Plant Molecular Biology

, Volume 25, Issue 1, pp 43–57

Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression

  • Marie-Françoise Gautier
  • Marie-Elisabeth Aleman
  • Anne Guirao
  • Didier Marion
  • Philippe Joudrier
Research Articles


From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthezised as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanism.

Key words

cDNA sequence cystine-rich proteins gene expression puroindolines tryptophan-rich domain wheat 


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  1. 1.
    Arondel V, Kader JC: Lipid transfer in plants. Experientia 46: 579–585 (1990).PubMedGoogle Scholar
  2. 2.
    Bednarek SY, Wilkins TA, Dombrowski JE, Raikhel NV: A carboxy-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell 2: 1145–1155 (1990).CrossRefPubMedGoogle Scholar
  3. 3.
    Blochet JE, Kaboulou A, Compoint JP, Marion D: Amphiphilic proteins from wheat flour: specific extraction, structure and lipid binding properties. In: Bushuk W, Thachuk R (eds), Gluten Proteins, pp. 314–325 (1991).Google Scholar
  4. 4.
    Blochet JE, Chevalier C, Forest E, Pebay-Peyroula E, Gautier MF, Joudrier P, Pézolet M, Marion D: Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X114 phase partitioning. FEBS Lett 329: 336–340 (1993).CrossRefPubMedGoogle Scholar
  5. 5.
    Buonocore V, DeBiasi M, Giardina P, Poerio E, Silano V: Purification and properties of an α-amylase tetrameric inhibitor from wheat kernel. Biochim Biophys Acta 831: 40–48 (1985).Google Scholar
  6. 6.
    Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloro-form extraction. Anal Biochem 162: 156–159 (1987).CrossRefPubMedGoogle Scholar
  7. 7.
    Désormeaux A, Blochet JE, Pézolet M, Marion D: Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed by infrared and raman spectroscopy. Role of disulfide bridges and phospholipids in the stabilization of the α-helix structure. Biochim Biophys Acta 1121: 137–152 (1992).PubMedGoogle Scholar
  8. 8.
    Dieryck W: Etude des gènes codant pour les protéines de transfert de lipides de 7 et 9 kDa de blé dur. Production de la protéine de transfert de lipides de 9 kDa dans E. coli. Thèse de Doctorat de l'Université Blaise Pascal, Clermont-Ferrand, 130 pp. (1993).Google Scholar
  9. 9.
    Dieryck W, Gautier MF, Lullien V, Joudrier P: Nucleotide sequence of a cDNA encoding a lipid transfer protein from wheat (Triticum durum Desf.) Plant Mol Biol 19: 707–709 (1992).PubMedGoogle Scholar
  10. 10.
    Fabijanski S, Chang SC, Dukiandjiev S, Bahramian MB, Ferrara P, Altosaar I: The nucleotide sequence of a cDNA for a major prolamin (avenin) in oat (Avena sativa L. cultivar Hinoat) which reveals homology with oat globulin. Biochem Physiol Pflanzen 183: 143–152 (1988).Google Scholar
  11. 11.
    Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P: Plant proteinaceous inhibitors of proteinases and α-amylases. Oxford Surv Plant Mol Cell Biol 4: 275–334 (1987).Google Scholar
  12. 12.
    Gatineau E, Toma F, Montenay-Garestier T, Takechi M, Fromageot P, Ménez A: A role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from naja nigricollis venom. Biochemistry 26: 8046–8055 (1987).PubMedGoogle Scholar
  13. 13.
    Gautier MF, Alary R, Joudrier P: Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein. Plant Mol Biol 14: 313–322 (1990).PubMedGoogle Scholar
  14. 14.
    Gautier MF, Alary R, Lullien V, Joudrier P: Nucleotide sequence of a cDNA encoding the wheat (Triticum durum Desf.) CM2 protein. Plant Mol Biol 16: 333–334 (1991).PubMedGoogle Scholar
  15. 15.
    Gerard GF, Miller K: Comparison of glyoxal and form-aldehyde gels for sizing rRNAs. Focus (Gibco BRL) 8: 5–6 (1986).Google Scholar
  16. 16.
    Greenwell P: Wheat starch granule proteins and their technological significance. In: Murray I (ed) Proceedings of the 37th Australian cereal chemistry conference, pp. 100–103. Cereal Chemistry Division Royal Australian Chemical Institute, Melbourne (1987).Google Scholar
  17. 17.
    Greenwell P: Biochemical studies of endosperm texture in wheat. 9th International Cereal and Bread Congress, Paris, 1–5 June, Abstract E4. Ind Céréales 77: 20 (1992).Google Scholar
