Progress 12/01/09 to 11/30/13
Outputs
OUTPUTS: Our preliminary data indicated that QPM starches swelled more in water than opaque2 (o2) starch. Our originally proposed model was that the number of open spaces in QPM starch granules could facilitate access of large macromolecules to the surface or interior of starch resulting in higher sensitivity to hydrolytic enzymes. Further testing using a recombinant inbred line population from a cross of a soft o2 and a hard kernel Quality Protein Maize (QPM) line showed that there was no significant difference between opaque lines and vitreous, modified lines in their uptake of large fluorescently labeled macromolecules. In addition, analysis of the gene expression of four candidate starch biosynthesis genes revealed no correlation of endosperm modification with the expression level of any of the four genes. Thermal analysis of the starch from the RILs revealed that the parental allele for two genes, pullulanase (Zpu1) and starch synthase III (SSIII), most highly influenced starch structure. The effect of these alleles on starch structure were opposite one another, RILs expressing the QPM allele for pullulanase had lower peak endotherm temperatures while RILs expressing the QPM allele of SSIII had higher peak endotherm temperatures. This indicated that pullulanase was associated with shorter amylopectin branches and SSIII resulted in starch with longer amylopectin branches. More detailed analysis quantifying enzyme activity for pullulanase showed a significant correlation between pullulanase activity and endosperm modification, which was consistent with highly modified lines having shorter average amylopectin branches. Additional analysis of sensitivity to hydrolysis by alpha-amylase showed that the starch from highly modified lines was more resistant to hydrolysis. This is the opposite result from what was predicted by the original model. In a separate study we measured global gene expression in wild type and o2 lines, which showed that several starch biosynthesis genes were altered in their expression. Western blot analysis showed that branching enzyme I and starch synthase IIa were highly expressed at the protein level and may explain the observed change in the thermal properties of o2 starch. Dissemination- the results from the supported research project were presented annually at the Maize genetics conference, including a symposium talk in 2010. The research was also presented at a regional AAAS meeting in 2010 and at the 2012 American Society for Plant Biology meeting in 2012. Two papers are in preparation and are nearly complete. These will be submitted to well regarded plant biology journals. PARTICIPANTS: PI- Bryan Gibbon summer salary allowed full time effort for this project during the summers. Research Technician- Salary was provided for Kasi Clay, who was responsible for most of the DSC and size-exclusion analysis of starch as well as analysis of starch-associated proteins. Graduate Student- Hao Wu is performing the assays for pullulanase and starch synthases. Graduate Student- Mo Jia has performed qPCR analysis of several starch synthase genes in opaque and modified opaque lines. Undergraduates- Ashley Whitmeyer, Pricila Delgado, Priscilla Hosein, Malcolm Ladines, Stephanie Simcox, Sara Nelson, Tyler Mappe, Anna Meese, Sterling Love, Kelsey Moriarty and Bethany Eason have done independent research projects over the course of the project related to characterization of QPM starches. TARGET AUDIENCES: Maize genetics community- project data will enhance understanding of the establishment and regulation of endosperm texture. University and High School student groups- The PD has presented the NIFA sponsored research to the tri-Beta Biology Honor Society, Baylor Undergraduate Research in Science and Technology student organization and a group of high school juniors from underrepresented minorities visiting the Baylor University campus. Local community- project collaborations with World Hunger Relief, Inc. provide the opportunity for community outreach and education at two "Farm Day" events that are hosted each year. In addition, the PD lectures twice per year to the interns at the farm on plant breeding and genetics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research funded by NIFA has greatly improved our understanding of the role of starch granule structure in the suppression of the opaque phenotype in opaque2 genetic backgrounds. Our originally proposed model was that the number of open spaces in QPM starch granules could facilitate amylase to get access to the surface or interior of starch resulting in higher sensitivity to hydrolytic enzymes. Therefore, higher endosperm modification was predicted to result in more open space, thus pullulanase activity should be positively correlated with the amylase sensitivity. We observed the opposite result. The mechanism of this effect on endosperm modification is not certain, but at least our previous model needs to be modified. We hypothesize that pullulanase could produce more short branches which might reduce the accessibility of amylase to the surface of the starch granule. We expect from the thermal analysis data that amylopectin branch length distributions will be shorter on average in lines with high pullulanase activity. With these data we could establish a new model on the relationship between pullulanase, starch structure and endosperm hardness, in which high pullulanase activity results in shorter glucan chains that resist digestion and promote formation of the previously reported attachments between starch granules in mature QPM endosperm. Understanding the mechanisms of endosperm maturation and kernel hardness is important for maintaining and improving seed quality. The change in knowledge enabled by this research project has demonstrated that there is an important role for pullulanase to influence the properties of starch granules that is also associated with the restoration of kernel hardness in QPM. Future application of these findings may include improving the stability of kernel hardness in other soft maize lines or in response to stress such as heat.
