Source: UNIVERSITY OF FLORIDA submitted to
INTEGRATING PHENOMICS AND MOLECULAR GENETICS TO ADVANCE MAIZE SEED BIOLOGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1006908
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2015
Project End Date
Jun 30, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Horticultural Science
Non Technical Summary
Corn is the largest yielding crop in the US with average grain yield of 171 bushels per acre and nearly 15 billion bushels produced in the US in 2014 (USDA-NASS). Since the 1930s, yield has steadily increased due to breeding hybrid lines for increased tolerance to high planting density and lengthening the growing season by planting earlier in the spring. Both of these trends impact maize seed biology. Higher planting density increases yield per acre of land but reduces the number of seed set per plant. Earlier planting increases the chance that seeds will experience cold stress immediately after planting and high quality seeds are paramount to ensure germination. This project is investigating the developmental mechanisms that control seed set, grain-fill, and seed quality at maturity. At a foundational level, the project will identify genes and biological processes that are important for seeds to commit to development and store the starch, protein, and oil that are the primary components of grain. The project will also determine how the composition of the kernel then impacts early seedling phenotypes such as germination. The long-term goal is to identify genes or specific traits expressed in the seed that can be targeted to improve seed set, grain-fill, and seed germination.
Animal Health Component
5%
Research Effort Categories
Basic
85%
Applied
5%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061510105030%
2011510108070%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
1) Identify genes that impact grain-fill and determine their potential as targets for crop improvement.2) Phenotype diverse maize germplasm to determine physiological relationships between mature kernel phenotypes, seed set, and early seedling growth characteristics.3) Train undergraduates, graduate students, and post-docs in phenomics and genetics.
Project Methods
The project will use molecular genetics, genomics, and quantitative genetics approaches to identify genes that impact seed and early seedling development in maize. Detailed developmental phenotyping is used to detemrine the biological functions of these genes. Biochemistry is employed to characterize protein function, while recombinant DNA technology is used to engineer alleles that may help improve plant tolerance to extreme weather or increase yield.

Progress 10/01/19 to 06/30/20

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists. Changes/Problems:Our lab activities during spring and summer of 2020were significantly impacted by the COVID19 global pandemic. In Florida, a mandatory stay-at-home order was in place for the entire month of April. Prior to the stay-at-home order, the University of Florida placed a pause on all data collection. These major limitations caused setbacks in planned laboratory work. We were able to get the majority of our planned field work completed; however, we did miss the opportunity to plant some of our delayed lines this field season. We missed out on a major opportunity for professional development for several lab members due to the cancellation of the 2020 Maize conference. By May, lab activities slowly began to resume with strict social distancing protocols in place significantly limiting the number of lab members able to work in the lab at anygiven time. We are hopeful that research activities will continue increasing at a gradual pace until we can resume full research efforts, though the timing of this will depend on the state of the pandemic in the coming months. With current restrictions in place limiting face-to-face contact, we are also unable to host new undergraduate summer interns for the 2020 season. This will cost us the opportunity to train underrepresented minority students from Florida A&M University as we have done yearly for several years now. What opportunities for training and professional development has the project provided?Student Training: PI served as the chair of the graduate committee for three doctoral students and served as a member of the committee for five additional doctoral students and one masters student. 2 doctoral students completed rotations in the lab; one decided to stay and complete PhD work with the lab. One undergraduate research internwastrained during this reporting period. Three post-doctoral students were also mentored. Conference Presentations: 2019 Joint International Sweet Corn Development Association and Corn Breeding Research Annual Meeting Chicago, Illinois Predicting Sugar and Starch in Mature Sweet Corn Kernels Hersh, Hope1*; Boehlein, Susan1; Gustin, Jeff1; Resende, Marcio1; Settles, A. Mark1 1University of Florida Abstract Sweet corn quality is dependent on sugar and starch content in the kernel at eating stage. Sweet corn lines with higher sugar levels at eating stage have a tendency for poor germination and emergence suggesting a trade-off between eating and seed quality. However, the relationship between eating stage constituents and mature kernel composition is not well understood. Quantifying sugar and starch levels analytically is time-consuming and expensive. To enable lower cost phenotyping, we developed a calibration for single kernel near infrared reflectance (skNIR) spectroscopy to predict sugar and starch in mature sweet corn kernels. A panel of 100 sweet corn varieties was assembled consisting of equal parts sugary1 (su1) and shrunken2 (sh2) mutant backgrounds. Single-kernel NIR spectra from 910 nm to 1670 nm were collected for 300 kernels. The individual kernels were milled into a fine powder and sugar and starch were quantified using enzymatic analytical chemistry. Surprisingly, mature su1 and sh2 kernels have extensive overlap of sugar and starch levels suggesting other genetic factors contribute to the final accumulation of these constituents in mature kernels. Predictive models were developed with Partial Least-Squares (PLS) regression of 67 genotypes. The models were then validated with the remaining 33 genotypes. Predictions for preliminary data using skNIR have a standard error of prediction for sugar of 33.2 mg g-1 fw and standard deviation of 49 mg g-1 for su1 and sh2 types. This error was substantially below the standard deviation of the populations suggesting that the PLS model can be used to bin kernels into high, moderate, and low sugar groups. Starch modeling is awaiting finalization of the analytical measurements. These results suggest that skNIR could be a rapid way of assessing sweet corn sugar content, which can help better define the relationships between eating quality, post-harvest shelf life, and mature seed germination. Expanding the Vision of Near-Infrared Spectroscopy to Create Novel Breeding Tools for Sweet Corn. Jeff Gustin1, John Williams1, John Baier1, Paul Armstrong2, Marcio Resende1, William Tracy2, Mark Settles1 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 USDA-ARS Grain Marketing & Production Research Center, Manhattan, KS 3 Department of Agronomy, University of Wisconsin, Madison, WI Abstract: Successful sweet corn hybrids possess numerous characteristics that optimize producer profit and satisfy consumer tastes. Breeders incorporate these numerous traits into their selection criterion when developing new inbred and hybrid lines. Phenotypic characterization can limit the number of evaluated lines if the trait is difficult or time consuming to measure, particularly if the data is collected at fresh eating stage. Therefore, tools that reduce data collection time can increase the number of lines evaluated and lead to faster genetic gain. Near-infrared spectroscopy (NIRS) is widely used for estimating the chemical composition of mature seeds and grains. We developed a custom NIRS platform that collects spectra from individual kernels. These near infrared (NIR) reflectance data can be translated into accurate predictions of chemical composition such as oil and protein content as well as physical characteristics such as mass and density. We will show recent advances for predicting relevant sweet corn kernel traits using single-kernel NIR. Specifically, we developed a prediction model for kernel pericarp thickness, an important trait contributing to consumer likability and pathogen resistance during imbibition and seedling emergence. Comparing Controlled Environmental Conditions to Simulate Field Trial Emergence in Sweet Corn Linda Dao1, Jeff Gustin1, Ryan Solemslie2, Edgar Spalding3, Nathan Miller3, Lindsey du Toit2, Marcio Resende1, A. Mark Settles1 1University of Florida 2University of Washington 3University of Wisconsin Abstract Environmental conditions are important in determining seedling emergence. For early spring plantings, unpredictable weather conditions necessitate cold tolerant varieties. This is particularly important for sweet corn, which have a recommended minimum planting temperature of 5.5? greater than field corn. We evaluated soil-based growth chamber assays to estimate genetic effects on cold tolerance. Our rationale was to reduce extraneous environmental factors from field trials, while maintaining a soil-based assay. Two cold environments were analyzed: an acute and chronic cold test. The acute test imbibes kernels at 4? for 5 days with emergence assayed over 7 days at 24?. The acute test uses a machine vision platform known as VIGOR, which automatically scores emergence with high accuracy. For the chronic cold test, seeds were sown and maintained in 14°C day and 10°C night, 14/10 h cycle to more closely simulate likely field conditions. Emergence was scored by visual counts on a daily basis from 14 to 30 days after sowing. An initial trial was conducted with 6 sugary1 and shrunken2 genotypes to compare the two cold conditions along with a 24°C control. The two cold treatments revealed similar trends in cold sensitivity for emergence percentage but had different scales of emergence time. For a second trial, we selected 14 sugary1 genotypes that had previously been shown to vary in emergence time and percentage in a field study in Central Washington. Comparisons between the field study and the growth chamber data are in progress. We anticipate that these experiments will help define a controlled temperature regime that can be used to find field-relevant alleles associated with sweet corn cold tolerance in a genome wide association study. How have the results been disseminated to communities of interest?Results have been disseminated through the publication of 5 peer review articles, 2 additional article submissions, several presentations to the scientific community, an interview with the press, and the development of a lab website. -In November of 2019 PI gave an invited talk at the 35th Annual Meeting of the American Society for Gravitational and Space Research in Denver, Colorado titled "Selecting for Chlamydomonas reinhardtii fitness in a liquid algal growth system on the International Space Station". -In December of 2019, a post-doc in the lab gave an invited seminar at the Hudson Alpha Institute for Biotechnology in Huntsville, Alabama. -In December of 2019, three lab members gave invited talks at the International Sweet Corn Developers Association Conference in Chicago Illinois. -In the April 2020 issue of Food Technology Magazine an article titled "The Sweet State of Corn" was published featuring an interview with Dr. Mark Settles regarding his efforts to lead a multi-disciplinary effort to improve sweet corn genetics. -During spring of 2020 we developed a Settles Lab website to effectively share our results with others. What do you plan to do during the next reporting period to accomplish the goals?Grant Applications: During this reporting period a total of 3 grant applications were submitted. In October of 2019 a graduate student fellowship proposal was submitted to the National Science foundation with a total award amount of $138,000. In March of 2020 a proposal was submitted to NASA titled "Multi-omics of Arthrospira platensis" with a total proposed budget of $749,995. In April of 2020 a proposal was submitted to the Florida Space Research Institute titled "OR-DRPD-SR12020: Determining microalgae resistance to ionizing radiation at a genome-wide scale" with a total proposed budget of $177,064. Additionally, we are in the processof submitting a new HATCH proposal. FAMU Student Visits: Traditionally, every summer we host undergraduates from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. COVID19 has disrupted these plans for 2020, but we do hope the program will resume in the summer of 2021. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Graduate Student Recruitments: Recruitment of graduate students from multiple programs at UF including Plant Molecular and Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued.

