Jay Thelen - US grants

Plant seeds are important, renewable sources of natural products such as oil, protein, starch and fiber. Though the biosynthetic pathways for these storage compounds are mostly known it is not clear how these pathways are regulated in oilseeds which produce higher quantities of oil and protein. Quantitation and identification of proteins expressed at multiple stages during embryo development of four diverse oilseeds will systematically reveal the dynamics of protein networks responsible for oil and protein accumulation in four diverse oilseeds. Public dissemination of these data in multiple, user-intuitive formats will provide new directions for the scientific community to engineer seed metabolism for the benefit of health and environment.

The website will be open for viewing within the first year of the proposal and will include both one- and two-dimensional gels of membrane and total proteins, respectively, from various stages of oilseed development (within first two-three years of the proposal). Identification of proteins will commence with the purchase of a tandem mass spectrometer in the third year of funding. Identities of the first 300 protein spots (project goal of 500-600) of Arabidopsis and oilseed rape will be posted to the website by the end of the third year support and prior to publication. Identification of 450-500 castor and soybean proteins will also commence in the third year of funding with a projected release date to begin in year four. Manual de novo sequencing of unidentified proteins and potential post-translationally modified proteins will be the subject of research for year five.

IDENTIFICATION AND ABSOLUTE QUANTITATION OF PROTEIN PHOSPHORYLATION NETWORKS IN OILSEEDS

PI: Jay J. Thelen, Department of Biochemistry, University of Missouri-Columbia
Collaborator: Dong Xu, Computer Science Department, University of Missouri Columbia

Plant seeds are important, renewable sources of natural products such as oil, protein, starch and fiber. Though the biosynthetic pathways for these storage compounds are mostly known, it is not clear how these pathways are regulated in oilseeds which produce higher quantities of oil and protein. Protein phosphorylation is an important regulatory mechanism for many metabolic enzymes and preliminary data reveal hundreds of phosphoproteins in developing oilseed rape and soybean. Identification and quantification of phosphoprotein control mechanisms in developing seed of three different oilseeds will provide the basis for understanding the regulatory networks involved in oilseed development and will help in designing new strategies for crop improvement. This proposal aims to unravel networks of phosphoproteins and protein kinases primarily involved in the regulation of metabolic processes during the oil and protein accumulation phase of seed development in three different oilseed plants, oilseed rape, soybean, and Arabidopsis thaliana. In addition to this fundamental scientific objective, the broader impacts of this proposal include: (1) organizing proteomics-themed workshops and developing web-based proteomics tutorials targeting High School teachers and students, respectively; (2) training plant scientists at the undergraduate through post-doctoral levels in the emerging discipline of proteomics; (3) recruiting under-represented students through University-sponsored undergraduate research programs and fellowships; (4) developing techniques for phosphopeptide identification and quantification using the emerging technology of absolute quantification (AQUA) peptides; (5) generating synthetic AQUA peptide tools for the plant protein phosphorylation community; and (6) public dissemination of phosphoproteomics data in multiple, user-intuitive formats to provide new directions for the scientific community to modify seed metabolism for the benefit of health and environment. For website see http://oilseedproteomics.missouri.edu

David R. Gang (PI), University of Arizona
Carol A. Soderlund (CoPI), University of Arizona
Jay Thelen (CoPI), University of Missouri

Summary of the Project
The rhizome is the original stem of the vascular plant lineage. Before plants developed upright stems, they grew horizontally via rhizomes. All extant primitive vascular plants and many advanced angiosperms still use rhizomes as their sole stem type. Rhizomes are responsible for the invasiveness, hardiness, and even harshness of many of the world's most significant weeds. The ability of these rhizomatous weedy plants to invade new territory via the underground has led to their great success world-wide, and is directly attributable to their rhizomes. In contrast, many of the world's most important medicinal plants, such as ginger and turmeric, produce their medicinally active components in their rhizomes. Despite their importance, very little is known about what genes are involved in the growth and function of rhizomes. The aim of this project is to identify genes and proteins that are expressed exclusively in the rhizome and to characterize the function of specific genes that play important roles in rhizome function from plants across the plant kingdom. Genes that are expressed exclusively or predominantly in rhizomes will be identified by constructing cDNA libraries from developing rhizomes from selected rhizomatous species and sequencing random clones from these libraries to provide a large set of expressed sequence tags (ESTs). Rhizome-specific expression of gene transcripts will be determined using DNA microarrays. Protein levels in the rhizomes versus other tissues will be determined using complementary proteomics technologies. These datasets will be integrated into one database, and will then be comparatively analyzed to identify rhizome-specific genes common to all species and specific to individual species. The roles of specific genes in rhizome differentiation, growth and development will then be validated using a number of approaches, including more detailed expression profiling and functional analysis by gene knock out or heterologous expression in other species. This project will impact our understanding of rhizome growth and function. Because the rhizome is the original vascular plant stem, and is still used by many species as their major stem type, this project will impact our understanding of the evolution of upright stems as well. Because many of the world?s worst weeds spread via their rhizomes and three of the most important of these weedy species will be the subject of investigation, this project will also produce information that could be used to better tailor efforts to control economically important weeds worldwide.

