Progress 06/01/20 to 11/30/24
Outputs
Target Audience:The long-term goal of this project is to expand and validate the applications of phytoglycogen as a plant product, and conversely develop sweet corn varieties selected to supply the needs for this application. Our primary target audience are stakeholders in the production of sweet corn as well as stakeholders involved in the use of plant products for biomedical applications. They include sweet corn breeding programs in seed companies, sweet corn growers, and companies interested in applications of phytoglycogen. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students and 1 undergraduate student was trained in the scope of this project. The undergraduate student has now been accepted to continue his training and will pursue graduate training. How have the results been disseminated to communities of interest?The results were communicated in different scientific conferences (Plant Science and Biomedical Sciences). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This report comprises a short no cost extension that was requested to finalize pending papers. Two manuscripts have now been submitted and are pending review. The results from each aim have now been completed and communicated.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2025
Citation:
Park J., Liu R., Boehlein S.K., Resende M.F.R., Hudalla G. Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. CSTAR 2025
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2025
Citation:
Mahon A., Suzuki N., Peixoto M.A., Leach K.A., Gustin J., Boehlein S.K., Resende M.F.R. Using Single-kernel Near-Infrared spectroscopy for prediction of phytoglycogen and other seed composition traits. Submitted
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2025
Citation:
Boehlein, S.K., Hennen-Bierwagen, T.A., Shuler, S.L., Tracy, W.F., Hannah, L.C., Resende, M.F.R, Myers, A.M. - Physical and regulatory interactions of two separately conserved (1-6) glycoside hydrolases that participate in maize endosperm starch biosynthesis. Submitted
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Park J., Liu R., Kim A.S., Cyr N.N., Boehlein S.K., Resende M.F.R., Savin D.A., Bailey L.S., Sumerlin B.S., Hudalla G. Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. SFB 2025
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Park J., Liu R., Kim A.S., Cyr N.N., Boehlein S.K., Resende M.F.R., Savin D.A., Bailey L.S., Sumerlin B.S., Hudalla G. Sweet Corn Phytoglycogen Dendrimers as a Lyoprotectant for Dry-State Protein Storage. BMES 2024
|
Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The long-term goal of this project is to expand and validate the applications of phytoglycogen as a plant product, and conversely develop sweet corn varieties selected to supply the needs for this application. Our primary target audience are stakeholders in the production of sweet corn as well as stakeholders involved in the use of plant products for biomedical applications. They include sweet corn breeding programs in seed companies, sweet corn growers, and companies interested in applications of phytoglycogen. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student funded and trained in the proposed scope of this project has presented the results in multiple scientific conferences. How have the results been disseminated to communities of interest?The results were presented (oral and poster presentations) in biomedical and plant science conferences. A peer reviewed publication was also published. What do you plan to do during the next reporting period to accomplish the goals?We have requested a brief no-cost extension to finalize the submission of pending peer-review manuscripts.
Impacts
What was accomplished under these goals?
Aim 1: The proposed research for this aim wasprevioulsy completed and finished. However, the yeast system that was established as part of this Aim (Boehleinet al.2023) led to the functional characterization between the interactions of maize ISA1 withZPU1, a Pullulanase-Type Starch-Debranching. This interaction affects phytoglycogen accumulation. We have expanded our understanding of the physical intercations of ISA1, ISA2 and ZPU1. Aim 2: In this reporting period, the proposed research research for aim 2 was completed. The results of the GWAS are currently being prepared for publication. Aim 3: We have continued to explore the observation thatphytoglycogen can stabilize the activity of various proteins during lyophilization. A paper was published where weshow that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake forming agent (Parket al.2024).