Publications
- Wu, H., Clay, K. L., Thompson, S. S. and Gibbon, B. C. (2013) High pullulanase activity is associated with endosperm modification in quality protein maize. In Preparation.
- Saladine, S. J., Jia, M. and Gibbon, B.C. (2013) Maize eIF5A binds to eEF1A and is associated with a cytoskeleton-rich fraction in maize endosperm. In Preparation.
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Progress 12/01/11 to 11/30/12
Outputs
OUTPUTS: In this final year of the granting period we have made significant progress to understand the biochemical basis for the influence of starch structure on kernel hardness in opaque2 (o2) mutants and in Quality Protein Maize (QPM) lines carrying suppressors of the soft kernel phenotype. A recombinant inbred line population has been used to understand the contribution of specific starch biosynthesis enzymes to changes in vitreousness and starch structure. Quantitative enzyme activity assays showed that the pullulanse activity of QPM was significantly higher than that of o2. Suprisingly, the pullulanase activity does not correlate well with Zpu1 gene expression, but instead is correlated with the parental allele of ZpuI that the RIL expresses. High pullulanase activity correlated well with both kernel vitreousness and hardness. Pullulanase activity is also associated with changes in starch thermal properties measured by differential scanning calorimetry (DSC), which reflect alterations in amylose or amylopectin structure. There is significant negative correlation between pullulanase activity and onset or peak endotherm temperature, but no significant correlation with the total enthalpy of gellation. Higher pullulanase activity may shorten the glucan chain length, resulting in lower onset and maximum temperature. We have also tested alpha-amylase sensitivity of the starch from these lines and discovered that pullulanase activity was negatively correlated with alpha-amylase hydrolysis (i.e. lines with high Zpu1 activity were resistant to digestion). In another study we tested expression of several starch biosynthesis genes varied between W64A and W64Ao2 based on gene expression analysis, 2D SDS-PAGE and western blotting. Because levels of several starch biosynthesis enzymes were altered in o2, in particular SSIIa and BEI, the properties of the starch from W64A and W64Ao2 mature kernels were analyzed by DSC to determine if these changes affected the starch structure. The onset and peak endotherm temperatures as well as the total enthalpy of gelatinization were significantly higher for W64Ao2. The higher values for these thermal properties in o2 are consistent with starch that has longer amylopectin branches and higher crystalline starch content. To further characterize the structure of the starch, the amylopectin branch length distributions of W64A and W64Ao2 were measured. The two genotypes had similar molar percent content of glucans, but the distribution of glucans from W64Ao2 was shifted toward a higher degree of polymerization. Western blot analysis showed that BEI protein accumulation is substantially higher in o2. High expression of BEI alters starch crystallinity and branching pattern, which may alter the association of the starch granules with endosperm proteins and thus cause a soft, opaque phenotype. The results of this NIFA sponsored research were disseminated at the 2012 Maize Genetics conference in Portland, OR and at the 2012 American Society for Plant Biology annual meeting in Austin, TX. A manuscript is in preparation to report the data on starch biosynthesis and kernel hardness that is planned to be submitted to Plant Physiology. PARTICIPANTS: PI- Bryan Gibbon summer salary allowed full time effort for this project during the summer. Research Technician- Kasi Clay is responsible for most of the DSC and size-exclusion analysis of starch as well as analysis of starch-associated proteins. She left employment in July and the salary was rebudgeted to support a graduate student. Graduate Student- Hao Wu is performed the assays for pullulanase and starch synthases. Graduate Student- Mo Jia has performed qPCR analysis of several starch synthase genes in opaque and modified opaque lines and shown that SBEII is more highly expressed in o2. Undergraduates- Anna Meese, Sterling Love, Kelsey Moriarty and Bethany Eason have done independent research projects over the last year related to characterization of QPM starches, starch biosynthesis genes or genes that regulate o2. World Hunger Relief, Inc. is collaborating on the project by providing field space, equipment, fertilizers and irrigation. TARGET AUDIENCES: Maize genetics community- project data will enhance understanding of the establishment and regulation of endosperm texture. University and High School student groups- The PD has presented the NIFA sponsored research to the tri-Beta Biology Honor Society, Baylor Undergraduate Research in Science and Technology student organization and a group of high school juniors from underrepresented minorities visiting the Baylor University campus. Local community- project collaborations with World Hunger Relief, Inc. provide the opportunity for community outreach and education at two "Farm Day" events that are hosted each year. In addition, the PD lectures twice per year to the interns at the farm on plant breeding and genetics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our previous model was that the number of open spaces in QPM starch granules could facilitate amylase to get access to the surface or interior of starch resulting in higher sensitivity to amylases. Therefore, higher pullulanase activity should have produced more or large open space, so that the