Plant Molecular Biology

, Volume 47, Issue 6, pp 727–738 | Cite as

A proteinase inhibitor II of Solanum americanum is expressed in phloem

  • Zeng-Fu Xu
  • Wen-Qing Qi
  • Xue-Zhi Ouyang
  • Edward Yeung
  • Mee-Len Chye


Although proteinase inhibitor proteins are known to confer insect resistance in transgenic plants, their endogenous roles remain undefined. Here, we describe the expression of a proteinase inhibitor II (PIN2) protein from Solanum americanum in phloem of stems, roots and leaves suggesting a novel endogenous role for PIN2 in phloem. The phloem consists of parenchyma cells, sieve elements (SE), and companion cells (CC) which are in close association with SE. We isolated two cDNAs encoding PIN2, SaPIN2a and SaPIN2b, from a S. americanum cDNA library using a tomato PIN2 cDNA as hybridization probe. SaPIN2a shows 73.6% identity to SaPIN2b. Southern blot analysis confirmed that two genes occur in S. americanum. Northern blot analysis showed that both are wound-inducible and are expressed in flowers. Unlike SaPIN2b and other previously characterized plant PIN2 proteins, SaPIN2a is abundantly expressed in stems. In situ hybridization studies on stem sections showed that SaPIN2a mRNA is expressed in CC and some SE, likely the immature developing SE, of external and internal phloem. Western blot analysis using SaPIN2a-specific antibodies showed SaPIN2a accumulation in stems, leaf midribs and fruits. Immunohistochemical localization, using these antibodies, revealed SaPIN2a expression in external and internal phloem of stem. Immunoelectron microscopy of stem, root and leaf sections further localized SaPIN2a to the CC and predominantly to the SE, particularly the parietal cytoplasm adjacent to the cell wall, the lumen and the sieve-area pores. These results suggest that, other than a possible role in plant defense, SaPIN2a could be involved in regulating proteolysis in the SE.