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Park J, Liu R, Kim AS, et al. Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage. J Biomed Mater Res. 2024; 112(12): 2026-2041. doi:10.1002/jbm.a.37761
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Resende, M. Understanding maize starch synthesis using synthetic biology and a yeast system. Oral presentation, UF synthetic biology working group.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Park J., Liu R., Boehlein S.K., Resende M.F.R., Hudalla G. Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. SFB 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Park J., Liu R., Boehlein S.K., Resende M.F.R., Hudalla G. Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. BMES 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Park J., Liu R., Kim A.S., Boehlein S.K., Resende M.F.R., Savin D.A., Hudalla G. Sweet Corn Phytoglycogen Dendrimers as a Lyoprotectant for Dry-State Protein Storage. KSEA CERC 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Park J., Liu R., Kim A.S., Cyr N.N., Boehlein S.K., Resende M.F.R., Savin D.A., Bailey L.S., Sumerlin B.S., Hudalla G. Sweet Corn Phytoglycogen Dendrimers as a Lyoprotectant for Dry-State Protein Storage. WBC 2024
|
Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The long-term goal of this project is to expand and validate the applications of phytoglycogen as a plant product, and conversely develop sweet corn varieties selected to supply the needs for this application. Our primary target audience are stakeholders in the production of sweet corn as well as stakeholders involved in the use of plant products for biomedical applications. They include sweet corn breeding programs in seed companies, sweet corn growers, and companies interested in applications of phytoglycogen. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The M.Sc student being funded and trained in the scope of this project has presented her research at the National Association of Plant Breeding conference.Junha Park has also presented an abstract on phytoglycogen as a protein stabilizing excipient. How have the results been disseminated to communities of interest?Results from this project have been presented at the National Association of Plant Breeding, the International Sweet Corn Development Association, and the Society for Biomaterials. What do you plan to do during the next reporting period to accomplish the goals?In our last reportingperiod, we plan to publish in peer reviewed journals the results that have already been obtained as part of Aims 1, 2, and 3. More especifically, we will finalize the comparison of the biochemical, molecular and phenotypic differences caused by the differentsugary1alleles, and identify the most probable candidates from our association panel that may be controlling PG accumulation. Sweet corn lines are still being grown in the field for validation of the experiments.
Impacts
What was accomplished under these goals?
AIM 1 - is now finalized and was completed with the help of a yeast system engineered with 16 maize genes involved in startch biosynthesis. The initial results were published in Boehleinet al.2023 (10.1093/plphys/kiad358). Using this system, we have now measured enzyme activity and carbohydrate synthesis of the differentsu1aleles. We have identified variable results dependent on the su1which affect phytoglycogen content and properties (Liu et al.2020 - https://doi.org/10.3390/molecules25030637). The results are currently being summarized into a publication. AIM 2 - The genome wide association study proposed on aim 2 is now also completed and has resulted in several candidates affecting phytoglycogen accumulation. The results support the hypothesis that PG biosynthesis is complex and regulated by other small effect genes (other thansu1).Twenty-five single nucleotide polymorphisms (SNPs) were found to be significantly associatedwith PG content in different developmental stages. We have initiated bioinformatic and comparative genomic analysis to generate further evidence about the candidate genes neighboring the significant SNPs. The results are currently being summarized into a publication. AIM 3 - We have identified specific phytoglycogen extracts that have low toxicity and are excellent excipients that can be used to stabilize protein activity. We report thatphytoglycogen can stabilize the activity of various proteins during lyophilization. Lyophilization of protein biopharmaceuticals is attractive because it can provide for long-term storage and transportation that is considerably less expensive than refrigerated "cold-chain" storage. We showed that phytoglycogen dendrimers are as good or better than commonly used industrial lyoprotectants. Using a library of phytoglycogen extracts from different maize sources, we show that lyoprotectant activity is a shared feature among phytoglycogens irrespective of source. However, as initially hypothesized in this proposal, we also show that the extracts from different starch mutants lead to differences in PG molecule diameter, citotoxicity, and efficiency of protein stabilization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Boehlein SK, Pfister B, Hennen-Bierwagen TA, Liu C, Ritter M, Hannah LC, Zeeman SC, Resende MFR, Myers AM. Soluble and insoluble ?-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm. Plant Physiol. 2023 Sep 22;193(2):1456-1478.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Junha Park, Renjie Liu, Boehlein Susan, Resende Marcio, Gregory A. Hudalla. Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. Society For Biomaterials, 2023
