Single-molecule Dynamics Of Glutamate Receptor GLR3.4 In Arabidopsis

Glutamate receptors play an important role in plant growth and development,metabolic regulation and signal transduction.AtGLRs is a glutamate receptor family in Arabidopsis thaliana,similar to iGluRs in animals.AtGLR3.4,a glutamate receptor from Arabidopsis thaliana,belongs to the third subgroup in the glutamate receptor family.The relationship between AtGLR3.4 and Ca2+ signal transduction has been reported in detail.AtGLR3.4 can respond to external stimuli such as mechanical stress and cold stress through Ca2+ signaling mechanism.However,most of the previous studies on AtGLR3.4 which were carried out in vitro fail to reflect the actual situation of protein in vivo.In addition,there are few studies on the dynamics of AtGLR3.4 protein at single-molecule level.This study used variable angle-total internal reflection fluorescence microscopy(VA-TIRFM)and laser scanning confocal microscopy(LSCM)techniques,in combination with other biochemistry techniques and cell biology methods,to study the dynamic behavior of AtGLR3.4 protein after amino acid treatment and the factors that affecting dynamics of AtGLR3.4 protein in Arabidopsis seedlings.The main results are as follows:(1)Treatment of Arabidopsis thaliana seedlings with Glu,GABA and GSH,and the phenotype of root were analyzed.The results showed that the root length of Arabidopsis thaliana seedlings became shorter after treated with different ligands Glu,GABA and GSH.(2)The dynamic characteristics of AtGLR3.4 in leaf cpidermal cells of Arabidopsis thaliana seedlings were observed by VA-TIRFM.The results showed that AtGLR3.4 was unevenly distributed on the plasma membrane,and there were four kinds of movement patterns of AtGLR3.4 by using MATLAB software,among which the directed movement was the most,accounting for 55%;Oligomerization analysis revealed that AtGLR3.4 existed on the membrane as a tetramer.After treatment with Glu and GABA,the motion range,diffusion coefficient,velocity and lifetime were increased;after treatment with different concentrations of GSH,the motion range,diffusion coefficient,and velocity were decreased,while lifetime were increased.The effect was concentration dependent.(3)The movement of AtGLR3.4 protein after LatB,Oryzalin,MβCD and cellulase treatment was tracked by MATLAB software to explore the effect of cell wall,lipid raft and cytoskeleton on protein movement.It was found that the most influential factor on the dynamics of AtGLR3.4 protein was the cell wall,followed by lipid raft,and the least affected was the cytoskeleton.In summary,this study used VA-TIRFM,laser confocal microscopy and other cell biology as well as biochemical methods to analyze the dynamics of AtGLR3.4 protein at the single-molecule level.The results showed that:(1)the oligomeric state and localization of AtGLR3.4 on the plasma membrane was highly dynamic;(2)After treatment with different ligands Glu,GABA and GSH,the movement characteristics of AtGLR3.4 protein changed obviously,however,the most significant change was found in the movement characteristics of AtGLR3.4 protein after GSH treatment,and the effect was concentration-dependent.;(3)among the factors affecting the movement of AtGLR3.4 protein,the cell wall was the most influential on GLR3.4 protein movement,followed by lipid raft,and the least affected was the cytoskeleton.These results provide a clear pattern of the dynamic changes of AtGLR3.4 protein treated with different ligands and the key factor affecting the dynamic characteristics of AtGLR3.4 protein,which provided a basis for further elucidating the dynamic regulation and signal transduction mechanism of cell membrane proteins.