The broader impacts of this project is that it will cross-train undergraduate and graduate students and postdoctoral researchers in multidisciplinary research approaches. This project will integrate research and education in two major ways: 1. Training of students in successful approaches to doing research in an interdisciplinary and geographically dispersed environment, which is becoming more important for success in the biological sciences; 2. Summer research experiences for undergraduate students and high school teachers. A strong emphasis will be placed on recruitment and training of underrepresented minorities and women, especially for teacher intern and undergraduate student positions, but also for the post-doctoral and graduate student positions. A web-based database of EST, microarray and proteomics data will be developed and made publicly accessible at the project web site: www.plant-rhizome.org.

PI: Jay Thelen (University of Missouri-Columbia)
CoPIs: Dong Xu, Dmitry Korkin (University of Missouri-Columbia)

Seeds are carbon- and nitrogen-dense structures that serve as the (plant) propagule and the (human) socioeconomic foundation for modern agriculture. Genetics and environmental conditions dictate the developmental program that ultimately determines both seed yield and composition, although the underlying regulatory proteins and mechanisms are only now beginning to be understood. Regulation of seed development has recently focused on transcriptional and epigenetic control of both embryogenesis and maturation. From a biochemical perspective, few regulatory processes take place within a cell without direct or indirect involvement of proteins, and protein-protein interactions. The nucleus and cytosol are the major locations for transcriptional, translational, and post-translational regulation. A quantitative inventory of the proteins involved in these processes and their interactome network, would be a valuable resource to develop testable models for regulatory control of embryo maturation. The main focus of this project is the parallel characterization of nuclear and cytosolic protein interactomes from developing embryos of soybean and rapeseed using a combination of biochemical, proteomic, and bioinformatic approaches. The major objectives of this project are to: 1) systematically and quantitatively analyze protein-protein interactions from cytosolic and nuclear fractions of developing embryos from soybean and rapeseed using a multi-dimensional biochemical strategy that includes protein crosslinking, co-sedimentation, and quantitative mass spectrometry; 2) perform bioinformatic and statistical analyses of potential interacting proteins using data from aim 1 for comparison between soybean and rapeseed, and develop generic protein complex identification tools for such data; and 3) disseminate software, proteomic and protein interaction data by deposition into extant databases (PRIDE, http://www.ebi.ac.uk/pride/; Database of Interacting Proteins, DIP, http://dip.doe-mbi.ucla.edu/; Soybean Knowledge Base, http://soykb.org) and develop a new web database.

While the research project targets the protein interactome in developing soybean and rapeseed embryos, it has broader significance for studying plants (especially crops) in general. This study will greatly enrich the data describing plant protein interactions, which is currently limited. These data can be used as a template for studying protein interactomes of other plants. Besides the data and their analyses, the techniques and bioinformatic tools that will be developed can also be applied to study interactomes of other species; in particular, experimental protocols and bioinformatic tools (including source code) will be made freely available to the public. The project will provide critical interdisciplinary training in plant biology, proteomics, and bioinformatics for undergraduates, graduate students, and postdoctoral scholars. Current discipline-based education provides very limited training on integrating biology, biotechnology, and bioinformatics, which is often needed in large-scale biological projects. This project provides a problem-based, interdisciplinary environment to provide training in this regard. Students and postdocs will be mentored in scientific writing, scientific presentations, publishing, grant applications, constructive peer review, project management, time management, and personnel management in an effort to direct the next generation of scientists towards the realistic and professional expectations of modern academic or commercial research. Efforts will be made to recruit people with diverse backgrounds to work on this project. Undergraduate researchers in life sciences and journalism will have opportunities to gain unique experience in computational biology and scientific communication through support from a recently awarded Howard Hughes Medical Institute grant.