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The long-term goal of this project is to expand and validate the applications of phytoglycogen as a plant product, and conversely develop sweet corn varieties selected to supply the needs for this application. Our primary target audience are stakeholders in the production of sweet corn as well as stakeholders involved in the use of plant products for biomedical applications. They include sweet corn breeding programs in seed companies, sweet corn growers, and companies interested in applications of phytoglycogen. Changes/Problems:Savin was on medical leave during the fall 2021 semester due to unanticipated open-heart surgery. What opportunities for training and professional development has the project provided?Junha Park presented an abstract on phytoglycogen as a protein stabilizing excipient at the 2022 NanoFlorida Meeting. How have the results been disseminated to communities of interest?Dan Savin presented about this project at University of Southern Mississippi, in a talk entitledMorphology Transitions in Responsive Block Copolymers: Incorporating Molecular Probes and Triggers. Sue Boehlein presented part of the results from Aim 1 at the 2021 Starch Roundtable. Marcio Resende presented part of the preliminary results from Aim 2 at the American Society of Horticultural Sciences. What do you plan to do during the next reporting period to accomplish the goals? Phytoglycogen structure will be analyzed from the series of inbred lines described above, both the su1- single mutant series and the parallel series of su1- isa2- double mutants. Phytoglycogen from the series of inbred lines, both single and double mutants will be isolated in quantity and used for cytotoxicity tests as described in the research proposal, towards the aim of identifying structural variants with optimal performance characteristics. Foundational sweet corn breeding lines will be initiated based on the su1-Bn2, isa2- double mutant combination. This allelic combination results in approximately the same amount of phytoglycogen in immature endosperm as does the su1-Ref allele that forms the basis of almost all extant phytoglycogen-containing sweet corn. The advantage of the su1-Bn2, isa2- combination is that the su1- allele is in the field corn haplotype of chromosome 4S rather than the sweet corn haplotype of su1-Ref. Phytoglycogen will also be produced in our yeast system for further studying and characterization The selected genotypes, mutants and yeast strains that are showing promising results will be used for in-vivo citotoxicity of different phytoglycogen variants. The results described in this report will be published in peer reviewed journals in one or more papers. The results will also be presented at national meetings of sweet corn breeders, for general information purposes but also to gauge potential interest in the approaches outlined here for development of novel sweet corn germplasm.
Impacts
What was accomplished under these goals?
AIM 1: Biochemical characterization of the different sugary1 alleles: Six different mutant alleles of thesu1-locus, and one non-mutant allele, were sequenced over the nearly complete transcribed region of the genomic locus. The alleles characterized weresu1?Ref,su1-am,su1-Bn2, andsu1-st,all of which have been described in the literature since the mid 20th century or earlier, andsu1-NCandsu1-SWthat were described in Tracy et al. (2006). Thesu1-Refallele is the same as that namedsu1-NEin the research proposal and in Tracy et al. (2006). One goal of this analysis was to discover or confirm the molecular lesion responsible for altering ISA1 activity in each of the mutant alleles. Novel results showedsu1-Bn2to be a missense mutation that changed ISA1 codon 628 from an Asn residue to Lys (N628K).su1-Refwas confirmed as the missense mutation W578R,su1-NCwas confirmed as the missense mutation R504C,su1?SWwas confirmed as the missense mutation N561S,su1-amwas confirmed as the missense mutation R308I, andsu1-stwas confirmed as a transposon insertion in exon 10. A second goal of the analysis was to identify thesu1locus haplotype containing each of the differentsu1-mutant alleles. As described in the research proposal, the possibility exists that unknown alleles linked to thesu1-mutation in sweet corn lines could influence plant characteristics including crop performance or vegetable quality. Almost all commercial sweet corn lines containsu1- mutations in the "sweet corn" haplotype typical of flint corn and related lines, not the "field corn" haplotype typical of dent corn lines. The results