Single-molecule And Vesicle Trafficking Analysis Of Ubiquitylation Involved The Activity Of AMMONIUM TRANSPORTER 1;3(AMT1;3) In Arabidopsis

Nitrogen?N?,as one of the essential macronutrients required for plants,is a constituent of nitrogen-containing compounds such as proteins,nucleic acids and cofactors.Ammonium is the preferential nitrogen source absorbed by root.In plant cells,the ammonium acquisition and transport is mediated by ammonium transporters?AMTs?,which play essential roles in the regulation of ammonium ion balance in all organisms.The AMT family comprises six isoforms in Arabidopsis thaliana,of which AMT1;3 is expressed only at the root and participate in the absorption and transport of ammonium ions in the root.Recent studies have shown that ubiquitylation not only regulates the degradation of proteins,but also participates in the sorting of proteins in endosomes and the endocytosis as the signal of membrane proteins internalization.However,whether ubiquitylation regulates the mechanism of endocytosis and vesicle trafficking of AMT1;3 and affects the dynamic characteristics of the protein are remained unclear.In this study,we cloned the Arabidopsis AMT1;3 gene and obtained the AMT1;3_K75RK233R-EGFP transgenic Arabidopsis plants.Laser scanning confocal microscope?LSCM?,variable-angle total internal reflection fluorescence microscopy?VA-TIRFM?and fluorescence correlation spectroscopy?FCS?techniques in combination with molecular biology and biochemistry methods were used to study the distribution of AMT1;3 proteins on the plasma membrane,exercise patterns,dynamics,endocytosis and vesicle transport under high-ammonium stress in AMT1;3_K75RK233R-EGFP mutant seedings.The main results are as follows:1.The analysed of degradation pathways of AMT1;3:?1?The AMT1;3 proteins was immunoprecipitated from AMT1;3-EGFP Arabidopsis thaliana.AMT1;3 immunoprecipitates was analyzed and revealed that AMT1;3 was ubiquitinated in vivo.?2?The degradation of AMT1;3 proteins in AMT1;3-EGFP Arabidopsis was analyzed,and it was found that AMT1;3 proteins was degradation in AMT1;3-EGFP transgenic plants under high-ammonium stress.When AMT1;3-EGFP transgenic Arabidopsis was co-treated with high-ammonium stress and 26S proteasome inhibitor MG132,the degradation of AMT1;3 proteins was inhibited,indicating that the ubiquitylation was participate in the degradation of AMT1;3 under high-ammonium stress.2.Obtained of AMT1;3_K75RK233R-EGFP mutants:According to analyzed the topological structure of AMT1;3 proteins,the lysine residues of the AMT1;3 that may be ubiquitinated was predicted and successfully performed site-directed mutagenesis on the two lysine residues.We got the AMT1;3_K75RK233R-EGFP mutants,which the lysine?K75 and K233?were replaced by arginine.3.The effect of ubiquitylation on the dynamic characteristics of AMT1;3 proteins:AMT1;3proteins had four motion patterns on plasma membrane,among which brownian was the main mode of exercise in control and mutant plants.This result indicated that ubiquitylation has no significant effect on the motion pattern of AMT1;3 on plasma membrane.The diffusion coefficient was significantly decreased,the percentage of long-distance motion markedly decreased and the velocity of AMT1;3proteins in AMT1;3_K75RK233R-EGFP was significantly increased compared with the control seedings under high-ammonium stress.These results showed that ubiquitylation affected the dynamic characteristics of AMT1;3 proteins on plasma membrane under high-ammonium stress.4.Ubiquitylation affected endocytosis of AMT1;3 proteins:There was no significant difference on the endocytosis of AMT1;3 proteins between AMT1;3-EGFP transgenic plants and AMT1;3_K75RK233R-EGFP mutant plants under N-sufficient condition.However,the endocytosis of AMT1;3 proteins was markedly inhibited in AMT1;3_K75RK233R-EGFP mutant plants after high-ammonium treatment compared with the AMT1;3-EGFP transgenic plants,indicating that ubiquitylation affected the endocytosis of AMT1;3 proteins under high ammonium stress.5.The effect of ubiquitylation on regulation of vesicle trafficking pathways of AMT1;3:To further analyzed the vesicle trafficking of AMT1;3,we generated the dual color lines expressing AMT1;3-EGFP,AMT1;3_K75RK233R-EGFP and VHA-a1-m RFP,At SNX1-m RFP,m RFP-SYP22,respectively,by crossings different transgenic lines.The results showed that the percentage of colocalization of AMT1;3 and VHA-al was increased and the percentage of AMT1;3 transfer into vacuolar for degradation was decreased in AMT1;3_K75RK233R-EGFP mutants,this result confirmed that the vesicle trafficking and the balance of degradation mechanism was influenced in AMT1;3_K75RK233R-EGFP mutants under high-ammonium stress.In conclusion,this study based on different live-cell microscopy together with molecular and biochemical biology methods to analyze the dynamic characteristics,endocytosis and vesicle trafficking pathways in elongation zone of AMT1;3 in AMT1;3_K75RK233R-EGFP mutants root under high-ammonium stress.Studies have revealed that ubiquitylation involved in the degradation of AMT1;3 proteins under high-ammonium stress;The movement of AMT1;3 proteins on the plasma membrane was highly dynamic and the ubiquitylation affected the absorption and transport of ammonium by changing the dynamic of AMT1;3 proteins;The defect of AMT1;3 ubiquitination inhibited the endocytosis of AMT1;3 proteins and led to the AMT1;3 proteins accumulated on the plasma membrane under high-ammonium stress;Accroding to investigate the intracellular trafficking of AMT1;3 proteins,the study showed that the protein degradation targeting to the vacuole was inhibited by ubiquitylation and the vesicle trafficking and balance of degradation of AMT1;3 in Arabidopsis thaliana was affected.These results indicateted that the dynamic characteristics,endocytosis and vesicle trafficking pathways of AMT1;3 proteins were changed in AMT1;3_K75RK233R-EGFP mutant plants after high-ammonium treatment.Taken together,these findings will lay foundation for the better understanding of the regulatory mechanisms of ubiquitination involved in the functional characterization of ammonium transporter proteins in plants.