This project will enhance our understanding of fatty acid synthesis in plants as an important step in developing new strategies for engineering designer oilseeds of the future. Plant oils are a valuable renewable resource for food, fuels and chemicals. The genes that encode the enzymes responsible for lipid synthesis have been identified, but regulation of these genes is still poorly understood. This project will take a multi-disciplinary approach to study the role of newly discovered proteins in regulating a key early step in fatty acid synthesis, which in turn controls total plant oil accumulation. The project will prepare two undergraduate students, including underrepresented minorities, and two graduate students for STEM careers. They will be trained in modern research approaches of plant biotechnology, genetics, and protein science. The undergraduates from University of Southern Mississippi (USM; an especially diverse student population) will travel to University of Missouri (MU) to join the MU undergraduates for a 9-week intensive summer research experience for undergraduates in the Thelen and Van Doren laboratories. The graduate students at MU and USM will acquire transferable skills from formal training in mentoring and science communication and by mentoring the undergraduate students in research.

This project leverages the expertise of a multi-disciplinary team to test the hypothesis that the newly discovered biotin attachment domain-containing (BADC) gene family and its counterpart biotin carboxyl carrier protein (BCCP) collectively act as a molecular rheostat to modulate acetyl CoA carboxylase (ACCase) activity and ultimately seed oil accumulation. Quantitative biophysical analyses will distinguish between two hypotheses for how BADC proteins intercalate into the holo-ACCase complex and reduce enzymatic activity. The analyses will test the alternatives of the BADC structure in solution versus its affinity for the BC subunit enabling BADC to compete with its functional, catalytic sibling protein, BCCP, for access to ACCase. The regulatory role of BADCs during seed development and induced regulation of fatty acid synthesis (FAS) will be characterized by quantifying gene expression and absolute protein levels of the BADC regulatory proteins relative to the ACCase enzyme, under various states of FAS. The rheostat hypothesis will be tested in planta by modulating BADC protein levels and quantifying carbon flux through ACCase and FAS and the effect on total oil accumulation. Additionally, the ability of BADC proteins to alleviate the FAS feedback inhibition that is induced in plants engineered to produce novel oils will be tested. This will clarify metabolic bottlenecks that limit oilseed engineering.

An award is made to the University of Missouri to acquire a high resolution mass spectrometer coupled with an ultra-high performance liquid chromatography system. The instrument will be used to help further our understanding of proteins in a variety of plants including soybean, corn, and elderberry. It will also be used to examine fertility loss in animals at the protein level in support of agriculture. The University is collaborating with Evans Analytical Group (EAG) Columbia (a GLP and CGMP-compliant product development and analytical testing services company based in Columbia, MO) in a continuing industry internship program for undergraduates. Each spring undergraduate interns participate in an intensive four-week training course. This in-depth course offers a select set of high-caliber undergraduates an opportunity to develop analytical skills, with an emphasis on separations and mass spectrometry, in preparation for being employed by EAG Columbia as summer interns. Biochemistry and Chemistry undergraduate students will also use the instrument in advanced lab classes to further their understanding of modern instrumentation.

Mass spectrometry is an essential method for determining the structure of large biomolecules such as proteins. It is also possible to use this method to examine modifications to proteins. The acquisition of the high resolution mass spectrometer will allow the investigators of the project to perform quantitative protein analyses, develop new analysis capabilities, and improve instrument access at the University of Missouri. The major users of the instrument have active research programs examining protein changes in a variety of plants and animals.

Plant oils are a renewable source of food, fuels, and chemicals; world output of plant oils must double by 2030 to meet projected demand. A major source of plant oils comes from seeds. While research has yielded improvements in seed oil quantity and quality, there are tradeoffs for other seed attributes such as protein content and yield. For example, plants that are bred or engineered to produce high seed oil often have decreased protein content or make fewer seeds. The molecular mechanism driving such tradeoffs remains unclear and creates a barrier for making improved crops with specific amounts of oil and protein content. This project seeks to unravel the genetic and molecular mechanisms related to the tradeoffs between oil, protein content, seed size, and seed set in order to enhance seed oil content of crops. By using an integrated approach, this project will investigate biochemical steps used by seeds to prioritize protein or oil production. Using this knowledge, this project will generate plants with improved seed oil production. In addition, this project promotes STEM education using the science of seed oils to train tomorrow's scientists as well as develops a science literacy program using hands-on research experience for students who are training to become future STEM teachers. The scientific and educational outcomes of this project will have broad impacts for both research and society through improved oil production in crop plants.

