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Plant Molecular Biology

, Volume 31, Issue 3, pp 465–478 | Cite as

A wound-inducible gene fromSalix viminalis coding for a trypsin inhibitor

  • Peter Saarikoski
  • David Clapham
  • Sara von Arnold
Regular Article

Abstract

A gene designatedswin1.1 has been isolated by screening aSalix viminalis genomic library with a heterologous probe,win3 fromPopulus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5′ end of the coding region is a sequence that encodes a hydrophobic region of 25–30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially withwin3 from wounded leaves ofPopulus. Southern blot analysis indicated thatswin1.1 is a member of a clustered gene family,swin1. An oligonucleotide corresponding to the putative hypervariable region to-wards the carboxyl end when used as a probe in Southern hybridization showed high specificity forswin1.1. Expression of theswin1.1 gene was enhanced in wounded leaves. Theswin1.1 coding region without the signal sequence was highly expressed inEscherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated fromSalix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translatedswin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of theswin1 gene family.

Key words

hypervariable region Kunitz family Salix viminalis serine protease inhibitor wound-induced 

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References

  1. 1.
    Bednarek SY and Raikhel NV: Intracellular trafficking of secretory proteins. Plant Molecular biology 20: 133–150 (1992).PubMedGoogle Scholar
  2. 2.
    Bernatzky R, Tanksley SD: Genetics of actin-related sequences in tomato. Theor Appl Genet 72: 314–321 (1986).CrossRefGoogle Scholar
  3. 3.
    Bishop PD, Markus DJ, Pearce G, Ryan CA: Proteinase inhibitor inducing activity in tomato leaves resides in oligosaccharides enzymatically released from cell walls. Proc Natl Acad Sci USA 78: 3536–3540 (1981).Google Scholar
  4. 4.
    Boulter D, Gatehouse AMR, Hilder V: Use of cowpea trypsin inhibitor (CpTI) to protect plants against insect predation. Biotech Adv 7: 489–497 (1989).CrossRefGoogle Scholar
  5. 5.
    Bradshaw HD, Hollick JB, Parsons TJ, Clarke HRG, Gordon MP: Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporamins and legume Kunitz trypsin inhibitors. Plant Mol Biol 14: 51–59 (1989).Google Scholar
  6. 6.
    Brown WE, Ryan CA: Isolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves. Biochemistry 23: 3418–3422 (1984).PubMedGoogle Scholar
  7. 7.
    Bryant J, Green TR, Gurusaddaiah S, Ryan CA: Proteinase inhibitor II from potatoes: isolation and characterization of its promoter components. Biochemistry 15: 3418–3424 (1976).PubMedGoogle Scholar
  8. 8.
    Chang, Puryear, Cairney: A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep II (2): 117–121 (1993).Google Scholar
  9. 9.
    Farmer EE, Ryan CA: Interplant communication: airborne methyl jasmonate induced synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci USA 87: 7713–7716 (1990).PubMedGoogle Scholar
  10. 10.
    Frangioni JV, Neel BG: Solubilization and purification of enzymatically active Glutathione S-Transferase (pGEX) fusion proteins. Analytical Biochemistry 210: 179–187 (1993).CrossRefPubMedGoogle Scholar
  11. 11.
    Gallie D: Posttranscriptional regulation of gene expression in plants. Annu Rev Physiol Plant Mol Biol 44: 77–105 (1993).CrossRefGoogle Scholar
  12. 12.
    Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P: Plant proteinaceous inhibitors of proteinases and a amylases. Oxf Surv Plant Mol Cell Biol 4: 275–334 (1987).Google Scholar
  13. 13.
    Geoffroy P, Legrand M, Fritig B: Isolation and characterization of a proteinaceous inhibitor of microbial proteinases induced during the hypersensitive reaction of tobacco to tobacco mosaic virus. Molecular Plant-Microbe Interactions vol 3 No 5: 327–333 (1990).PubMedGoogle Scholar
