1. Analysis of Cell Wall Proteinsduring Xylem Vessel SecondaryCell Wall Formation in CellCulture Students:Gurung Jyoti MohanDwivedi Gaurav DuttaLinlin GaoSupervisors:Irene GranlundEdouard Pesquet

2. OutlineINTRODUCTIONMATERIALS & METHODSRESULTS & DISCUSSIONACKNOWLEDGEMENT 3. Aim of the study Objective of the present study is to accomplish fractionation of cell wall from normal cells and cells that has secondary cell wall to identify the different proteins involved in the growing of secondary cell wall and lignification. After the formation of the secondary cell walls, the identification of cell wall proteins and the quality of cell wall fractionation was achieved by using MS/MS. 4. IntroductionSecondary cell walls are themajor constituent oftracheary elements (TEs) andfibers in wood, which is themost abundant biomassproduced by plants.The secondary cell wallsprovide strong mechanicalstrength to trachearyelements and fibers, andultimately to plant organs. 5. Components of secondary cell wallThe principal componentsof secondary walls are:CelluloseHemicelluloseLignin. 6. HemicelluloseI s i n t he f or mof het er opol ym s (m r i x pol ysacchar i des), such aser atar abi noxyl ans, pr esent al ong w t h cel l ul ose i n al m al l pl ant cel l w l s. i ost al 7. Lignin synthesis in tracheary elements (TEs)Lignin is found in the secondary cell wall of tracheary elements and xylemfibers.The tracheary elements are water and mineral conducting structures in wood.They undergo programmed cell death (PCD) to mature and form a hollow tubewith a lignified secondary cell wall.The lignified secondary cell wall provides a mechanicalstability, hydrophobicity and pathogenic defense. 8. Lignifications in secondary cell wall 9. Lignifications in secondary cell wall 10. Limitations arises during theextraction of cell wall proteins Proteins may be confined in the polysaccharide matrix ofcellulose, hemi-cellulose and pectin. Some proteins are difficult to solubilize. Some proteins undergo post-translational modifications. Lack of surrounding membrane may result in a loss ofcell wall proteins. 11. Suspension cell culture of A. thalianaCell induction for TE differentiationBasal cells without any hormone inductioHarvesting of cell culture with vacuum filtrationCell wall preperation by tissue grinding Subsequent washes in increasing concentration of sucroseProtein extraction by different concentration of salts (NP40 + CaCl2) SDS-PAGE and Western Blotting Protein identification by LC-MS/MS 12. Tracheary Elements (TEs) Differentiation System in vitroNormal CellsNormal Cells Hormones Basal Basal 13. Tracheary Elements (TEs) Differentiation System in vitro Normal Cells TrachearyBasalelements (TEs) Induced 14. Cells harvest by Vacuum filter 15. Freezer mill 16. Sonication 17. Medium mill 18. Cell Wall PreparationSolubize in 150mM NaCl and 10% glycerol in 100mM Acetate Buffer pH 4.61000 g, 4C, 15 min, 3 acc. Supernatant Pellet Solubilize in 0.4M sucrose in acetate buffer pH4.61000 g, 4C, 15 min, 3 accSupernatant PelletSolubilize in 0.6M sucrose in acetate buffer pH4.61000 g, 4C, 15 min, 3 accSupernatant Pellet Solubilize in 1M sucrose in acetate buffer pH4.61000 g, 4C, 15 min, 3 accSupernatant Pellet 19. Solubilise in 5mM MES-KOH pH 5.6 with 5 mM MgCl21000 g, 4C, 15 min, 3 acc SupernatantPellet 20 000 g, 4C, 10 minFiltrate and freeze in liquid nitrogenPelletWash two times with 5 mM MES-KOH pH 5.6 with 5 mM MgCl2 with centrifugation in between CW4 Freeze in liquid nitrogen and grind.Solubilise in 0.05% NP40 + 10% DMSO in 5mM MES-KOH pH 5.6 with 5mM MgCl2 20 000 g, 4C, 10 min NP40 extraciton PelletWash with 5 mM MES-KOH pH 5.6 with 5 mM MgCl2 solubilise in 0.1M, 0.5M and 2M and 4MCaCl2 in 5mM MES-KOH pH 5.6 with 5mM MgCl20.1M CaCl2 0.5M CaCl2 2M CaCl24M CaCl220 000 g, 4C, 10 min Pellet0.1 M Extraction0.5M Extraction2M Extraction 4M Extraction CW5 20. LC-MS/MS 21. Mascot search 22. Data analysiswww.arabidopsis.orgwww. plantenergy.uma.edu.au 23. Cell culture, harvest andhomogenizationAfter 7 days of cell culture, approx. 15-20% ofinduced cells had transformed into Tracheryelement (TE) formation.Cell disruption was carried out by either of thethree methods: medium mill, sonication or freezermill.Freezer mill method was the most efficient amongthe three. 24. Cell disruption by different methods Before sonication grindingSonication Medium mill Medium millFreezer mill 25. SDS-PAGE and Western blotting Proteins were seperated using SDS-PAGE. Western blotting provided the purity of cell wallisolated. It was carried out using anti-tubulin antibody asthe primary antibody. Tubulin are the proteins that make upmicrotubule, a cellular component that lie beneath the secondary cell walls. 26. Western blotting using anti-tubulinantibody as the primary antibodyBasal samples Induced samples 27. LC-MS/MS and bioinformatic analysis We chosed 0.1M CaCl2 extraction (Induced and basal) andCW5 pellet (Induced and basal). We identified 79 proteins from CW5-pellet (Induced) and94 proteins from CW5-pellet (basal). Out of these, 44.3% were CWPs in induced sample and39.3% were CWPs in basal sample. Notably, both the induced and basal CW5-pellet revealedthe presence of some protein contaminants. Conversely, in case of 0.1M CaCl2 extract, we identified47.1% of CWPs in basal supernatant compared to 31.1% ofCWPs in induced supernatant. 28. Association between induction hormones and cell wall proteinsThis implies that majority of cell wall proteins of basalsample were released during the extraction point.Two possible reasons: CWPs in basal sample with no TEs were loosely boundto the cell wall. Some CWPs are tightly associated with the cell wallduring secondary cell wall formation. 29. Concluding remarks Thismethod of preparing cell wall throughmechanical disruption, fractionation and extraction ofproteins with different salt concentration provides agood cell wall preperation technique. In fact, the principle of this technique can offer a stagefor studying cell wall proteome.