, Volume 225, Issue 4, pp 1001–1017 | Cite as

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways

  • Maria Rosaria Leucci
  • Gian-Pietro Di Sansebastiano
  • Massimiliano Gigante
  • Giuseppe Dalessandro
  • Gabriella PiroEmail author
Original Article


The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (secGFP) and secreted rat preputial β-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries.


Cell-wall polysaccharide and protein secretion Dominant-negative mutant effect Green-fluorescent protein Secretory pathway SNARE proteins Tobacco 



Brefeldin A






Green-fluorescent protein


Rat preputial β-glucuronidase


Syntaxin in plant 1; syntaxin homologue NtSyr1 (SYP121)


Soluble variant of Sp1



We would like to thank Prof. M.R. Blatt (Glasgow University) for providing transgenic seeds. Special thanks to ISPA-CNR section of Lecce for the use of Zeiss confocal microscope. This work was supported by a grant from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST- PRIN 2005).


  1. Aoyama T, Chua NH (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612PubMedCrossRefGoogle Scholar
  2. Baluska F, Hlavacka A, Samaj J, Palme K, Robinson DG, Matoh T, McCurdy DW, Menzel D, Volkmann D (2002) F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments. Plant Physiol 130:422–431PubMedCrossRefGoogle Scholar
  3. Baluska F, Samaj J, Wojtaszek P, Volkmann D, Menzel D (2003) Cytoskeleton-plasma membrane cell wall continuum in plants. Emerging links revisited. Plant Physiol 133:482–491PubMedCrossRefGoogle Scholar
  4. Bar-Peled M, Raikhel N (1997) Characterization of AtSec12 and AtSar1, proteins likely involved in endoplasmic reticulum and Golgi transport. Plant Physiol 114:315–324PubMedCrossRefGoogle Scholar
  5. Battey NH, James NC, Greenland AJ, Brownlee C (1999) Exocytosis and endocytosis. Plant Cell 11:643–659PubMedCrossRefGoogle Scholar
  6. Blatt MR (2002) Toward understanding vesicle traffic and the guard cell model. New Phytol 153:405–413CrossRefGoogle Scholar
  7. Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 3:243–293PubMedCrossRefGoogle Scholar
  8. Carpita NC, McCann MC (2002) The functions of cell wall polysaccharides in composition and architecture revealed through mutations. Plant Soil 247:71–80CrossRefGoogle Scholar
  9. Carrol AD, Moyen C, VanKesteren P, Tooke F, Battey NH, Brownlee C (1998) Ca2+, annexins, and GTP modulate exocytosis from maize root cap protoplasts. Plant Cell 10:1267–1276CrossRefGoogle Scholar
  10. Dalessandro G, Piro G, Northcote DH (1988) A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues. Planta 175:60–70CrossRefGoogle Scholar
  11. Delmer DP (1999) Cellulose biosynthesis: exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50:245–276PubMedCrossRefGoogle Scholar
  12. Di Sansebastiano G-P (1999) Targeting of soluble proteins to two different vacuoles in plant cells. PhD thesis, University of Neuchatel, Neuchatel, SwitzerlandGoogle Scholar
  13. Di Sansebastiano GP, Paris N, Marc-Martin S, Neuhaus JM (1998) Specific accumulation of GFP in a non-acid vacuolar compartment via a C-terminal propeptide-mediated sorting pathway. Plant J 15:449–457PubMedCrossRefGoogle Scholar
  14. Doblin MS, Kurek I, Jacob-Wilk D, Delmer DP (2002) Cellulose biosynthesis in plants: from genes to rosettes. Plant Cell Physiol 43:1407–1420PubMedCrossRefGoogle Scholar
  15. Driouich A, Zhang GF, Staehelin LA (1993) Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. Plant Physiol 101:1363–1373PubMedCrossRefGoogle Scholar
  16. Dulubova I, Yamaguchi T, Arac D, Li H, Huryeva I, Min SW, Rizo J, Sudhof TC (2003) Convergence and divergence in the mechanism of SNARE binding by Sec1/Munc 18-like proteins. Proc Natl Acad Sci USA 100:32–37PubMedCrossRefGoogle Scholar
  17. Feingold DS, Barber GA (1990) Nucleotide sugars. Methods Plant Biochem 2:39–78Google Scholar
  18. Freydl E, Meins FJ, Boller T, Neuhaus J-M (1995) Kinetics of prolyl hydroxylation, intracellular transport and C-terminal processing of the tobacco vacuolar chitinase. Planta 147:250–256Google Scholar
  19. Geelen D, Leyman B, Batoko H, Di Sansebastiano G-P, Moore I, Blatt MR (2002) The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain. Plant Cell 14:387–406PubMedCrossRefGoogle Scholar
  20. Giddings THJ, Brower DL, Staehelin LA (1980) Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary walls. J Cell Biol 84:327–339PubMedCrossRefGoogle Scholar
  21. Haigler CH, Brown RMJ (1986) Transport of rosettes from the Golgi apparatus to the plasma membrane in isolated mesophyll cells of Zinnia elegans during differentiation to tracheary elements in suspension culture. Protoplasma 134:111–120CrossRefGoogle Scholar
  22. Hanton SL, Matheson LA, Brandizzi F (2006) Seeking a way out: export of proteins from the plant endoplasmic reticulum. Trends Plant Sci 11:335–343PubMedCrossRefGoogle Scholar
  23. Hawes CR, Bandizzi F, Andreeva AV (1999) Endomembranes and vesicle trafficking. Curr Opin Plant Biol 2:454–461PubMedCrossRefGoogle Scholar
  24. Homann U, Tester M (1997) Ca2+-indipendent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell. Proc Natl Acad Sci USA 94:6565–6570PubMedCrossRefGoogle Scholar