Impacts
What was accomplished under these goals? Settles A.M. (2020) EMS mutagenesis of maize pollen. Methods Mol Biol. 2122:25-33. Effective mutagenesis is critical for connecting traits of interest to specific plant genes. The development of site-directed mutagenesis and sequenced-indexed genetics resources in maize allows for targeted analysis of individual genes. These reverse genetics approaches have the potential for confirmation bias by only studying candidate genes for association with traits of interest. Genetic screens of induced, random mutations are important for identifying novel loci as well as interacting factors for known mutant loci. Chemical mutagenesis provides very high mutation rates and can be used for a variety of screen designs. This chapter provides an updated protocol for ethyl methanesulfonate (EMS) mutagenesis of maize pollen using paraffin or mineral oil. Mutagenesis occurs in mature pollen causing nonconcordant endosperm and embryo genotypes as well as sectored M1 plants. Considerations for these factors in genetic screens are discussed. Gillmor, C.S., et al. (2020) Genetic screens to target embryo and endosperm pathways in Arabidopsis and maize. Methods Mol Biol. 2122:3-14.The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet.Arabidopsisand maize have evolved different types of seeds, opening a range of experimental opportunities. Development of theArabidopsisembryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: inArabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments. Zhang, J., et al.(2020) Competitive growth assay of mutagenizedChlamydomonas reinhardtiicompatible with the International Space Station Veggie Plant Growth Chamber.Front. Plant Sci.A biological life support system for spaceflight would capture carbon dioxide waste produced by living and working in space to generate useful organic compounds. Photosynthesis is the primary mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting light, water, and carbon dioxide into biomass, particularly under limiting, artificial light conditions that are a necessity in space photosynthetic production. Although there is great promise in developing algae for chemical or food production in space, most spaceflight algae growth studies have been conducted on solid agar-media to avoid handling liquids in microgravity. Here we report that breathable plastic tissue culture bags can support robust growth ofChlamydomonas reinhardtiiin the Veggie plant growth chamber, which is used on the International Space Station (ISS) to grow terrestrial plants. Live cultures can be stored for at least 1 month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested using a competitive growth assay with mutations induced by short-wave ultraviolet light (UVC) mutagenesis in either wild-type (CC-5082) orcw15mutant (CC-1883) strains at the start of the assay. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and non-synonymous mutations relative to natural variants among laboratory strains. Genes with mutations indicating positive selection were enriched for information processing genes related to DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. These data suggest that modification of DNA repair, signal transduction, and metabolite transport may be needed to improve growth rates in this spaceflight production system. Hacisalihoglu, G., et al. (2020) Protein, weight, and oil determination by single-seed near-infrared spectroscopy for selection of seed quality and yield traits in pea (Pisum sativum).J. Science Food Agriculture.Pea (Pisum sativum) is a prevalent cool?season crop that produces seeds valued for their high protein content. Modern cultivars have incorporated several traits that improved harvested yield. However, progress toward improving seed quality has received less emphasis, in part due to the lack of tools for easily and rapidly measuring seed traits. In this study we evaluated the accuracy of single?seed near?infrared spectroscopy (NIRS) for measuring pea?seed weight, protein, and oil content. A total of 96 diverse pea accessions were analyzed using both single?seed NIRS and wet chemistry methods. To demonstrate field relevance, the single?seed NIRS protein prediction model was used to determine the impact of seed treatments and foliar fungicides on the protein content of harvested dry peas in a field trial.External validation of partial least squares (PLS) regression models showed high prediction accuracy for protein and weight (R2= 0.94 for both) and less accuracy for oil (R2= 0.74). Single?seed weight was weakly correlated with protein and oil content in contrast with previous reports. In the field study, the single?seed NIRS predicted protein values were within 10 mg g−1of an independent analytical reference measurement and were sufficiently precise to detect small treatment effects. The high accuracy of protein and weight estimation show that single?seed NIRS could be used in the dual selection of high?protein, high?weight peas early in the breeding cycle, allowing for faster genetic advancement toward improved pea nutritional quality. Ribeiro, C., et al. (2020) Engineering 6-phosphogluconate dehydrogenase to improve heat tolerance in maize seed development.BioRxiv, pre-print.Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2 that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing theWaxy1chloroplast targeting peptide coding sequence to thePgd1andPgd2open reading frames. These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm specific promoter increased 6PGDH activity with enhanced heat stabilityin vitro. WPGD1 and WPGD2 transgenes complement thepgd3defective kernel phenotype indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high nighttime temperature conditions by increasing kernel number. These results provide insight on subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress.

Publications

  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Settles A.M. (2020) EMS mutagenesis of maize pollen. Methods Mol Biol. 2122:25-33. (https://doi/10.1007/978-1-0716-0342-0_3).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gillmor C.S., Settles A.M., Lukowitz W. (2020) Genetic screens to target embryo and endosperm pathways in Arabidopsis and maize. Methods Mol Biol. 2122:3-14. (https://doi/10.1007/978-1-0716-0342-0_1).
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Gustin J.L., Frei U.K., Baier J., Armstrong P., L�bberstedt T., Settles A.M. (2020) Maize (Zea mays subsp. mays) Haploid Classification using Single Kernel Near-Infrared Spectroscopy. Plant Breeding, In revision.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, J., Muller, B.S., Tyre, K.N., Hersh, H.L., Bai, F., Hu, Y., Resende, M.F.R. Jr., Rathinasabapathi, B., Settles, A.M. (2020) Competitive growth assay of mutagenized Chlamydomonas reinhardtii compatible with the International Space Station Veggie Plant Growth Chamber. Front. Plant Sci. (https://doi.org/10.3389/fpls.2020.00631)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hacisalihoglu, G., Freeman J., Armstrong P.R., Seabourn B.W., Porter L.D., Settles A.M., Gustin J.L. (2020) Protein, weight, and oil determination by single-seed near-infrared spectroscopy for selection of seed quality and yield traits in pea (Pisum sativum). J. Science Food Agriculture.(https://doi.org/10.1002/jsfa.10389)
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Ribeiro, C., Hennen-Bierwagen, T.A., Myers, A.M., Cline, K., Settles, A. M. (2020) Engineering 6-phosphogluconate dehydrogenase to improve heat tolerance in maize seed development. BioRxiv, pre-print. (https://doi.org/10.1101/2020.05.21.108985)
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Hacisalihoglu, G., Beisel, N.S., Settles, A.M. (2020) Characterization of pea seed nutritional value within a diverse collection of 96 pea (Pisum sativum) genotypes using ionomics. In preparation.


