A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax
2017; Elsevier BV; Volume: 16; Issue: 9 Linguagem: Inglês
10.1074/mcp.m116.063727
ISSN1535-9484
AutoresMalika Chabi, Estelle Goulas, Céline C. Leclercq, Isabelle de Waele, Christophe Rihouey, Ugo Cenci, Arnaud Day, Anne‐Sophie Blervacq, Godfrey Neutelings, Ludovic Duponchel, Patrice Lerouge, Jean-François Hausman, Jenny Renaut, Simon Hawkins,
Tópico(s)Microbial Metabolites in Food Biotechnology
ResumoExperimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes. Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity. Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes. Flax (Linum usitatissimum L.) is an economically important species that is grown for its cellulose-rich bast fibers used in textiles (linen) and composites, as well as for its seeds that are used as animal feed and a source of oils containing unsaturated fatty acids such as Alpha linolenic acid (ALA) (1Singh K.K. Mridula D. Rehal J. Barnwal P. Flaxseed: A potential source of food, feed and fiber.Crit. Rev. Food Sci. Nutr. 2011; 51: 210-222Crossref PubMed Scopus (267) Google Scholar). Flax seeds also contain high amounts of the lignan secoisolariciresinol diglucoside (SDG) that shows several biological activities of interest for human health (2Touré A. Xueming X. Flaxseed lignans: Source, biosynthesis, metabolism, antioxidant activity, Bio-active components, and health benefits.Compr. Rev. Food Sci. Food Saf. 2010; 9: 261-269Crossref PubMed Scopus (230) Google Scholar). As in other plant species improvement in the production and quality of products obtained from flax, as well as a better tolerance to abiotic stress and pathogens requires a more thorough understanding of many aspects of flax biology. Over the last decade a number of genomics, transcriptomics and functional studies have made significant contributions to our knowledge on flax, but proteomic studies have been generally limited to certain organs and/or cell types and a more comprehensive overview of the flax proteome is as yet lacking (3Wang Z. Hobson N. Galindo L. Zhu S. Shi D. McDill J. Yang L. Hawkins S. Neutelings G. Datla R. Lambert G. Galbraith D.W. Grassa C.J. Geraldes A. Cronk Q.C. Cullis C. Dash P.K. Kumar P.A. Cloutier S. Sharpe A.G. Wong G.K.-S. Wang J. Deyholos M.K. The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.Plant J. 2012; 72: 461-473Crossref PubMed Scopus (297) Google Scholar, 4Huis R. Morreel K. Fliniaux O. Lucau-Danila A. Fenart S. Grec S. Neutelings G. Chabbert B. Mesnard F. Boerjan W. Hawkins S. Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.PLANT Physiol. 2012; 158: 1893-1915Crossref PubMed Scopus (73) Google Scholar, 5Roach M.J. Mokshina N.Y. Badhan A. Snegireva A.V. Hobson N. Deyholos M.K. Gorshkova T.A. Development of cellulosic secondary walls in flax fibers requires -galactosidase.Plant Physiol. 2011; 156: 1351-1363Crossref PubMed Scopus (86) Google Scholar, 6Mokshina N. Gorshkova T. Deyholos M.K. Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers.PLoS ONE. 2014; 9: e97949Crossref PubMed Scopus (41) Google Scholar, 7Chantreau M. Grec S. Gutierrez L. Dalmais M. Pineau C. Demailly H. Paysant-Leroux C. Tavernier R. Trouvé J.-P. Chatterjee M. Guillot X. Brunaud V. Chabbert B. Van Wuytswinkel O. Bendahmane A. Thomasset B. Hawkins S. PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.BMC Plant Biol. 2013; 13Crossref PubMed Scopus (26) Google Scholar, 8Chantreau M. Portelette A. Dauwe R. Kiyoto S. Crônier D. Morreel K. Arribat S. Neutelings G. Chabi M. Boerjan W. Yoshinaga A. Mesnard F. Grec S. Chabbert B. Hawkins S. Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.Plant Cell Online. 2014; 26: 4462-4482Crossref PubMed Scopus (39) Google Scholar, 9Chantreau M. Chabbert B. Billiard S. Hawkins S. Neutelings G. