Iron (Fe) is an essential co-factor in ubiquitous metabolic processes, but it is also potentially toxic to cells. To meet organismal needs while avoiding toxicity, the physiological availability of Fe must be closely regulated. This is achieved by proteins that sense iron status and regulate a cascade of signaling activity to balance iron uptake from diet, storage in tissues, and transport from organ to organ. Errors in the choreography of this Fe-handling system lead to diseases of iron deficiency (e.g. anemia) or Fe excess (e.g. hemochromatosis). In studies of Fe homeostasis, physiological indicators of organism health are routinely observed, but the levels of Fe transporter proteins and details of their regulatory interactions are not readily quantified in situ. Reductionist approaches identify the function of individual proteins and their immediate signaling partners, but these approaches do not explain the complex, interdependent, and multiscale phenomenon of systemic homeostasis. By coupling computational systems modeling with in vivo experiments, however, existing knowledge about Fe handling at the cellular, organ, and organism levels can be integrated. This makes it possible to assess the validity of potential biological mechanisms and identify profitable experimental strategies that efficiently pin down causal molecular phenomena. Furthermore, mathematical models can be used to learn from model systems and then generalize to analogous functional relationships across organisms. This is fortuitous as the model plant Arabidopsis thaliana exhibits modes of Fe regulation that are analogous to those in humans, and Arabidopsis offers a particularly versatile tool for studying cell-type and organ-specific features of iron signaling. Ubiquitin ligases are a particularly important class of Fe-sensor proteins conserved between plants and animals. These intracellular actors bind Fe at specific domains. Fe binding alters their stability and modulates their ability to post-translationally regulate Fe deficiency response proteins. We previously identified BRUTUS (BTS) to be the iron-binding ubiquitin ligase in Arabidopsis and demonstrated that its absence leads to systemic Fe excess. The goal of this project is to use mathematical models and the model organism Arabidopsis to delineate the molecular rules that allow BTS, an enzyme confined to the plant vasculature, to mediate systemic Fe homeostasis. Based on our preliminary mathematical model, we propose the novel hypothesis that BTS plays opposing roles in the plant root and shoot, participating in tissue-specific signaling pathways that enable sensing of Fe status at the principal site of Fe usage (shoot) and communicate Fe need to the site of Fe uptake (root). Our specific Aims are: Aim 1 - Determine how Fe binding modulates the stability, localization and regulatory activity of BTS. Aim 2 - Characterize how oligomerization regulates the mobility, stability, and regulatory activity of ILR3, and, consequently, Fe deficiency response in the root. Aim 3 - Identify the molecular mechanism of BTS function in the shoot and its impact on shoot-to-root signaling.

"Project is in support of PSI" Plant disease resistance proteins of the nucleotide binding leucine-rich repeat (NLR) type are activated and induce a strong defense response known as effector-triggered immunity or ETI, upon recognition of specific pathogen-derived effector proteins. The effectiveness of this system depends on its inactivity when the cognate pathogen is not present, rapid induction when a pathogen is recognized followed by a rapid suppression after induction. The ubiquitin-proteasome pathway, mediated by the sequential actions of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) and E3 (ubiquitin ligase) enzymes is a major protein modification process found in all eukaryotes. Our preliminary data indicates that maize ZmCER9 E3-ligase mediates degradation of the Rp1-D disease resistance protein specifically after its activation. We have further evidence that CER9 may act in a similar way to degrade other plant resistance proteins once activated. This appears to be a previously undescribed mechanism that mediates the deactivation of the defense response after activation. Based on its homology, ZmCER9 appears to be a component of the endoplasmic reticulum associated degradation (ERAD), a fundamental eukaryotic quality-control system that degrades incorrectly folded proteins. In plants this pathway has been relatively poorly characterized and there are no known substrates of the branch of the pathway mediated by CER9. Activated Rp1-D may represent the first known substrate of this branch of the ERAD pathway in plants. We hypothesize that ERAD-Mediated Degradation of Activated NLRs (EMDAN) is a general mechanism for the deactivation of ETI in plants. We propose to use a range of molecular, genomics and cell biology techniques to characterize the role of CER9, ERAD and related pathways involved in ubiquitin/proteasome associated processes in controlling ETI in maize and Arabidopsis.

Minimizing crop loss and increasing output, across the food supply chain, will increase the economic viability of US growers and the global economic competitiveness of industry and stakeholder partners. We have assembled a diverse team across different National and International Universities with faculty that have track records of convergent research, education, and outreach. We will be well positioned to implement a Networks of Networks with diverse backgrounds, ethnicities, genders, experiences, and disciplines to drive research and innovation. Students and postdocs will be exposed to hands-on learning, on-farm technology training, cooperative extension, commercialization, industry engagement, and transdisciplinary education to create a highly trained workforce that is equipped to address food security and safety challenges.

