, Volume 254, Issue 1, pp 367–377 | Cite as

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells

  • Kae AkitaEmail author
  • Megumi Kobayashi
  • Mayuko Sato
  • Natsumaro Kutsuna
  • Takashi Ueda
  • Kiminori Toyooka
  • Noriko Nagata
  • Seiichiro Hasezawa
  • Takumi Higaki
Original Article


In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.


Arabidopsis thaliana Exocytosis Microscopic image analysis Paramural body Pavement cell ST-mRFP 



We thank Dr. Mamiko Sato of the Japan Women’s University and Ms. Mayumi Wakazaki of the RIKEN Center for Sustainable Resource Sciences for microscopic observation. We thank Dr. Haruko Ueda and Prof. Ikuko Hara-Nishimura of Kyoto University for kind comments. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI to K.A. (26891006), N.K. (24770038), T.U. (24114003), K.T. (24687007 and 23657051), N.N. (23120526), S.H. (24114007 and 25291056), and T.H. (25711017).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11:36–42Google Scholar
  2. Akita K, Higaki T, Kutsuna N, Hasezawa S (2015) Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells. Plant Signal Behav 10, e1024396CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ambrose C, DeBono A, Wasteneys G (2013) Cell geometry guides the dynamic targeting of apoplastic GPI-linked lipid transfer protein to cell wall elements and cell borders in Arabidopsis thaliana. PLoS One 8, e81215CrossRefPubMedPubMedCentralGoogle Scholar
  4. An Q, Ehlers K, Kogel KH, van Bel AJE, Hückelhoven R (2006a) Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus. New Phytol 172:563–576CrossRefPubMedGoogle Scholar
  5. An Q, Hückelhoven R, Kogel KH, van Bel AJE (2006b) Multivesicular bodies participate in a cell wall associated defense response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbiol 8:1009–1019CrossRefPubMedGoogle Scholar
  6. Armour WJ, Barton DA, Law AM, Overall RL (2015) Differential growth in periclinal and anticlinal walls during lobe formation in Arabidopsis cotyledon pavement cells. Plant Cell 27:2484–2500CrossRefPubMedPubMedCentralGoogle Scholar
  7. Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447CrossRefPubMedGoogle Scholar
  8. Chardin P, McCormick F (1999) Brefeldin A: the advantage of being uncompetitive. Cell 97:153–155CrossRefPubMedGoogle Scholar
  9. Chen J, Wang F, Zheng S, Xu T, Yang Z (2015) Pavement cells: a model for non-transcriptional auxin signaling and crosstalks. J Exp Bot 66:4957–4970CrossRefPubMedPubMedCentralGoogle Scholar
  10. Denzer K, Kleijmeer MJ, Heijnen HFG, Stoorvogel W, Geuze HJ (2000) Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci 113:3365–3374PubMedGoogle Scholar
  11. Elsner J, Michalski M, Kwiatkowska D (2012) Spatiotemporal variation of leaf epidermal cell growth: a quantitative analysis of Arabidopsis thaliana wild-type and triple cyclinD3 mutant plants. Ann Bot 109:897–910CrossRefPubMedPubMedCentralGoogle Scholar
  12. Falbel TG, Koch LM, Nadeau JA, Segui-Simarro JM, Sack FD, Bednarek SY (2003) SCD1 is required for cell cytokinesis and polarized cell expansion in Arabidopsis thaliana. Development 130:4011–4024CrossRefPubMedGoogle Scholar
  13. Frank MJ, Smith LG (2002) A small, novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells. Curr Biol 12:849–853CrossRefPubMedGoogle Scholar
  14. Frank MJ, Cartwright HN, Smith LG (2003) Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis. Development 130:753–762CrossRefPubMedGoogle Scholar
  15. Fu Y, Li H, Yang Z (2002) The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis. Plant Cell 14:777–794CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fu Y, Gu Y, Zheng Z, Wasteneys G, Yang Z (2005) Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis. Cell 120:687–700CrossRefPubMedGoogle Scholar
  17. Fu Y, Xu T, Zhu L, Wen M, Yang Z (2009) A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis. Curr Biol 19:1827–1832CrossRefPubMedPubMedCentralGoogle Scholar
  18. Geitmann A, Ortega JK (2009) Mechanics and modeling of plant cell growth. Trends Plant Sci 14:467–478CrossRefPubMedGoogle Scholar
  19. Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B (2003) Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol 13:1378–1387CrossRefPubMedGoogle Scholar
  20. Hardham AR, Takemoto D, White RG (2008) Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack. BMC Plant Biol 8:63CrossRefPubMedPubMedCentralGoogle Scholar