Progress 07/01/15 to 06/30/20

Outputs
Target Audience:The target audience reached consisted of undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community, and seed company scientists. Changes/Problems:Our lab activities during spring and summer of 2020 were significantly impacted by the COVID19 global pandemic. In Florida, a mandatory stay-at-home order was in place for the entire month of April. Prior to the stay-at-home order, the University of Florida placed a pause on all data collection. These major limitations caused setbacks in planned laboratory work. We were able to get the majority of our planned field work completed; however, we did miss the opportunity to plant some of our delayed lines this field season. We missed out on a major opportunity for professional development for several lab members due to the cancellation of the 2020 Maize Genetics Conference. By May, lab activities slowly began to resume with strict social distancing protocols in place significantly limiting the number of lab members able to work in the lab at any given time. We are hopeful that research activities will continue increasing at a gradual pace until we can resume full research efforts, though the timing of this will depend on the state of the pandemic in the coming months. With current restrictions in place limiting face-to-face contact, we are also unable to host new undergraduate summer interns for the 2020 season. This will cost us the opportunity to train underrepresented minority students from Florida A&M University as we have done yearly for several years now. What opportunities for training and professional development has the project provided?Student Training 6 post-doctoral associates, 5 doctoral graduate students, 16undergraduate students, and 2 high school students were mentored and trained throughout the duration of this HATCH project. Opportunities for Professional Development During the duration of this HATCH project, a total of 44 presentations (posters andtalks)were given. Details ofeach presentation were reported individually in earlier progress reports. -PI gave invited talk at FASEB Mechanisms in Plant Development conference (2015) -1 post-doc talk and 11 posters at 58th annual Maize genetics conference (2016) -1 graduate student talk at the PMCB Annual Retreat (2016) -2 student talks and 7 posters presented at 58th annual Maize genetics conference (2017) -3 posters presented at the 59th annual Maize genetics conference (2018) -PI gave an invited talk at the University of Florida- HZAU Bilateral Conference on Horticulture in Wuhan, China (2018) -PI was co-author for talk at Phenome 2019 in Tuscon, Arizona (2019) -One student talk and 8 posters were presented at the 61st Annual Maize Genetics Conference in St. Lois, Missouri (2019) -PI gave a plenary talk at the 4th Maize Biology Conference of China in Zhengzhou, China (2019) -A post-doctoral student gave an invited talk at the CROPS Conference in Huntsville, Alabama (2019) -PI was a co-author for a presentation given at the FASEB Mechanisms in Plant Development Conference in Olean, New York (2019) -PI gave an invited talk at the 35th Annual Meeting of the American Society for Gravitational and Space Research in Denver, Colorado (2019) -Three lab members gave invited talks at the International Sweet Corn Developers Association Conference in Chicago Illinois (2019) How have the results been disseminated to communities of interest?In addition to the 17 publications and 44 conference presentations reported already, 17 invited seminars were given throughout the duration of this HATCH project. Details of each seminar were reported in earlier progress reports. Additionally, we have developed a Settles Lab website to effectively share our results with any interested community. PI has also participated in a large number of interviews with the press to share results of major lab projects with the general public. Total seminars/invited talks = 17 -PI gave an invited seminar at the Plant Science Institute at Iowa State University in Ames (2015) -PI gave an invited lecture within the Department of Plant Biology at the Carnegie Instuition for Science in Stanford, California (2016) -PI gave an invited seminar for the Departamento de Fitotecnia at the Universidade Federal de Viçosa in Vicosa, Brazil (2016) -PI gave an invited seminar for the Interdisciplinary Plant Group at the University of Missouri in Columbia, Missouri (2016) -PI gave an invited seminar at the KWS Saat Gateway Research Center in St. Lois, Missouri (2017) -PI gave an invited seminar at NSF Integrative Organismal Systems in Washington, D.C (2017) -PI gave an invited seminar at Oakland University in Rochester, Michigan (2017) -PI was a contributing author to an invited talk at the Phenome conference (2018) -PI gave a seminar for the RNA Club Seminar Series at the University of Florida in Gainesville, Florida (2018) -PI gave an NSF Outreach Presentation at Florida A&M University for the Department of Biology (2018) -PI gave an invited seminar at the Key Laboratory of Plant Cell Engineering and Germplasm Innovation for Shandong University in Qingdao, China (2018) -PI gave an invited seminar at the Key Laboratory of Wheat and Maize Crops Science at Henan Agricultural University in Zhengzhou, China (2018) -PI gave an outreach presentation to a FIRST Lego League Team in Gainesville, Florida (2018) -PI gave a talk for the Horticultural Sciences Department Seminar Series at the University of Florida in Gainesville, Florida (2018) -PI gave a seminar for the Biotechnology Research Institute at the Chinese Academy of Agricultural Services in Beijing, China (2019) -PI gave a seminar for the Department of Botany and Plant Pathology at Oregon State University (2019) -A post-doc in the lab gave an invited seminar at the Hudson Alpha Institute for Biotechnology in Huntsville, Alabama (2019) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A total of 17 peer-reviewed manuscripts were published regarding results related to Objectives 1 and 2 of this HATCH project. Publication abstracts and details were reported individually in earlier progress reports as they were published. Accomplishments related to Objective 3 are summarized separatelyin subseqeunt questionsof this report.

