interaction 14-3-3 proteins and PM H+-ATPase by adding FC and AMP to dian superior 35under Al stress were involved in the regulation of aluminum tolerance and nitrogen uptake.Co-immunoprecipitation analysis showed that FC promoted the interaction of 14-3-3 proteins and phosphorylated PM H+-ATPase,which increase the activity of PM H+-ATPase(about 1-fold),enhance H+-pump activity and ability of H+ efflux and improving nitrogen uptake in rice under Al stress.However,AMP reduced the interaction of 14-3-3 proteins and phosphorylaed PM H+-ATPase,which decrease PM H+-ATPase activity(about 0.67-fold),reduce significant activity of H+-pump and ability of H+ efflux,and descend nitrogen uptake in rice under Al stress.These results further confirmed that the interaction of 14-3-3 proteins and PM H+-ATPase were involved in the regulation of nitrogen uptake in the dian superior 35 under Al stress.In summary,nitrogen uptake in rice under Al stress was decreased by reducing the interaction level of 14-3-3 protein and PM H+-ATPase and weaking PM H+-ATPase activity and H+ pump activity Adding exogenous AsA and specific activator FC could reduce the oxidative damage of A1 toxicity and enhance the interaction of 14-3-3 protein and PM H+-ATPase,which improve PM H+-ATPase and H+ pump activity for increasingnitrogen uptake of rice.Add PM H+-ATPase inhibitor AMP could increase oxidative damage of aluminum toxicity and reduce the interaction of 14-3-3 protein and PM H+-ATPase,which weaking PM H+-ATPase activity and H+-pump activity and the ability of N-uptake by rice.It suggested that the change of PM H+-ATPase and H+ pump activity of rice under Al stress could be obtained by altering the interaction level of e 14-3-3 protein and PM H+-ATPase,which affect the ability of N-uptake by rice.and changed nitrogen absorption ability in rice under aluminum stress.The mechanism of PM ATPase in the regulation of nitrogen uptake by rice was further confirmed at molecular level.This provided the genetic resources and strategies for improving the efficiency of N-uptake by rice under aluminum stress. |
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