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.Plant Biotechnol. J. 2015; 13: 1312-1324Crossref PubMed Scopus (31) Google Scholar, 10Ibragimova N.N. Mokshina N.E. Gorshkova T.A. Cell wall proteins of flax phloem fibers.Russ. J. Bioorganic Chem. 2012; 38: 117-125Crossref Scopus (7) Google Scholar, 11Hotte N.S.C. Deyholos M.K. A flax fibre proteome: identification of proteins enriched in bast fibres.BMC Plant Biol. 2008; 8: 52Crossref PubMed Scopus (34) Google Scholar, 12Barvkar V.T. Pardeshi V.C. Kale S.M. Kadoo N.Y. Giri A.P. Gupta V.S. Proteome profiling of flax (Linum usitatissimum) seed: Characterization of functional metabolic pathways operating during seed development.J. Proteome Res. 2012; 11: 6264-6276Crossref PubMed Scopus (20) Google Scholar, 13Day A. Fénart S. Neutelings G. Hawkins S. Rolando C. Tokarski C. Identification of cell wall proteins in the flax (Linum usitatissimum) stem.Proteomics. 2013; 13: 812-825Crossref PubMed Scopus (24) Google Scholar). This represents an important gap in our knowledge because although transcriptomics, for example, provides information about transcript accumulation in a biological sample, other factors (e.g. translation efficiency, protein turnover, post-translational modifications etc.) can also affect the final quantity and biological activity of the corresponding protein (14Haider S. Pal R. Integrated analysis of transcriptomic and proteomic data.Curr. Genomics. 2013; 14: 91-110Crossref PubMed Scopus (245) Google Scholar, 15Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Crossref PubMed Scopus (3185) Google Scholar). Proteomics is therefore an important complementary tool that has been successfully used in plant biology to improve our understanding of many different processes (16Rossignol M. Peltier J.B. Mock H.P. Matros A. Maldonado A.M. Jorrín J.V. Plant proteome analysis: A 2004–2006 update.Proteomics. 2006; 6: 5529-5548Crossref PubMed Scopus (144) Google Scholar, 17Cui S. Hu J. Guo S. Wang J. Cheng Y. Dang X. Wu L. He Y. Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.J. Exp. Bot. 2012; 63: 711-726Crossref PubMed Scopus (62) Google Scholar). Given their economic interest, most proteomics studies in flax have focused on stems and seeds (11Hotte N.S.C. Deyholos M.K. A flax fibre proteome: identification of proteins enriched in bast fibres.BMC Plant Biol. 2008; 8: 52Crossref PubMed Scopus (34) Google Scholar, 12Barvkar V.T. Pardeshi V.C. Kale S.M. Kadoo N.Y. Giri A.P. Gupta V.S. Proteome profiling of flax (Linum usitatissimum) seed: Characterization of functional metabolic pathways operating during seed development.J. Proteome Res. 2012; 11: 6264-6276Crossref PubMed Scopus (20) Google Scholar, 13Day A. Fénart S. Neutelings G. Hawkins S. Rolando C. Tokarski C. Identification of cell wall proteins in the flax (Linum usitatissimum) stem.Proteomics. 2013; 13: 812-825Crossref PubMed Scopus (24) Google Scholar, 18Gábrišová D. Klubicová K. Danchenko M. Gömöry D. Berezhna V.V. Skultety L. Miernyk J.A. Rashydov N. Hajduch M. Do cupins have a function beyond being seed storage proteins?.Front. Plant Sci. 2016; 6: 1-9Crossref Scopus (14) Google Scholar). In the case of stems, the interest is mainly directed toward the cellulose-rich bast fibers that are in the outer stem tissues. Nevertheless, the inner stem tissues (shives) are not without interest as they represent a potentially interesting by-product of bast fiber extraction that can be used for composites, animal bedding and bioenergy. In both cases industrial quality is closely related to the structure of the corresponding plant cell walls that are produced and modified during the life of the plant via the action of a complex battery of enzymes involved in the biosynthesis and the remodeling of different cell wall polymers (e.g. cellulose, hemicelluloses, pectin, lignin). An