Glycoproteome of Elongating Cotton Fiber Cells

Abstract

Cotton ovule epidermal cell differentiation into long fibers primarily depends on wall-oriented processes such as loosening, elongation, remodeling, and maturation. Such processes are governed by cell wall bound structural proteins and interacting carbohydrate active enzymes. Glycosylation plays a major role in the structural, functional, and localization aspects of the cell wall and extracellular destined proteins. Elucidating the glycoproteome of fiber cells would reflect its wall composition as well as compartmental requirement, which must be system specific. Following complementary proteomic approaches, we have identified 334 unique proteins comprising structural and regulatory families. Glycopeptide-based enrichment followed by deglycosylation with PNGase F and A revealed 92 unique peptides containing 106 formerly N-linked glycosylated sites from 67 unique proteins. Our results showed that structural proteins like arabinogalactans and carbohydrate active enzymes were relatively more abundant and showed stage- and isoform-specific expression patterns in the differentiating fiber cell. Furthermore, our data also revealed the presence of heterogeneous and novel forms of structural and regulatory glycoproteins. Comparative analysis with other plant glycoproteomes highlighted the unique composition of the fiber glycoproteome. The present study provides the first insight into the identity, abundance, diversity, and composition of the glycoproteome within single celled cotton fibers. The elucidated composition also indirectly provides clues about unicellular compartmental requirements underlying single cell differentiation. Cotton ovule epidermal cell differentiation into long fibers primarily depends on wall-oriented processes such as loosening, elongation, remodeling, and maturation. Such processes are governed by cell wall bound structural proteins and interacting carbohydrate active enzymes. Glycosylation plays a major role in the structural, functional, and localization aspects of the cell wall and extracellular destined proteins. Elucidating the glycoproteome of fiber cells would reflect its wall composition as well as compartmental requirement, which must be system specific. Following complementary proteomic approaches, we have identified 334 unique proteins comprising structural and regulatory families. Glycopeptide-based enrichment followed by deglycosylation with PNGase F and A revealed 92 unique peptides containing 106 formerly N-linked glycosylated sites from 67 unique proteins. Our results showed that structural proteins like arabinogalactans and carbohydrate active enzymes were relatively more abundant and showed stage- and isoform-specific expression patterns in the differentiating fiber cell. Furthermore, our data also revealed the presence of heterogeneous and novel forms of structural and regulatory glycoproteins. Comparative analysis with other plant glycoproteomes highlighted the unique composition of the fiber glycoproteome. The present study provides the first insight into the identity, abundance, diversity, and composition of the glycoproteome within single celled cotton fibers. The elucidated composition also indirectly provides clues about unicellular compartmental requirements underlying single cell differentiation. Cotton fibers are single-cell epidermal seed trichomes that undergo major developmental changes involving overlapping stages of growth including initiation, elongation, and maturation (1Kim H.J. Triplett B.A. Cotton fiber growth in planta and in vitro. models for plant cell elongation and cell wall biogenesis.Plant Physiol. 2001; 127: 1361-1366Crossref PubMed Scopus (494) Google Scholar, 2Qin Y.M. Zhu Y.X. How cotton fibers elongate: a tale of linear cell-growth mode.Curr. Opin. Plant Biol. 2011; 14: 106-111Crossref PubMed Scopus (197) Google Scholar). Matured fibers contain ∼95% cellulose and 1.8% protein (3Wakelyn P.J. French A.D. Cotton fiber chemistry and technology. 2007Google Scholar), whereas elongating fibers contain 22% protein in its primary cell wall (4Singh B. Avci U. Eichler Inwood S.E. Grimson M.J. Landgraf J. Mohnen D. Sørensen I. Wilkerson C.G. Willats W.G. Haigler C.H. A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.Plant Physiol. 2009; 150: 684-699Crossref PubMed Scopus (71) Google Scholar). The relatively increased protein content and its consistency throughout the elongation phase correlates with its stage-specific compartmental requirement (5Yang Y.W. Bian S.M. Yao Y. Liu J.Y. Comparative proteomic analysis provides new insights into the fiber elongating process in cotton.J. Proteome Res. 2008; 7: 4623-4637Crossref PubMed Scopus (77) Google Scholar). Elongation is the most active and vigorous phase, during which the cell extends between 2 to 6 cm in length at a rate of >2 mm/day (1Kim H.J. Triplett B.A. Cotton fiber growth in planta and in vitro. models for plant cell elongation and cell wall biogenesis.Plant Physiol. 