Proteome analysis of two soft-grained wheats of different processing quality: cultivar-specific proteins
D. J. Skylas A B F , S. J. Cordwell A , G. Craft A , B. McInerney A , M. J. Wu B C , J. Chin B C , C. W. Wrigley B D and P. J. Sharp B EA Australian Proteome Analysis Facility, Macquarie University, NSW 2109, Australia.
B Value Added Wheat CRC Ltd, North Ryde, NSW 1670, Australia.
C NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, NSW 2570, Australia.
D Food Science Australia, North Ryde, NSW 1670, Australia.
E Plant Breeding Institute, University of Sydney, Camden, NSW 2570, Australia.
F Corresponding author. Email: dskylas@proteome.org.au
Australian Journal of Agricultural Research 56(2) 145-155 https://doi.org/10.1071/AR04114
Submitted: 17 May 2004 Accepted: 20 December 2004 Published: 28 February 2005
Abstract
Proteome analysis was conducted on the grain of 2 closely related soft biscuit-making wheats (Bowie and Rosella cultivars) differing in processing quality. Comparisons between these wheat cultivars were carried out on total wholemeal proteins, extracts with enriched starch granule proteins, and extracts enriched with gliadin storage proteins, with the intention of characterising, identifying, and cataloguing cultivar-specific proteins that could be used for segregation purposes. Initially, 2-dimensional gel electrophoresis was carried out on total wholemeal proteins using a broad range pH 3–10 immobilised pH gradient for the first dimension. Further screening was carried out using a combination of mid to narrow range immobilised pH gradients, including pH 4–7, 5.5–6.7, 5–8, 6–9, and 6–11. Best cultivar-specific protein fractionation was provided by the pH 5–8 range. Altogether, 4 unique cultivar-specific protein spots were excised from the pH 5–8 gels and identified by means of peptide mass fingerprinting, tandem mass spectrometry, or N-terminal sequencing. Starch granule protein extracts were prepared and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A western blot was performed and probed with an anti-puroindoline-a antibody. Further to this, extracts enriched in gliadin storage proteins were isolated using 70% ethanol and analysed by 2-dimensional gel electrophoresis. The resulting gliadin protein maps showed 18 unique cultivar-specific gliadins. They were excised from the pH 6–9 gels and submitted for N-terminal amino acid sequencing. Overall, this study identified 23 proteins that could be used to distinguish between these closely related cultivars and may provide information on the molecular basis for the differences in processing exhibited by these wheats. The findings reported also contribute to a longer term objective of developing a broad and comprehensive knowledge base of commercial wheats, in regard to protein composition and their inherent processing qualities.
Additional keywords: wheat, wholemeal, proteomics, cultivar-discrimination.
Acknowledgments
This research has been facilitated by access to the Australian Proteome Analysis facility (APAF) established under the Australian Governments’ Major National Research Facilities program. This study was funded by the Value Added Wheat CRC. Daniel J. Skylas is a Post-doctoral Fellow funded by the Value Added Wheat CRC.
Amiour N,
Merlino M,
Leroy P, Branlard G
(2002) Proteomic analysis of amphiphilic proteins of hexaploid wheat kernels. Proteomics 2, 632–641.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
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 Letters 329, 336–340.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Clarke BC,
Hobbs M,
Skylas D, Appels R
(2000) Genes active in developing wheat endosperm. Functional and Integrated Genomics 1, 44–55.
Cornish GB,
Bekes F,
Allen HM, Martin DJ
(2001a) Flour proteins linked to quality traits in an Australian doubled haploid wheat population. Australian Journal of Agricultural Research 52, 1339–1348.
| Crossref | GoogleScholarGoogle Scholar |
Cornish GB,
Skylas DJ,
Siriamornpun S,
Bekes F,
Wrigley CW, Wootton M
(2001b) Grain proteins as markers of genetic traits in wheat. Australian Journal of Agricultural Research 52, 1161–1171.
| Crossref | GoogleScholarGoogle Scholar |
Cozzolino R,
Di Giorgi S,
Fisichella S,
Garozzo D,
Lafiandra D, Palermo A
(2001) Proteomics of gluten: mapping of subunit 1 Ax2* in Cheyenne cultivar by matrix-assisted laser desorption/ionisation. Rapid Communications in Mass Spectrometry 15, 1129–1135.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Finnie C,
Melchior S,
Roepstorff P, Svensson B
(2002) Proteome analysis of grain filling and seed maturation in barley. Plant Physiology 129, 1308–1319.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Gallardo K,
Job C,
Groot SP,
Puype M,
Demol H,
Vandekerckhove J, Job D
(2001) Proteomic analysis of arabidopsis seed germination and priming. Plant Physiology 126, 835–848.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Gobom J,
Nordhoff E,
Mirgorodskaya E,
Ekman R, Roepstorff P
(1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionisation mass spectrometry. Journal of Mass Spectrometry 34, 105–116.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Granier F
(1988) Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 9, 712–718.