improved knowledge of these enzymes, as well as of other structural (nonenzymatic) cell wall proteins, will contribute to a better understanding of how cell wall assembly (and hence quality) is regulated in fibers and shives. In a previous proteomics study of the whole flax stem (i.e. inner-and outer-stem tissues NOT separated) we identified 152 cell wall proteins based on the use of TargetP, Predator, and WoLF PSORT algorithms (13Day A. Fénart S. Neutelings G. Hawkins S. Rolando C. Tokarski C. Identification of cell wall proteins in the flax (Linum usitatissimum) stem.Proteomics. 2013; 13: 812-825Crossref PubMed Scopus (24) Google Scholar). Although this work provided an interesting insight into flax stem cell wall metabolism it did not consider the very different structures of cell walls from stem inner- and outer-tissues (19Morvan C. Andème-Onzighi C. Girault R. Himmelsbach D.S. Driouich A. Akin D.E. Building flax fibres: More than one brick in the walls.Plant Physiol. Biochem. 2003; 41: 935-944Crossref Scopus (193) Google Scholar, 20Day A. Ruel K. Neutelings G. Crônier D. David H. Hawkins S. Chabbert B. Lignification in the flax stem: Evidence for an unusual lignin in bast fibers.Planta. 2005; 222: 234-245Crossref PubMed Scopus (123) Google Scholar). The fiber cell wall has been extensively investigated by different authors and is characterized by an extremely thick secondary layer that is sometimes referred to as a gelatinous layer (G-layer) 1The abbreviations used are: AA, Auxilliary activities; AGPs, Arabinogalactan proteins; AIR, Alcohol insoluble residue; ALA, Alpha linolenic acid; CAZy, Carbohydrate Active enZyme; CBM, Carbohydrate binding modules; CE, Carbohydrate esterases; CSC, Cellulose Synthase complex; CWP, Cell wall proteins; FT-IR, Fourier Transform Infrared; GH, Glycosyl hydrolases; G-layer/fibers, Gelatinous layer/fibers; GT, Glycosyl transferases; ID, Proteins with interacting domains; LM, Lipid metabolism; M, Miscellaneous; NCP, Noncellulosic polysaccharides; OR, Oxidoreductases; P, Proteases; PAC, Proteins acting on carbohydrates; PIC, Protease inhibitor cocktail; PL, Polysaccharide lyases; PVPP, Polyvinylpolypyrrolidone; S, Signaling; SDG, secoisolariciresinol diglucoside; SP, Structural proteins; UF, Proteins with unknown functions; UGT, UDP-glycosyltransferase; XEH, Xyloglucan endo-hydrolase; XET, Xyloglucan endotransglycosylase; XTH, Xyloglucan endo-transglycosylase/hydrolase. 1The abbreviations used are: AA, Auxilliary activities; AGPs, Arabinogalactan proteins; AIR, Alcohol insoluble residue; ALA, Alpha linolenic acid; CAZy, Carbohydrate Active enZyme; CBM, Carbohydrate binding modules; CE, Carbohydrate esterases; CSC, Cellulose Synthase complex; CWP, Cell wall proteins; FT-IR, Fourier Transform Infrared; GH, Glycosyl hydrolases; G-layer/fibers, Gelatinous layer/fibers; GT, Glycosyl transferases; ID, Proteins with interacting domains; LM, Lipid metabolism; M, Miscellaneous; NCP, Noncellulosic polysaccharides; OR, Oxidoreductases; P, Proteases; PAC, Proteins acting on carbohydrates; PIC, Protease inhibitor cocktail; PL, Polysaccharide lyases; PVPP, Polyvinylpolypyrrolidone; S, Signaling; SDG, secoisolariciresinol diglucoside; SP, Structural proteins; UF, Proteins with unknown functions; UGT, UDP-glycosyltransferase; XEH, Xyloglucan endo-hydrolase; XET, Xyloglucan endotransglycosylase; XTH, Xyloglucan endo-transglycosylase/hydrolase. because of the similarity with the corresponding layer in tension wood G-fibers (21van Hazendonk J.M. Reinerik E.J.M. de Waard P. van Dam J.E.G. Structural analysis of acetylated hemicellulose polysaccharides from fibre flax (Linum usitatissimum L.).Carbohydr. Res. 1996; 291: 141-154Crossref Scopus (106) Google Scholar, 22Baley C. Analysis of the flax fibres tensile behaviour and analysis of the tensile stiffness increase.Compos. - Part A Appl. Sci. Manuf. 