Journal of Plant Research

, Volume 128, Issue 6, pp 975–986 | Cite as

Monoclonal antibody-based analysis of cell wall remodeling during xylogenesis

  • Naoki ShinoharaEmail author
  • Koichi Kakegawa
  • Hiroo Fukuda
Regular Paper


Xylogenesis, a process by which woody tissues are formed, entails qualitative and quantitative changes in the cell wall. However, the molecular events that underlie these changes are not completely understood. Previously, we have isolated two monoclonal antibodies, referred to as XD3 and XD27, by subtractive screening of a phage-display library of antibodies raised against a wall fraction of Zinnia elegans xylogenic culture cells. Here we report the biochemical and immunohistochemical characterization of those antibodies. The antibody XD3 recognized (1→4)-β-d-galactan in pectin fraction. During xylogenesis, the XD3 epitope was localized to the primary wall of tracheary-element precursor cells, which undergo substantial cell elongation, and was absent from mature tracheary elements. XD27 recognized an arabinogalactan protein that was bound strongly to a germin-like protein. The XD27 epitope was localized to pre-lignified secondary walls of tracheary elements. Thus these cell-wall-directed monoclonal antibodies revealed two molecular events during xylogenesis. The biological significance of these events is discussed in relation to current views of the plant cell wall.


Arabinogalactan protein Cell wall Germin-like protein Pectic galactan Monoclonal antibody Xylogenesis 



We thank Tadashi Ishii for help with sugar-linkage analysis; Jin Nakashima for help with cell sectioning; Kuninori Iwamoto for searching ZeGLP1 cDNA sequence; and Maaike de Jong for critical reading of the manuscript. This work was supported partly by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (NC-CARP project) and from the Japan Society for the Promotion of Science (23227001) to HF.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10265_2015_758_MOESM1_ESM.docx (89 kb)
Supplementary material 1 (DOCX 89 kb)