  14. 14.
    Green RT, Ryan CA: Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175: 776–777 (1972).Google Scholar
  15. 15.
    Gullberg U, Ryttman H: Genetics of field resistance toMelampsora inSalix viminalis. European J Forest Pathol 23 (2): 75–84 (1993).Google Scholar
  16. 16.
    Hattori T, Nakamura K: Genes encoding for the major tuberous root protein of sweet potato: Indentification of putative regulatory sequence in the 5′ upstream region. Plant Mol Biol 11: 417–426 (1988).Google Scholar
  17. 17.
    Hattori T, Yoshida N, Nakamura K: Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein. Plant Mol Biol 13: 563–572 (1989).Google Scholar
  18. 18.
    Hill RE, Hastie ND: Accelerated evolution in the reactive centre regions of serine protease inhibitors. Nature 326: 96–99 (1987).CrossRefPubMedGoogle Scholar
  19. 19.
    Hill RE, Shaw PH, Boyd PA, Baumann H, Hastie ND: Plasma protease inhibitors in mouse and man: divergence within reactive centre regions. Nature 311: 175–177 (1984).PubMedGoogle Scholar
  20. 20.
    Hollick JB, Gordon MP: A poplar tree proteinase inhibitor-like gene promoter is responsive to wounding in transgenic tobacco. Plant Mol Biol 22: 561–572 (1993).Google Scholar
  21. 21.
    Hung CH, Lee MC, Lin JY: Nucleotide sequence of cDNA forAcacia confusa trypsin inhibitor and implication of post-translation processing. Biochem Biophys Res Commun 184: 1524–1528 (1992).PubMedGoogle Scholar
  22. 22.
    Jofuku KD, Goldberg RB: Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants. Plant Cell 1: 1079–1093 (1989).CrossRefPubMedGoogle Scholar
  23. 23.
    Joubert FJ, Dowdle EBD: The primary structure of the inhibitor of tissue plasminogen activator found in the seeds ofErythrina caffra. Thromb Haemost 57: 356–360 (1987).PubMedGoogle Scholar
  24. 24.
    Kernan A, Thornburg RW: Auxin levels regulate the expression of a wound-inducible proteinase inhibitor II-chloramphenicol acetyl transferase gene fusion in vitro and in vivo. Plant Physiol 91: 73–78 (1989).Google Scholar
  25. 25.
    Kimura M, Kouzuma Y, Yamasaki N: Amino acid sequence of chymotrypsin inhibitor ECI from seeds ofErythrina variegata (Linn.) var. Orientalis. Biosci Biotechnol Biochem 57: 102–106 (1993).PubMedGoogle Scholar
  26. 26.
    Kyte J, Doolitte RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132 (1982).PubMedGoogle Scholar
  27. 27.
    Laemmli UK: Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).PubMedGoogle Scholar
  28. 28.
    LaskowskiJr M, Kato I: Protein inhibitors of proteinases. Annu Rev Biochem 49: 593–626 (1980).CrossRefPubMedGoogle Scholar
  29. 29.
    Lipman DJ, Pearson WR: Rapid and sensitive protein similarity searches. Science 227: 1435–1441 (1985).PubMedGoogle Scholar
  30. 30.
    Maniatis T, Fritsch EF, Sambrook J: Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  31. 31.
    Mares M, Meloun B, Palvik M, Kostka V, Baudys M: Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family. Febs Lett 251: 94–98 (1989).CrossRefPubMedGoogle Scholar
  32. 32.
    Nakamura K, Matsuoka K: Protein targetting to the vacuole in plant cells. Plant Physiol 101: 1–5 (1993).CrossRefPubMedGoogle Scholar
  33. 33.
    Ohtsubo KI, Richardson M: The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran of rice (Oryza sativa L:) seeds. Febs Lett 309: 68–72 (1992).CrossRefPubMedGoogle Scholar
  34. 34.
    Pautot V, Holzer FM, Walling LL: Differential expression of tomato proteinase inhibitor I and II genes during bacterial pathogen invasion and wounding. Mol. Plant Microbe Inter 4: 284–292 (1991).Google Scholar
  35. 35.
    Pearce G, Strydom D, Johnson S, Ryan CA: A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253: 895–898 (1991).Google Scholar
  36. 36.