Publications

  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Settles A.M. (2020) EMS mutagenesis of maize pollen. Methods Mol Biol. 2122:25-33. (https://doi/10.1007/978-1-0716- 0342-0_3).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gillmor C.S., Settles A.M., Lukowitz W. (2020) Genetic screens to target embryo and endosperm pathways in Arabidopsis and maize. Methods Mol Biol. 2122:3-14. (https://doi/10.1007/978-1-0716-0342-0_1).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gustin J.L., Frei U.K., Baier J., Armstrong P., L�bberstedt T., Settles A.M. (2020) Maize (Zea mays subsp. mays) Haploid Classification using Single Kernel Near-Infrared Spectroscopy. Plant Breeding, In revision.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, J., Muller, B.S., Tyre, K.N., Hersh, H.L., Bai, F., Hu, Y., Resende, M.F.R. Jr., Rathinasabapathi, B., Settles, A.M. (2020) Competitive growth assay of mutagenized Chlamydomonas reinhardtii compatible with the International Space Station Veggie Plant Growth Chamber. Front. Plant Sci. (https://doi.org/10.3389/fpls.2020.00631)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hacisalihoglu, G., Freeman J., Armstrong P.R., Seabourn B.W., Porter L.D., Settles A.M., Gustin J.L. (2020) Protein, weight, and oil determination by single-seed near-infrared spectroscopy for selection of seed quality and yield traits in pea (Pisum sativum). J. Science Food Agriculture.(https://doi.org/10.1002/jsfa.10389)
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Ribeiro, C., Hennen-Bierwagen, T.A., Myers, A.M., Cline, K., Settles, A. M. (2020) Engineering 6-phosphogluconate dehydrogenase to improve heat tolerance in maize seed development. BioRxiv, pre-print. (https://doi.org/10.1101/2020.05.21.108985)
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Hacisalihoglu, G., Beisel, N.S., Settles, A.M. (2020) Characterization of pea seed nutritional value within a diverse collection of 96 pea (Pisum sativum) genotypes using ionomics. In preparation.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?61st Annual Maize Genetics Conference St. Lois, Missouri The maize maternal rough endosperm1 (mre1) mutant is a parent-of-origin effect locus that disrupts endosperm development Full Author List: Bai, Fang1; Barbazuk, W. Brad2; Davenport, Ruth2; Zhang, Junya1; Hu, Ying1; Bagadion, Alyssa1; Settles, A. Mark1 1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida, USA 32611 2 Department of Biology and Genetics Institute, University of Florida, Gainesville, Florida, USA, 32611 Evolutionary conservation of protein-protein interaction involved in the splicing of U12-type introns Full Author List: Levine, Laurel A1; Corll, Jacob B1; Bai, Fang2; Mier, Dominic D1; Settles, A. Mark2; Lal, Shailesh1 1 Department of Biological Sciences, Oakland University, Rochester Hills, MI 48309 2 Department Of Horticultural Sciences, University of Florida, Gainesville, FL 32611 Exploring the use of NIRS to predict starch in whole seed pea Full Author List: Hacisalihoglu, Gokhan1; Freeman, Jelani1; Gustin, Jeff2; Settles, Mark2 1 Florida A&M University, Biological Sciences Dept, Tallahassee, FLORIDA 32307 2 Univ. of Florida, Gainesville, FL Maize and human RNA binding motif protein 48 have an evolutionarily conserved essential role in U12-dependent intron splicing Full Author List: Siebert, Amy E.1; Corll, Jacob B.1; Gronevelt, J. Paige1; Levine, Laurel A.1; Davenport, Ruth3; Westrick, Randal J.1; Barbazuk, W. Brad3; Settles, A. Mark2; Madlambayan, Gerard1; Lal, Shailesh1 1 Department of Biological Sciences, Oakland University, Rochester Hills, MI 48309 2 Department Of Horticultural Sciences, University of Florida, Gainesville, FL 32611 3 Department of Biology, University of Florida, Gainesville, FL 32611 Priming modulation of early maize seedling performance under cold stress using vigor: A machine vision HT seedling emergence assay Full Author List: Hacisalihoglu, Gokhan1; Miller, Nathan2; Gustin, Jeff3; Settles, Mark3 1 Florida A&M University, Biological Sciences Dept, Tallahassee, FLORIDA 32307 2 University of Wisconsin, Madison, WI 3 University of Florida, Gainesville, FL RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation Full Author List: Corll, Jacob1; Bai, Fang2; Shodja, Donya N.1; Davenport, Ruth3; Feng, Guanqiao3; Mudunkothge, Janaki2; Brigolin, Christian J.1; Martin, Federico2; Spielbauer, Gertraud2; Tseung, Chi-Wah2; Siebert, Amy E.1; Barbazuk, W. Brad1 3; Lal, Shailesh1; Settles, A. Mark2 1 Department of Biological Sciences, Oakland University, Rochester, Michigan, 48309, USA 2 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida, 32611, USA 3 Department of Biology and Genetics Institute, University of Florida, Gainesville, Florida, 32611, USA Genome-wide association analyses of maize kernel traits in the Wisconsin diversity (WiDiv) panel Full Author List: Mu?ller, Bárbara S. F.1; Gustin, Jeffery L.1; Baier, John1; Kaeppler, Shawn M.2; de Leon, Natalia2; Settles, A. Mark1 1 University of Florida, Horticultural Sciences Department, 1253 Fifield Hall, Gainesville, FL 32611. 2 University of Wisconsin-Madison, Department of Agronomy, 1575 Linden Drive, Madison, WI 53706. QTL analysis of maize seedling cold tolerance using Vigor: a machine vision assay for seedling emergence. Full Author List: Dao, Linda2; Gustin, Jeffery L2; Settles, A. Mark2; Baier, John E2; Spalding, Edgar P1; Miller, Nathan D1 1 Department of Botany; University of Wisconsin; Madison, WI, 53706 2 Horticultural Sciences Department; University of Florida; Gainesville, FL, 32611 Ovary abortion is prevalent in diverse maize inbred lines and is under genetic control. Full Author List: Gustin, Jeffery L1; Boehlein, Susan K1; Shaw, Janine R1; Junior, Weschester3; Settles, A. Mark1; Webster, Ashley2; Tracy, William F2; Hannah, L. Curtis1 1 Horticultural Sciences Department; University of Florida; Gainesville, FL, 32611 2 Department of Agronomy; University of Wisconsin; Madison, WI, 53706 3 Florida Agricultural and Mechanical University; Tallahassee, FL, 32307 How have the results been disseminated to communities of interest?Results have been disseminated through publication of 3 peer reviewed scientific papers, in addition to a multitude of presentations to the scientific community. In December 2018, PI gave an invited seminar at the Key Laboratory of Plant Cell Engineering and Germplasm Innovation for Shandong University in Qingdao, China titled "Maize endosperm mutants reveal minor spliceosome factors". Also in December 2018, PI gave invited seminars both at the Key Laboratory of Wheat and Maize Crops Science at Henan Agricultural University in Zhengzhou, China and at the University of Florida- HZAU Bilateral Conference on Horticulture in Wuhan, China titled "Sweet CAP: Integrated technologies to improve sweet corn production and marketability". In September 2018 PI gave a talk for the Horticultural Sciences Department Seminar Series at the University of Florida in Gainesville, Florida titled "Maize endosperm mutants reveal minor spliceosome factors". Additionally in September of 2018, PI gave an outreach presentation to a FIRST Lego League Team in Gainesville, Florida titled "Deep space exploration: Earth-based analogs and International Space Station Research". In February 2019, PI was co-author for talk at Phenome 2019 in Tuscon, Arizona titled "VIGOR: A Controlled Environment-based Machine Vision Assay of Seedling Emergence from Soil." In March 2019, one student talk and 8 posters were presented at the 61st Annual Maize Genetics Conference in St. Lois, Missouri. In April of 2019, PI gave a plenary talk at the 4th Maize Biology Conference of China in Zhengzhou, China titled "Endosperm cell differentiation requires efficient splicing of minor introns". Additionally in April of 2019, PI gave a seminar for the Biotechnology Research Institute at the Chinese Academy of Agricultural Services in Beijing, China titled "Multiple development mechanisms for parent-of-origin effect mutations in maize". In May of 2019 PI gave a seminar for the Department of Botany and Plant Pathology at Oregon State University titled "Endosperm cell differentiation requires efficient splicing of minor introns". In June 2019, a post-doctoral student was invited to give a talk at the CROPS 2019 Conference in Huntsville, Alabama titled "Genome-wide association studies of kernel composition and root gravitropism in maize". PI was a co-author for a presentation given in July of 2019 at the FASEB Mechanisms in Plant Development Conference in Olean, New York titled "Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia effects in maize". What do you plan to do during the next reporting period to accomplish the goals?Grant Applications: During this reporting period, a total of 2 grant applications were completed and submitted. Dr. Settles, in role of Co-PI, submitted a proposal titled "A maize genetic model to interrogate the conserved factors and roles of U12" to NSF-MCB for a total budget of $1,874,590. In the role of Senior Personnel, Dr. Settles was involved in submitting a pre-proposal titled "Science and Technology Center for Optimization of Cereal Grain" to NSF-OIA on 6/25/19. FAMU Student Visits Every summer, we host undergraduate students from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Graduate Student Recruitment: Recruitment of graduate students from multipleprograms at UF including: Plant Molecular & Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued.

Impacts
What was accomplished under these goals? Science Reported in Literature: Zhang J., Wu S., Boehlein S.K., McCarty D.R., Myeras A., Settles A.M. (2019) Maize defective kernel5 (dek5) is homolgous to bacterial TamB and required for chloroplast envelope biogenesis. J. Cell Biol., 218(8):2638-2658. Abstract Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maizedefective kernel5(dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number indek5with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4,Arabidopsis thalianaEMB2410/TIC236, andEscherichia colitamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars indek5developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses showdek5reduces levels of Toc75 and chloroplast envelope transporters. Moreover,dek5chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins. Boehlein S., Liu P., Webster A., Ribeiro C., Suzuki M., Wu S., Guan J.C., Stewart J., Tracy W., Settles A.M., McCarty D., Koch K., Hannah L.C., Hennen-Bierwagen T., Myers A. (2019) Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill. Plant J., 99(1):23-40. (https://doi/10.1111/tpj.14283). Abstract Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night (38/28°C])) or elevated day, normal night (38/17°C), were compared with those from controls grown under normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not when daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either upregulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development. Bai F., Corll J., Shodja D., Davenport R., Feng G., Mudunkothge J., Brigolin C.J., Martin F., Spielbauer G., Tseung C.W., Seibert A.E., Barbazuk W.B., Lal S., Settles A.M. (2019) RNA Binding Motif Protein48 is required for U12 splicing and maize endosperm differentiation. Plant Cell 31(3):715-733. (https://doi.org/10.1101/341917). Abstract The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize (Zea mays) RNA binding motif protein 48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, zinc finger CCCH-type, RNA binding motif, and Ser/Arg rich 2/Rough endosperm 3 (RGH3). Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors required for intron recognition form complexes with RBM48. Human RBM48 interacts with armadillo repeat containing 7 (ARMC7). Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize. Student Training PI served as the chair on the PhD committee of two doctoral students, and as a member on the graduate committee of four additional doctoral students. A total of six undergraduate students were trained in the lab during this reporting period. PI also mentored three post-doctoral students during this reporting period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang J., Wu S., Boehlein S.K., McCarty D.R., Myeras A., Settles A.M. (2019) Maize defective kernel5 (dek5) is homolgous to bacterial TamB and required for chloroplast envelope biogenesis. J. Cell Biol., 218(8):2638-2658.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Boehlein S., Liu P., Webster A., Ribeiro C., Suzuki M., Wu S., Guan J.C., Stewart J., Tracy W., Settles A.M., McCarty D., Koch K., Hannah L.C., Hennen-Bierwagen T., Myers A. (2019) Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill. Plant J., 99(1):23-40. (https://doi/10.1111/tpj.14283).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Bai F., Corll J., Shodja D., Davenport R., Feng G., Mudunkothge J., Brigolin C.J., Martin F., Spielbauer G., Tseung C.W., Seibert A.E., Barbazuk W.B., Lal S., Settles A.M. (2019) RNA Binding Motif Protein48 is required for U12 splicing and maize endosperm differentiation. Plant Cell 31(3):715-733. (https://doi.org/10.1101/341917).


