, Volume 195, Issue 1, pp 80–87 | Cite as

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein

  • F. Favaron
  • R. D'Ovidio
  • E. Porceddu
  • P. Alghisi


A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endoPGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.

Key words

Fungal polygalacturonases Glycine Inhibitor purification Polygalacturonase inhibitor 





polymerase chain reaction




polygalacturonase-inhibiting protein


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abu-Gougk, A.A., Strand, L.L., Labavitch, J.M. (1983a) Development-related changes in decay susceptibility and polygalacturonase inhibitor content of “Bartlett” pear fruit. Physiol. Plant Pathol. 23, 101–109Google Scholar
  2. Abu-Goukh, A.A., Greve, L.C., Labavitch, J.M. (1983) Purification and partial characterization of “Bartlett” pear fruit polygalacturonase inhibitors. Physiol. Plant Pathol. 23, 111–122Google Scholar
  3. Abu-Goukh, A.A., Labavitch, J.M. (1983) The in vivo role of “Bartlett” pear fruit polygalacturonase inhibitors. Physiol. Plant Pathol. 23, 123–135Google Scholar
  4. Albersheim, P., Anderson, A.J. (1971) Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc. Natl. Acad. Sci. USA 68, 1815–1819Google Scholar
  5. Barnmore, C.R., Nguyen, T.K. (1985) Polygalacturonase inhibition in rind of Valencia orange infected with Diploidia natalensis. Phytopathology 75, 446–449Google Scholar
  6. Benhamou, N., Lafitte, C., Barthe, J.P., Esquerré-Tugayé, M.T. (1991) Cell surface interactions between bean leaf cells and Colletotrichum lindemuthianum. Cytochemical aspects of pectin breakdown and fungal endopolygalacturonase accumulation. Plant Physiol. 97, 234–244Google Scholar
  7. Bishop, P.D., Makus, D.J., Pearce, G., Ryan, C.A. (1981) Proteinase inhibitor-inducing factor activity in tomato leaves residues in oligosaccharides enzymically released from cell walls. Proc. Natl. Acad. Sci. USA 78, 3536–3540Google Scholar
  8. Bradford, M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254CrossRefPubMedGoogle Scholar
  9. Brown, A.E., Adikaram, N.K.B. (1982) The differential inhibition of pectic enzymes from Glomerella cingulata and Botrytis cinerea by a cell wall protein from Capsicum annuum fruit. Phytopath. Z. 105, 27–38Google Scholar
  10. Bruce, R.J., West, C.A. (1989) Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. Plant Physiol. 89, 889–897Google Scholar
  11. Cervone, F., DeLorenzo, G., Degra, L., Salvi, G., Bergami, M. (1987) Purification and characterization of a polygalacturonaseinhibiting protein from Phaseolus vulgaris L. Plant Physiol. 85, 631–637Google Scholar
  12. Cervone, F., Hahn, M.G., DeLorenzo, G., Darvill, A., Albersheim, P. (1989) Host-Pathogen Interactions. XXXIII. A plant protein converts a fungal pathogenesis factor into an elicitor of plant defence responses. Plant Physiol. 90, 542–548Google Scholar
  13. Colbert, J.T., Hershey, H.P., Quail, P.H. (1983) Autoregulatory control of translable phytochrome mRNA levels. Proc. Natl. Acad. Sci. USA 80, 2248–2252Google Scholar
  14. Collmer, A., Keen, N.T. (1986) The role of pectic enzymes in plant pathogenesis. Annu. Rev. Phytopathol. 24, 383–409Google Scholar
  15. Cooper, R.M. (1984) The role of cell wall degrading enzymes in infection and damage. In: Plant diseases: Infection, damage and loss, pp. 261–281, Wood, R.K.S., Jellis, G.J., eds. Blackwell, OxfordGoogle Scholar
  16. Davis, K.R., Darvill, A.G., Albersheim, P., Dell, A. (1986) Hostpathogen interactions. XXX. Characterization of elicitors of phytoalexin accumulation in soybean released from soybean cell walls by endopolygalacturonic acid lyase. Z. Naturforsch. 41c, 39–48Google Scholar
  17. Davis, K.R., Hahlbrock, K. (1987) Induction of defense responses in cultured parsley cells by plant cell wall fragments. Plant Physiol. 85, 1286–1290Google Scholar
  18. Degrà, L., Salvi, G., Mariotti, D., DeLorenzo, G., Cervone, F. (1988) A polygalacturonase-inhibiting protein in alfalfa callus cultures. J. Plant Physiol. 133, 364–366Google Scholar
  19. D'Ovidio, R., Scarascia Mugnozza, G.T., Tanzarella, O.A. (1991) rDNA cloning and rapid hybrid identification in Populus spp. (Salicaceae). Plant Syst. Evol. 177, 165–174Google Scholar
  20. D'Ovidio, R., Tanzarella, O.A., Porceddu, E. (1992) Isolation of an alpha-type gliadin gene from Triticum durum Desf. and genetic polymorphysm at the Gli-2 loci. J. Genet. Breed. 46, 41–48Google Scholar
  21. D'Ovidio, R., Anderson, O.D. (1994) PCR analysis to distinguish between alleles of a member of a multigene family correlated with wheat quality. Theor. Appl. Genet., in pressGoogle Scholar