    Pena-Cortes H, Sanchez-Serrano JJ, Mertens R, Willmitzer L, Prat S: Abscisic acid is involved in the wound-induced expression of the proteinase inhibitor II gene in potato and tomato. Proc Natl Acad Sci USA 86: 9851–9855 (1989).Google Scholar
  37. 37.
    Plunkett G, Senear DF, Zuroske G, Ryan CA: Proteinase inhibitors I and II from leaves of wounded tomato plants: purification and properties. Arch Biochem Biophys 213: 463–472 (1982).PubMedGoogle Scholar
  38. 38.
    Reed KC: Nucleic acid hybridizations with positive charge-modified nylon membrane. Methods in Gene Technology Vol 1: 127–160 (1991).Google Scholar
  39. 39.
    Richardson M, Campos FAP, Xavier-Filho J, Macedo MLR, Maia GMC, Maeda K: The complete amino-acid sequence of the endogenous alpha-amylase inhibitor in wheat. Biochim Biophys Acta 871: 250–256 (1986).Google Scholar
  40. 40.
    Rickauer M, Fournier J, Esquerré-Tougayé M-T: Induction of proteinase inhibitors in tobacco cell suspension culture by elicitors ofPhytophthora parasitica var. nicotianae. Plant Physiol 90: 1065–1070 (1989).Google Scholar
  41. 41.
    Ryals J, Uknes S, Ward E: Systematic Acquired Resistance. Plant Physiol 104: 1109–1112 (1994).PubMedGoogle Scholar
  42. 42.
    Ryan CA, Moloshok T, Pearce G, An G, Thornburg RW, Hall G, Johnson R, Farmer EE, Palm C: Engineering proteinase inhibitor genes for plant defense against predators. Jour Iowa Acad Sci 97 (1): 9–14 (1990).Google Scholar
  43. 43.
    Ryan CA: The Herbivores, their interactions with secondary plant metabolites Rosenthal A and Janzen DH (eds): Academic Press, New York: 599–618 (1979).Google Scholar
  44. 44.
    Saarikoski P, von Arnold S, Clapham D, Gullberg U: Genetic differences in wound-induced ethylene production among different clones ofSalix viminalis L. Silvae Genetica 42: 121–126 (1993).Google Scholar
  45. 45.
    Shumway LK, Yang VV, Ryan CA: Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells. Planta 129: 161–165 (1976).Google Scholar
  46. 46.
    Strong D, Larsson S, Gullberg U: Discontinuous genetic variation in resistance ofSalix viminalis L to a gall-midge attack. Evolution 47 (1): 291–300 (1993).Google Scholar
  47. 47.
    Tai H, Mchenry L, Fritz PJ, Furtek DB: Nucleic acid sequence of a 21 kDa cocoa seed protein with homology to the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Plant Mol Biol 16: 913–915 (1991).CrossRefPubMedGoogle Scholar
  48. 48.
    Theerasilp S, Hitotsuya H, Nakajo S, Nakaja K, Nakamura Y, Kurihara Y: Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin. J Biol Chem 264: 6655–6659 (1989).PubMedGoogle Scholar
  49. 49.
    Von Heijne G: A new method for predicting signal sequence cleavage sites. Nucleic Acids 14: 4683–4690 (1986).PubMedGoogle Scholar
  50. 50.
    Von Heijne G: Patterns of amino acids near signal sequence cleavage site. Eur J Biochem 133: 17–21 (1983).PubMedGoogle Scholar
  51. 51.
    Wildon DC, Thain JF, Minchin PEH, Gubb IR, Reilly AJ, Skipper YD, Doherty HM, O'Donnell PJ, Bowles DJ: Electrical signalling and systemic proteinase inhibitor induction in the wounded plant. Nature 360: 62–65 (1992).CrossRefGoogle Scholar
  52. 52.
    Yamamoto M, Hara S, Ikenaka T: Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L) DC). J Biochem 94: 849–863 (1983).PubMedGoogle Scholar
  53. 53.
    Yarwood A: The amino acid sequence and reactive (inhibitory) site of the major trypsin isoinhibitor (DE5) isolated from seeds of the Brazilian Carolina tree (Adenanthera pavonina L.). Biochim Biophys Acta 872: 134–140 (1986).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Peter Saarikoski
    • 1
  • David Clapham
    • 1
  • Sara von Arnold
    • 1
  1. 1.Department of Forest Genetics, The Genetic CenterSwedish University of Agricultural SciencesUppsalaSweden

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