confirmedsu1-Ref,su1-NC, andsu1-SWare all in the sweet corn haplotype. In contrast, novel results from the project demonstrated thatsu1-standsu1?Bn2are in the field corn haplotype. Thus, use of these two alleles in future sweet corn breeding offers a potential means of separating the su1- alleles that are foundational for sweet corn from unknown, possibly deleterious linked alleles present in current sweet corn lines. This aspect of the project offers an alternative to separatingsu1-mutations from the chromosome 4S linkage group in whichsu1-Refresides. The research proposal suggested a transgenic means of creatingsu1-Refor othersu1-mutations in lines that possess the field corn haplotype at that region of chromosome 4. The new results suggest thatsu1-Bn2can serve this role without the need for complex transgenic manipulations, which is beneficial for future breeding of non-GMO foods. For this reason, the project has elected not to pursue further the transgenic objectives of the proposal, and to turn instead to utilization of alternativesu1-alleles that originated in the field corn haplotype. The rationale of the analysis of allele structure described above suggests that different alleles of su1 will affect starch biosynthesis differently and result in phytoglycogen with different molecular structures. If so, these variant forms of phytoglycogen can be tested for utility as drug delivery agents as described in other sections of the proposal, i.e., in toxicity tests in cultured cells. To address these objectives, a series of near-isogenic maize lines were generated in the W64A inbred background that each contains a different allele of the su1 locus, particularlysu1-Ref,su1-am,su1-Bn2, andsu1-st. Phytoglycogen content in endosperm from each of the inbred lines was determined. Mature seed can now be provided to partner team members for isolation of phytoglycogen and performance testing of that material. Further variation in the biochemical determinants that lead to phytoglycogen accumulation in maize endosperm was engendered by creating double mutant lines involving each of the four alleles listed above that lack the ISA2 protein. ISA2 assembles in a complex with ISA1, the product of su1, to form the debranching enzyme necessary to prevent phytoglycogen accumulation in non-mutant, i.e., completely starchy, maize lines. Further alteration of this enzyme activity owing to elimination of ISA2, beyond the changes engendered by thesu1-mutation affecting ISA1, is proposed to further alter the resultant phytoglycogen content and structure. This material offers further variation in phytoglycogen that can be functionally tested for cellular toxicity and other application characteristics. AIM 2: Quantification of PG in a diverse sweet corn germplasm and identification of regulatory regions controlling PG production: We continue to explore different methods to perform the genome wide association analysis, including FarmCPU and GEMMA packages. In parallel, QTL populations have been created by crossing sweet corn lines with low phytoglycogen content with lines with high amount. We expect to validate the GWAS results using this population. AIM 3: Evaluation of the bio-compatibility and physical characteristics of sweet corn phytoglycogen: We have continued to make progress characterizing the cytocompatibility of different phytoglycogen extracts in vitro. These efforts are serving to narrow the pool of candidates that will be screened for biocompatibility in vivo to complete Aim 3. We continued to perform particle size characterization of phytoglycogen samples using multi-angle dynamic light scattering (MA-DLS). The average particle sizes of 14 phytoglycogen samples obtained from Aims 1 and 2 were measured. In general, all particles were around 100 nm with only a couple outlying samples. Some of the outlying samples had lower solubility, signified by lower scattering count rates. All distributions were broad, with PDIs > 0.1. In contrast to typical particle sizing, MA-DLS gives additional information about the distribution of particles in solution. As an example, in a broad or bimodal distribution of particle sizes, each size contributes to the scattering profile differently. Larger particles have a greater relative contribution to the forward scattering (i.e., lower angles of scattering) and are largely bleached out at higher angles. If there is a bimodal distribution of particles, the relative intensity of each mode will change with angle. Similarly, broad, unimodal distributions will shift to lower sizes at higher scattering angle. Distributions of particle sizes were determined using the constrained nonlinear regularization routine (CONTIN), and the particle size distributions at three angles for 14 phytoglycogen samples were also obtained. As expected for a broad or bimodal distribution, the relative intensity of the larger particles is more dominant at forward angles (45°, red curves) compared with backward angles (135°, black curves). There is a gradual shift between these two extremes when considering an intermediate scattering angle (90°, blue curves.)
