Structural And Molecular Mechanism Analysis Of EXLA1 And CESA7 In Cotton Cell Wall

Cotton fiber is a kind of special single cell trichome differentiated from outer epidermal cells of the seed.Cotton fiber is the main raw material for clothing and quilts,and also an important raw material for paper and textile industry.The characteristics of cotton fiber cell wall determine the qualities of cotton fiber,and cell wall proteins and membrane proteins play important roles in fiber development.Hence,analysis of these proteins’ structures provide insights into the molecular mechanisms of cotton fiber development.The development of cotton fiber can be divided into five stages: fiber initiation stage,fiber elongation stage,transition stage from primary wall to secondary wall,secondary wall synthesis stage,dehydration and maturation stage.Expansin,that is expressed preferentially during fiber elongation,encodes cell wall relaxation proteins that relax cell walls by breaking hydrogen bonds between cell wall polysaccharides and can induce irreversible cell wall elongation under acidic conditions.Studies in our laboratory had shown that the fiber of transgenic upland cotton with overexpression of Expansin gene GbEXPATR became longer,thinner and stronger,but its molecular mechanism had not been uncovered yet.Cellulose,the most abundant biopolymer synthesized on land,is made of linear chains of β(1-4)linked D-glucose and is the main component of mature fiber.Cellulose synthase complex(CSC)is responsible for the synthesis of cellulose microfibrils and their secretion into the cell wall.Previous studies had shown that CSC was located in the plasma membrane visualized as six-fold symmetrical rosettes,which was composed of multiple cellulose synthase(CesA)subunits.Despite a series of advances in the study of CesA function,the conformation of CesA in cotton together with its catalytic mechanism for cellulose synthesis remains unclear.In this study,we focused on the structure and function of cotton Expansin and CesA,the progresses are listed below:1.Two eukaryotic secreted expression systems of Pichia pastoris and insect cells in vitro were established.The plant cell wall protein Expansin is a secreted protein.In order to express Expansin protein with correct folding and post-translational modification,GFP protein was used as a positive control.GFP was successfully secreted out of the cell and purified by modifying the expression construct,optimizing the expression and purification methods.These progress provide possibilities of Expansin expression in vitro.2.GbEXLA1 with high concentration and purity was acquired by screening Expansin homologs in secretory expression of insect cells and its high resolution crystal was successfully obtained.GbEXLA1 with high expression and purity was obtained by codon optimization and screening of 70 GbEXPATR homologous proteins in island cotton.By optimizing buffer,purifying of cation exchange and gel filtration,the solubility and protein behavior was improved and successfully crystallized.With PEG3350 and Tacsimate as crystallization conditions,a GbEXLA1 crystal structure with resolution of2.8 (?) was obtained.It was also proved that GbEXLA1 did not have enzymatic activity by in vitro hydrolysis of polysaccharides.3.Homotrimeric structure of GhCesA7 was determined using single particle cryo-electron microscopy.Firstly,the expression of all CesA members in upland cotton was screened and a high expression protein GhCesA7 was successfully identified.By optimizing pH buffer system,detergent,protein truncations and cryo-electron microscope sample preparation conditions,the samples that can be used for 300 k V cryo-electron microscope data collection were obtained.2,715,441 single particles from 5116 images were used for 2d classification.Then 2,245,002 single particles were selected for 3D reconstruction and model construction.Finally,the GhCesA7 homologous trimer structure with resolution of 3.5 (?) was determined.4.Structural details and biochemical activity of GhCesA7 were analyzed.Aβ-1,4-glucan can be observed in each GhCesA7 protomer.This inner space formed by TM1-TM6 should be the glucan translocating channel.The helix together with IF1-IF3 formed the entrance of the glucan translocating channel.Compared to the wild type GhCesA7,The cellulose synthase activity of three mutants(D540N,D742 N,and W784A)were severely disrupted,.The sequence alignment revealed that these active sites were conserved in both eukaryotes and prokaryotes,suggesting that eukaryotes and prokaryotes may share a set of catalytic mechanisms.The trimeric conformation of GhCesA7 was mainly stabilized by PCR domain and TM7.As PCR domain is also conserved among all CesAs of eukaryotes.Therefore,it is speculated that CesA in plants can form not only homotrimers but also heterotrimers.These results lay a solid foundation for crystal structure analysis of GbEXLA1.Besides,the successful structural analysis of homotrimeric GhCesA7 provide an important basis for the study of cellulose synthesis mechanism,which provide a clue for further studies on the molecular mechanisms of fiber development.