The College of Agriculture and Life Sciences (CALS) at NCState trains students to address challenges in agricultural productivity, the safety and nutrition of the food supply, and the application of plant and animal products to disparate uses such as biofuels. This grant will provide tuition for a total of about 15 Masters students in Biochemistry over 5 years. Graduates of the program will be prepared to find good-paying jobs in the agricultural and food industry in North Carolina, or to continue their academic training. Currently16% of the jobs in NC are based in agriculture (NC Department of Agriculture), with over 80 agbiotech companies in the state. Training in a basic science such as biochemistry will provide students the flexibility to adapt to changes in the job market. The goal of this proposal is two-fold: 1) to diversify the workforce by providing educational opportunities for academically-talented low-income and minority students, and 2) to promote interdisciplinary research and training in biochemistry applied to problems in agriculture and human health. The grant is focused on research areas in nutrition and metabolic regulation that link biochemistry to applications in the Departments of Food, Bioprocessing and Nutrition Sciences; Plant and Microbial Biology; and Animal Science. Students will be recruited from throughout NC. The Masters program is less diverse than the undergraduate program at NCState, justifying the need to promote minority recruitment. The grant will provide tuition for in-coming students to complete a thesis Masters degree. Students will be responsible for paying living expenses. A curriculum and research experience will be devised for each student according to their background and personal goals. During their first year, new students will develop a strong background in biochemistry by completing the 3 core biochemistry classes. The remaining 5 courses will be based on the student's interests. A Fall seminar series will introduce students to research opportunities with the 25 faculty members from the 4 departments in CALS as well as industry opportunities in the RTP area. This seminar will promote interdisciplinary interactions between diverse labs, all requiring knowledge of metabolic pathways to address contemporary problems in microbes, plants, animals and human nutrition. During the Spring semester, students will select 2 labs to investigate further for a month each before choosing a lab for their Masters research. We anticipate the average time to degree will be 2.5 years. During the second year students will participate in professional development discussions to prepare for post-graduation. Overall this grant will provide training for low-income and minority students to contribute to solving challenges in agriculture and human health including environmental changes due to global warming, increased population pressures, and the relationship between nutrition and health.

We propose an Engineering Research Center for Green and Climate Resilient Built Environments (Green CriBs), which will drive innovation, engineering and widespread adoption of novel transparent envelope window and building materials to provide extreme thermal insulation together with dynamic and responsive light admittance for the built environment, its occupants and activities. Doing so will maximize the climate resilience of society, enhance environmental justice, reduce greenhouse gas emissions and accelerate grid decarbonization.

Plant development is frequently celebrated for its plastic nature. The cell fates in the plant tissue are not irreversibly fixed but they are prone to change their identity depending upon intracellular and extracellular signals. Although this concept is well established, we know remarkably little about cell differentiation mechanisms. In the proposed study we aim to i) identify gene regulatory networks involved in specification and differentiation from stem cells to the final stage of differentiation and; ii) predict the cellular strategies used to cope with intrinsic and extrinsic cues, specifically iron availability. The development of phloem sieve elements (SE) is a powerful model for this kind of study as the specification and differentiation occur relatively rapidly (in the course of ~20 cells) and the final termination stage is dramatically characterized by programmed nuclear degradation. Iron availability has been shown to be critical for differentiation and enucleation of human erythroblasts (REF). However, little is known about how nutrient availability affects differentiation of plants cells.

Agriculture is the primary economic activity undergirding human survival and quality of life and global economic development. To grow agricultural productivity we will establish an interdisciplinary graduate training program to address Plant Production within the Targeted Expertise Shortage Area (TESA) of Food Production. The goals of this program are: 1) comprehensively train three PhD fellows, each in a core discipline within plant production with cross-training in complementary areas; 2) provide experiential training within a technology rich, multidisciplinary research and Extension platform; and 3) graduate students proficient at integrating computational, environmental, biological and physical data into decision tools for increased yield and economic sustainability. This will be achieved through: recruitment of top tier, diverse Fellows; intensive advising and mentoring by exemplary faculty; outstanding academic, international, and industry-based research opportunities; leadership and professional development training, and internships with local Agbiotech companies. Fellows������������������ research will be grounded in the innovative research platform (AMPLIFY), a strategic industry-academia- producer partnership conducting interdisciplinary multi-scale systems research to advance high- yield sustainable agriculture to meet our world������������������s growing food requirements. Success will be measured by: 1) diversity of recruits; 2) presentations at professional conferences and publication in refereed journals; 3) timely degree completion; and 4) successful placements in industry, academia, or government appropriate to TESA. This NNF is relevant to the USDA/NIFA Challenge Area, Plant Production. Measurable impacts on TESAs include a more diverse scientific workforce trained in skills necessary to address complex challenges facing agriculture.

