2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This CRIS unit contributes to the goals of NP107, Human Nutrition, through Component 6 (Health Promoting Properties of Plant and Animal Foods) and Component 7 (Bioavailability of Nutrients and Food Components), and will have direct relevance to Performance Goal 3.1.3 (Nutritious plant and animal products: Develop more nutritious plant and animal products for human consumption) and will also contribute to NP302, Plant Biological and Molecular Processes. This project contributes to ARS Strategic Goal 4: Improve the Nation's Nutrition and Health; Objectives 4.1.1 and 4.1.2. The unit contains three individual projects:. 1)Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability;. 2)Genetic engineering of vacuolar H+/metal antiport activity; and. 3)Investigations into calcium oxalate formation in plants. Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability Our knowledge is incomplete regarding the mechanisms and regulatory processes responsible for the transport, partitioning, and storage of minerals, or the biosynthesis of phytochemicals, in crop plants. This is unfortunate, because the integration of these mechanisms and processes helps to determine the nutritional composition of edible plant organs, and may influence our ability to utilize nutrients from foods. A better understanding of these processes is needed, in order to design biotechnological or breeding strategies for improving the nutritional quality of our food supply. In studies using both model plant species and agronomic crops, we will investigate the role and influence of various molecular, membrane localized, cellular, and whole-plant processes on the movement and partitioning of minerals (especially calcium, iron, and zinc) in plants and develop methods to incorporate labeled compounds in crop plants, such that the absorption of various phytonutrients from plant foods can be easily studied in humans. This project is important because the nutritional health and well-being of humans are entirely dependent on plant foods. Plants are critical components of the dietary food chain in that they provide almost all essential mineral and organic nutrients to humans, either directly, or indirectly when plants are first consumed by animals. Unfortunately, not all plant foods contain the full complement of essential nutrients needed for human health, nor do they usually contain given nutrients in sufficiently concentrated amounts to meet daily dietary requirements in a single serving. In addition, because of low intakes of fruit and vegetables, many Americans are not consuming the recommended dietary intake of many nutrients, especially minerals such as calcium. Inadequate intake of essential nutrients is a prime factor in a number of diet-related illnesses such as osteoporosis, certain cardiovascular diseases, and macular degeneration. Besides the negative impact these illnesses have on quality of life, they also put a tremendous burden on our national economy through avoidable health-care costs. To avoid these problems and to ensure the adequate nutrition of all US citizens, efforts are needed to improve the nutritional quality of plants, with respect to both nutrient concentration and composition. Project 2: Genetic engineering of vacuolar H+/metal antiport activity Plants can not run from environmental stresses; they are forced to adapt in order to survive. Our researchers are studying the mechanisms by which plants sequester nutrients and toxic metals into the plant vacuole to cope with environmental challenges. At the molecular level, our goals are to understand the structure, biological function, and regulation of transporter proteins that control trafficking into and out of the plant vacuole; to learn how to manipulate the expression and function of these transporters to increase the nutritional content of crop plants, improve plant productivity, and cleanse polluted soils. These objectives are integral components of the global agricultural movement whose aim is to end world hunger by developing innovative ways of increasing grain yields, particularly via the use of genetically improved food plant varieties. The model systems utilized in our laboratory include the yeast Saccharomyces cerevisiae and the plant Arabidopsis thaliana. We employ a combination of molecular and cellular approaches in these systems to characterize the expression and physiological function of vacuolar ion transporters. Additionally, we are using these ion transporters as "bait" in yeast and plant systems to identify the molecules that interact with these transporters and, thus, regulate ion homeostasis. Once we have characterized and identified the ensemble of ion transporters and their regulatory molecules, we can begin to manipulate ion storage, signal transduction events, and the environmental constraints of traditional agricultural practices. Our lab believes it is important to determine the fundamental mechanisms of plant ion homeostasis using genetic model systems, and researchers are applying our basic research in yeast and Arabidopsis to the manipulation of agriculturally important crops such as tomatoes, potatoes, rice, and carrots. We are assessing whether transgenic carrots and potatoes that express Arabidopsis calcium transporters will have increased levels of bioavailable calcium and whether the expression of Arabidopsis vacuolar metal transporters in various transgenic plants can be used for phytoremediation to "mine" toxic metals out of polluted soils. Project 3: Investigations into calcium oxalate formation in plants Basic knowledge is lacking regarding the mechanisms and regulatory processes responsible for calcium oxalate formation in crop plants. Oxalate is an antinutrient and toxin that is present in many edible plants. Oxalate binds calcium in a state that renders it unavailable for human absorption and direct absorption of free oxalate from plant foods has been shown to be a problem for many prone to urinary stone formation. Over 