  22. Favaron, F., Alghisi, P., Marciano, P., Magro, P. (1988) Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin. Physiol. Mol. Plant Pathol. 33, 385–395Google Scholar
  23. Favaron, F., Alghisi, P., Marciano, P. (1992) Characterization of two Sclerotinia sclerotiorum polygalacturonases with different abilities to elicit glyceollin in soybean. Plant Sci. 83, 7–13Google Scholar
  24. Favaron, F., Castiglioni, C., DiLenna, P. (1993) Inhibition of some rot fungi polygalacturonases by Allium cepa L. and Allium porrum L. extracts. J. Phytopathol. 139, 201–206Google Scholar
  25. Favaron, F., Peretto, R., Bonfante, P., Alghisi, P. (1993) Differential absorption and localization of two Sclerotinia sclerotiorum endo-polygalacturonases in soybean hypocotyls. Physiol. Mol. Plant Pathol. 43, 353–364Google Scholar
  26. Fielding, H. (1981) Natural inhibitors of fungal polygalacturonases in infected fruits. J. Gen. Microbiol. 123, 377–381Google Scholar
  27. Frediani, M., Cremonini, R., Salvi, G., Caprari, C., Desiderio, A., D'Ovidio, R., Cervone, F., DeLorenzo, G. (1993) Cytological localization of the PGIP genes in the embryo suspensor cells of Phaseolus vulgaris L. Theor. Appl. Genet. 87, 369–373Google Scholar
  28. Hoffman, R.M., Turner, J.G. (1982) Partial purification of proteins from pea leaflets that inhibit Ascochyta pisi endopolygalacturonase. Physiol. Plant Pathol. 20, 173–187Google Scholar
  29. Hoffman, R.M., Turner, J.G. (1984) Occurrence and specificity of an endopolygalacturonase inhibitor in Pisum sativum. Physiol. Plant Pathol. 24, 49–59Google Scholar
  30. Johnston, D.J., Ramanathan, V., Williamson, B. (1993) A protein from immature raspberry fruits which inhibits endopolygalacturonases from Botrytis cinerea and other micro-organisms. J. Exp. Bot. 262, 971–976Google Scholar
  31. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 222, 680–685Google Scholar
  32. Lawton, M.A., Lamb, C.J. (1987) Transcriptional activation of plant defence genes by fungal elicitor, wounding and infection. Mol. Cell. Biol. 7, 335–341Google Scholar
  33. Legendre, L., Heinstein, P.F., Low, P.S. (1992) Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells. J Biol. Chem. 267, 20140–20147Google Scholar
  34. Milner, Y., Avigad, G. (1967) A copper reagent for the determination of hexuronic acids and certain ketohexoses. Carbohydr. Res. 4, 359–361Google Scholar
  35. Nothnagel, E.A., McNeil, M., Albersheim, P., Dell, A. (1983) Hostpathogen interactions. XXII. A galacturonic acid oligosaccharide from plant cell walls elicits phytoalexins. Plant Physiol. 71, 916–926Google Scholar
  36. Robertsen, B. (1986) Elicitors of the production of lignin-like compounds in cucumber hypocotyls. Physiol. Mol. Plant Pathol. 28, 137–148Google Scholar
  37. Ryder, T.B., Cramer, C.L., Bell, J.N., Robbins, M.P., Dixon, R.A., Lamb, C.J. (1984) Elicitor rapidly induces chalcone synthase mRNA in Phaseolus vulgaris cells at the onset of the phytoalexin defence response. Proc. Natl. Acad. Sci. USA 81, 5724–5728Google Scholar
  38. Salvi, G., Giarizzo, F., DeLorenzo, G., Cervone, F. (1990) A polygalacturonase-inhibiting protein in the flowers of Phaseolus vulgaris L. J. Plant Physiol 136, 513–518Google Scholar
  39. Sanger, F., Nicklen, S., Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467Google Scholar
  40. Showalter, D.B., Sommer, S.S. (1989) The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Anal. Biochem. 177, 90–94Google Scholar
  41. Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–509Google Scholar
  42. Stotz, H.U., Powell, A.L.T., Damon, S.E., Greve, C.G., Bennet, A.B., Labavitch, J.M. (1993) Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv. Bartlett. Plant Physiol. 102, 133–138Google Scholar
  43. Toubart, P., Desideri, A., Salvi, G., Cervone, F., Daroda, L., DeLorenzo, G., Bergmann, C., Darvill, A.G., Albersheim, P. (1992) Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. Plant J. 2, 367–373Google Scholar
  44. Whal, G.M., Stern, M., Stark, G.R. (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethal-paper and rapid hybridization by using dextransulphate. Proc. Natl. Acad. Sci. USA 76, 3683–3685Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • F. Favaron
    • 1
  • R. D'Ovidio
    • 2
  • E. Porceddu
    • 2
  • P. Alghisi
    • 1
  1. 1.Istituto di Patoiogia vegetalePadovaItaly
  2. 2.Dipartimento diAgrobiologia e AgrochimicaViterboItaly

Personalised recommendations