2002; 33: 939-948Crossref Scopus (814) Google Scholar, 23Mellerowicz E.J. Gorshkova T.A. Tensional stress generation in gelatinous fibres: A review and possible mechanism based on cell-wall structure and composition.J. Exp. Bot. 2012; 63: 551-565Crossref PubMed Scopus (158) Google Scholar, 19Morvan C. Andème-Onzighi C. Girault R. Himmelsbach D.S. Driouich A. Akin D.E. Building flax fibres: More than one brick in the walls.Plant Physiol. Biochem. 2003; 41: 935-944Crossref Scopus (193) Google Scholar). Analyses of the flax fiber cell wall shows that it is made up of ∼70% cellulose, 5–15% noncellulosic polysaccharides (NCPs) consisting of beta-1,4-galactans and arabinogalactans, as well as extremely low amounts of lignin (20Day A. Ruel K. Neutelings G. Crônier D. David H. Hawkins S. Chabbert B. Lignification in the flax stem: Evidence for an unusual lignin in bast fibers.Planta. 2005; 222: 234-245Crossref PubMed Scopus (123) Google Scholar, 24Davis E.A. Derouet C. Herve Du Penhoat C. Morvan C. Isolation and an N.M.R. study of pectins from flax (linum usitatissimum L.).Carbohydr. Res. 1990; 197: 205-215Crossref Scopus (74) Google Scholar, 25Girault R. Bert F. Rihouey C. Jauneau A. Morvan C. Jarvis M. Galactans and cellulose in flax fibres: Putative contributions to the tensile strength.Int. J. Biol. Macromol. 1997; 21: 179-188Crossref PubMed Scopus (62) Google Scholar, 26Gorshkova T. Morvan C. Secondary cell-wall assembly in flax phloem fibres: Role of galactans.Planta. 2006; 223: 149-158Crossref PubMed Scopus (108) Google Scholar). In contrast, the walls of xylem (shive) cells contain lower amounts of cellulose and much higher amounts (approx. 30%) of lignin (20Day A. Ruel K. Neutelings G. Crônier D. David H. Hawkins S. Chabbert B. Lignification in the flax stem: Evidence for an unusual lignin in bast fibers.Planta. 2005; 222: 234-245Crossref PubMed Scopus (123) Google Scholar). Because cell wall structure is the result of the combined action of cell wall biosynthesis and remodeling enzymes it is of interest to compare the cell wall proteomes of these two contrasted tissues. Further information contributing to a better understanding of the link between cell wall structure and the cell wall proteome can also be obtained by analyzing other organs that contain different/similar cell types (e.g. leaves that contain a majority of cells with primary cell walls and roots that have a similar structure to whole stems). Even though cell wall polymers are localized in the cell wall, many of the proteins associated with their biosynthesis are not localized in this compartment. For example, the phenylpropanoid enzymes involved in lignin monomer (monolignol) biosynthesis are localized in the cytosol and the cellulose synthase enzymes are associated with the cellulose synthase complex (CSC) in the plasma membrane (27Boerjan W. Ralph J. Baucher M. Lignin biosynthesis.Annu. Rev. Plant Biol. 2003; 54: 519-546Crossref PubMed Scopus (3361) Google Scholar, 28McFarlane H.E. Döring A. Persson S. The cell biology of cellulose synthesis.Annu. Rev. Plant Biol. 2014; 65: 69-94Crossref PubMed Scopus (348) Google Scholar). Similarly, glycosyltransferases (GTs) associated with hemicellulose and pectin biosynthesis are located in Golgi membranes (29Scheller H.V. Ulvskov P. Hemicelluloses.Annu. Rev. Plant Biol. 2010; 61: 263-289Crossref PubMed Scopus (1830) Google Scholar, 30Atmodjo M.a. Hao Z. Mohnen D. Evolving views of pectin biosynthesis.Annu. Rev. Plant Biol. 2013; 64: 747-779Crossref PubMed Scopus (368) Google Scholar). In contrast, glycosylhydrolases (GHs) involved in cell wall remodeling are indeed located in the cell wall (31Franková L. Fry S.C. Biochemistry and physiological roles of enzymes that "cut