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Student Training:PI served as the chair of the graduate committee for two doctoral students and served as a member of the committee for seven additional doctoral students and one masters student. Nine undergraduate research interns were trained during this reporting period. Three post-doctoral students were also mentored. 60th Annual Maize Genetics Conference, St. Malo, France Soil-based machine vision seedling emergence assay for studying cold tolerance in maize Full Author List: Miller, Nathan D.1; Settles, A. Mark2; Baier, John W.2; Spalding, Edgar P.1; Gustin, Jeffery L.2 1 Department of Botany, University of Wisconsin, Madison, WI; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL Maize RNA Binding Motif Protein48 (RBM48) is required for minor intron splicing and promotes endosperm cell differentiation Full Author List: Corll, Jacob1; Bai, Fang2; Shodja, Donya1; Brigolin, Christian J.1; Davenport, Ruth3; Feng, Guanqiao2; Martin, Federico2; Spielbauer, Gertraud2; Steinhauer, Tyler1; Tseung, Chi-Wah2; Barbazuk, W. Brad3; Lal, Shailesh K.1; Settles, A. Mark2 1 Department of Biological Sciences, Oakland University, Rochester, MI 48309; 2 Horticural Sciences Department, University of Florida, Gainesville, FL 32611; 3 Department of Biology, University of Florida, Gainesville, FL 32611 Modulation of cold sensitivity and early seedling performance by priming in 27 maize NAM parental inbred lines Full Author List: Hacisalihoglu, Gokhan1; Gustin, Jeff2; Miller, Nathan3; Kantanka, Sarfo1; Settles, Mark2 1 Florida Agricultural and Mechanical University, Biological Sciences Department, Tallahassee, FL 32307; 2 Horticultural Science Department, University of Florida, Gainesville, FL 32611; 3 Department of Botany, University of Wisconsin, Madison, WI 53706 How have the results been disseminated to communities of interest?Results have been disseminated through the publication of 5 peer review scientific papers, submission of 3 additional papers currently under review, and several presentations to the scientific community. In October of 2017 PI gave an invited seminar at Oakland University in Rochester, Michigan titled "HERA: an Earth-based NASA analog to study human factors for deep space exploration". In February 2018, PI was a contributing author to one talk titled "Modulation of Cold Sensitivity and Early Seedling Performance by Priming in Maize NAM Parental Inbred Lines" at the Phenome conference held in Tuscon, Arizona. In March 2018, three poster presentations were given at the 60th Annual Maize Genetics Conference in St. Malo, France. Also in March of 2018, PI gave a seminar for the RNA Club Seminar Series at the University of Florida in Gainesville, Florida titled "Maize endosperm mutants reveal minor spliceosome factors". In April 2018, PI gave a NSF Outreach Presentation at Florida A&M University for the Department of Biology titled "Genetics and phenotyping technologies to understand maize seed biology". What do you plan to do during the next reporting period to accomplish the goals?Grant Applications: During this reporting period a total of 3 grant applications were submitted. On 11/20/17 a proposal was submitted to NSF-MCB for $1,425,249 for Defining the U12 Splicing Component of the Maize Endosperm Switch from Cell Proliferation to Differentiation. On 12/8/17 a pre-proposal was submitted to NIFA-SCRI for $8,000,000 on Sweet CAP: Integrated Technologies to Improve Sweet Corn Production and Marketability. On 6/21/18 a proposal was submitted to TRISH-BRASH for $795,460 for Synthetic Biology Tools for Algal Production of Common Foods. FAMU Student Visits: Every summer, we host undergraduate students from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Graduate Student Recruitment: Recruitment of graduate students from multiples programs at UF including: Plant Molecular & Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued.

