Regulation Of G Protiens And WNK Protein Kinases On Nitrate Uptake And Utilization In Arabidopsis Thaliana

Heterotrimeric GTP binding proteins complex function as membrane signal transduction components that mediate the corresponding response to cell external stimulus in all eukaryotes.Plant G protein is involved in regulating many physiological and biochemical processes,including plant root and shoot morphology,cell proliferation,stomatal movement,photoperiod,biotic and abiotic stress and nitrogen use efficiency,etc.The previous protein interaction interactome did not showed physical interaction between G protein and NRTs,but several candidates linked these two groups,including WNK kinase.Plant WNK kinase family play important roles in regulating plant photoperiod and osmotic stress.WNK kinase were also responsible for the internalization process induced by glucose in RGS1.WNK8 physically interacts with nitrate transport protein NRT1.1,so we speculate that G protein signaling system and WNK kinases may be involved in regulating nitrogen absorption and utilization.In this paper,we used Petri dish and hydroponics method to treat G protein and WNK kinase mutants with different concentrations of nitrate and observed the differences in their root system and leaf growth at seedling stage,growth situation,nitrogen absorption and transport,related enzyme activity and gene expression at reproductive stage.The main results and conclusions are as follows:1.The results of root phenotype analysis of G protein mutants(agb1-2 and rgs1-2)showed that the primary root length of wild-type Col was inhibited by high nitrogen concentration.Different from wild-type,the primary root growth of mutant agb1-2 was directly proportional to the concentration of nitrogen Col in seedling stage,and the number of lateral roots was significantly higher than that of wild-type,and the root-shoot ratio of agb1-2 was significantly higher in reproductive stage with high nitrogen.The phenotype of rgs1-2 is opposite to that of agb1-2.2.In G protein mutants(agb1-2,gpa1-3 and rgs1-2),the expression of NRT1.1 under nitrate stimulation showed the similar trend as the wild type,so does NRT2.1 and NRT3.1,which indicated that G protein did not directly mediate nitrate signal transduction at the transcription level.In WNK kinase mutants(wnk8-1 and wnk8-2),the expression levels of NRT1.1 and NRT2.2 after nitrate treatment were significantly lower than those of wild type,which indicated that WNK kinases played an important role in the primary nitrate responses,and no significant differences were detected in NRT related gene expression in other WNK mutants,indicating that WNK8 had specificity in NRT gene expression regulation.3.The plate experiment showed that under low nitrogen condition,double mutant wnk8/10 and triple mutant wnk1/8/10 showed inhibited roots and shoot growth compared to wild type at seeding stage.Under high nitrogen condition,the root system of mutant was developed in seedling stage,the growth deficiency of leaf area under the condition of low nitrogen had disappeared during the reproductive stage.The long-term nitrogen treatment experiment showed that under low nitrogen treatment,the biomass and GS activity of WNK kinase mutants decreased significantly compared to the wild type,and the silique number also decreased probably because the mutation of the WNK kinase delayed the flowering time.Under high nitrogen condition,the root GS activity of the WNK mutants increased significantly compared to the wild type,while the whole biomass and silique biomass was significantly reduced.4.Isotope tracer experiments showed that the nitrogen translocation ratio of wnk1/8/10 was significantly lower than that of wild type after low nitrogen treatment,and wnk1 was higher than that of wild type.The translocation ratio of wnk1/8/10 increased significantly compared to wild-type under high nitrogen conditions,which showed that WNK kinase was involved in the nitrogen translocation from root to shoot.Unlike the double mutants wnk8/10 and triple mutants wnk1/8/10,the single mutants of these three genes did not show uniform phenotype.Above all,G proteins and WNK protein kinases play an important role in nitrogen regulation of plant growth.G proteins respond to external nitrogen signals by influencing the root system architecture of Arabidopsis thaliana.At WNK1,AtWNK8 and AtWNK10 play a positive role in regulating nitrogen uptake and utilization of plants under low nitrogen conditions.Nitrogen uptake by plants in seedling stage will not be affected under high nitrogen condition,and WNK kinases participate in nitrogen transport and assimilation at reproductive stage.This paper explained the regulation mode of G protein and WNK kinases on absorption and utilization of nitrogen in Arabidopsis thaliana,which provided a new idea for the molecular regulation mechanism of plant response to nitrogen signal.