pullulanase activity should be positively correlated with the amylase sensitivity. The mechanism of this result is not certain, but at least, our previous model needs to be modified. We hypothesize that pullulanase could produce more short branches which might reduce the accessibility of amylase to the surface of the starch granule. However, this hypothesis needs to be further confirmed, and the measurement of glucan chain length is important. We expect from the prior data that amylopectin branch length distributions will be shorter on average in lines with high pullulanase activity, with these data we could establish a new model on the relationship between pullulanase, starch structure and endosperm hardness. Understanding the mechanisms of endosperm maturation and kernel hardness is important for maintaining and improving seed quality. The research over the past year has shown that there is an important role for pullulanase to influence the properties of starch granules that is also associated with the restoration of kernel hardness in QPM. Future application of these findings may include improving the stability of kernel hardness in other soft maize lines or in response to stress such as heat.
Publications
- Jia, M., Clay, K. L., Wu, H., Jung, R., Larkins, B. A. and Gibbon, B. C. (2013) Identification of Lysine-Rich Proteins and Starch Biosynthesis Gene Expression in the opaque2 Mutant by Transcriptional and Proteomic Analysis. BMC Plant Biol. Revision submitted.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: Objective 1) Analysis of Starch Biosynthesis and structure in QPM- In the past year we have performed an extensive analysis of the gene expression of the 4 starch biosynthesis genes that we discovered had distinct QPM-associated alleles. We analyzed 19 recombinant inbred lines (RILs) that varied in the extent of modification, and that were homozygous for different combinations of the parental alleles at the 4 loci. Correlation analysis showed that there was no statistically significant correlation of expression level of any of the genes with kernel vitreousness, hardness or density. Likewise gene expression levels did not correlate significantly with the thermal properties of the starch from these lines. This is possibly due to relatively high variance in gene expression from line to line. Analysis of the correlation of the specific parental allele carried by individual RILs showed that there was a very strong effect of the QPM allele for starch synthase III and the pullulanase type debranching enzyme on the thermal properties of the starch. The main effect of both loci was to decrease the total enthalpy of melting, which is generally associated with lower crystallinity. This structural change may explain part of the observation that QPM is generally more digestible than normal maize. Objective 2) Starch granule porosity and differential association of proteins with QPM starch- Analysis of the permeability of purified starches from inbred and RIL lines shows that QPM starches have a higher size exclusion limit for high molecular weight dextrans than o2 mutants. We have shown that o2 mutants have alterations in the expression of starch synthesis genes and that they have starch with thermal properties generally associated with more crystalline starches. We know that some cytosolic proteins can permeate QPM starches and hypothesized that QPM starch would preferentially associate with certain endosperm proteins. Our initial global analysis of proteins by 2D-SDS PAGE have been inconclusive and we are going to more specifically test the most likely candidate, which is the 27 kDa gamma-zein. This protein was recently shown to be necessary for restoration of vitreous endosperm. Ongoing work will test the influence of the higher porosity on starch digestibility. Two manuscripts are in preparation, one reporting the effect of the o2 mutation on protein and starch quality and another on the structural and biochemical changes observed in QPM lines and our RIL population. PARTICIPANTS: PI- Bryan Gibbon summer salary allows full time effort for this project during the summers. Research Technician- Kasi Clay is responsible for most of the DSC and size-exclusion analysis of starch as well as analysis of starch-associated proteins. Graduate Student- Hao Wu is performing the assays for pullulanase and starch synthases. Graduate Student- Mo Jia has performed qPCR analysis of several starch synthase genes in opaque and modified opaque lines. Undergraduates- Ashley Whitmeyer, Stephanie Simcox, Sara Nelson and Tyler Mappe have done independent research projects over the last year related to characterization of QPM starches. TARGET AUDIENCES: Maize genetics community- project data will enhance understanding of the establishment and regulation of endosperm texture. Local community- project collaborations with World Hunger Relief, Inc. provide the opportunity for community outreach and education at two "Farm Day" events that are hosted each year. In addition, the PD teaches 1-2 lectures per year to the interns at the farm on plant breeding and genetics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Understanding the mechanisms of endosperm maturation and kernel hardness is important for maintaining and improving seed quality. The research has shown that starch may not play a large role in restoring kernel hardness in QPM, however it has demonstrated that QPM specific alleles of starch synthesis enzymes are associated with the low crystallinity, more porous starch structure previously observed in QPM lines. This more porous starch structure may create a more digestible endosperm, which would be valuable for animal feeds.