Impacts
What was accomplished under these goals? Springer N.M., Anderson S.N.,...Settles A.M., et al. (2018) The W22 genome: a foundation for maize functional genomics and transposon biology. Nature Genetics 50(9):1282-1288. The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands ofMutator(Mu) andDissociation(Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome. Gabay-Laughnan S., Settles A.M., et al. (2018) Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize. G3: Genes, Genomes, Genetics. 8(1):291-302. Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) ofZea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclearrestorer-of-fertility(restorer) mutations rescue pollen function. To better understand these genetic interactions, we screenedActivator-Dissociation(Ac-Ds),Enhancer/Suppressor-mutator(En/Spm), andMutator(Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered fromAc-DsandEn/Spmstocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-SMutransposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously describedrfl2-1mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development Lappe R.R., Baier J.W.,...Settles A.M., et al. (2018) Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm. Proc Natl Acad Sci U.S.A. 115(1):E24-E33. Maizeopaque2(o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded byo2includepdk1andpdk2that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill.pdk1andpdk2were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi.pdk2accounts for the large majority of endosperm PPDK, whereaspdk1specifies the abundant mesophyll form. Thepdk1-mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned byo2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal thato2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression. Hacisalihoglu G., Burton A.L.,...Settles A.M., et al. (2018) Quantitative trait loci associated with soybean seedweight and composition under different phosphorus levels.J. Integr. Plant Biol.doi:10.1111/jipb.12612. Seed size and composition are important traits in food crops and can be affected by nutrient availability in the soil. Phosphorus (P) is a non-renewable, essential macronutrient, and P deficiency limits soybean (Glycine max) yield and quality. To investigate the associations of seed traits in low- and high-P environments, soybean recombinant inbred lines (RILs) from a cross of cultivars Fiskeby III and Mandarin (Ottawa) were grown under contrasting P availability environments. Traits including individual seed weight, seed number, and intact mature pod weight were significantly affected by soil P levels and showed transgressive segregation among the RILs. Surprisingly, P treatments did not affect seed composition or weight, suggesting that soybean maintains sufficient P in seeds even in low-P soil. Quantitative trait loci (QTLs) were detected for seed weight, intact pods, seed volume, and seed protein, with five significant QTLs identified in low-P environments and one significant QTL found in the optimal-P environment. Broad-sense heritability estimates were 0.78 (individual seed weight), 0.90 (seed protein), 0.34 (seed oil), and 0.98 (seed number). The QTLs identified under low P point to genetic regions that may be useful to improve soybean performance under limiting P conditions. Gustin J.L., Boehlein S.K.,...Settles A.M., et al. (2018)Ovary abortion is prevalent in diverse maize inbred lines and is under genetic control. Scientific Reports. 8:13032. Crop improvement programs focus on characteristics that are important for plant productivity. Typically genes underlying these traits are identified and stacked to create improved cultivars. Hence, identification of valuable traits for plant productivity is critical for plant improvement. Here we describe an important characteristic for maize productivity. Despite the fact mature maize ears are typically covered with kernels, we find that only a fraction of ovaries give rise to mature kernels. Non-developed ovaries degenerate while neighboring fertilized ovaries produce kernels that fill the ear. Abortion occurs throughout the ear, not just at the tip. We show that the fraction of aborted ovaries/kernels is genetically controlled and varies widely among maize lines, and low abortion genotypes are rare. Reducing or eliminating ovary abortion could substantially increase yield, making this characteristic a new target for selection in maize improvement programs.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Zhang J., Wu S., Boehlein S.K., McCarty D.R., Myers A., Settles A.M. (2018) Maize defective kernel5 (dek5) is homologous to bacterial TamB and required for chloroplast envelope biogenesis. J. Cell Biol., In revision. (https://doi.org/10.1101/372045).
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bai F., Corll J., Shodja D., Davenport R., Feng G., Mudunkothge J., Brigolin C.J., Martin F., Spielbauer G., Tseung C.W., Seibert A.E., Barbazuk W.B., Lal S., Settles A.M. (2018) RNA Binding Motif Protein48 is required for U12 splicing and maize endosperm differentiation. Nucleic Acids Res., In review (https://doi.org/10.1101/341917).
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Hacisalihoglu G., Kantanka S., Miller N., Gustin J.L., Settles A.M. (2018) Modulation of early seedling performance via priming under sub-optimal temperatures. PLOS One, In review.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Springer N.M., Anderson S.N., Andorf C.M., Ahern, K.R., Bai F., Barad O., Barbazuk W.B., Bass H.W., Baruch K., Ben-Zvi G., Buckler E.S., Bukowski R., Campbell M.S., Cannon E.K.S., Chomet P., Dawe R.K., Davenport R., Dooner H.K., Du L.H., Du C., Easterling K.A., Gault C., Guan J.C., Hunter C.T., Jander G., Jiao Y., Koch K.E., Kol G., Kollner T., KudoT., Li Q., Lu F., Mayfield-Jones D., Mei W., McCarty D.R, Noshay J., Portwood J.L., Ronen G., Settles A.M., Shem-Tov D., Shi J., Soifer I., Stein J.C., Suzuki M., Vera D.L., Vollbrecht E., Vrebalov J.T., Ware D., Wei S., Wimalanathan K., Woodhouse M.R., Xiong W., Brutnell T.P. (2018) The W22 genome: a foundation for maize functional genomics and transposon biology. Nature Genetics 50(9):1282-1288.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gabay-Laughnan S., Settles A.M., Hannah L.C., Porch T.G., Becraft P.W., McCarty D.R., Koch K.E., Zhao L., Kamps T.L., Chamusco K.C., Chase C.D. (2018) Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize. G3: Genes, Genomes, Genetics. 8(1):291-302.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lappe R.R., Baier J.W., Boehlein S.K., Huffman R., Lin Q., Settles A.M., Hannah L.C., Stewart J.D., Scott M.P., Myers A.M., Hennen-Bierwagen T.A. (2018) Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm. Proc Natl Acad Sci U.S.A. 115(1):E24-E33.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hacisalihoglu G., Burton A.L., Gustin J.L., Eker S., Asikli S., Heybet E.H., Ozturk L., Cakmak I., Yazici A.,Burkey K.O., Orf J., Settles A.M. (2018) Quantitative trait loci associated with soybean seed weight and composition under different phosphorus levels.J. Integr. Plant Biol.doi:10.1111/jipb.12612.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gustin J.L., Boehlein S.K., Shaw J.R., Junior W., Settles A.M., Webster A., Tracy W.F. Hannah L.C. (2018)Ovary abortion is prevalent in diverse maize inbred lines and is under genetic control. Scientific Reports. 8:13032.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?59th Annual Maize Genetics Conference, St. Louis, Missouri Student Talk - Engineering amyloplast 6-phosphogluconate dehydrogenase activity to improve heat stability of the oxidative pentose phosphate pathway Full Author List: Ribeiro, Camila2; Myers, Alan M.3; Hennen-Bierwagen, Tracie3; Cline, Kenneth C.1 2; Tracy, William F.4; Boehlein, Susan D1; Hannah, L. Curtis1 2; Settles, A. Mark1 2 1 Horticultural Sciences Department, University of Florida, Gainesville, Florida, 32611; 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32611; 3 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, 50011; 4 Department of Agronomy, University of Wisconsin, Madison, Wisconsin, 53706 Student Talk - Evolutionarily conserved role of a novel RNA binding protein involved in the splicing of U12-type introns Full Author List: Siebert, Amy E.1; Gronevelt, J. Paige1; Kenney, Catalina V.1; Davenport, Ruth2; Barbazuk, W. Brad2; Westrick, Randal J.1; Settles, A. Mark2; Lal, Shailesh K.1; Madlambayan, Gerard J.1 1 Department of Biological Sciences, Oakland University, Rochester, MI, 48309; 2 Department of Horticultural Sciences, University of Florida, Gainesville, FL, 32611? Poster - Machine vision seedling emergence assay for maize seed biology Full Author List: Miller, Nathan1; Gustin, Jeff2; Baier, John2; Spalding, Edgar1; Settles, Mark2 1 Department of Botany, University of Wisconsin-Madison, Madison, WI: 2 Horticultural Sciences Department, University of Florida, Gainesville, FL Poster - Engineering amyloplast 6-phosphogluconate dehydrogenase activity to improve heat stability of the oxidative pentose phosphate pathway Full Author List: Ribeiro, Camila2; Myers, Alan M.3; Hennen-Bierwagen, Tracie3; Cline, Kenneth C.1 2; Tracy, William F.4; Boehlein, Susan D1; Hannah, L. Curtis1 2; Settles, A. Mark1 2 1 Horticultural Sciences Department, University of Florida, Gainesville, Florida, 32611; 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32611; 3 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, 50011; 4 Department of Agronomy, University of Wisconsin, Madison, Wisconsin, 53706 Poster - Cold maize phenotyping: Effect of seed priming on maize seedling emergence Full Author List: Hacisalihoglu, Gokhan1; Gustin, Jeff2; Kantanka, Sarfo1; Settles, A. Mark2 1 Department of Biological Sciences, Florida A&M University, Tallahassee, FL 32307; 2 Horticultural Science Department, University of Florida, Gainesville, FL 3261 Poster -Protein-protein interaction of splicing factors involved in the processing of U12 introns in maize Full Author List: Corll, Jacob1; Brigolin, Christian J.1; Shodja, Donya1; Martin, Federico2; Settles, A. Mark2; Lal, Shailesh1 1 Department of Biological Sciences, Oakland University, Rochester Hills, MI 48309; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611 Poster - Anomalous splicing of U12-type introns underlies the developmental defects of a maize mutant in a novel RNA binding motif protein 48 (rbm48) Full Author List: Lal, Shailesh K.1; Bai, Fang2; Shodja, Donya1; Brigolin, Christian J.1; Martin, Federico2; Tseung, Chi-Wah2; Barbazuk, W. Brad2; Settles, A. Mark2 1 Department of Biological Sciences, Oakland University, Rochester Hills, MI 48309; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611 Poster - The dosage-effect defective kernel1 (ded1) transcription factor locus intersects genome dosage and imprinting regulation of endosperm development Full Author List: Mudunkothge, Janaki S.1; Char, Si Nian2; Zhang, Junya1; Spielbauer, Gertraud1; Baier, John1; Yang, Bing2; Settles, A. Mark1 1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611; 2 Department of Genetics, Development and Cell Biology, Iowa State University Ames, IA 50011 Poster - Parent-of-origin effect rough endosperm mutants alter cellular development of the endosperm in maize Full Author List: Bai, Fang1; Daliberti, Mary1; Bagadion, Alyssa1; Davenport, Ruth1; Xu, Miaoyun2; Li, Yubing1; Evans, Mathew M. S.3; Barbazuk, W. Brad1; Settles, A. Mark1 1 University of Florida, Horticultural Sciences Department, Gainesville, FL, USA 32611; 2 Chinese Academy of Agricultural Sciences, BeiJing, P. R. China; 3 Carnegie Institution for Science, Stanford, CA, USA 94305 2017 University of Florida Plant Molecular & Cellular Biology Retreat Daytona Beach, Florida Student talk - Engineering amyloplast 6-phosphogluconate dehydrogenase activity to improve heat stability of the oxidative pentose phosphate pathway. 2Camila Ribeiro, 3Alan M. Myers, 3Tracie Hennen-Bierwagen, 1,2 Kenneth C. Cline, 4William F. Tracy, 1Susan D. Boehlein, 1,2 Curt Hannah, 1,2 A. Mark Settles 1Horticultural Sciences Department, University of Florida, Gainesville, FL; 2Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL; 3Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University; 4Department of Agronomy, University of Wisconsin How have the results been disseminated to communities of interest?Results have been disseminated by publishing 2 peer reviewed scientific papers, submission of 5 additional manuscripts under review, as well as through several presentations to the to the scientific community. In December 2016 PI gave an invited seminar for the Interdisciplinary Plant Group at the University of Missouri in Columbia, Missouri titled "Maize endosperm cell differentiation requires the minor spliceosomes". In January 2017 PI gave an invited seminar at the KWS Saat Gateway Research Center in St. Lois, Missouri titled "Phenotype discovery from maize seed phenomics". In March 2017 PI gave an invited seminar at NSF Integrative Organismal Systems in Washington, D.C. titled "Transdisciplinary studies of maize kernel biology". Also, in March of 2017 two student talks and seven posters were presented at the 59th Annual Maize Genetics Conference in St. Lois, Missouri. What do you plan to do during the next reporting period to accomplish the goals?Grant Applications: A total of three grant applications were completed during this reporting period. On 10/24/16 a proposal for $175,860 was submitted to UF-CASIS on Domesticating Algae for Sustainable Production of Feedstocks in Space. On 11/15/16 a pre-proposal was submitted to NIFA-SCRI for $6,200,000 on Sweet CAP: Integrated technologies to improve sweet corn production and marketability. On 1/9/17 a proposal was submitted to NSF-IOS on The Ded1 Transcription Factor Intersects Genome Dosage and Imprinting Regulation of Endosperm Development. FAMU Student Visits: Every summer, we host undergraduate students from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Graduate Student Recruitment: Recruitment of graduate students from multipleprograms at UF including: Plant Molecular &Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued.