and paste" plant cell-wall polysaccharides.J. Exp. Bot. 2013; 64: 3519-3550Crossref PubMed Scopus (142) Google Scholar). A global view of the 'cell wall proteome' should therefore consider not only those proteins that are physically located in the cell wall, but also relevant proteins that are present in other compartments. We therefore used protein extractions that were designed at producing fractions enriched in membrane proteins, as well as soluble and cell wall proteins (21van Hazendonk J.M. Reinerik E.J.M. de Waard P. van Dam J.E.G. Structural analysis of acetylated hemicellulose polysaccharides from fibre flax (Linum usitatissimum L.).Carbohydr. Res. 1996; 291: 141-154Crossref Scopus (106) Google Scholar, 33Fenart S. Ndong Y.-P.A. Duarte J. Rivière N. Wilmer J. van Wuytswinkel O. Lucau A. Cariou E. Neutelings G. Gutierrez L. Chabbert B. Guillot X. Tavernier R. Hawkins S. Thomasset B. Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.BMC Genomics. 2010; 11: 592Crossref PubMed Scopus (46) Google Scholar, 34Goulas E. Dily F. Le Teissedre L. Corbel G. Robin C. Ourry A. Vegetative storage proteins in white clover (Trifolium repens L.): Quantitative and qualitative features.Ann. Bot. 2001; 88: 789-795Google Scholar). Altogether our results represent an important contribution to our understanding of cell wall biology in flax. This knowledge will provide a clearer vision of bast fiber construction and the link between cell wall structure and quality, not only in flax, but also in other commercially important species such as hemp and jute. In addition, a better understanding of the molecular mechanisms that enable plant cells to build cellulose-rich walls will provide fundamental knowledge necessary to engineer plant species for the production of more efficient lignocellulosic biomass for biofuels. Seeds of the flax (Linum usitatissimum L.) cultivar Diane were germinated on moistened paper for 24 h at 25 °C in the dark, and then transferred to hydroponic culture and grown for a total of 70 d (corresponding to the vegetative stage of growth when cell wall formation is active) on increasing concentrations of M&S nutrient solution in the absence of sucrose and agar according to the following regime (32Murashige T. Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures.Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53695) Google Scholar, 33Fenart S. Ndong Y.-P.A. Duarte J. Rivière N. Wilmer J. van Wuytswinkel O. Lucau A. Cariou E. Neutelings G. Gutierrez L. Chabbert B. Guillot X. Tavernier R. Hawkins S. Thomasset B. Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.BMC Genomics. 2010; 11: 592Crossref PubMed Scopus (46) Google Scholar). Days 0–30 (1/20 M&S renewed every 2 d, photoperiod 12/11 h day/night, temperature 14/12 °C day/night); days 30–60 (1/15 M&S renewed every 1 d, photoperiod 13/11 h day/night, temperature 16/14 °C day/night); days 60–70 (1/10 M&S renewed every 1 d, photoperiod 14/10 h day/night, temperature 18/16 °C day/night). Light intensity was 210.18 μmol.s/m2, and hygrometry was 60%. Leaves, roots and stems were harvested separately after 70 d and rapidly frozen in liquid nitrogen, and stored at −80 °C. For the stems, outer tissues and inner tissues were separated as described previously (20Day A. Ruel K. Neutelings G. Crônier D. David H. Hawkins S. Chabbert B. Lignification in the flax stem: Evidence for an unusual lignin in bast fibers.Planta. 