  1. Badjić JD, Nelson A, Cantrill SJ, Turnbull WB, Stoddart JF (2005) Multivalency and cooperativity in supramolecular chemistry. Acc Chem Res 38:723–732. doi: 10.1021/ar040223k CrossRefPubMedGoogle Scholar
  2. Barr DP, Aust SD (1994) Effect of superoxide and superoxide dismutase on lignin peroxidase-catalyzed veratryl alcohol oxidation. Arch Biochem Biophys 311:378–382. doi: 10.1006/abbi.1994.1251 CrossRefPubMedGoogle Scholar
  3. Bernier F, Berna A (2001) Germins and germin-like proteins: plant do-all proteins. But what do they do exactly? Plant Physiol Bioch 39:545–554. doi: 10.1016/S0981-9428(01)01285-2 CrossRefGoogle Scholar
  4. Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3:1–30. doi: 10.1111/j.1365-313X.1993.tb00007.x CrossRefPubMedGoogle Scholar
  5. Carter C, Thornburg RW (2000) Tobacco nectarin I—purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defense of floral reproductive tissues. J Biol Chem 275:36726–36733. doi: 10.1074/Jbc.M006461200 CrossRefPubMedGoogle Scholar
  6. Chaffey N, Cholewa E, Regan S, Sundberg B (2002) Secondary xylem development in Arabidopsis: a model for wood formation. Physiol Plant 114:594–600. doi: 10.1034/j.1399-3054.2002.1140413.x CrossRefPubMedGoogle Scholar
  7. Chen Y et al (1999) Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. J Mol Biol 293:865–881. doi: 10.1006/jmbi.1999.3192 CrossRefPubMedGoogle Scholar
  8. Davidson RM, Reeves PA, Manosalva PM, Leach JE (2009) Germins: a diverse protein family important for crop improvement. Plant Sci 177:499–510. doi: 10.1016/J.Plantsci.08.012 CrossRefGoogle Scholar
  9. Dean GH et al (2007) The Arabidopsis MUM2 gene encodes a β-galactosidase required for the production of seed coat mucilage with correct hydration properties. Plant Cell 19:4007–4021. doi: 10.1105/tpc.107.050609 PubMedCentralCrossRefPubMedGoogle Scholar
  10. Dolan L, Roberts K (1995) Secondary thickening in roots of Arabidopsis thaliana: anatomy and cell surface changes. New Phytol 131:121–128. doi: 10.1111/J.1469-8137.1995.Tb03061.X CrossRefGoogle Scholar
  11. Dunwell JM, Gibbings JG, Mahmood T, Naqvi SMS (2008) Germin and germin-like proteins: evolution, structure, and function Crit Rev. Plant Sci 27:342–375. doi: 10.1080/07352680802333938 CrossRefGoogle Scholar
  12. Dyson RJ, Band LR, Jensen OE (2012) A model of crosslink kinetics in the expanding plant cell wall: yield stress and enzyme action. J Theor Biol 307:125–136. doi: 10.1016/j.jtbi.2012.04.035 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi: 10.1093/nar/gkh340 PubMedCentralCrossRefPubMedGoogle Scholar
  14. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 77:305–319. doi: 10.1016/0022-1759(85)90044-4 CrossRefPubMedGoogle Scholar
  15. Fukuda H, Komamine A (1980) Establishment of an experimental system for the study of tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans. Plant Physiol 65:57–60. doi: 10.1104/pp.65.1.57 PubMedCentralCrossRefPubMedGoogle Scholar
  16. Godfrey D, Able AJ, Dry IB (2007) Induction of a grapevine germin-like protein (VvGLP3) gene is closely linked to the site of Erysiphe necator infection: a possible role in defense? Mol Plant Microbe Interact 20:1112–1125. doi: 10.1094/MPMI-20-9-1112 CrossRefPubMedGoogle Scholar
  17. Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224. doi: 10.1093/molbev/msp259 CrossRefPubMedGoogle Scholar
  18. Gucciardo S, Wisniewski JP, Brewin NJ, Bornemann S (2007) A germin-like protein with superoxide dismutase activity in pea nodules with high protein sequence identity to a putative rhicadhesin receptor. J Exp Bot 58:1161–1171. doi: 10.1093/jxb/erl282 CrossRefPubMedGoogle Scholar
  19. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. doi: 10.1093/sysbio/syq010 CrossRefPubMedGoogle Scholar
  20. Hirst EL, Jones JK, Walder WC (1947) Pectic substances; the constitution of the galactan from Lupinus albus. J Chem Soc 25:1225–1229. doi: 10.1039/JR9470001225 CrossRefPubMedGoogle Scholar
  21. Hornick CL, Karuch F (1972) Antibody affinity-III the role of multivalance. Immunochemistry 9:325–340. doi: 10.1016/0019-2791(72)90096-1 CrossRefPubMedGoogle Scholar
  22. Hurkman WJ, Lane BG, Tanaka CK (1994) Nucleotide sequence of a transcript encoding a germin-like protein that is present in salt-stressed barley (Hordeum vulgare L.) roots. Plant Physiol 104:803–804. doi: 10.1104/pp.104.2.803 PubMedCentralCrossRefPubMedGoogle Scholar
  23. Jarvis MC, Hall MA, Threlfall DR, Friend J (1981) The polysaccharide structure of potato cell walls: chemical fractionation. Planta 152:93–100. doi: 10.1007/BF00391179 CrossRefPubMedGoogle Scholar
  24. Jones L, Seymour GB, Knox JP (1997) Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1→4)-β-d-galactan. Plant Physiol 113:1405–1412. doi: 10.1104/pp.113.4.1405 PubMedCentralPubMedGoogle Scholar