In this proposal, we present a novel paradigm for identifying putative cis-regulatory promoter targets that control the regulation of stress responses in plants. This paradigm will also be used to identify critical regulatory components that differentiate the regulatory stress response across different cell types. We first develop the computational and analytical infrastructure needed to build a dynamic model of the gene regulatory network from time-course transcription profile data that quantifies the stress response. Novel analytical model refinement techniques are proposed to reduce the space of feasible solutions, generate specifications for model validation experiments, and test functional redundancy in the response. Parallel computing architectures will be used to scale the implementation of these model refinement approaches to the size and complexity associated with gene regulatory networks. The dynamic model of the gene regulatory network will be used to identify relationships between genes, build corresponding functional modules, and identify putative cis-regulatory promoter targets and regulatory components that can be used to alter responses to biotic and abiotic stresses in plants. Previous cell-specific transcription profiling has indicated that cell types have distinct expression profiles and respond differently to stress. We will generate cell-specific time-course transcription profiles using experiment specifications derived from the dynamic gene regulatory network. These data will be used to create a cell-specific dynamic gene regulatory network for identifying regulators that are key in differentiating the stress response between cell types.

Intellectual Merit: The ability to respond to fluctuations in nutritional availability is critical for all living cells. Multicellular organisms can respond to such challenges by altering a number of physiological, developmental and molecular processes that are often controlled at the level of transcription. While conventional molecular biology and physiological approaches have revealed the importance of genes involved in nutrient uptake and transport, few transcriptional regulators have been identified that coordinate organismal responses to nutrient availability. This research focuses on the following questions: What transcription factors regulate response to iron deprivation in plants? How do these transcription factors interact with each other and other proteins to regulate gene expression? Is there a protein complex that acts as an iron sensing mechanism in plant roots that reacts and leads to the classic response to low iron? Since plant iron transporters translocate other metals nonspecifically, studying this mechanism will further inform us about homeostasis of other metals. Moreover, the protein-protein interactions that we will examine shares features with an iron sensing and response mechanism recently described in mammalian cells and would, therefore, shed new light on conserved iron sensing and response mechanisms. Broader Impacts: Iron deficiency, which causes anemia, is the most prevalent nutritional disorder in the world. Approximately 30% of the world?s population is iron deficient, which results in increased maternal mortality and infant loss, impaired growth and cognitive development and decreased immune response. In addition, iron deficiency is a global problem for the growth of major crops grown in calcareous soils. This project proposes to increase our understanding of how plants respond to iron deprivation with the long term goal of producing plants with enhanced nutritional capacity and tolerance of nutrient-poor soils, thus increasing our ability to address increasing global nutritional needs. In addition to agricultural and nutritional contributions, this project will create resources for the scientific community at large, including a new model for how iron content is sensed within cells. Moreover, the project will provide unique research opportunities for students, in particular, underrepresented minority groups attending smaller historically black colleges and universities. Thus, the project will help to increase the dept and breath of molecular and plant biology researchers.

Plant Systems Biology resides at the intersection of biology and engineering, where practitioners integrate multi-scale systems engineering approaches with biological data. Systems biology is used to model, understand, and control combinatorial biological interactions across physiological, structural, and biomolecular processes. Directed focus of systems biology on plants will facilitate the exploration of the governing mechanisms behind complex biological processes, culminating in the design of whole plant systems; specifically the control of factors that impact plant metabolism, development, and adaptation. Successful Plant Systems Biology research and educational programs will also be at the forefront of the transformation of plant biology in the post-genomic era. While there exists reasonable efforts in plant systems biology that has been established in both the United States and in Europe, consolidated efforts in this field for modeling and controlling whole plant systems remains elusive. With all parties having similar long term goals, it would benefit all parties if efforts were taken to standardize approaches, format data repositories, and clarify policies around coordinated approaches for addressing common objectives. We request funds to support a Plant Systems Biology workshop that will provide a venue for 30 national and international researchers with interests in plant systems biology to discuss and highlight limitations and focus on critical questions associated with feasibility and necessary technological and experimental advances for large-scale design of plant systems. The specific workshop objectives are as follows: 1) Explore and identify areas of complementary interests, needs, and opportunities associated with the growing discipline of Plant Systems Biology. 2) Identify the primary research grand challenges and explore potential limits of the field from both the biological, computational, and engineering perspectives. 3) Formulate funding strategies for future collaborations.