Progress 09/01/07 to 08/31/12
Outputs
OUTPUTS: The most significant outputs of this project were the creation and characterization of new grain hardness alleles. The wheat grain hardness locus is functionally comprised of the two genes puroindoline a and b. In completing this project we were able to identify a large number of EMS derived puroindoline alleles. The characterization of these alleles demonstrated that the central region of the protein is in fact the most important and the active site of each protein. Due to varying levels of function remaining among the different alleles we were able to select defined levels of grain hardness among progeny lines. These results were disseminated via publications and by presentation at scientific meetings. PARTICIPANTS: PI, Mike Giroux, provided direction to graduate students and overall direction. co-PI, John Martin, contributes direction. Postdoc Kyung-Hee Kim project completed an initial analysis of how the Puroindolines modify seed polar lipid content. Research Associate Petrea Hofer focused upon milling and baking quality of the new puroindoline alleles. TARGET AUDIENCES: The target audience for this project is wheat breeders and geneticists and the results have been disseminated to them via publication in refereed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcome of this project is in two areas. First, we created a relatively large number of new puroindoline alleles that may prove useful in terms of improving wheat end product quality. Second, we demonstrated that the central tryptophan-rich region is the most sensitive region of puroindoline and mutation. This demonstrated that a focus upon this region may prove most fruitful in further studies designed to determine how the puroindolines control grain texture. This study could impact wheat breeding by demonstrating the ability to modify grain hardness and end product quality via selection of novel Ha loci. We had two publications in 2012. One focused on the role of puroindolines in wheat seed fungal resistance while the other on the stage of development at which puroindolines impact wheat seed lipid levels.
Publications
- Kim, K.-H., L. Feiz, J.M. Martin, and M.J. Giroux (2012) Puroindolines are associated with decreased polar lipid breakdown during wheat seed development. J Cereal Science 56(2):142-146.
- Kim, K-H., L. Feiz, A.T. Dyer, W. Grey, A.C. Hogg, J.M. Martin, and M.J. Giroux (2012) Increased resistance to Penicillium seed rot in transgenic wheat over-expressing puroindolines. J Phytopathology 160 (5):243-247.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: We have continued to focus on the characterization of new grain hardness alleles. The goal of this research was first to identify regions of the puroindolines that are most important for function of the proteins. The secondary goal was to identify individual puroindoline alleles and grain hardness levels ideal for different end product quality applications. The majority of the alleles we are working with came from an EMS treated hexaploid wheat population. The identification of these new alleles is being followed up on by each new allele upon wheat grain hardness and end product quality. We completed a complementary study examining whether the puroindolines could be used to decrease leaf fungal resistance in corn. PARTICIPANTS: PI, Mike Giroux, provided direction to graduate students and overall direction. co-PI, John Martin, contributes direction. M.S. graduate student Nicholas Reynolds completed his work on synthetic wheats. Postdoc Kyung-Hee Kim project is completing an initial analysis of how the Puroindolines modify seed polar lipid content. TARGET AUDIENCES: The target audiences for this project are wheat breeders and geneticists and the results have been disseminated to them via publication in refereed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcome of this project is in two areas. First, we created a relatively large number of new puroindoline alleles that may prove useful in terms of improving wheat end product quality. Second, we demonstrated that the central tryptophan-rich region is the most sensitive region of puroindoline and mutation. This demonstrated that a focus upon this region may prove most fruitful in further studies designed to determine how the puroindolines control grain texture. This study could impact wheat breeding by demonstrating the ability to modify grain hardness and end product quality via selection of novel Ha loci.