| PubMed |
Hill RD, Walker-Simmons M, Robertson M
(1988) Effect of environment and ABA on the synthesis of alpha-amylase inhibitor in cereal grains during development and germination. ‘Proceedings of the 5th International Symposium on Pre-harvest Sprout Cereals’. Norway. (Ed. K Ringlund ,
E Mosleth ,
DJ Mares )
pp. 130–138. (Westview Press: Boulder, CO)
Kersten B,
Bürkle L,
Kuhn EJ,
Giavalisco P,
Konthur Z,
Lueking A,
Walter G,
Eickhoff H, Schneider U
(2002) Large-scale plant proteomics. Plant Molecular Biology 48, 133–141.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Komatsu S,
Muhammad A, Rakwal R
(1999) Separation and characterisation of proteins from green and etiolated shoots of rice (Oryza sativa L.): towards a rice proteome. Electrophoresis 20, 630–636.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Kussmann M,
Nordhoff E,
Rahbek-Nielsen H,
Haebel S, Rossel-Larsen M , et al.
(1997) Matrix-assisted laser desorption/ionisation mass spectrometry sample preparation techniques designed for various peptide and protein analytes. Journal of Mass Spectrometry 32, 593–601.
| Crossref | GoogleScholarGoogle Scholar |
Larsen MR,
Cordwell SJ, Roepstorff P
(2002) Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionisation-mass spectrometry. Proteomics 2, 1277–1287.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Marques K,
Sarazin B,
Chane-Favre L,
Zivy M, Thiellement H
(2001) Comparative proteomics to establish genetic relationships in the Brassicaceae family. Proteomics 1, 1457–1462.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Masojć P, Larsson-Raźnikiewicz M
(1991) Variations of the levels of α-amylase and endogenous α-amylase inhibitor in rye and triticale grain. Swedish Journal of Agricultural Research 21, 3–9.
Masojć P,
Zawistowski J,
Howes NK,
Aung T, Gale MD
(1993) Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat. Theoretical and Applied Genetics 85, 1043–1048.
Matsudaira P
(1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. Journal of Biological Chemistry 262, 10 035–10 038.
Molloy MP
(2000) Two-dimensional electrophoresis of membrane proteins using immobilised pH gradients. Analytical Biochemistry 280, 1–10.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Østergaard O,
Melchior S,
Roepstorff P, Svensson B
(2002) Initial proteome analysis of mature barley seeds and malt. Proteomics 2, 733–739.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Payne PI,
Nightingale MA,
Krattiger AF, Holt LM
(1987) The relationship between the HMW glutenin subunit composition and the bread-making quality of British grown wheat varieties. Journal of the Science of Food and Agriculture 40, 51–65.
Rakwal R,
Agrawal GK, Yonekura M
(1999) Separation of proteins from stressed rice (Oryza sativa L.) leaf tissues by two dimensional polyacrylamide gel electrophoresis: induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride. Electrophoresis 20, 3472–3478.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Rakwal R, Komatsu S
(2000) Role of jasmonate in the rice (Oryza sativa L.) self-defence mechanism using proteome analysis. Electrophoresis 21, 2492–2500.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Roberts JKM
(2002) Proteomics and a future generation of plant molecular biologists. Plant Molecular Biology 48, 143–154.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Skylas DJ,
Cordwell SJ,
Hains PG,
Larsen MR,
Basseal DJ,
Walsh BJ,
Blumenthal C,
Rathmell W,
Copeland L, Wrigley CW
(2002) Heat shock of wheat during grain filling: proteins associated with heat-tolerance. Journal of Cereal Science 35, 175–188.
| Crossref | GoogleScholarGoogle Scholar |
Skylas DJ,
Mackintosh JA,
Cordwell SJ,
Basseal DJ,
Walsh BJ,
Harry J,
Blumenthal C,
Copeland L,
Wrigley CW, Rathmell W
(2000) Proteome approach to the characterisation of protein composition in the developing and mature wheat-grain endosperm. Journal of Cereal Science 32, 169–188.
| Crossref | GoogleScholarGoogle Scholar |
Skylas DJ,
Walsh BJ,
Copeland L,
Rathmell W, Wrigley CW
(2001) The wheat-grain proteome as a basis for more efficient cultivar identification. Proteomics 1, 1542–1546.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Stacy RAP,
Munthe E,
Steinum T,
Sharma B, Aalen RB
(1996) A peroxiredoxin antioxidant is encoded by a dormancy-related gene, Per1, expressed during late development in the aleurone and embryo of barley grains. Plant Molecular Biology 31, 1205–1216.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Thiellement H,
Bahrman N,
Damerval C,
Plomion C,
Rossignol M,
Santoni V,
de Vienne D, Zivy M
(1999) Proteomics for genetic and physiological studies in plants. Electrophoresis 20, 2013–2026.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vawser M, Cornish GB, Shepherd KW
(2002) Rheological dough properties of Aroona isolines differing in glutenin subunit composition. ‘Proceedings of the 52nd Australian Cereal Chemistry Conference’. New Zealand. (Ed. CK Black ,
JF Panozzo ,
CW Wrigley ,
IL Batey ,
N Larsen )
pp. 53–58. (Royal Australian Chemical Institute: Melbourne, Vic)
Wrigley CW
(1994) Developing better strategies to improve grain quality for wheat. Australian Journal of Agricultural Research 45, 1–17.
| Crossref | GoogleScholarGoogle Scholar |