2005; 222: 234-245Crossref PubMed Scopus (123) Google Scholar). Leaves, roots, outer stems, and inner stems from 40 plants were ground independently in liquid nitrogen to a fine homogenized powder using a grinder and then pooled to constitute 2 biological replicates, each consisting of material from 20 plants. Two g powder were used for the extraction of soluble and cell wall proteins and 7 g were used to obtain a fraction enriched in membrane proteins. Altogether 24 samples were obtained: 4 organs/tissues × 3 sub-fractions × 2 biological replicates. Two technical replicates were performed for each biological replicate. Only proteins identified in both biological replicates were retained for further analyses. Soluble proteins were extracted according as previously described (34Goulas E. Dily F. Le Teissedre L. Corbel G. Robin C. Ourry A. Vegetative storage proteins in white clover (Trifolium repens L.): Quantitative and qualitative features.Ann. Bot. 2001; 88: 789-795Google Scholar). Briefly, 2 g ground sample were further ground for 5 min in 10 ml Tris-HCl buffer (50 mm, 0.06% PIC (w/v, Protease inhibitor mixture), pH 7.5; Sigma-Aldrich) and then centrifuged (10 min at 4 °C, 16,000 × g). The pellet was recovered and stored at −80 °C for cell wall protein (CWP) extraction (see below). The supernatant was recovered and incubated (15 min, room temperature, gentle agitation) with protamine sulfate (1 mg/1 ml) before being centrifuged (10 min, 18,000 × g). The resulting supernatant was recovered and soluble proteins (S) were precipitated by incubation with 72% TCA (w/v, trichloroacetic acid, 1 h, −20 °C). The pellet was washed once with cold acetone and dried at room temperature (5 min) before being stored at −20 °C. CWPs were extracted from the CWP pellet as previously described (35Feiz L. Irshad M. Pont-Lezica R.F. Canut H. Jamet E. Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls.Plant Methods. 2006; 2: 10Crossref PubMed Scopus (134) Google Scholar). Briefly, the pellet was washed with 500 ml sodium acetate buffer (5 mm, pH 4.6) and filtered through a nylon membrane (40 × 40 microns, Millipore Corporation, Bedford, MA). The pellet was washed again with sodium acetate buffer (5 mm, pH 4.6) and then incubated with 20 ml 1.5 m NaCl (30 min at 4 °C with gentle agitation). The solid cell wall residue was then further extracted by (1) incubation with 20 ml CaCl2 buffer (5 mm sodium acetate, 200 mm CaCl2, 30min at 4 °C) and (2) incubation with 20 ml LiCl buffer (5 mm sodium acetate, 2 m LiCl, 30min at 4 °C). The liquid fractions from all CWP extractions were combined and CWPs precipitated by adding 10% TCA (w/v). Membrane (M) proteins were extracted from 7 g ground sample according to Song et al. (58Song D. Xi W. Shen J. Bi T. Li L. Characterization of the plasma membrane proteins and receptor-like kinases associated with secondary vascular differentiation in poplar.Plant Mol. Biol. 2011; 76: 97-115Crossref PubMed Scopus (29) Google Scholar, 59Day A. Fénart S. Neutelings G. Hawkins S. Rolando C. Tokarski C. Identification of cell wall proteins in the flax (Linum usitatissimum) stem.Proteomics. 2013; 13: 812-825Crossref PubMed Scopus (28) Google Scholar). Briefly, a small amount of 1% PVPP (polyvinylpolypyrrolidone) was added to ground material before being incubated in 50 ml extraction buffer (0.5 m Tris-HCl, pH 8.5, 0.7 m sucrose, 0.1 m KCl, 50 mm EDTA, 1 mm PMSF (phenylmethylsulfonyl fluoride), 2% (v/v) β-mercaptoethanol, 0.1% w/v PIC), 5 min at 4 °C). Next, the homogenate was centrifuged (10 min, 12,000 × g, 4 °C) and filtered. The liquid fraction was diluted by an equal volume of ice-cold water and centrifuged (150,000 × g, 40 min). The resulting pellet was then washed three times with ice-cold water and dissolved in SDS buffer (0.5 m Tris-HCl pH 8.5, 2% (v/v) β-mercaptoethanol, 30% (v/v) glycerol, 4% SDS, 1 mm PMSF, 0.1% PIC) and heated for 5 min at 80 °C, before being centrifuged (12,000 × g 30 min at room temperature). The supernatant was extracted three times with an equal volume of water-saturated phenol. Proteins were precipitated at −20 °C overnight from the phenol phase by adding 5 volumes of cold methanol containing 0.1 m