  25. Hoson T (1998) Apoplast as the site of response to environmental signals. J Plant Res 111:167–177PubMedCrossRefGoogle Scholar
  26. Jahn R, Lang T, Sudhof TC (2003) Membrane fusion. Cell 112:519–533PubMedCrossRefGoogle Scholar
  27. Kaiser CA, Gimeno RE, Shaywitz DA (1997) Protein secretion, membrane biogenesis and endocytosis. In: Pringle JB Jr, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 91–227Google Scholar
  28. Kaul S, Koo HL, Jenkins J, Rizzo M, Rooney T, Tallon LJ, Feldblyum T, Nierman W, Benito MI, Lin XY, et al (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  29. Laemmli UK, Favre M (1972) Maturation of the head of bacteriophage T4. J Mol Biol 80:575–599CrossRefGoogle Scholar
  30. Lanubile R, Piro G, Dalessandro G (1997) Effect of brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings. J Exp Bot 1997:1925–1933Google Scholar
  31. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I, Lukowitz W, Jurgens G (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 139:1485–1493PubMedCrossRefGoogle Scholar
  32. Leyman B, Geelen D, Blatt MR (2000) Localization and control of expression of Nt-Syr1, a tobacco SNARE protein. Plant J 24:369–381PubMedCrossRefGoogle Scholar
  33. Leyman B, Geelen D, Quintero FJ, Blatt MR (1999) A tobacco syntaxin with the role in hormonal control of guard cell ion channels. Science 283:537–540PubMedCrossRefGoogle Scholar
  34. McCann MC, Bush M, Milioni D, Sado P, Stacey NJ, Catchpole G, Defernez M, Carpita NC, Hofte H, Ulvskov P, Wilson RH, Roberts K (2001) Approches to understanding the functional architecture of the plant cell wall. Phytochemistry 57:811–821PubMedCrossRefGoogle Scholar
  35. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant Mol Biol 15:473–497Google Scholar
  36. Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455Google Scholar
  37. Nebenführ A, Staehelin LA (2001) Mobile factories: Golgi dynamics in plant cells. Trends Plant Sci 6:160–176PubMedCrossRefGoogle Scholar
  38. Neuhaus J-M, Sticher L, Meins F, Boller T (1991) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88:10362–10366PubMedCrossRefGoogle Scholar
  39. Northcote DH (1979) The involvement of the Golgi apparatus in the biosynthesis and secretion of glycoproteins and polysaccharides. Biomembranes 10:51–76PubMedGoogle Scholar
  40. Pacoda D, Montefusco A, Piro G, Dalessandro G (2004) Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells. J Plant Physiol 161:1143–1156PubMedCrossRefGoogle Scholar
  41. Picton JM, Steer MW (1981) Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D. J Cell Sci 49:261–272PubMedGoogle Scholar
  42. Piro G, Zuppa A, Dalessandro G, Northcote DH (1993) Glucomannan synthase in pea epicotyls: the mannose and glucose transferases. Planta 190:206–220PubMedCrossRefGoogle Scholar
  43. Powell PP, Kyle JW, Miller RD, Pantano J, Grubb JH, Sly WS (1988) Rat liver beta-glucuronidase: cDNA cloning, sequence comparison and expression of a chimeric protein in COS cells. Biochem J 250:547–555PubMedGoogle Scholar
  44. Ritzenthaler C, Nebenführ A, Movafeghi A, Stussi-Garaud C, Behnia L, Pimpl P, Staehelin LA, Robinson DG (2002) Reevaluation of the effects of brefeldin A on plant cells using tobacco bright yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera. Plant Cell 14:237–261PubMedCrossRefGoogle Scholar
  45. Sanderfoot AA, Assaad FF, Raikhel NV (2000) The Arabidopsis genome. An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol 124:1558–1569PubMedCrossRefGoogle Scholar
  46. Sanderfoot AA, Pilgrim M, Adam L, Raikhel NV (2001) Disruption of individual members of Arabidopsis syntaxin gene families indicates each has essential functions. Plant Cell 13:659–666PubMedCrossRefGoogle Scholar
  47. Schindler T, Bergfeld R, Hohl M, Schopfer P (1994) Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth, cell-wall extensibility and secretion of cell-wall proteins. Planta 192:404–413CrossRefGoogle Scholar
  48. Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475PubMedCrossRefGoogle Scholar
  49. Shipley JM, Grubb JH, Sly WS (1993) The role of glycosylation and phosphorylation in the expression of active human β-glucuronidase. J Biol Chem 268:12193–12198PubMedGoogle Scholar
  50. Staehelin LA, Moore I (1995) The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu Rev Plant Physiol Plant Mol Biol 46:261–288Google Scholar
  51. Takeuchi M, Tada M, Saito C, Yashiroda H, Nakano A (1998) Isolation of a tobacco cDNA encoding Sar1 GTPase and analysis of its dominant mutations in vesicular traffic using a yeast complementation system. Plant Cell Physiol 39:590–599PubMedGoogle Scholar
  52. Ueda T, Matsuda N, Anai T, Tsukaya H, Uchimiya H, Nakano A (1996) An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase. Plant Cell 8:2979–2091CrossRefGoogle Scholar
  53. Vitale A, Denecke J (1999) The endoplasmic reticulum-gateway of the secretory pathway. Plant Cell 11:615–628PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Maria Rosaria Leucci
    • 1
  • Gian-Pietro Di Sansebastiano
    • 1
  • Massimiliano Gigante
    • 1
  • Giuseppe Dalessandro
    • 1
  • Gabriella Piro
    • 1
    Email author
  1. 1.Di.S.Te.B.A.Università di LecceLecceItaly

Personalised recommendations