Impacts
What was accomplished under these goals? Science Reported in Literature: Gault C.M., Martin F., Mei W., Bai F., Black J.B., Barbazuk W.B., Settles A.M. (2017) Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development. Proc. Natl. Acad. Sci. U.S.A., In press. Abstract RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2.ZRSR2mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maizergh3mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced inrgh3Genes affected inrgh3andZRSR2mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. Thergh3mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. Hacisalihoglu G., Settles A.M. (2017) Quantification of seed ionome variation in 90 diverse soybean (Glycine max) lines. J. Plant Nutrition. In Press. Abstract Climate change and rising carbon dioxide (CO2) levels are expected to reduce the mineral nutrient content of soybean seeds. The main objective of this study was to survey diverse soybean germplasm for variation in seed elemental concentrations and their relationships between elements, protein content, and individual seed weight. Seeds from 90 soybean genotypes were weighed and subjected to inductively coupled plasma-mass spectrometry (ICP-MS) ionomics analysis and Carbon/Nitrogen (C/N) analysis to determine protein. The results demonstrated substantial variation with the possibility of significantly improving most mineral nutrients, especially selenium (Se), copper (Cu), iron (Fe), and manganese (Mn). This diverse survey identifies genotypes that can complement existing soybean breeding programs for improving seed nutritional quality. Correlation analysis identified two clusters of co-variant elements: zinc (Zn), phosphorus (P), and sulfur (S) as well as Zn, Cu, Se, and rubidium (Rb) were positively correlated with each other. Tolerable upper limits of Rb intake are not defined for humans illustrating the need to monitor trace elements along with desirable nutrients. Student Training: PI served as chair on the Phd Committee of one doctoral student, and as a member on the PhD Committee of 6 additional doctoral students. Three post-doctoral students were mentored, and six undergraduate research interns were trained during this reporting period.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Gault C.M., Martin F., Mei W., Bai F., Black J.B., Barbazuk W.B., Settles A.M. (2017) Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development. Proc. Natl. Acad. Sci. U.S.A., In press.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Hacisalihoglu G., Settles A.M. (2017) Quantification of seed ionome variation in 90 diverse soybean (Glycine max) lines. J. Plant Nutrition. In Press.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Settles A.M., Martin F., Tseung C.W., Moses J.D., Karoblyte V., Bagadion A.M., Ferradaz E., Patel A., Altshuler S., Baier J. (201X) Bulked segregant analysis (BSA) for large-scale mapping of rough endosperm (rgh) seed mutants in maize. G3: Genes, Genomes, Genetics, In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Springer N.M., Anderson S.N., Andorf C.M., Ahern, K.R., Bai F., Barad O., Barbazuk W.B., Bass H.W., Baruch K., Ben-Zvi G., Buckler E.S., Bukowski R., Campbell M.S., Cannon E.K.S., Chomet P., Dawe R.K., Davenport R., Dooner H.K., Du L.H., Du C., Easterling K.A., Gault C., Guan J.C., Hunter C.T., Jander G., Jiao Y., Koch K.E., Kol G., Kollner T., KudoT., Li Q., Lu F., Mayfield-Jones D., Mei W., McCarty D.R, Noshay J., Portwood J.L., Ronen G., Settles A.M., Shem-Tov D., Shi J., Soifer I., Stein J.C., Suzuki M., Vera D.L., Vollbrecht E., Vrebalov J.T., Ware D., Wei S., Wimalanathan K., Woodhouse M.R., Xiong W., Brutnell T.P. (201X) The W22 genome: a foundation for maize functional genomics and transposon biology. Nature Genetics. In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Huang B., Ratushna V., Baier J., Gustin J.L., Settles A.M., James M.G., Hennen-Bierwagen T.A., Myers A.M. (201X) Regulatory interactions of the starch synthase III N terminal domain in vitro and in vivo in Zea mays endosperm. Plant Physiol., In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Lappe R.R., Baier J.W., Boehlein S.K, Huffman R., Lin Q., Settles A.M., Hannah L.C., Stewart J.D., Scott M.P., Hennen-Bierwagen T.A., Myers A.M. (201X) Functions of maize genes encoding pyruvate orthophosphate dikinase in developing endosperm. Proc. Natl. Acad. Sci. U.S.A., In revision.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?58th Annual Maize Genetics Conference Jacksonville, Florida The maize dosage-effect defective kernel1 (ded1) locus encodes a MYB transcription factor controlling endosperm development and grain-fill. Authors: Mudunkothge, Janaki S.1; Zhang, Junya1; Spielbauer, Gertraud1; Baier, John1; Char, Si N.2; Yang, Bing2; Settles, A. Mark1 1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611; 2 Department of Genetics, Development and Cell Biology, Iowa State University Ames, IA 50011 Conserved gene targets between the maize RGH3 and human ZRSR2 RNA splicing factors Authors: Settles, A. Mark1; Gault, Christine2; Martin, Federico3; Bai, Fang1; Mei, Wenbin1; Davenport, Ruth1;Barbazuk, W. Brad1 1 University of Florida, Gainesville, FL, USA 32611; 2 Cornell University, Ithaca, NY, USA 14853; 3 Colorado State University, Fort Collins, CO, USA 80523 Sequence, assembly and annotation of the maize W22 genome Full Author List: Andorf, Carson8; Ahern, Kevin9; Bai, Fang10; Barad, Omer6; Barbazuk, W. Brad10; Bass, Hank W.4;Baruch, Kobi6; Ben-Zvi, Gil6; Buckler, Edward S.8 9; Bukowski, Robert9; Davenport, Ruth10; Dooner, Hugo K.7; HeDu, Limei7; Du, Chunguang5; Easterling, Katherine A.4; Gault, Christine M.9; Guan, Jiahn-Chou10; Jander, Georg1;Jiao, Yinping2; Koch, Karen10; Kol, Guy6; Kudo, Toru10; Li, Qing11; Lu, Fei9; Mayfield-Jones, Dustin3; Mei, Wenbin10; McCarty, Don10; Portwood, John8; Ronen, Gil6; Settles, Mark A.10; Shem-Tov, Doron6; Soifer, Ilya6; Springer, Nathan M.11;Suzuki, Masaharu10; Vera, Daniel L.4; Vollbrecht, Erik12; Vrebalov, Julia T.1; Ware, Doreen2; Wimalanathan,Kokulapalan12; Xiong, Wenwei5; Brutnell, Thomas P.3 1 Boyce Thompson Institute for Plant Research, Tower Road, Cornell Campus, Ithaca, NY 14853; 2 Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724; 3 Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, MO 63132; 4 The Florida State University, Dept. of Biological Sciences, 319 Stadium Drive, Tallahassee, FL 32306; 5 Montclair State University, 1 Normal Avenue, Montclair, NJ 07043; 6 NRGene, Energin .R Technologies, 3 Golda Meir Street, Ness Ziona, Israel; 7 Rutgers University, Dept. of Plant Science, Waksman Institute, Busch Campus, Piscataway, NJ 08854; 8 USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA 50011; 9 Cornell University, Institute for Genomic Diversity, 159 Biotechnology Bldg, Ithaca, NY 14853; 10 University of Florida, Horticultural Sciences Dept., 1376 Mowry Road, Gainesville, FL 32611; 11 University of Minnesota, College of Biological Sciences, 1475 Gortner Avenue, St. Paul, MN 55108; 12 Iowa State University, College of Agriculture & Life Sciences, 2206 Molecular Biology, Ames, IA 50011 Accurate Calibration Transfer between NIR Spectrometers for Prediction of Single Seed Composition and Shape Traits Authors: Hacisalihoglu, Gokhan1; Gustin, Jeff2; Armstrong, Paul3; Peter, Gary4; Settles, A. Mark2 1 Florida A&M University, Department of Biological Sciences, Tallahassee, FL 32307; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611; 3 USDA-ARS, Center for Grain & Animal H. Research, Manhattan, KS 66502; 4 School of Forest Res. & Conserv., University of Florida, Gainesville, FL 32611 Engineering amyloplast 6-phosphogluconate dehydrogenase to improve heat stability of the oxidative pentose phosphate pathway in maize seed development Authors: Ribeiro, Camila1; Boehlein, Susan K.2; Cline, Kenneth C.1 2; Myers, Alan M.3; Tracy, William F.4; Hannah, L. Curtis1 2; Settles, A. Mark1 2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA, 32611; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL, USA, 32611; 3 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA, 50011; 4 Department of Agronomy, University of Wisconsin, Madison, WI, USA, From Maize to Medicine: Analysis of the human ortholog of a novel RNA Binding Motif Protein involved in disruption of maize cell differentiation and proliferation Authors: Siebert, Amy E.1; Gronevelt, J. Paige1; Pino, Alexis1; Bai, Fang2; Battistuzzi, Fabia U.1; Barbazuk, W. Brad2; Westrick, Randal J.1; Settles, A. Mark2; Lal, Shailesh K.1; Madlambayan, Gerard J.1 1 Department of Biological Sciences, Oakland University, Rochester Hills, MI 48309; 2 Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611 The maize RNA Binding Motif48 (rbm48) locus controls endosperm cell differentiation and proliferation Full Author List: Brigolin, Christian J.1; Bai, Fang2; Shodja, Donya1; Martin, Federico2; Tseung, Chi-Wah2; Davenport, Ruth2; Riegel, Annaliese M.1; Jankulovski, Elizabeth1; Barbazuk, W. Brad2; Settles, A. Mark2; Lal, Shailesh1 1 Department of Biological Sciences, Oakland University, Rochester, MI 48309; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611 Finding the Haploid Needle in a Hybrid Haystack: Discrimination of Haploid Maize Kernels by Single Kernel Near-Infrared Spectroscopy Authors: Gustin, Jeffery1; Frei, Ursula2; Baier, John1; Settles, A. Mark1; Lubberstedt, Thomas2 1 Horticultural Sciences Department, University of Florida, Gainesville, FL; 2 Department of Agronomy, Iowa State University, Ames, IA Quantitative genetic analysis of the NC350 x B73 recombinant inbred line (RIL) population using single-kernel near infrared spectroscopy (NIR) Full Author List: Stutts, Lauren1; Baier, John1; Settles, A. Mark1; Gustin, Jeffrey L1 1 University of Florida; Gainesville, Florida, United States 32611 Mapping and genetic transmission of maternal rough endosperm (mre) mutants that display parent-of-origin effects in maize kernel development Authors: Daliberti, Mary1; Bai, Fang1; Settles, A. Mark1 1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611 Parent-of-origin effect mutants regulating endosperm cellular development in the maize seed Full Author List: Bai, Fang1; Daliberti, Mary1; Xu, Miaoyun2; Bagadion, Alyssa1; Li, Yubing1; Baier, John1; Tseung, Chi?Wah1; Evans, Matthew M.3; Settles, A. Mark1 1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611; 2 Biotechnology Research Institute, National Key Facility for Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94025 The maize defective kernel5 (dek5) locus encodes a chloroplast-localized protein required for plastid division, membrane stability, and starch accumulation. Authors: Zhang, Junya1; Wu, Shan2; Barkan, Alice3; Cline, Kenneth1 2; McCarty, Donald1 2; Settles, A. Mark1 2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611; 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611; 3 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 University of Florida Plant Molecular & Cellular Biology Annual Retreat Daytona Beach, Florida Engineering amyloplast 6-phosphogluconate dehydrogenase to improve heat stability of the oxidative pentose phosphate pathway in maize seed Development 2Camila Ribeiro, 1Susan D. Boehlein, 1,2 Kenneth C. Cline, 3Alan M. Myers, 4William Tracy, 1,2 Curt Hannah, 1,2 A. Mark Settles 1 Horticultural Sciences Department, University of Florida, Gainesville, FL; 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University; 4 Department of Agronomy, University of Wisconsin How have the results been disseminated to communities of interest?Results have been disseminated by publishing two peer reviewed scientific papers, submission of four additional manuscripts currently under review, as well as through several presentations to the scientific community. PI gave an invited seminar at the Plant Science Institute at Iowa State University in Ames, Iowa titled "Phenotype discovery from a maize seed and seedling phenomics pipeline" in November of 2015. In March of 2016, one post-doc student gave a talk and eleven posters were presented at the 58th Annual Maize Genetics Conference in Jacksonville, Florida. In May of 2016, one graduate student presented at the University of Florida Plant Molecular and Cellular Biology Annual Retreat in Daytona Beach, Florida. In June of 2016, PI gave an invited lecture within the Department of Plant Biology at the Carnegie Instuition for Science in Stanford, California titled "Maize endosperm cell differentiation requires the minor spliceosome". In August of 2016, PI gave an invited seminar for the Departamento de Fitotecnia at the Universidade Federal de Viçosa in Vicosa, Brazil titled "Phenotype discovery from a maize seed and seedling phenomics pipeline". What do you plan to do during the next reporting period to accomplish the goals?Grant Applications: PI applied for 3 additional grants during this reporting period. On 6/30/16 a cooperative agreement proposal was submitted for a $40,000 grant from USDA-ARS for developing automated solutions for maize haploid classification. On 8/10/16 two grant proposals, just under $500,000 each, were submitted to NIFA-AFRI for either improving maize kernel yield through the Ded1 dosage-effect Myb transcription factor or preventing kernel loss through mitigations of ovule abortion. FAMU Student Visits: Every summer, we host undergraduate students from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Graduate Student Recruitment: Recruitment of graduate students from multipleprograms at UF including: Plant Molecular & Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued.

