Single Molecule Analysis Of PHT1;1 On Plasma Membrane In Living Arabidopsis Cell

As the key member of the phosphorus transporter family in plants,PHT1;1 has important biological function in regulating plant uptake of phosphorus.It has been shown that low phosphorus treatment will lead to the accumulation of PHT1;1 on the plasma membrane and its intracellular trafficking is regulated by a variety of biological mechanisms.However,the information about dynamic behaviors of PHT1;1 on the plasma membrane is still unclear.In this paper,we used variable angle total internal reflection fluorescence microscope(VA-TIRFM)with single particle tracking(SPT)and photo bleating methods to observe the dynamic changes of PHT1;1 tagged with green fluorescent protein on the plasma membrane in Arabidopsis seedlings under different phosphorus concentrations.We performed fluorescence targeting experiments in Arabidopsis using PHT1;1-GFP fusions.Fluorescent images showed that PHT1;1-GFP was mainly targeted to the plasma membrane in Arabidopsis root.The images under VA-TIRFM shows the protein density of PHT1;1 on plasma membrane under low phosphorus treatment was higher than of control.While,the density was reduced by high phosphorus treatment.We further analysed the movement patterns of PHT1;1-GFP by means of VA-TIRFM and SPT.The results indicated that PHT1;1-GFP displayed four kinds of movement patterns,including brownian diffusion,directed diffusion,restricted diffusion and mixed trajectory under normal growth condition.In addition,the kinetics parameters of PHT1;1 on PM were analyzed by Origin software and Gaussian function.We found that the life time of PHT1 under low phosphorus treatment is longer than control,in contrast to the results obtained under high phosphorus treatment.The diffusion coefficient of PHT1;1 exhibited two fitting peaks characterized with long and short distance motion under normal growth condition.However,the fitting peak with short distance motion disappeared under low phosphorus treatment,and two fitting peaks under high phosphorus treatment was samller than that of controls.Furthermore,the motion range decreased under low phosphorus treatment compared with the control,while higher motion rangeunder high phosphorus treatment was observed.Thus,the movement speed of PHT1;1 on the plasma membrane under low phosphorus treatment is faster in comparison with control,but high phosphorus treatment slowed its movement.To better understand the communication between the PHT1;1 dynamics and microdomains on PM,M?CD was used to disturb the microdomains.It was found that the life time,the diffusion coefficient and the motion range of PHT1;1 on the PM were larger than those of controls.We also analyzed the oligomerizationof PHT1;1 by PIF(Progressive Idealization and Filtering).We found that PHT1;1 on the plasma membrane mainly exist as monomers and dimers,but trimers may be present under high phosphorus treatment.In summary,we investigated the dynamic motion of PHT1;1 under different phosphorus concentrations on single-molecule level by using VA-TIRFM.The major findings from this study are noteworthy:(1)PHT1;1-GFP is located on the plasma membrane where PHT1;1-GFP displayed four kinds of movement patterns;(2)PHT1;1 has different dynamic parameters associated with phosphorus levels,and the speed of protein movement is accelerated under low phosphorus concentration;(3)Kinetic parameters of PHT1;1 increased after M?CD treatment independ of phosphorus levels;(4)PHT1;1 transporter mainly presented monomers and dimers on the plasma membrane.In this study,the distribution,dynamic changes and oligomerization of PHT1;1 on the plasma membrane were analyzed at the single-molecule level for the first time in plant living cells,which provided new insights into the investigations of phosphorus transport mechanisms via PHT1;1.