Quantitative, comparative profiling of high-oil soybean, Camelina, and Arabidopsis will reveal the coordinated metabolic response to de-regulated de novo FAS. These findings will reveal the next metabolic constraint for engineering seed oil content, discover new genes involved in acyl lipid metabolism, and provide the plant lipid community with characterized, high-oil germplasm for gene stacking and comparative genomics. This project will integrate comparative transcriptomics, proteomics, and translatomics during three stages of seed development of FAS-engineered, high-oil plants to develop correlative and kinetic models to make metabolic predictions, and discover coregulated gene networks. Flux analyses and clustering models will be used to link high-oil germplasm to causal effects within combined central and lipid metabolic networks and used to make hypotheses for next-generation crop improvements. Aspects of in vivo FAS rates, the uncoupling of FAS from TAG synthesis that can lead to futile cycles of FAS and breakdown; and acyl fluxes through the lipid metabolic network will be investigated to elucidate new bottlenecks for TAG accumulation as a result of de-regulating FAS. Web resources (fatplants.net) developed as a part of this project will serve to educate the community and provide an interactive tool to integrate metabolism and gene regulation Integrated tools are necessary to inspire tomorrow's scientists and engineers and enable multidisciplinary training of all ages. This project will also develop a hands-on science literacy program for future educators of science and technology (Sci-FEST) and high school science teaching modules on plant metabolism and biotechnology will be created by the program participants.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This project will improve understanding of an important class of proteins, the so-called receptor-like kinases. Like cellular sentries, receptor-like kinases have crucial functions in sensing and orchestrating responses to environmental cues. Kinases perceive molecular signals from other organisms (like pathogens) or from neighboring cells and pass signals along to other proteins to initiate defense responses or other changes, such as halting growth. Consistent with the importance of these functions, kinases are one of the most abundant plant protein families; however, kinases have also proven to be difficult to study. New advances in both understanding and technology will be used in this project to enable matching of plant kinases to the signals that they detect and to the downstream ?recipients? of the information, thereby revealing the function of these proteins. Improvements in knowledge of kinases will open new avenues for improving plant production for food, feed, fiber, and energy. In addition, the project will train at least five young scientists to use advanced technologies and will provide plant science educational opportunities to ~60 teachers via professional development workshops, introduce ~30 Native American high school and undergraduate students to research via week-long plant signaling research project workshops, and provide ~40 summer and academic year undergraduate research opportunities that meld plant and data science.

This research will characterize the functions and substrates of receptor-like kinases (RLKs) of Arabidopsis, rice, and soybean. Objective 1 aims to reveal RLK functions by combing quantification of phosphorylation and S-acylation (e.g., palmitoylation) as indicators of involvement of an RLK in a biological process. This analysis will take advantage of new results that suggest that these posttranslational modifications are antagonistic. The project will use proteomics technology to deeply and quantitatively sample total and plasma membrane-enriched proteins during rice internode development, with and without drought stress; and soybean and rice root responses to conserved putative small secreted peptides. The relatively well-understood Arabidopsis- Pseudomonas syringae interaction will be examined to optimize methods. The focus on peptide hormone ligands and root growth will survey broadly across predicted small secreted peptide families. Objective 2 takes a complementary approach to revealing RLK function, by creating deep (12k) peptide libraries for each species and screening them against purified protein kinases with the goal of identifying kinase clients for at least 25 novel kinases. The kinase client assay accelerates assignment of downstream signaling and its usefulness has been demonstrated by rapid community adoption. In Objective 3, experimental results will be extended using machine learning approaches to predict plant phosphorylation sites and specific kinase clients. Proteome data will be used for molecular network reconstruction and for comparative analysis to identify patterns in RLK signaling. Results and predictions will be used to enhance previously established databases, P3DB and SoyKB, which are already well-used by the community, amplifying project value.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.