Impacts
What was accomplished under these goals? Science Reported in Literature: Hacisalihoglu G., Gustin J.L., Louisma J., Armstrong P., Peter G.F., Walker A.R., Settles A.M. (2016) Enhanced single seed trait predictions in soybean (Glycine max) and robust calibration model transfer with near infrared reflectance spectroscopy. J. Agr. Food Chem. 64: 1079-1086 Single seed near-infrared reflectance (NIR) spectroscopy predicts soybean (Glycine max) seed quality traits of moisture, oil, and protein. We tested the accuracy of transferring calibrations between different single seed NIR analyzers of the same design by collecting NIR spectra and analytical trait data for globally diverse soybean germplasm. X-ray microcomputed tomography (μCT) was used to collect seed density and shape traits to enhance the number of soybean traits that can be predicted from single seed NIR. Partial least-squares (PLS) regression gave accurate predictive models for oil, weight, volume, protein, and maximal cross-sectional area of the seed. PLS models for width, length, and density were not predictive. Although principal component analysis (PCA) of the NIR spectra showed that black seed coat color had significant signal, excluding black seeds from the calibrations did not impact model accuracies. Calibrations for oil and protein developed in this study as well as earlier calibrations for a separate NIR analyzer of the same design were used to test the ability to transfer PLS regressions between platforms. PLS models built from data collected on one NIR analyzer had minimal differences in accuracy when applied to spectra collected from a sister device. Model transfer was more robust when spectra were trimmed from 910 to 1679 nm to 955-1635 nm due to divergence of edge wavelengths between the two devices. The ability to transfer calibrations between similar single seed NIR spectrometers facilitates broader adoption of this high-throughput, nondestructive, seed phenotyping technology. Bai F., Daliberti M., Bagadion A., Xu M., Li Y., Baier J., Tseung C.W., Evans M.M., Settles A.M. (2016) Parent-of-origin-effect rough endosperm mutants in maize. Genetics. 204: 221-231. Parent-of-origin effect loci have non-Mendelian inheritance in which phenotypes are determined by either the maternal or paternal allele alone. In angiosperms, parent-of-origin effects can be caused by loci required for gametophyte development or by imprinted genes needed for seed development. Few parent-of-origin effect loci have been identified in maize (Zea mays) even though there are a large number of imprinted genes known from transcriptomics. We screened rough endosperm (rgh) mutants for parent-of-origin effects using reciprocal crosses with inbred parents. Six maternal rough endosperm (mre) and three paternal rough endosperm (pre) mutants were identified with three mre loci mapped. When inherited from the female parent, mre/+ seeds reduce grain-fill with a rough, etched, or pitted endosperm surface. Pollen transmission of pre mutants results in rgh endosperm as well as embryo lethality. Eight of the mutants had significant distortion from the expected one-to-one ratio for parent-of-origin effects. Linked markers for mre1, mre2, and mre3 indicated that the mutant alleles have no bias in transmission. Histological analysis of mre1, mre2, mre3, and pre*-949 showed altered timing of starch grain accumulation and basal endosperm transfer cell layer (BETL) development. The mre1 locus delays BETL and starchy endosperm development, while mre2 and pre*-949 cause ectopic starchy endosperm differentiation. We conclude that many parent-of-origin effects in maize have incomplete penetrance of kernel phenotypes and that there is a large diversity of endosperm developmental roles for parent-of-origin effect loci. Student Training: PI served as the chair on the PhD committee of two doctoral students, and as a member on the PhD committee of 5 additional doctoral students. Six undergraduate research interns and one high school student intern were trained during this reporting period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hacisalihoglu G., Gustin J.L., Louisma J., Armstrong P., Peter G.F., Walker A.R., Settles A.M. (2016) Enhanced single seed trait predictions in soybean (Glycine max) and robust calibration model transfer with near infrared reflectance spectroscopy. J. Agr. Food Chem. 64: 1079-1086
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Bai F., Daliberti M., Bagadion A., Xu M., Li Y., Baier J., Tseung C.W., Evans M.M., Settles A.M. (2016) Parent-of-origin-effect rough endosperm mutants in maize. Genetics. 204: 221-231.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Gault C.M., Martin F., Mei W., Black J.B., Barbazuk W.B., Settles A.M. (2015) Aberrant U12 intron splicing due to the hypomorphic rough endosperm3 splicing factor locus. Nature Communications, In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Settles A.M., Martin F., Tseung C.W., Moses J.D., Karoblyte V., Bagadion A.M., Ferradaz E., Patel A., Altshuler S., Baier J. (2015) Bulked segregant analysis (BSA) for large-scale mapping of rough endosperm (rgh) seed mutants in maize. G3: Genes, Genomes, Genetics, In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Huang B., Ratushna V., Baier J., Gustin J.L., Settles A.M., James M.G., Hennen-Bierwagen T.A., Myers A.M. (201X) Regulatory interactions of the starch synthase III N terminal domain in vitro and in vivo in Zea mays endosperm. Plant Physiol., In revision.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Lappe R.R., Baier J.W., Boehlein S.K, Huffman R., Lin Q., Settles A.M., Hannah L.C., Stewart J.D., Scott M.P., Hennen-Bierwagen T.A., Myers A.M. (201X) Functions of maize genes encoding pyruvate orthophosphate dikinase in developing endosperm. Proc. Natl. Acad. Sci. U.S.A., In revision.


Progress 07/01/15 to 09/30/15

Outputs
Target Audience:The target audience is undergraduate students, graduate students, post-doctoral researchers, the maize genetics scientific community and seed company scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?In August of 2015, PI gave an invited talk at the FASEB Mechanisms in Plant Development Conference in Saxton's River, Vermont titled "Maize endosperm cell differentiation requires the minor spliceosome." What do you plan to do during the next reporting period to accomplish the goals?Every summer, we host undergraduate students from Florida Agricultural and Mechanical University (FAMU) to intern in our lab. FAMU is a historically African-American college, therefore with these student internships we aim to help increase diversity in the academic scientific sector. Recruitment of graduate students from multiple programs at UF including: Plant Molecular & Cellular Biology, Horticultural Sciences, Genetics & Genomics, Bioinformatics will be pursued

Impacts
What was accomplished under these goals? PI served as a chair on the PhD committee of two doctoral graduate students. Additionally, PI served as a member on the PhD committee of three doctoral graduate students. PI also served as a mentor to 3 post-doctoral students. One undergraduate and one high school intern were also mentored by the PI during this reporting period.

Publications