  21. Harding C, Heuser J, Stahl P (1983) Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 97:329–339CrossRefPubMedGoogle Scholar
  22. Higaki T, Sano T, Hasezawa S (2007) Actin microfilament dynamics and actin side-binding proteins in plants. Curr Opin Plant Biol 10:549–556CrossRefPubMedGoogle Scholar
  23. Higaki T, Kutsuna N, Hosokawa Y, Akita K, Ebine K, Ueda T, Kondo N, Hasezawa S (2012) Statistical organelle dissection of Arabidopsis guard cells using image database LIPS. Sci Rep 2:405CrossRefPubMedPubMedCentralGoogle Scholar
  24. Higaki T, Kutsuna N, Hasezawa S (2013) LIPS database with LIPService: a microscopic image database of intracellular structures in Arabidopsis guard cells. BMC Plant Biol 13:81CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ito E, Fujimoto M, Ebine K, Uemura T, Ueda T, Nakano A (2012a) Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging. Plant J 69:204–216CrossRefPubMedGoogle Scholar
  26. Ito Y, Uemura T, Shoda K, Fujimoto M, Ueda T, Nakano A (2012b) cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells. Mol Biol Cell 23:3203–3214CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jacques E, Verbelen JP, Vissenberg K (2014) Review on shape formation in epidermal pavement cells of the Arabidopsis leaf. Funct Plant Biol 41:914–921CrossRefGoogle Scholar
  28. Jaillais Y, Fobis-Loisy I, Miège C, Gaude T (2008) Evidence for a sorting endosome in Arabidopsis root cells. Plant J 53:237–247CrossRefPubMedGoogle Scholar
  29. Jones MA, Shen JJ, Fu Y, Li H, Yang Z, Grierson CS (2002) The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth. Plant Cell 14:763–776CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jung JY, Kim YW, Kwak JM, Hwang JU, Young J, Schroeder JI, Hwang I, Lee Y (2002) Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements. Plant Cell 14:2399–2412CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lancelle SA, Hepler PH (1992) Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma 167:215–230CrossRefGoogle Scholar
  32. Latijnhouwers M, Hawes C, Carvalho C, Oparka K, Gillingham AK, Boevink P (2005) An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1. Plant J 44:459–470CrossRefPubMedGoogle Scholar
  33. Li H, Lin Y, Heath RM, Zhu MX, Yang Z (1999) Control of pollen tube tip growth by a ROP GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11:1731–1742PubMedPubMedCentralGoogle Scholar
  34. Marchant R, Peat A, Banbury GH (1967) The ultrastructural basis of hyphal growth. New Phytol 66:623–629CrossRefGoogle Scholar
  35. McCloud TG, Burns MP, Majadly FD, Muschik GM, Miller DA, Poole KK, Roach JM, Ross JT, Lebherz WB 3rd (1995) Production of brefeldin-A. J Ind Microbiol 15:5–9CrossRefPubMedGoogle Scholar
  36. McMichael CM, Reynolds GD, Koch LM, Wang C, Jiang N, Nadeau J, Sack FD, Gelderman MB, Pan J, Bednarek SY (2013) Mediation of clathrin-dependent trafficking during cytokinesis and cell expansion by Arabidopsis stomatal cytokinesis defective proteins. Plant Cell 25:3910–3925CrossRefPubMedPubMedCentralGoogle Scholar
  37. Naito S, Hirai MY, Chino M, Komeda Y (1994) Expression of a soybean (Glycine max [L.] Merr.) seed storage protein gene in transgenic Arabidopsis thaliana and its response to nutritional stress and to abscisic acid mutations. Plant Physiol 104:497–503CrossRefPubMedPubMedCentralGoogle Scholar
  38. Negi J, Moriwaki K, Konishi M, Yokoyama R, Nakano T, Kusumi K, Hashimoto-Sugimoto M, Schroeder JI, Nishitani K, Yanagisawa S, Iba K (2013) A Dof transcription factor, SCAP1, is essential for the development of functional stomata in Arabidopsis. Curr Biol 23:479–484CrossRefPubMedPubMedCentralGoogle Scholar
  39. Nishitani K, Tominaga R (1991) In vitro molecular weight increase in xyloglucans by an apoplastic enzyme preparation from epicotyls of Vigna angularis. Physiol Plant 82:490–497CrossRefGoogle Scholar
  40. Panteris E, Galatis B (2005) The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments. New Phytol 167:721–732CrossRefPubMedGoogle Scholar
  41. Panteris E, Apostolakos P, Galatis B (1993) Microtubules and morphogenesis in ordinary epidermal cells of Vigna sinensis leaves. Protoplasma 174:91–100CrossRefGoogle Scholar
  42. Panteris E, Apostolakos P, Galatis B (1994) Sinuous ordinary epidermal cells: behind several patterns of waviness, a common morphogenetic mechanism. New Phytol 127:771–780CrossRefGoogle Scholar
  43. Parton RM, Fischer-Parton S, Watahiki MK, Trewavas AJ (2001) Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes. J Cell Sci 114:2685–2695PubMedGoogle Scholar