Publications
- Zhang, J., J.M. Martin, P. Balint-Kurti, L. Huang, and M.J. Giroux (2011) The wheat puroindoline genes confer fungal resistance in transgenic corn. Phytopathology 159:188-190.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Our recent efforts have continued to focus on the effects of variation in wheat hardness upon end product quality or the identification of new alleles. This area of research has proven fruitful in wheat in that we have identified many new alleles of both puroindoline genes. Several new puroindoline alleles were derived from synthetic wheats having a different D genome donor than that found in standard hexaploid wheat. However, all synthetic wheats conferred soft wheat texture in crosses with hexaploid wheat cultivars. Further, all synthetic wheat derived Hardness loci were either identical in function to the wild type Hardness locus found in hexaploid wheat or slightly less functional. The slightly less functional haplotypes conferred soft wheat texture that is about 8 units harder than soft hexaploid wheat varieties. This result was reported in a refereed journal article. The majority of the new Puroindolines we have identified have come from the screening of an EMS treated hexaploid wheat population. The identification of these new alleles was followed quite recently by an initial characterization of the effect of each new allele upon wheat grain hardness. The grain hardness values were used to directly predict the level of remaining gene function and demonstrated that the tryptophan rich domain of each puroindoline protein was the functional site. This study was also the first demonstration in planta of using mutagenesis to define the active site of a protein. We completed several complementary studies examining whether the puroindolines would bind to corn starch lipids and modify corn seed quality. We demonstrated that constitutive expression of the puroindolines is associated with increased embryo size and seed oil content in corn. PARTICIPANTS: PI, Mike Giroux, provided direction to graduate students and overall direction. co-PI, John Martin, contributes direction. M.S. graduate student Nicholas Reynolds completed his work on synthetic wheats. Postdoc Kyung-Hee Kim project is completing an initial analysis of how the Puroindolines modify seed polar lipid content. Research Associate Petrea Hofer has focused upon milling and baking quality of the new puroindoline alleles. TARGET AUDIENCES: The target audience for this project are wheat breeders and geneticists and the results have been disseminated to them via publication in refereed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcome of the puroindoline a and b (Pina and Pinb, respectively) mutagenesis project was two fold. First, we created a relatively large number of new puroindoline alleles that may prove useful in terms of improving wheat end product quality. Second, we demonstrated that the central tryptophan-rich region is the most sensitive region of puroindoline a and b to mutation. This demonstrated that a focus upon this region may prove most fruitful in further studies designed to determine how the puroindolines control grain texture. The major outcome of the synthetic wheat survey studies were in demonstrating that the majority of synthetic wheat derived Hardness loci impart increased grain hardness relative to the standard soft hexaploid wheat Hardness locus. This study could impact wheat breeding by demonstrating nthe ability to modify grain hardness and end product quality via selection of novel Ha loci.
Publications
- Zhang, J., J.M. Martin, B. Beecher, C. Lu, L.C. Hannah, M.L. Wall, I. Altosaar, and M.J. Giroux. (2010) The ectopic expression of the wheat puroindoline genes increase germ size and seed oil content in transgenic corn. Plant Molecular Biology 74:353-365.
- Reynolds, N.P., J.M. Martin, and M.J. Giroux. (2010) Novel Ha locus, Pina-D1c/Pinb-D1h, affects soft white spring wheat milling and baking. Cereal Chemistry 87(3):237-242.
- Finnie, S.M., R. Jeannotte, C.F. Morris, M.J. Giroux, and J.M. Faubion. (2010) Variation in polar lipids located on the surface of wheat starch. Journal of Cereal Science 51:73-80.
- Feiz, L., B.S. Beecher, J.M. Martin, and M.J. Giroux. (2009c) In planta mutagenesis determines the functional regions of the wheat puroindoline proteins. Genetics 183(3):853-860.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Activities: We created F2 populations of 46 unique puroindoline a and b missense alleles we created via EMS mutagenesis by crossing each back to the parent variety Alpowa. Grain hardness of the 46 EMS mutations was then measured using segregating F2:F3 populations. The method used was disseminated via presentation in a peer reviewed paper and the information on all alleles and their grain hardness is contained within another peer reviewed paper (in press). We also measured seed polar lipid content in wheat genotypes varying in puroindoline content. The results indicate that puroindoline expression is associated with increased seed polar lipid levels. The final project was the completion of experiments to assess the effects of puroindoline transgene expression upon corn starch quality. Corn starch wet milling yields were increased by puroindoline expression. The corn starch results were disseminated via a peer reviewed paper. PARTICIPANTS: PI, Mike Giroux, provided direction to graduate students and overall direction. co-PI, John Martin, contributes direction. M.S. graduate student Nicholas Reynolds is working on synthetic wheats. Ph.D. graduate student Leila Feiz's project was screening the EMS population and analysis of the new Pin alleles. She has also quantified membrane lipids in wheats varying in puroindoline content. Research Associate Petrea Hofer has focused upon milling and baking quality of the new puroindoline alleles. Undergraduate students Lindsay Elmore and Anna Snapp have helped with various aspects of this project. TARGET AUDIENCES: Target audiences have included colleagues at Montana State University and collaborators at Washington State University. We have also presented our results in two talks at national meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The puroindoline a and b (Pina and Pinb, respectively) mutagenesis project major outcome was that we were able to quantify the impact of individual missense alleles upon PIN function, as measured by grain hardness. The effect of the mutations ranged from neutral (74 %) to intermediate to function-abolishing. In comparisons of the effect of mutations within Pina and Pinb, we found that the percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB which indicated that PINB is more critical to overall Ha function. Further, the results demonstrated that the Tryptophan-rich region is the most critical to overall protein function. The study involving the relationship of puroindoline expression to seed polar lipid content led to the hyphothesis that PINs impact grain texture via stabilization of polar lipids in developing seeds. The corn starch wet milling project impact would be the ability to improve starch wet milling yields via transgenic expression of the wheat puroindolines.