  25. Kabat EA (1976) Structural concepts in immunology and immunochemistry, 2nd edn. Holt, Rinehart and Winston, New YorkGoogle Scholar
  26. Kishi-Kaboshi M, Muto H, Takeda A, Murata T, Hasebe M, Watanabe Y (2014) Localization of tobacco germin-like protein 1 in leaf intercellular space. Plant Physiol Biochem 85:1–8. doi: 10.1016/j.plaphy.2014.10.005 CrossRefPubMedGoogle Scholar
  27. Knox JP (2008) Revealing the structural and functional diversity of plant cell walls. Curr Opin Plant Biol 11:308–313. doi: 10.1016/j.pbi.2008.03.001 CrossRefPubMedGoogle Scholar
  28. Knox JP, Linstead PJ, Peart J, Cooper C, Roberts K (1991) Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation. Plant J 1:317–326. doi: 10.1046/j.1365-313X.1991.t01-9-00999.x CrossRefPubMedGoogle Scholar
  29. Lane BG (2000) Oxalate oxidases and differentiating surface structure in wheat: germins. Biochem J 349:309–321PubMedCentralCrossRefPubMedGoogle Scholar
  30. Lane BG et al (1992) Germin isoforms are discrete temporal markers of wheat development. Eur J Biochem 209:961–969. doi: 10.1111/j.1432-1033.1992.tb17369.x CrossRefPubMedGoogle Scholar
  31. Lane BG, Dunwell JM, Ray JA, Schmitt MR, Cuming AC (1993) Germin, a protein marker of early plant development, is an oxalate oxidase. J Biol Chem 268:12239–12242PubMedGoogle Scholar
  32. Larson PR (1994) The vascular cambium: development and structure. Springer series in wood science. Springer-Verlag, BerlinCrossRefGoogle Scholar
  33. Liwanag AJM et al (2012) Pectin biosynthesis: GALS1 in Arabidopsis thaliana is a β-1,4-galactan β-1,4-galactosyltransferase. Plant Cell 24:5024–5036. doi: 10.1105/Tpc.112.106625 PubMedCentralCrossRefPubMedGoogle Scholar
  34. Macquet A, Ralet MC, Loudet O, Kronenberger J, Mouille G, Marion-Poll A, North HM (2007) A naturally occurring mutation in an Arabidopsis accession affects a β-d-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed mucilage. Plant Cell 19:3990–4006. doi: 10.1105/tpc.107.050179 PubMedCentralCrossRefPubMedGoogle Scholar
  35. McCartney L, Ormerod AP, Gidley MJ, Knox JP (2000) Temporal and spatial regulation of pectic (1→4)-β-d-galactan in cell walls of developing pea cotyledons: implications for mechanical properties. Plant J 22:105–113. doi: 10.1046/j.1365-313x.2000.00719.x CrossRefPubMedGoogle Scholar
  36. McCartney L, Steele-King CG, Jordan E, Knox JP (2003) Cell wall pectic (1→4)-β-d-galactan marks the acceleration of cell elongation in the Arabidopsis seedling root meristem. Plant J 33:447–454. doi: 10.1046/J.1365-313x.2003.01640.X CrossRefPubMedGoogle Scholar
  37. Michalak M, Thomassen LV, Roytio H, Ouwehand AC, Meyer AS, Mikkelsen JD (2012) Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans. Enzyme Microb Technol 50:121–129. doi: 10.1016/j.enzmictec.2011.11.001 CrossRefPubMedGoogle Scholar
  38. Motose H, Sugiyama M, Fukuda H (2004) A proteoglycan mediates inductive interaction during plant vascular development. Nature 429:873–878. doi: 10.1038/Nature02619 CrossRefPubMedGoogle Scholar
  39. Nakashima J, Endo S, Fukuda H (2004) Immunocytochemical localization of polygalacturonase during tracheary element differentiation in Zinnia elegans. Planta 218:729–739. doi: 10.1007/s00425-003-1167-4 CrossRefPubMedGoogle Scholar
  40. Nakata M, Watanabe Y, Sakurai Y, Hashimoto Y, Matsuzaki M, Takahashi Y, Satoh T (2004) Germin-like protein gene family of a moss, Physcomitrella patens, phylogenetically falls into two characteristic new clades. Plant Mol Biol 56:381–395. doi: 10.1007/s11103-004-3475-x CrossRefPubMedGoogle Scholar
  41. Ogawa K, Kanematsu S, Asada K (1997) Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification. Plant Cell Physiol 38:1118–1126CrossRefPubMedGoogle Scholar
  42. Ohdaira Y, Kakegawa K, Amino S, Sugiyama M, Fukuda H (2002) Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements. Planta 215:177–184. doi: 10.1007/s00425-001-0731-z CrossRefPubMedGoogle Scholar
  43. Ohmiya A (2002) Characterization of ABP19/20, sequence homologues of germin-like protein in Prunus persica L. Plant Sci 163:683–689. doi: 10.1016/S0168-9452(02)00231-5 CrossRefGoogle Scholar
  44. Ohmiya A, Tanaka Y, Kadowaki K, Hayashi T (1998) Cloning of genes encoding auxin-binding proteins (ABP19/20) from peach: significant peptide sequence similarity with germin-like proteins. Plant Cell Physiol 39:492–499CrossRefPubMedGoogle Scholar
  45. Park YB, Cosgrove DJ (2012) A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases. Plant Physiol 158:1933–1943. doi: 10.1104/pp.111.192880 PubMedCentralCrossRefPubMedGoogle Scholar