2001; 127: 1361-1366Crossref PubMed Scopus (494) Google Scholar, 2Qin Y.M. Zhu Y.X. How cotton fibers elongate: a tale of linear cell-growth mode.Curr. Opin. Plant Biol. 2011; 14: 106-111Crossref PubMed Scopus (197) Google Scholar). Increase in length involve the expansive deformation of the cell wall, including loosening, expansion, and remodeling. These processes collectively determine the cell wall's yielding properties and are governed by the cellulose microfibril-matrix network and associated factors, such as wall bound structural proteins and interacting enzymes (6Masuda Y. Auxin-induced cell elongation and cell wall changes.Bot. 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Transcript profiling during fiber development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fiber quality.Plant Cell Physiol. 2009; 50: 1364-1381Crossref PubMed Scopus (100) Google Scholar). Furthermore, experimental data from other plant systems highlight the roles of carbohydrate active enzymes (CAZymes) 1The abbreviations used are:CAZymesCarbohydrate active enzymesCon AConcanavalin ALACLectin affinity chromatographyAGPsArabinogalactan proteinsFLAFasciclin-like arabinogalactan proteinsGPAsGlycosylphosphatidylinositol anchored proteinsXETs/XTHsXyloglucan endotransglycosylases/hydrolasesPNGasePeptide N-glycoamidaseMWCOMolecular weight cut offFASPFilter aided sample preparationSpCSpectral counts. 1The abbreviations used are:CAZymesCarbohydrate active enzymesCon AConcanavalin ALACLectin affinity chromatographyAGPsArabinogalactan proteinsFLAFasciclin-like arabinogalactan proteinsGPAsGlycosylphosphatidylinositol anchored proteinsXETs/XTHsXyloglucan endotransglycosylases/hydrolasesPNGasePeptide N-glycoamidaseMWCOMolecular weight cut offFASPFilter aided sample preparationSpCSpectral counts., such as xyloglucan endotransglycosylases/hydrolases (XETs/XTHs) (10Darley C.P. 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In addition to CAZymes, genes encoding structural proteins, such as arabinogalactans (AGPs) and fasciclin-like arabinogalactans (FLAs), have been shown to play crucial roles in fiber development (17Li Y. Liu D. Tu L. Zhang X. Wang L. Zhu L. Tan J. Deng F. Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber.Plant Cell Rep. 2010; 2: 193-202Crossref Scopus (52) Google Scholar). AGPs are also known to act as signaling molecules, modulators of cell wall mechanics, pectin plasticizers (18Lamport D.T. Kieliszewski M.J. Showalter A.M. Stress upregulates periplasmic arabinogalactan-proteins.Plant Biosys. 2005; 139: 60-64Crossref Scopus (12) Google Scholar), and stimulators of XET activity (19Takeda T. Fry S.C. Control of xyloglucan endotransglucosylase activity by salts and anionic polymers.Planta. 2004; 219: 722-732Crossref PubMed Scopus (34) Google Scholar) and are also involved in K. The biology of arabinogalactan Rev. Plant Biol. 2007; PubMed Scopus Google Scholar). their roles M. J. at the cell Physiol. 2010; PubMed Scopus Google Scholar), experimental the and abundance that the roles of the is active enzymes A affinity proteins arabinogalactan proteins anchored proteins endotransglycosylases/hydrolases weight cut aided sample counts. active enzymes A affinity proteins arabinogalactan proteins anchored proteins endotransglycosylases/hydrolases weight cut aided sample counts. to understand fiber development of events based on the expression of abundant and proteins from fiber (5Yang Y.W. Bian S.M. Yao Y. Liu J.Y. Comparative proteomic analysis provides new insights into the fiber elongating process in cotton.J. Proteome Res. 2008; 7: 4623-4637Crossref PubMed Scopus (77) Google Scholar, C.Y. Wang H. Pang Y. Xu C. Jiao Y. Qin Y.M. Western T.L. Yu S.X. Zhu Y.X. Comparative proteomics indicates that biosynthesis of pectic precursors is important for cotton fiber and arabidopsis root hair elongation.Mol. Cell. Proteomics. 2010; 9: 2019-2033Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, K. M. Zhang C. Yao L. J. Zhang T. Comparative proteomic analysis the cotton fiber differentiation and Proteomics. PubMed Scopus Google Scholar). insight into the cell wall bound enzymes and structural proteins have been proteomic The of such and structural proteins are known to be destined for the cell wall and N-linked glycosylated in S.J. F. The plant and associated Plant Sci. PubMed Scopus Google Scholar). The of such proteins to or which in is for to the cell wall and the of the cell wall J.P. The role of carbohydrate in in Physiol. PubMed Google Scholar). the glycoproteome indirectly the composition of plant cell and may also reflect the system functional properties of the wall S.J. F. The plant and associated Plant Sci. PubMed Scopus Google Scholar, Zhang S. for and of the plant Plant Sci. PubMed Scopus Google Scholar). the glycoproteome of cotton fibers may also clues about the single cell compartmental and the probable role of proteins during development. our such have not been in cotton fibers. 