ammonium acetate. After precipitation, the proteins were pelleted by centrifugation (12,000 × g, 10 min at 4 °C) and washed three times with 90% cold methanol, and once with 90% cold acetone before being vacuum dried (5 min). Samples were subsequently prepared for analyses by suspension in rehydration buffer (7 m Urea, 2 m Thiourea, 100 mm DTT, 2% w/v CHAPS). Protein content was determined using a reducing agent compatible (BioRad) and a detergent compatible (Bradford) assay. Twenty-five μg total proteins per sample were partially separated by electrophoresis on a precast ready gel according to the manufacturer's instructions (CriterionTM XT precast 1D gel, 4–12% Bis-Tris, 1 mm x 12 wells, Bio-Rad). The gel was stained with Instant Blue (Gentaur BVBA, Kampenhout, Belgium) and 5 bands were excised from each sample lane, cut into 1–2 mm cubes and transferred into a microplate. Proteins were reduced with 10 mm DTT (in 100 mm ammonium bicarbonate) for 30 min at 56 °C, then alkylated with 55 mm iodoacetamide (in 100 mm ammonium bicarbonate) for 20 min at room temperature. Finally, gel pieces were de-stained and then digested overnight by modified trypsin enzyme (sequencing mass grade, Promega). Peptides were recovered and separated on a NanoLC™-2D System (Eksigent, Sciex, Belgium) coupled to a TripleTOF® 5600+ mass spectrometer (Sciex, Belgium). Peptide desalting and enrichment were achieved using a pre-column (C18 PepMapTM, 5 μm, 5 mm × 300 μm i.d., Thermo scientific, Bremen, Germany). Peptides were separated and eluted on a C18 reverse phase column (PepMapTM 100, 3 μm, 100Å, 75 μm i.d. × 15 cm, Thermo scientific) using a linear binary gradient (solvent A: 0.1% FA (formic acid); solvent B: 80% ACN 0.1% FA; 5 min 5% B, 40 min 5% to 55% B, flow rate of 300 nl/min). The peptides were injected into the TripleTOF® 5600+ with a NanoSpray III source using a 10 μm i.d. emitter (New Objective, Woburn, MA). The source parameters used vary depending on optimized conditions on each day, the values were for gas1 = [1–6], gas2 = 0, curtain gas = [20–30], the ion spray voltage ∼2.2keV. For each sample 2 biological and 2 technical replicates were randomly analyzed. MS analysis was performed in information-dependent acquisition mode. MS spectra were acquired using 250 ms accumulation time per spectrum with a mass range of 300–1250 Da. The top 20 precursor ions were selected in each MS scan for subsequent MS/MS scans with high sensitivity during 250 ms of accumulation time (range from m/z 100–1250 Da) and the voltage was automatically adjusted with the system of rolling collision energy. The dynamic exclusion was set at 10 s. Systems were controlled by Analyst software (version TF1.7, Sciex). An automatic mass recalibration was performed using a beta-galactosidase digest during the sequence of samples. CID spectra were processed by Mascot (version 2.4.2) using Protein Pilot (version 4.5, Sciex) by searching against the Linum usitatissimum database (v1.0, http://www.phytozome.net released on 10th December 2014, 43 484 entries). The searches were performed with the following parameters: enzyme: trypsin, 2 missed cleavages, mass accuracy precursor: 20 ppm, mass accuracy fragments: 0.3 Da, fixed modifications: Carbamidomethyl (C), dynamic modifications: Dioxidation (W), Oxidation (HW), Trp-> Kynurenin (W), Oxidation (M). Three supplemental filters were applied to Mascot results: (1) a peptide confidence (p > 0.05), (2) an individual ion score (calculated by Mascot) of the considered research, and (3) a minimum of two significant peptides per protein. Only proteins fulfilling these criteria were retained. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (36Vizcaino J.A. Csordas A. Del-Toro N. Dianes J.A. Griss J. Lavidas I. Ma
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