  46. Passioura JB, Fry SC (1992) Turgor and cell expansion: beyond the Lockhart equation. Aust J Plant Physiol 19:565–576. doi: 10.1071/PP9920565 CrossRefGoogle Scholar
  47. Pattathil S et al (2010) A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiol 153:514–525. doi: 10.1104/pp.109.151985 PubMedCentralCrossRefPubMedGoogle Scholar
  48. Randall RC, Phillips GO, Williams PA (1989) Fractionation and characterization of gum from Acacia senegal. Food Hydrocoll 3:65–75. doi: 10.1016/S0268-005X(89)80034-7 CrossRefGoogle Scholar
  49. Ryser U, Schorderet M, Guyot R, Keller B (2004) A new structural element containing glycine-rich proteins and rhamnogalacturonan I in the protoxylem of seed plants. J Cell Sci 117:1179–1190. doi: 10.1242/jcs.00966 CrossRefPubMedGoogle Scholar
  50. Schier R et al (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 263:551–567. doi: 10.1006/jmbi.1996.0598 CrossRefPubMedGoogle Scholar
  51. Schindler T, Bergfeld R, Schopfer P (1995) Arabinogalactan proteins in maize coleoptiles: developmental relationship to cell death during xylem differentiation but not to extension growth. Plant J 7:25–36. doi: 10.1046/J.1365-313x.1995.07010025.X CrossRefPubMedGoogle Scholar
  52. Seifert GJ, Roberts K (2007) The biology of arabinogalactan proteins. Annu Rev Plant Biol 58:137–161. doi: 10.1146/annurev.arplant.58.032806.103801 CrossRefPubMedGoogle Scholar
  53. Shinohara N, Fukuda H (2002) Isolation of monoclonal antibodies recognizing rare and dominant epitopes in plant vascular cell walls by phage display subtraction. J Immunol Methods 264:187–194. doi: 10.1016/S0022-1759(02)00088-1 CrossRefPubMedGoogle Scholar
  54. Shinohara N, Demura T, Fukuda H (2000) Isolation of a vascular cell wall-specific monoclonal antibody recognizing a cell polarity by using a phage display subtraction method. Proc Natl Acad Sci USA 97:2585–2590. doi: 10.1073/pnas.050582197 PubMedCentralCrossRefPubMedGoogle Scholar
  55. Sievers F et al (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi: 10.1038/msb.2011.75 PubMedCentralCrossRefPubMedGoogle Scholar
  56. Sojar HT, Bahl OP (1987) Chemical deglycosylation of glycoproteins. Methods Enzymol 138:341–350. doi: 10.1016/0076-6879(87)38029-2 CrossRefPubMedGoogle Scholar
  57. Stacey NJ, Roberts K, Carpita NC, Wells B, McCann MC (1995) Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements. Plant J 8:891–906. doi: 10.1046/j.1365-313X.1995.8060891.x CrossRefGoogle Scholar
  58. Ulvskov P et al (2005) Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes. Planta 220:609–620. doi: 10.1007/s00425-004-1373-8 CrossRefPubMedGoogle Scholar
  59. Willats WGT, Steele-King CG, Marcus SE, Knox JP (1999) Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation. Plant J 20:619–628. doi: 10.1046/J.1365-313x.1999.00629.X CrossRefPubMedGoogle Scholar
  60. Woo EJ, Dunwell JM, Goodenough PW, Marvier AC, Pickersgill RW (2000) Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities. Nat Struct Biol 7:1036–1040. doi: 10.1038/80954 CrossRefPubMedGoogle Scholar
  61. Woodward MP, Young WW Jr, Bloodgood RA (1985) Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods 78:143–153. doi: 10.1016/0022-1759(85)90337-0 CrossRefPubMedGoogle Scholar
  62. Yamahara T et al (1999) Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss, Barbula unguiculata. J Biol Chem 274:33274–33278. doi: 10.1074/jbc.274.47.33274 CrossRefPubMedGoogle Scholar
  63. York WS, Darvill AG, Mcneil M, Stevenson TT, Albersheim P (1986) Isolation and characterization of plant cell walls and cell wall components. Methods Enzymol 118:3–40. doi: 10.1016/0076-6879(86)18062-1 CrossRefGoogle Scholar
  64. Zhong R, Ripperger A, Ye ZH (2000) Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants. Plant Physiol 123:59–70. doi: 10.1104/pp.123.1.59 PubMedCentralCrossRefPubMedGoogle Scholar
  65. Zykwinska AW, Ralet MC, Garnier CD, Thibault JF (2005) Evidence for in vitro binding of pectin side chains to cellulose. Plant Physiol 139:397–407. doi: 10.1104/pp.105.065912 PubMedCentralCrossRefPubMedGoogle Scholar
  66. Zykwinska A, Thibault JF, Ralet MC (2007) Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged. J Exp Bot 58:1795–1802. doi: 10.1093/jxb/erm037 CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2015

Authors and Affiliations

  • Naoki Shinohara
    • 1
    • 3
    Email author
  • Koichi Kakegawa
    • 2
  • Hiroo Fukuda
    • 1
  1. 1.Department of Biological Sciences, Graduate School of ScienceUniversity of TokyoTokyoJapan
  2. 2.Department of Biomass ChemistryForestry and Forest Products Research InstituteTsukubaJapan
  3. 3.Department of Developmental Biology and Neurosciences, Graduate School of Life SciencesTohoku UniversitySendaiJapan

Personalised recommendations