Theoretical and Applied Genetics

, Volume 110, Issue 3, pp 470–478 | Cite as

Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a

Original Paper


Puroindoline a (Pin-a) and puroindoline b (Pin-b), two basic isoforms encoded by the Pina-D1 and Pinb-D1 loci respectively, involved in controlling grain texture in wheat, were isolated from starch granules of soft wheat cultivars using three different extraction procedures, and fractionated by acidic polyacrylamide gel electrophoresis (A-PAGE). Tris buffer containing 1% Triton X-114 extracted Pin-a and small amounts of Pin-b, whereas 1% SDS preferably extracted Pin-b. Large amounts of both puroindolines were isolated by a solution containing 50% propan-2-ol and 50 mM NaCl. This solution extracted reduced amounts of Pin-b and no traces of Pin-a from starch granules of 20 hard common wheats containing the null allele Pina-D1b. The absence of Pin-a was confirmed by immunostaining with an anti-Pin-a antiserum. With the exception of two cultivars, null Pin-a cultivars gave no PCR fragment with three primer pairs specific to either the coding region or the promoter region of Pina-D1a, suggesting that major changes had occurred at the Pina-D1 locus in these genotypes. Cultivars Fortuna and Glenman were unique in giving size-specific PCR fragments with all primer pairs for the allele Pina-D1a and showed a cytosine deletion at position 267 in the coding region of the Pin-a gene, which resulted in a TGA stop codon at position 361. However, there was no evidence of a mutated protein in the A-PAGE or SDS-PAGE patterns of Fortuna and Glenman. The novel gene, provisionally named Pina-D1c, is the first null allele due to a point mutation that has been identified at the Pina-D1 locus.