Publications
- Holding, D.R, Hunter, B.G., Klingler, J.P. , Wu, S., Guo, X., Gibbon, B.C. , Wu, R., Schulze, J.-M., Jung, R. and Larkins, B.A. (2011) Characterization of opaque2 modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred. Theoret. Appl. Genet. 122:783-794. http://dx.doi.org/10.1007/s00122-010-1486-3
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: Good progress has been made on the qualitative characterization of starch synthase activities by zymogram anlaysis and the examination of starch granule porosity by high molecular weight fluorescent dextrans, as described in Objective 1 approach 1 and Objective 2 approach 3. Additionally, the analysis of a large number of starch samples from quality protein maize and starch mutant lines has been completed, as described in objective 1 approach 3. Additional data related to the expression of starch biosynthesis genes in opaque2 mutants has also been gathered. Some of these results have been reported at local and international scientific meetings as follows: Symposium talk- Suppression of opaque2 Phenotypes by Altered Starch Granule Structure. Salighedar, MM; Throneberry, K; Nguyen, HH; Medlin, R; Gibbon, BC. 2010. Maize Genetics Conference Abstracts. 52:Talk 17, Riva del Garda, Italy 3/20/10 Poster Presentation- Analysis of TOR kinase Pathway Gene Expression in Developing Maize Kernels and Opaque Mutants. (2010) Jia, M; Gibbon, BC.. Maize Genetics Conference Abstracts. 52:P57 Riva del Garda, Italy 3/18/10-3/20/10 Throneberry, Kyle, Mitra M. Salighedar, Hien H. Nguyen, Rachel Medlin and Bryan Gibbon (Baylor University) SUPPRESSION OF OPAQUE2 PHENOTYPES BY ALTERED STARCH GRANULE STRUCTURE. AAAS SW Regional Meeting, Houston, TX 4/10/10 PARTICIPANTS: PI- Bryan Gibbon summer salary allows full time effort for this project during the summers. Research Technician- Kasi Clay, an agronomy major from Texas A&M. This position allowed her to return to an Agricultural/Agronomy related job from an administrative position. Graduate Student- Hao Wu is setting up the assays for pullulanase and starch synthases. Graduate Student- Mo Jia has performed qPCR analysis of several starch synthase genes in opaque and modified opaque lines. Undergraduates- Ashley Whitmeyer, Pricila Delgado, Priscilla Hosein and Malcolm Ladines have done independent research projects over the last year related to characterization of QPM starches. TARGET AUDIENCES: Maize genetics and seed development community- project data will enhance understanding of the establishment and regulation of endosperm texture. Local community- project collaborations with World Hunger Relief, Inc. provide the opportunity for community outreach and education at two "Farm Day" events that are hosted each year. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The support of this project by NIFA enabled travel by the PI and his students to share results with other maize scientists at the maize genetics conference. In addition the fieldwork for this project is performed at a local non-profit farm that trains interns interested in international agricultural development and food security. This relationship has enabled the PI to provide extension outreach and educate the interns about developing maize lines with improved nutritional quality. From the data gathered so far we believe that changes in starch synthase II activity may be a less important factor in modification of opaque2 endosperm than originally indicated by our preliminary data. The analysis of a number of opaque2 mutant lines, starch biosynthesis mutants and modified opaque2 lines by differential scanning calorimetry showed that the thermal properties of starch from modified opaque2 lines was most similar to pullulanase and starch synthase III mutants. Therefore we are redirecting more effort to characterize these biosynthetic activities first, rather than starch synthase II.
Publications
- No publications reported this period
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