black nightshade companion cell immunolocalization in situ hybridization sieve-area pore sieve element 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Atkinson, A.H., Heath, R.L., Simpson, R.J., Clarke, A.E. and Anderson, M.A. 1993. Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5: 203–213.Google Scholar
  2. Balachandran, S., Xiang, Y., Schobert, C., Thompson, G.A. and Lucas, W. 1997. Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata. Proc. Natl. Acad. Sci. USA 94: 14150–14155.Google Scholar
  3. Balandin, T., van der Does C., Albert, J.-M.B., Bol J.F. and Linthorst, H.J.M. 1995. Structure and induction pattern of anovel proteinase inhibitor class II gene of tobacco. Plant Mol. Biol. 27: 1197–1204.Google Scholar
  4. Bostwick, D.E., Dannenhoffer, J.M., Skaggs, M.I., Lister, R.M., Larkins, B.A. and Thompson, G.A. 1992. Pumpkin phloem lectin genes are specifically expressed in companion cells. Plant Cell 4: 1539–1548.Google Scholar
  5. Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.Google Scholar
  6. Brandstadter, J., Rossbach, C. and Theres, K. 1996. Expression of genes for a defensin and a proteinase inhibitor in specific areas of the shoot apex and the developing flower in tomato. Mol. Gen. Genet. 252: 146–154.Google Scholar
  7. Bryant, J., Green, T.R., Gurusaddaiah, T. and Ryan, C.A. 1976. Proteinase inhibitor II from potatoes: isolation and characterization of the promoter components. Biochemistry 15: 3418–3424.Google Scholar
  8. Brzin, J. and Kidric, M. 1995. Proteinases and their inhibitors in plants: role in normal growth and in response to various stress conditions. Biotechnol. Genet. Eng. Rev. 13: 420–467.Google Scholar
  9. Choi, D., Park, J.-A., Seo, Y.S., Chun, Y.J. and Kim, W.T. 2000. Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L. Biochim. Biophys. Acta. 1492: 211–215.Google Scholar
  10. Chye, M.-L., Huang, B.-Q. and Zee, S.Y. 1999. Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product. Plant J. 18: 205–214.Google Scholar
  11. Clark, A.M., Jacobsen, K.R., Bostwick, D.E., Dannenhoffer, J.M., Skaggs, M.I. and Thompson, G.A. 1997. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima. Plant J. 12: 49–61.Google Scholar
  12. Cox, K.H. and Goldberg, R.B. 1988. Analysis of plant gene expression. In: C.H. Shaw (Ed.) Plant Molecular Biology: A Practical Approach, IRL Press, Oxford, pp. 1–35.Google Scholar
  13. Crawford, K.M. and Zambryski, P.C. 1999. Phloem transport: are you chaperoned? Curr. Biol. 9: 281–285.Google Scholar
  14. Dannenhoffer, J.M., Schulz, A., Skaggs, M.I., Bostwick, D.E. and Thompson, G.A. 1997. Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits. Planta 201: 405–414.Google Scholar
  15. Dannenhoffer, J.M., Suhr, R.C. and Thompson, G.A. 2001. Phloem-specific expression of the pumpkin fruit trypsin inhibitor. Planta 212: 155–162.Google Scholar
  16. Dellaporta, S.L., Wood, J. and Hicks, J.B. 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1: 19–21.Google Scholar
  17. Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, D. and Wu, R. 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnol. 14: 494–498.Google Scholar
  18. Esau, K. 1977. Anatomy of Seed Plants, 2nd ed. John Wiley, New York.Google Scholar
  19. Evert, R.F. 1990. Dicotyledons. In: H.-D. Behnke and R.D. Sjolund (Eds.) Sieve Elements: Comparative Structure, Induction and Development, Springer-Verlag, Berlin, pp. 103–137.Google Scholar
  20. Gadea, J., Mayda, M.E., Conejero, V. and Vera, P. 1996. Characterization of defense-related genes ectopically expressed in viroid-infected tomato plants. Mol. Plant-Microbe Interact. 9: 409–415.Google Scholar
  21. Graham, J.S., Hall, G. and Ryan, C.A. 1986. Regulation of synthesis of proteinase inhibitors I and II mRNAs in leaves of wounded tomato plants. Planta 169: 399–405.Google Scholar
  22. Graham, J.S., Pearce, G., Merryweather, J., Titani, K., Ericsson, L.H. and Ryan, C.A. 1985. Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II. J. Biol. Chem. 260: 6561–6564.Google Scholar
  23. Greenblatt, H.M., Ryan, C.A. and James, M.N.G. 1989. Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-I from Russet Burbank potato tubers at 2.1 Å resolution. J. Mol. Biol. 205: 201–228.Google Scholar
  24. Gustafson, G. and Ryan, C.A. 1976. Specificity of protein turnover in tomato leaves. Accumulation of proteinase inhibitors induced with the wound hormone, PIIF. J. Biol. Chem. 251: 7004–7010.Google Scholar
  25. Habu, Y., Fukushima, H., Sakata, Y., Abe, H. and Funada, R. 1996. A gene encoding a major Kunitz proteinase inhibitor of storage organs of winged bean is also expressed in the phloem of stems. Plant Mol. Biol. 32: 1209–1213.Google Scholar
  26. Hendriks, T., Vreugdenhil, D. and Stiekema, W.J. 1991. Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol. Biol. 17: 385–394.Google Scholar
  27. Jameson, B.A. and Wolf, H. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput. Appl. Biosci. 4: 181–186.Google Scholar
  28. Johnson, R., Narvaez, J., An, G. and Ryan, C. 1989. Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae. Proc. Natl. Acad. Sci. USA 86: 9871–9875.Google Scholar
  29. Klopfenstein, N.B., Allen, K.K., Avila, F.J., Heuchelin, S.A., Martinez, J., Carman, R.C., Hall, R.B., Hart, E.R. and McNabb, H.S. 1997. Proteinase inhibitor II gene in transgenic poplar: chemical and biological assays. Biomass Bioenergy 12: 299–311.Google Scholar
  30. Kuhn, C., Franceschi, V.R., Schulz, A., Lemoine, R. and Frommer, W.B. 1997. Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements. Science 275: 1298–1300.Google Scholar
  31. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.Google Scholar