Publications
- Feiz, L., H.W. Wanjugi, C.W. Melnyk, I. Altosaar, J.M. Martin, and M.J. Giroux. (2009a) Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content. J Cereal Science 50:91-98.
- Feiz, L., J.M. Martin, and M.J. Giroux. (2009b) Creation and functional analysis of new Puroindoline alleles in Triticum aestivum. Theoretical and Applied Genetics, 118:247-257.
- Zhang, J., J.M. Martin, B. Beecher, C.F. Morris, L.C. Hannah, and M.J. Giroux (2009) Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields. Plant Biotechnology 7:733-743.
- Turuspekov, Y., J.M. Martin, J.G.P. Bowman, B.S. Beecher, and M.J. Giroux. (2008) Associations between Vrs1 alleles and grain quality traits in spring barley Hordeum vulgare L. Cereal Chemistry, 85(6):817-823.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We completed the screening of an EMS wheat population consisting of 2000 lines and identified 50 total missense puroindoline alleles. The information has been disseminated via presentation at a national cereal chemistry meeting as well as in a peer reviewed paper (in press). These new alleles of puroindoline a and b, Pina and Pinb, respectively, are currently being analyzed as to their effects upon grain hardness. They will further be used to test their effects upon starch granule binding and starch lipid levels. We will ultimately also measure milling and baking quality. Our approach has proven successful in that testing of the new alleles has shown they vary widely in terms of function as determined by their effects upon grain hardness. This indicates that the new Pina and Pinb alleles will be useful in determining the structure/function relationships for both PINA and PINB. We have also developed methods to quantify lipids on the surface of starch granules and are in the process of determining individual lipids that are PINA or PINB specific. PARTICIPANTS: PI, Mike Giroux, provided direction to graduate students and overall direction. co-PI, John Martin, contributes direction. M.S. graduate student Nicholas Reynolds is working on synthetic wheats. Ph.D. graduate student Leila Feiz's project has been screening the EMS population and analysis of the new Pin alleles. She has also quantified membrane lipids in wheats varying in puroindoline content. Undergraduate students Lindsay Elmore and Anna Snapp have helped with various aspects of this project. TARGET AUDIENCES: Target audiences have included colleagues at Montana State University, collaborators at Washington State University. We have also presented our results in two talks at national meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our findings demonstrate that it is feasible to create new puroindoline alleles to enhance the range of grain hardness available to wheat breeders. More generally, our results demonstrate a mutagenesis followed by direct sequencing approach would be useful for many wheat genes in terms of increasing available allelic variation.
Publications
- Turuspekov, Y., B. Beecher, Y. Darlington, J. Bowman, T. K. Blake, and M. J. Giroux. (2008) Hardness Locus Sequence Variation and Endosperm Texture in Spring Barley. Crop Sci 48 (3):1007-1019.
- Lacerenza, J.A., J.M. Martin, L.E. Talbert, S.P. Lanning, and M.J. Giroux. (2008) Relationship of ethanol yield to agronomic and seed quality characteristics of small grains. Cereal Chemistry 85(3):322-328.
- Martin, J.M., J. D. Sherman, S. P. Lanning, L. E. Talbert, and M. J. Giroux. (2008). Effect of Variation in Amylose Content and Puroindoline Composition on Bread Quality in a Hard Spring Wheat Population. Cereal Chemistry 85 (2):266-269.
- Feiz, L., J.M. Martin, and M.J. Giroux. (2008) The relationship between wheat (Triticum aestivum L.) grain hardness and wet-milling quality. Cereal Chemistry 85(1):44-50.
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