Dynamic Localization And Endocytosis Of Sucrose Transporter StSUT1 And AtSUC2

In higher plants,photosynthesis products are usually synthesized in mesophyll cells,and most of them are transported via phloem through apoplast pathway to the site where the plant needs growth and development in the form of sucrose.And sucrose transporter plays a very important role in the loading and unloading of sucrose.Sucrose transporter is localized in the membrane,especially in the plasma membrane,and depends on the plasma membrane to function.However,due to the existence of plant cell walls and the raft-like microdomain on the cell plasma membrane is too small to observe,most of the researches on location and endocytosis are in the yeast.Therefore,this project focuses on studying the effects of membrane structure and redox on the localization and function of sucrose transporter in plants,and further exploring its endocytic mechanism.We supposed that plasma membrane,especially raft-like microdomain would influence the localization,function and internalization,so we first used reagents to destroy the cell membrane structure.Use BFA to block vesicle movement and make the recycle process of endocytosis impossible.It was found that the transporting capacity of sucrose transporter decreased,which indicated that the sucrose transporter had an endocytic circulation system.Then myriocin was used to inhibit the synthesis of sphingolipids and destroy the raft-like microdomain of plasma membrane.It was found that the transport capacity of sucrose transporter was decreased.The quenching experiment further revealed that the absence of sphingolipids would lead to a decrease in the sucrose transporter distribution on the plasma membrane.It is shown that sucrose transporter exists in the raft-like microdomain,and there may have clathrin-independent endocytosis.It was found that the loss of some sterols would cause an increase the transport capacity of the sucrose transporter after inhibiting the synthesis of sterols by m?CD.Reduced transport capacity.After using Fenpropimorph to change the conformation of sterols on the plasma membrane of the cells,compared with the results of m?CD treatment,it was found that the change in the amount of sterols is a necessary factor to cause the quaternary structure of proteins.In addition,FRAP was used to detect that under the m?CD concentration we used,the change in sterol content had no effect on the lateral transport of sucrose transporter on the cell membrane.Finally,a single collection of Dronpa fluorescent spots co-expressed with AtSUC2 was performed using variable angle illumination technique technology.The generated super-analytical images confirmed that the reduction of a small amount of sterols would transform AtSUC2 from a state of aggregation in a part of the plasma membrane to a more uniform distribution.To study the effect of the redox environment on the function and localization of the sucrose transporters AtSUC2 and St SUT1,esculin experiments and fluorescence quenching experiments were used.Through the treatment with redox reagents,we found that the oxidants H2O2 and GSSH can cause their functions to be improved without changing the number of sucrose transporters in the plasma membrane,but sucrose transporters have significantly aggregated on the plasma membrane.The reducing agents DTT and GSH do not cause changes in the distribution of sucrose transporters on the plasma membrane,but they cause a decrease in transport capacity and a decrease in the number of proteins on the membrane.To explore the mechanism of endocytosis in plants,a Rab5 i Arabidopsis mutant was selected in this experiment.Rab5 i protein can interact with Rab5 protein,which plays a key role in clathrin-mediated endocytosis.Western blot experiments and phloem sap collection experiments were performed on Arabidopsis mutants to find that Rab5 i maintains the function of Stability plays an important role.After transient expression of Rab5 i and AtSUC2 in tobacco,the esculin fluorescence imaging results after m?CD treatment showed that Rab5 i can offset the change in the ability of sucrose transporter caused by some m?CD.This characteristic is most likely to be improved by increasing the endocytosis of AtSUC2.Recycling is achieved.