  32. Lorberth, R., Dammann, C., Ebneth, M., Amati, S. and Sanchez-Serrano, J.J. 1992. Promoter elements involved in environmentaland developmental control of potato proteinase inhibitor II expression. Plant J. 2: 477–486.Google Scholar
  33. Miller, E.A., Lee, M.C.S., Atkinson, A.H.O. and Anderson, M.A. 2000. Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata. Plant Mol. Biol. 42: 329–333.Google Scholar
  34. Nagy, F., Kay, S.A. and Chua, N.-H. 1988. Analysis of gene expression in transgenic plants. In: S.B. Gelvin and R.A. Schilperoort (Eds) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. B4: 1–29.Google Scholar
  35. Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1–6.Google Scholar
  36. Oparka, K.J. and Turgeon, R. 1999. Sieve elements and companion cells-traffic control centers of the phloem. Plant Cell 11: 739–750.Google Scholar
  37. Pearce, G., Johnson, S. and Ryan, C.A. 1993. Purification and characterization from tobacco (Nicotiana tabacum)leaves ofsix small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family. Plant Physiol. 102: 639–644.Google Scholar
  38. Pearce, G., Ryan, C.A. and Liljegren, D. 1988. Proteinase inhibitor I and II in fruit of wild tomato species: transient components of a mechanism for defense and seed dispersal. Planta 175: 527–531.Google Scholar
  39. Pena-Cortes, H., Sanchez-Serrano, J., Rocha-Sosa, M. and Willmitzer, L. 1988. Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding. Planta 174: 84–89.Google Scholar
  40. Pena-Cortes, H., Willmitzer, L. and Sanchez-Serrano, J.J. 1991. Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell 3: 963–972.Google Scholar
  41. Richardson, M. 1979. The complete amino acid sequence and trypsin reactive (inhibitory) site of the major proteinase inhibitor from the fruits of aubergine (Solanum melongena L.). FEBS Lett. 104: 322–326.Google Scholar
  42. Rosahl, S., Eckes, P., Schell, J. and Willmitzer, L. 1986. Organ-specific gene expression in potato: isolation and characterization of tuber-specific cDNA sequences. Mol. Gen. Genet. 202: 368–373.Google Scholar
  43. Ryan, C.A. 1989. Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. BioEssays 10: 20–24.Google Scholar
  44. Ryan, C.A. and Walker-Simmons, M. 1981. Plant Proteinases. In: A. Marcus (Ed.) The Biochemistry of Plants, vol. 6, Academic Press, New York, pp. 321–350.Google Scholar
  45. Sabnis, D.D. and Hart, J.W. 1978. The isolation and some properties of a lectin (haemagglutinin) from Curcubita phloem exudates. Planta 142: 97–101.Google Scholar
  46. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY.Google Scholar
  47. Sanchez-Serrano, J., Schmidt, R., Schell, J. and Willmitzer, L. 1986. Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol. Gen. Genet. 203: 15–20.Google Scholar
  48. Schilling, E.E., Ma, Q. and Andersen, R.N. 1992. Common names and species identification in black nightshades, Solanum sect. Solanum (Solanaceae). Econ. Bot. 46: 223–225.Google Scholar
  49. Schulz, A. 1990. Wound-sieve elements. In: H.-D. Behnke and R.D. Sjolund (Eds) Sieve Elements: Comparative Structure, Induction and Development, Springer-Verlag, Berlin, pp. 199–217.Google Scholar
  50. Schulz, A. 1998. Phloem. Structure related to function. In: H.-D. Behnke, K. Esser, J.W. Kadereit, U. Luttge and M. Runge (Eds) Progress in Botany, vol. 59, Springer-Verlag, Berlin, pp. 429–475.Google Scholar
  51. Schulz, A., Alosi, M.C., Sabnis, D.D. and Park, R.B. 1989. A phloem-specific, lectin-like protein is located in pine sieve-element plastids by immunocytochemistry. Planta 179: 506–515.Google Scholar
  52. Smith, L.M., Sabnis, D.D. and Johnson, R.P.C. 1987. Immunocy-tochemical localisation of phloem lectin from Cucubita maxima using peroxidase and colloidal-gold labels. Planta 170: 461–470.Google Scholar
  53. Solomon, M., Belenghi, B., Delledonne, M., Menachem, E. and Levine, A. 1999. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11: 431–443.Google Scholar
  54. Stiekema, W.J., Heidekamp, F., Dirkse, W.G., van Beckum, J., de Haan, P., ten Bosch, C. and Louwerse, J.D. 1988. Molecular cloning and analysis of four potato tuber mRNAs. Plant Mol. Biol. 11: 255–269.Google Scholar
  55. Taylor, B.H., Young, R.J. and Scheuring, C.F. 1993. Induction of a proteinase inhibitor II-class gene by auxin in tomato roots. Plant Mol. Biol. 23: 1005–1014.Google Scholar
  56. Thompson, G.A. and Schulz, A. 1999 Macromolecular trafficking in the phloem. Trends Plant Sci. 4: 354–360.Google Scholar
  57. Thornburg, R.W., An, G., Cleveland, T.E., Johnson, R. and Ryan, C.A. 1987. Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyl-transferase gene fusion in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 84: 744–748.Google Scholar
  58. von Heijne, G. 1983. Patterns of amino acids near signal-sequence cleavage site. Eur. J. Biochem. 133: 17–21.Google Scholar
  59. Wu, Y., Llewellyn, D., Mathews, A. and Dennis, E.S. 1997. Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol. Breed. 3: 371–380.Google Scholar
  60. Xoconostle-Cazares, B., Xiang, Y., Ruiz-Medrano, R., Wang, H.-L., Monzer, J., Yoo, B.-C., McFarland, K.C., Franceschi, V.R. and Lucas, W.J. 1999. Plant paralog to viral movement protein that potentiates transport of mRNA into the phloem. Science 283: 94–98.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Zeng-Fu Xu
    • 1
  • Wen-Qing Qi
    • 2
  • Xue-Zhi Ouyang
    • 2
  • Edward Yeung
    • 3
  • Mee-Len Chye
    • 2
  1. 1.Biotechnology Research CenterZhongshan UniversityGuangzhouChina
  2. 2.Department of Botanythe University of Hong KongHong KongChina
  3. 3.Department of Biological SciencesUniversity of Calgary, CalgaryAlbertaCanada

Personalised recommendations