75% of all urinary stones contain calcium oxalate as their primary component. A better understanding of these processes is needed, in order to design strategies for improving the nutritional quality of our food supply. As a step toward elucidating the mechanisms and regulatory processes controlling calcium oxalate formation, researchers plan to utilize novel calcium oxalate defective mutants that range in oxalate content. These mutants will be used to identify genes that affect calcium sequestration and oxalate biosynthesis and metabolism. Analysis of these mutants will also help scientists determine the pathway(s) of oxalate biosynthesis and where this biosynthesis occurs with in a cell. Studies investigating the linkage of oxalate biosynthesis to other metabolic processes are being studied to determine the affects of altering calcium oxalate formation for plant growth and development. The potential impact is to better understand how calcium oxalate is made, thus giving us the ability to alter the production of oxalate in plants. This will allow for alterations in a plant's mineral bioavailability, thus making it more nutritious for consumers. It is important to know if a plant that normally has oxalate and calcium oxalate crystals will be affected by the change (gain or loss) in its ability to form crystals. This is important because if the plant is adversely affected then it will not be marketable even though it is potentially more nutritious. This research will lead to improved nutritional quality of the US food supply by enhancing the overall content or by improving the bioavailability of essential minerals in important food crops. Understanding how calcium and oxalate are handled by plants will also contribute to our knowledge on manipulating these items. 2.List by year the currently approved milestones (indicators of research progress) Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability Year 1 (2005) Complete the whole-plant iron partitioning and root reductase studies in diverse pea lines. Initiate growth of Arabidopsis RILs for seed harvest and Fe analysis. Complete the development of the Medicago truncatula RIL population (to F7 generation). Obtain Medicago truncatula metal homeostasis mutants (from TILLING Project). Initiate developmental characterization of Ca accretion in chickpea and pea. Conduct selenate uptake studies in wheat and broccoli. Year 2 (2006) Perform QTL analysis for seed Fe concentration and begin fine mapping of candidate loci in Arabidopsis. Analyze seeds of Medicago RIL population (from France) and conduct QTL analysis. Conduct functional studies with Medicago metal homeostasis mutants and initiate microarray studies. Measure apoplastic Ca in chickpea and pea pod tissues. Conduct timing studies for selenate isotope application in wheat and broccoli. Year 3 (2007) Continue fine mapping of candidate seed Fe loci in Arabidopsis. Continue QTL analysis and initiate fine mapping of Medicago RIL population. Continue functional studies and microarray analyses with Medicago metal homeostasis mutants. Measure Ca diffusivity of funicular tissues in chickpea. Improve heavy-water labeling procedures for tomato, pepper, and cantaloupe. Year 4 (2008) Complete the fine mapping of candidate seed Fe loci in Arabidopsis. Continue fine mapping of candidate seed metal loci in Medicago RIL population. Continue functional studies with Medicago metal homeostasis mutants. Conduct Ca influx studies and Ca-oxalate analyses in chickpea and Medicago pod tissues. Execute Se and heavy-water intrinsic labelings of various crops for collaborative bioavailability projects. Year 5 (2009) Develop Arabidopsis and Medicago truncatula transgenic lines (altered Fe-related genes) to conduct additional functional analyses. Use bioinformatic analyses to identify orthologues of Ca-related genes in Medicago and chickpea; develop macroarrays and study gene expression profiles in pod tissues. Project 2: Genetic engineering of vacuolar H+/metal antiport activity Year 1 (2005) Generate plant expression vectors and create CAX2 transformants. Characterize CAX2 transgenic lines and initiate transport studies and phenotype analysis. Begin studies investigating CAX2 autoinhibition. Begin CAX2 mutagenesis. Screen for plant gene products which activate CAX2. Begin phenotype analysis of CAX2 mutants. Isolate additional CAX2 alleles and begin transport studies. Isolate CAX5 and CAX6 alleles. Clone CAX6 cDNA. Begin studies using yeast heterologous expression. Year 2 (2006) Complete characterization of CAX2 transgenic lines, including phenotype analysis and transport studies. Continue studies investigating CAX2 autoinhibition. Complete CAX2 mutagenesis. Identify CAX2 regulatory proteins. Complete phenotype analysis of CAX2 mutants. Begin characterization of CAX5 and CAX6 regulation. Finish transport studies in yeast. Year 3 (2007) Complete CAX2 autoinhibition studies. Begin preliminary investigations of CAX2 regulatory proteins. Complete transport studies with CAX2 mutants. Generate CAX2 double mutants. Initiate phenotype analysis of CAX5 and CAX6 alleles. Begin analysis of plant transport properties of CAX5 and CAX6 alleles. Year 4 (2008) Continue investigations of CAX2 regulatory proteins. Analyze phenotype of CAX2 double mutants. Conclude CAX5 and CAX6 regulation studies. Complete phenotype analysis of CAX5 and CAX6 alleles. Year 5 (2009) Complete investigations of CAX2 regulatory proteins. Conduct transport studies with CAX2 double mutants. Complete the analysis of plant transport properties of CAX5 and CAX6 alleles. Project 3: Investigations into calcium oxalate formation in plants Year 1 (2005) Cross cod mutant to A20 mapping line, grow F1 and bulk F2 seeds Measure ascobate levels in selected Medicago truncatula lines Measure starch, oxalate, and chlorophyll levels in leaves of various mutant lines Grow, harvest, fix and embed plant tissues for microscopic examination Year 2 (2006) Screen F2 mapping population for calcium oxalate phenotype and generate