Wheat Cultivar Starch Granule Chinese Spring Grain Texture Bread Wheat Cultivar 


  1. Blochet J-E, Chevalier C, Forest E, Pebay-Peyroula E, Gautier M-F, Joudrier P, Pezolet M, Marion D (1993) Complete amino acid sequence of puroindoline, a new basic and cysteine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning. FEBS Lett 329:336–340CrossRefPubMedGoogle Scholar
  2. Branlard G, Amiouur N, Igrejas G, Gaborit T, Herbette S, Dardevet M, Marion D (2003) Diversity of puroindolines as revealed by two-dimensional electrophoresis. Proteomics 3:168–174PubMedGoogle Scholar
  3. Capparelli R, Borriello G, Giroux MJ, Amoroso MG (2003) Puroindoline A-gene expression is involved in association of puroindolines to starch. Theor Appl Genet 107:1463–1468PubMedGoogle Scholar
  4. Corona V, Gazza L, Boggini G, Pogna NE (2001a) Variation in friabilin composition as determined by A-PAGE fractionation and PCR amplification, and its relationship to grain hardness in bread wheat. J Cereal Sci 34:243–250Google Scholar
  5. Corona V, Gazza L, Zanier R, Pogna NE (2001b) A tryptophan-to-arginine change in the triptophan-rich domain of puroindoline b in five French bread wheat cultivars. J Genet Breed 55:187–189Google Scholar
  6. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21Google Scholar
  7. Dubreil L, Compoint JP, Marion D (1997) Interaction of puroindoline with wheat flour polar lipids determines their foaming properties. J Agric Food Chem 45:108–116CrossRefGoogle Scholar
  8. Dubreil L, Gaborit B, Bouchet B, Gallant DJ, Broekaert L, Quillien L, Marion D (1998) Spatial and temporal distribution of the major isoforms of puroindolines (puroindoline-a and puroindoline-b) and non specific lipid transfer protein (ns-LTP1e1) of Triticum aestivum seeds. Relationships with their in vitro antifungal properties. Plant Sci 138:121–135CrossRefGoogle Scholar
  9. Gautier MF, Aleman ME, Guirao D, Marion D, Joudrier P (1994) Triticum aestivum puroindolines, two basic cysteine-rich seed proteins: cDNA sequence analysis and developmental gene expression. Plant Mol Biol 25:43–57PubMedGoogle Scholar
  10. Giroux M, Morris CF (1997) A glycine to serine change in puroindoline b is associated with grain hardness and low levels of starch-surface friabilin. Theor Appl Genet 95:857–864CrossRefGoogle Scholar
  11. Giroux MJ, Morris CF (1998) Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b. Proc Natl Acad Sci USA 95:6262–6266Google Scholar
  12. Giroux MJ, Talbert L, Habernicht DK, Lanning S, Hemphill A, Martin JM (2000) Association of puroindoline sequence type and grain hardness in hard red spring wheat. Crop Sci 40:370–374Google Scholar
  13. Greenwell P, Schofield JD (1986) A starch granule protein associated with endosperm softness in wheat. Cereal Chem 63:379–380Google Scholar
  14. Greenwell P, Schofield JD (1989) The chemical basis of grain hardness and softness. In: Helsinki H, Salovaara (eds) Wheat end-use properties, University of Helsinki and Lahti Research Training Center, pp 59–72Google Scholar
  15. Hogg AC, Sripo T, Beecher B, Martin JM, Giroux MJ (2004) Wheat puroindolines interact to form friabilin and control wheat grain hardness. Theor Appl Genet 108:1089–1097PubMedGoogle Scholar
  16. Krishnamurthy K, Giroux MJ (2001) Expression of wheat puroindoline genes in transgenic rice confers grain softness. Nat Biotechnol 19:162–166Google Scholar
  17. Krishnamurthy K, Balconi C, Sherwood JE, Giroux MJ (2001) Wheat puroindolines enhance fungal disease resistance in transgenic rice. Mol Plant Microbe Interact 14:1255–1260PubMedGoogle Scholar
  18. Igrejas G, Leroy P, Charmet G, Gaborit T, Marion D, Branlard G (2002) Mapping QTLs for grain hardness and puroindoline content in wheat (Triticum aestivum L). Theor Appl Genet 106:19–27PubMedGoogle Scholar
  19. Igrejas G, Gaborit T, Oury F-X, Chiron H, Marion D, Branlard G (2001) Genetic and environmental effects on puroindoline-a and puroindoline-b content and their relationships to technological parameters in French bread wheats. J Cereal Sci 34:37–47Google Scholar
  20. Law CN, Young CF, Brown JWS, Snape JW, Worland AJ (1978) The study of grain protein control in wheat using whole chromosome substitution lines. In: Seed protein improvement by nuclear techniques. International Atomic Energy Agency, Vienna, pp 483–490Google Scholar
  21. Lillemo M, Morris CF (2000) A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe. Theor Appl Genet 100:1100–1107CrossRefGoogle Scholar
  22. Lillemo M, Simeone MC, Morris CF (2002) Analysis of puroindoline a and b sequences from Triticum aestivum cv. “Penawawa” and related diploid taxa. Euphytica 126:321–331CrossRefGoogle Scholar
  23. Mattern PJ, Morris R, Schmidt JW, Johnson VA (1973) Location of genes for kernel properties in the wheat variety “Cheyenne” and chromosome substitution lines. In: Sears ER, Sears LMS (eds) Proceedings of the 4th international wheat genetic symposium, University of Missouri Press, Columbia, pp 703–707Google Scholar
  24. Morris CF, Greenblatt GA, Bettge AD, Malkawi HI (1994) Isolation and characterization of multiple forms of friabilin. J Cereal Sci 21:167–174CrossRefGoogle Scholar
  25. Pogna NE, Gazza L, Corona V, Zanier R, Niglio A, Mei E, Palumbo M, Boggini G (2002) Puroindolines and kernel hardness in wheat species. In: Ng PKW, Wrygley CW (eds) Wheat quality elucidation. AACC, St. Paul, pp 155–169Google Scholar
  26. Turnbull KM, Gaborit T, Marion D, Rahman S (2000) Variation in puroindoline polypeptides in Australian cultivars in relation to grain hardness. Aust J Plant Physiol 27:153–158Google Scholar
  27. Turnbull KM, Marion D, Gaborit T, Appels R, Rahman S (2003) Early expression of grain hardness in the developing wheat endosperm. Planta 216:699–706PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • L. Gazza
    • 1
  • F. Nocente
    • 1
  • P. K. W. Ng
    • 2
  • N. E. Pogna
    • 1
  1. 1.Istituto Sperimentale per la CerealicolturaRomeItaly
  2. 2.Department of Food Science and Human NutritionMichigan State UniversityEast LansingUSA

Personalised recommendations