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Finegan et al. 2022 - Genetic Perturbation of the Starch Biosynthesis in Maize Endosperm Reveals Sugar-Responsive Gene Networks. Front Plant Sci. 2021; 12: 800326.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Junha et al. 2022 - Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient. Biomedical Engineering Society
|
Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The long-term goal of this project is to expand and validate the applications of phytoglycogen as a plant product, and conversely develop sweet corn varieties selected to supply the needs for this application. Our primary target audience are stakeholders in the production of sweet corn as well as stakeholders involved in the use of plant products for biomedical applications. They include sweet corn breeding programs in seed companies, sweet corn growers, and companies interested in applications of phytoglycogen. Changes/Problems:During the previous reporting period, our field tests were impacted by Covid restrictions. Hence, we were not able to grow the sweet corn plants with the different su1 mutants (Experiment 1.1). These plants are currently being grown. Furthermore, the Iowa State University Plant Transformation Facility is closing, and is no longer accepting new maize transformation projects. We had originally planned to use this facility in Experiment 1.3. Alternative transformation facilities will be investigated for suitability for this project. Alternatives are available at the University of Wisconsin or the University of Nebraska. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The current results are still preliminary. An oral presentationis scheduled for the 2021 Starch Roundtable, aninternational event well attended by the stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Aim1: During the next reporting period, we will expand the yeast system to include ISA2 and evaluate the properties of the resulting water soluble glucan. work will proceed on generation of transgenic maize lines that will test for linkage drag affecting the phytoglycogen phenotype, as described in Experiment 1.3. Aim 2: Genome wide association analysis will be conducted using at least two different methods. Aim 3: In the next reporting period, phytoglycogen from an expanding number of sources will be analyzed for glucan chain length distribution. These samples will be provided by the Resende laboratory, and the source of the material is specified in the Project Narrative (Experiment 3.1). Samples characterized in experiment 3.1 will also be evaluated for cytotoxicity in experiment 3.2
Impacts
What was accomplished under these goals?
AIM 1:Biochemical characterization of the differentsugary1alleles: Phytoglycogen (PG) chain length distribution has been determined from a collection of mutant maize lines that carry mutations other than the standard su1-Ref allele used as the foundation of phytoglycogen-type sweet corn varieties. In several instances the chain length distribution and the quantity of phytoglycogen varies from that standard lines. This material is amenable to further physical characterization, including particle size distribution and hydration water structure. The material is also amenable to cytotoxicity assays as described in the project narrative. Furthermore, Saccharomyces cerevisiae purged from its 5 native glycogen producing enzymes (Pfister et al. 2016) was replaced with the maize biosynthetic enzymes. These included thestarch synthases, starch branching enzymes, and ISA1.Both soluble polyglucans (SPG) and insoluble polyglucans (IPG) were obtained using this approach. The ISA1 from maize was shown to be active by both converting a proportion of SPG to IPG and from the accumulation of malto-oligosaccharides (MOS). We then replaced the ISA gene with each of the sugary1 allele genes and looked forthe ability to convert SPG to IPG and the formation of MOS. None of the mutant alleles showed either activity suggesting that the point mutation in each of the alleles causes the gene product to be inactive. AIM 2:Quantification of PG in a diverse sweet corn germplasm and identification of regulatory regions controlling PG production: We have quantified PG content in our sweet corn diversity panel which is composed of 700 diverse lines. Quantification was performed in kernels harvested 21 days after pollination (DAP) as well as grains maintained at room temperature for 7 days after harvest at 21 DAP (21+7). Best linear unbiased predictions (BLUPs) were generated to adjust the estimates for the experimental design. Results showed a large amount of variability in PG content and in the change of PG content from 21 DAP to the content in the 21+7 samples. Heritabilities ranged from 0.83 for PG content at 21 DAP to 0.45 for the difference between the two collections. Preliminary GWAS analysis indicate significant hits outside of the sugary1 region, as was originally hypothesized. AIM 3:Evaluation of the bio-compatibility and physical characteristics of sweet corn phytoglycogen: We performed initial physical characterization of the phytoglycogen particles extracted from the diversity population. Six samples were selected, three in each tail of the distribution for PG concentration. Hydrodynamic radius of the particles varied from genotype to genotype, but the polydispersity index was consistent across samples. The experiment will be repeated in the future and more genotypes will be evaluated. In addition, we have also evaluated the cytotoxicity of the water soluble polyglucan extracted from yeast. Results showed minimal toxicity towards NIH 3T3 fibroblasts and a mild level of toxicity over THP-1 monocytes only when SPG was used in high concentrations.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2021
Citation:
Boehlein et al. Synthesis of maize endosperm soluble and insoluble polyglucans in yeast. 2021 Starch Round Table
|
|