  18. 18.
    Greenwell P, Schofield JD: A starch granule protein associated with endosperm softness in wheat. Cereal Chem 63: 379–380 (1986).Google Scholar
  19. 19.
    Greenwell P, Schofield JD: The chemical basis of grain hardness and softness. In: Wheat End-Use Properties. Proceedings from ICC Symposium 89. University of Helsinki and Lahti Research Training Center, Helsinki, pp. 59–72 (1989).Google Scholar
  20. 20.
    Grosset J, Marty I, Chartier Y, Meyer Y: mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible. Plant Mol Biol 15: 485–496 (1990).PubMedGoogle Scholar
  21. 21.
    Gutierrez C, Sanchez-Monge R, Gomez L, Ruiz-Tapiador M, Castanera P, Salcedo G: α-amylase activities of agricultural insect pests are specifically affected by different inhibitor preparation from wheat and barley endosperm. Plant Sci 72: 37–44 (1990).CrossRefGoogle Scholar
  22. 22.
    Holwerda BC, Galvin NJ, Baranski TJ, Rogers JC: In vitro processing of aleurain, a barley vacuolar thiol protease. Plant Cell 2: 1091–1106 (1990).CrossRefPubMedGoogle Scholar
  23. 23.
    Jolly CJ, Rahman S, Kortt AA, Higgins TJV: Characterization of the wheat Mr 15 000 ‘grain softness protein’ and analysis of the relationship between its accumulation in the whole seed and grain softness. Theor Appl Genet 86: 589–597 (1993).Google Scholar
  24. 24.
    Joshi CP: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15: 6643–6653 (1987).PubMedGoogle Scholar
  25. 25.
    Kashlan N, Richardson M: The complete amino acid sequence of a major wheat protein inhibitor of α-amylase. Phytochemistry 20: 1781–1784 (1981).CrossRefGoogle Scholar
  26. 26.
    Kornfeld S, Mellman I: The biogenesis of lysosomes. Annu Rev Cell Biol 5: 483–525 (1989).PubMedGoogle Scholar
  27. 27.
    Kroczek R, Siebert E: Optimization of northern analysis by vacuum-blotting, RNA transfer visualisation and ultraviolet fixation. Anal Biochem 184: 90–95 (1990).PubMedGoogle Scholar
  28. 28.
    Le Guernevé C: Propriétés physico-chimiques des lipides polaires en relation avec les caractéristiques des pâtes boulangères. Mise en évidence des interactions entre une protéine de blé et les lipides polaires. Thèse de l'Université de Nantes, 159 pp. (1992).Google Scholar
  29. 29.
    Lullien V, Alary R, Joudrier P, Gautier MF: Characterization of cDNA clone encoding the Triticum aestivum L. CM16 protein: homology with the Triticum durum Desf. sequence. Plant Mol Biol 16: 373–374 (1991).PubMedGoogle Scholar
  30. 30.
    Lullien V, Alary R, Guirao A, Joudrier P, Gautier MF: Isolation and nucleotide sequence of a cDNA clone encoding the bread wheat (Triticum aestivum L.) CM17 protein. Plant Mol Biol 17: 1081–1082 (1991).PubMedGoogle Scholar
  31. 31.
    Lundgard R, Svensson B: The four major forms of barley β-amylase. Purification, characterization and structural relationship. Carlsberg Res Commun 52: 313–326 (1987).Google Scholar
  32. 32.
    Maeda K, Kakabayashi S, Matsubara H: Complete amino acid sequence of an α-amylase inhibitor in wheat kernel (0.19 inhibitor). Biochim Biophys Acta 828: 213–221 (1985).PubMedGoogle Scholar
  33. 33.
    Matsuoka K, Nakamura K: Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proc Natl Acad Sci USA 88: 834–838 (1991).PubMedGoogle Scholar
  34. 34.
    Messing J, Geraghty D, Heidecker G, Hu NT, Kridl J, Rubenstein I: Plant gene structure. In: Kosuge T, Meredith CP, Hollaender A (eds), Genetic Engineering of plants, pp. 211–227. Plenum Press, New York (1983).Google Scholar
  35. 35.
    Monnet FP: Caractérisation d'une protéine de fixation de lipides de blé dur, purification, séquençage, ADN complémentaire; relations aux protéines végétales de transfert de lipides et aux inhibiteurs d'α-amylase/trypsine des céréales. Thèse de l'Université de Montpellier, 121 pp. (1990).Google Scholar
  36. 36.
    Mullis KB, Faloona FA: Specific synthesis of DNA in-vitro via a polymerase catalysed chain reaction. Meth Enzymol 155: 335–350 (1987).PubMedGoogle Scholar
  37. 37.