  44. Parton RM, Fischer-Parton S, Trewavas AJ, Watahiki MK (2003) Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension. J Cell Sci 116:2707–2719CrossRefPubMedGoogle Scholar
  45. Qiu JL, Jilk R, Marks MD, Szymanski DB (2002) The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development. Plant Cell 14:101–118CrossRefPubMedPubMedCentralGoogle Scholar
  46. Robards AW, Kidwai P (1969) Vesicular involvement in differentiating plant vascular cells. New Phytol 68:343–349CrossRefGoogle Scholar
  47. Robatzek S, Chinchilla D, Boller T (2006) Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev 20:537–542CrossRefPubMedPubMedCentralGoogle Scholar
  48. Sampathkumar A, Krupinski P, Wightman R, Milani P, Berquand A, Boudaoud A, Hamant O, Jönsson H, Meyerowitz EM (2014) Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells. eLife 3, e01967CrossRefPubMedPubMedCentralGoogle Scholar
  49. Samuels AL, Giddings TH Jr, Staehelin LA (1995) Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J Cell Biol 130:1345–1357CrossRefPubMedGoogle Scholar
  50. Séveno M, Bardor M, Paccalet T, Gomord V, Lerouge P, Faye L (2004) Glycoprotein sialylation in plants? Nat Biotechnol 22:1351–1352CrossRefPubMedGoogle Scholar
  51. Shah MM, Fujiyama K, Flynn CR, Joshi L (2003) Sialylated endogenous glycoconjugates in plant cells. Nat Biotechnol 21:1470–1471CrossRefPubMedGoogle Scholar
  52. Smith LG, Oppenheimer DG (2005) Spatial control of cell expansion by the plant cytoskeleton. Annu Rev Cell Dev Biol 21:271–295CrossRefPubMedGoogle Scholar
  53. Stefano G, Renna L, Moss T, McNew JA, Brandizzi F (2012) In Arabidopsis, the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the C-terminal domain of RHD3, a non-essential GTPase. Plant J 69:957–966CrossRefPubMedGoogle Scholar
  54. Tanchak MA, Griffing LR, Mersey BG, Fowke LC (1984) Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts. Planta 162:481–486CrossRefPubMedGoogle Scholar
  55. Toyooka K, Sato M, Kutsuna N, Higaki T, Sawaki F, Wakazaki M, Goto Y, Hasezawa S, Nagata N, Matsuoka K (2014) Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells. Plant Cell Physiol 55:1544–1555CrossRefPubMedGoogle Scholar
  56. Tse YC, Mo B, Hillmer S, Zhao M, Lo SW, Robinson DG, Jiang L (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16:672–693CrossRefPubMedPubMedCentralGoogle Scholar
  57. Tse YC, Lo SW, Hillmer S, Dupree P, Jiang L (2006) Dynamic response of prevacuolar compartments to brefeldin A in plant cells. Plant Physiol 142:1442–1459CrossRefPubMedPubMedCentralGoogle Scholar
  58. Uemura T, Kim H, Saito C, Ebine K, Ueda T, Schulze-Lefert P, Nakano A (2012) Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants. Proc Natl Acad Sci U S A 109:1784–1789CrossRefPubMedPubMedCentralGoogle Scholar
  59. Wang F, Shang Y, Fan B, Yu JQ, Chen Z (2014) Arabidopsis LIP5, a positive regulator of multivesicular body biogenesis, is a critical target of pathogen-responsive MAPK cascade in plant basal defense. PLoS Pathog 10, e1004243CrossRefPubMedPubMedCentralGoogle Scholar
  60. Xu T, Wen M, Nagawa S, Fu Y, Chen JG, Wu MJ, Perrot-Rechenmann C, Friml J, Jones AM, Yang Z (2010) Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143:99–110CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zhang C, Halsey LE, Szymanski DB (2011) The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells. BMC Plant Biol 11:27CrossRefPubMedPubMedCentralGoogle Scholar
  62. Zhao L, Sack FD (1999) Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves. Am J Bot 86:929–939CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Kae Akita
    • 1
    Email author
  • Megumi Kobayashi
    • 2
  • Mayuko Sato
    • 3
  • Natsumaro Kutsuna
    • 1
    • 4
  • Takashi Ueda
    • 5
    • 6
  • Kiminori Toyooka
    • 3
  • Noriko Nagata
    • 2
  • Seiichiro Hasezawa
    • 1
  • Takumi Higaki
    • 1
  1. 1.Department of Integrated Biosciences, Graduate School of Frontier SciencesThe University of TokyoKashiwaJapan
  2. 2.Faculty of ScienceJapan Women’s UniversityTokyoJapan
  3. 3.RIKEN Center for Sustainable Resource SciencesYokohamaJapan
  4. 4.Research and Development DivisionLPixel Inc.TokyoJapan
  5. 5.Department of Biological Sciences, Graduate School of ScienceThe University of TokyoTokyoJapan
  6. 6.Division of Cellular DynamicsNational Institute for Basic BiologyOkazakiJapan

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