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C. to the of the plant cell wall 2011; PubMed Scopus Google Scholar). major the protein were highlight a the of AGPs and in the fiber glycoproteome with AGPs and were relatively in the other data containing enzymes that are carbohydrate were from the other data that is a unique of the cotton fiber glycoproteome. of the and proteins with and were relatively in the fiber glycoproteome. the protein whereas relatively in fiber and The development of cotton seed epidermal cells into long fibers been and factors, such as structural proteins, CAZymes, and factors, have been for the roles that play in the differentiation systems level and of protein is and of analysis understand their roles in development. yielding properties the differentiation events to the length and proteins are known to be glycosylated in S.J. F. The plant and associated Plant Sci. PubMed Scopus Google Scholar), and in contribute to such yielding glycoproteome be to study the proteins in S.J. F. The plant and associated Plant Sci. PubMed Scopus Google Scholar). and analysis of the cotton fiber glycoproteome revealed that the of the proteins were destined for the cell wall or were extracellular in our results suggested that the structural proteins were relatively with the and other enzymes In by complementary proteomic approaches, we have been to the of the fiber glycoproteome. a based revealed the presence of unique and peptides to protein from molecular weight whereas analysis showed protein with In deglycosylation PNGase F and A showed overlapping and unique in the N-linked to protein of the presence of protein and in cotton The presence of the forms of the protein the requirement for proteins to functions at developmental and and The abundance and of the identified clues about the of the cotton fiber Y. T. J. M. M.J. D. abundance profiling of the 2008; 9: PubMed Scopus Google Scholar). The were relatively more abundant and with other analysis revealed that most of the and carbohydrate or interacting of Comparative analysis with plant glycoproteomes C. S. of the glycoproteome of A affinity and 2011; PubMed Scopus Google Scholar, L. M. L. A of on A is in cell wall 2007; PubMed Scopus (105) Google Scholar, L. C. H. B. M. M. of the of a content in 2011; PubMed Scopus Google Scholar, Y. M. B. C. to the of the plant cell wall 2011; PubMed Scopus Google Scholar) revealed that enzymes and containing carbohydrate were in cotton which the unique of the fiber glycosylated enzymes. In a cell such as a cotton which is in structural and the presence of and carbohydrate interacting in enzymes would be as the activity by or enzymes in to its F. C. B. P.M. H.J. How and carbohydrate to Sci. 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J. as structure and of and 2011; 12: PubMed Scopus Google Scholar). data the abundance, and associated functional roles of such in plant systems are In to the processes occurring during elongation, the fiber cell wall to contain structural Plant AGPs and are to play major and roles in cell development plant 47: Scopus Google proteins were in the glycoproteome In the we and abundant of the and play major roles as structural in cotton fiber in their molecular and were and may to in and be the major of the functional for such The of proteins play a major role during by as molecular Cell wall also enzymes such as as in in K. The biology of arabinogalactan Rev. Plant Biol. 2007; PubMed Scopus Google Scholar). Our earlier showed that encoding arabinogalactans and were in a G. M. S. K. B. S. analysis of cotton of genes and pathways involved in elongation and of Biol. PubMed Scopus Google Scholar). protein encoding were during of K. G. M. S. H. S. of of L. genes and pathways involved in cotton initiation and PubMed Scopus Google Scholar), play major roles in fiber development and In we have a major to the cotton fiber glycoproteome and have revealed cell wall destined structural and proteins. Our analysis identified the abundance and of fiber such as and other glycoproteins. Such structural proteins and play roles throughout fiber development and The and heterogeneous of the identified the of G. by the A and based functional showed that the fiber glycoproteome followed a to other plant were unique to cotton The of protein and their abundance shown in study reflect the major of the structural and functional the wall yielding properties of the cotton to and for their during with