    Neuhaus JM, Sticher L, Meins F, Boller T: A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88: 10362–10366 (1991).PubMedGoogle Scholar
  38. 38.
    Rahman S, Jolly CJ, Higgins TJ: The chemistry of wheat-grain hardness. Chem Australia, September: 397 (1991).Google Scholar
  39. 39.
    Rasmussen SK, Welinder KG, Hejgaard J: cDNA cloning, characterization and expression of an endosperm-specific barley peroxidase. Plant Mol Biol 16: 317–327 (1991).PubMedGoogle Scholar
  40. 40.
    Rogers SO, Bendich AJ: Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5: 69–76 (1985).Google Scholar
  41. 41.
    Russell PL, Gough BM, Greenwell P, Fowler A, Munro HS: A study by ESCA of the surface of native and chlorine-treated wheat starch granules. J Cereal Sci 5: 83–100 (1987).Google Scholar
  42. 42.
    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).PubMedGoogle Scholar
  43. 43.
    Schiffer M, Chang CH, Stevens FJ: The functions of tryptophan residues in membrane proteins. Protein Engin 5: 213–214 (1992).Google Scholar
  44. 44.
    Schofield JD, Greenwell P: Wheat starch granule proteins and their technological significance. In: Morton ID (ed), Cereals in a European Context, pp. 407–420. Ellis Horwood, Chichester (1987).Google Scholar
  45. 45.
    Selsted ME, Levy JN, VanAbel RJ, Cullor JS, Bontems RJ, Barany G: Purification, characterization, synthesis and cDNA cloning of indolicidin: A tryptophan-rich microbicidal tridecapeptide from neutrophils. In: Smith JA, Rivier JE (eds), Peptides: Chemistry and Biology, pp. 905–907. ESCOM Science Publishers, Leiden, Netherlands (1991).Google Scholar
  46. 46.
    Shewry PR, Field JM, Kirkman MA, Faulks AJ, Miflin BJ: The extraction, solubility, and characterization of two groups of barley storage polypeptides. J Exp Bot 31: 393–407 (1980).Google Scholar
  47. 47.
    Silano V, Furia M, Gianfreda L, Macri A, Palescandolo R, Rab A, Scardi V, Stella E, Valfre F: Inhibition of amylases from different origins by albumins from the wheat kernel. Biochim Biophys Acta 391: 170–178 (1975).PubMedGoogle Scholar
  48. 48.
    Sogaard M, Olsen FL, Svensson B: C-terminal processing of barley α-amylase 1 in malt, aleurone protoplasts, and yeast. Proc Natl Acad Sci USA 88: 8140–8144 (1991).PubMedGoogle Scholar
  49. 49.
    Sommer R, Tautz D: Minimal homology requirements for PCR primers. Nucl Acids Res 16: 6749 (1989).Google Scholar
  50. 50.
    Stephen CJ, Jones C, Schofield JP: A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucl Acids Res 18: 7463–7464 (1990).PubMedGoogle Scholar
  51. 51.
    Stuart LS, Harris TH: Bactericidal and fungicidal properties of a crystalline protein isolated from unbleached wheat flour. Cereal Chem 19: 288–300 (1942).Google Scholar
  52. 52.
    Terras FRG, Goderis IJ, VanLeuven F, Vanderleyden J, Cammue PA, Broekaert WF: In vitro antifungal activity of a radish (Raphanus sativus L.) seed protein homologous to nonspecific lipid transfer proteins. Plant Physiol 100: 1055–1058 (1992).Google Scholar
  53. 53.
    Van denBulcke M, Bauw G, Castresana C, VanMontagu M, Vandekerckhove J: Characterization of vacuolar and extracellular β-1,3-glucanases of tobacco: evidence for a strictly compartementalized plant defense system. Proc Natl Acad Sci USA 86: 2673–2677 (1989).Google Scholar
  54. 54.
    Vitale A, Chrispeels MJ: Sorting of proteins to the vacuoles of plant cells. BioEssays 14: 151–160 (1992).PubMedGoogle Scholar
  55. 55.
    Von-Heijne G: A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14: 4683–4690 (1986).PubMedGoogle Scholar
  56. 56.
    Welinker KG: Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence and some structural characteristics of horseradish peroxidase c. Eur J Biochem 96: 483–502 (1979).PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Marie-Françoise Gautier
    • 1
  • Marie-Elisabeth Aleman
    • 1
  • Anne Guirao
    • 1
  • Didier Marion
    • 2
  • Philippe Joudrier
    • 1
  1. 1.Laboratoire de Biochimie et Biologie Moléculaire des CéréalesINRAMontpellier Cédex 01France
  2. 2.Laboratoire de Biochimie et Technologie des ProtéinesINRANantes Cédex 03France

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