seed bulks from crosses to new mapping lines (French). Initiate feeding studies with radiolabeled asorbate and other potential oxalate precursors Grow plants under different light conditions to assess oxalate levels Conduct biomass measurements of plants grown under different light regimes Determine calcium availability of a plant with a genetic reduction in oxalate and calcium oxalate formation (additional milestone). Year 3 (2007) Isolate genomic DNA, conduct PCR, analyze products on agarose gels, and assign cod mutation to chromosome. Screen F2 mapping population for calcium oxalate phenotype from other mapping lines Process radiolabeled tissues for micorautoradiography Section embedded tissues for microscopic examination Year 4 (2008) Continue refinement of mutation map position using available markers and initiate complementation analysis. Complete microautoradiography studies. Initiate photosynthetic measurements under different light regimes. Conduct TEM analysis of cod mutants and wild type plants. Year 5 (2009) Complete photosynthetic study Complete TEM study Complete complementation analysis 4a.List the single most significant research accomplishment during FY 2006. Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability Iron Reduction in Leaves Contributes to Iron Delivery to Seeds Iron is an essential nutrient for humans, which sometimes is limiting in the food supply; thus, researchers have been interested in identifying ways to increase iron concentration in plant foods, and especially in seed crops. Iron, as ferric citrate, is transported to plant leaves yet we don't know if this iron needs to be changed in order for it to be redistributed from leaves to more important developing seeds, which would be food for human consumption. Children's Nutrition Research Center researchers studied a line of soybean in which the expression of a gene from another plant allowed for enhanced iron reduction in the soybean leaves. Iron analysis of different above-ground tissues revealed increased iron concentrations in the transgenic plants, including up to a 100% increase in leaves, a 60% increase in pod walls, and a 10% increase in seeds. Although only a modest increase in seed iron levels was found, the results suggest that leaf iron reduction plays a role in the overall process of iron delivery to seeds and these results will help molecular biologists design better strategies to elevate the iron concentration of plant seeds. More iron in edible plant foods will benefit all consumers by helping to ensure that recommended daily iron intakes are achieved. [NP107 - Component 7 (Bioavailability of Nutrients and Food Components)] 4b.List other significant research accomplishment(s), if any. Project 2: Genetic engineering of vacuolar H+/metal antiport activity Increasing Dietary Calcium in Plant Foods A lack of adequate calcium in the diet has been associated with osteoporosis, bone fractures, and others; thus, identifying a means to increase intake is important. Researchers at the Children's Nutrition Research Center have generated novel crops using molecular breeding technology that show increased concentrations of calcium. Such accomplishments should alleviate the lack of calcium in many worldwide individuals' diets. These results were accomplished by using molecular breeding technology, specifically a transporter from plants was expressed at high levels in these crops. The impact of these novel crops will be in their improved nutritional status for consumers. [NP107 - Component 6 (Health Promoting Properties of Plant and Animal Foods)] Project 3: Genetic investigation of calcium oxalate formation Maximizing Absorbable Calcium in Plant Foods Basic knowledge is lacking regarding the mechanisms and regulatory processes responsible for calcium oxalate formation in crop plants. Oxalate is an antinutrient and toxin that is present in many edible plants. Researchers at the Children's Nutrition Research Center at Houston, TX, discovered that by genetically decreasing the amount of calcium locked-up in calcium oxalate crystals found in plants, researchers could increase the amount of available calcium for nutritional absorption. Our findings proved that using an in vitro dialysis system that mimics the human digestion and absorption processes, it is feasible to genetically alter edible plants to increase their nutritional value in terms of calcium. This finding is significant especially when one considers the reliance of worldwide populations on plant foods as their main source of calcium as well as the failure of many of us to meet the recommended daily amount for calcium intake. [NP107 - Component 7 (Bioavailability of Nutrients and Food Components)] 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. This CRIS unit contributes to the goals of NP107, Human Nutrition, through Component 6 (Health Promoting Properties of Plant and Animal Foods) and Component 7 (Bioavailability of Nutrients and Food Components), and will have direct relevance to Performance Goal 3.1.3 (Nutritious plant and animal products: Develop more nutritious plant and animal products for human consumption) and will also contribute to NP302, Plant Biological and Molecular Processes. This project contributes to ARS Strategic Goal 4: Improve the Nation's Nutrition and Health; Objectives 4.1.1 and 4.1.2. Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability Root iron reductase activity and seed iron concentration were measured in 20 diverse genotypes of pea, in order to evaluate the role of this root process on ultimate seed iron concentration. No correlation was found between these parameters, indicating that other processes are responsible for achieving higher seed iron levels. Additionally, the results show that iron reductase activity should not be targeted as a strategy to enhance seed iron levels, as has been previously suggested. An elevated iron level in foods for humans is an important goal, which will ensure adequate iron intake from the food supply. Two recombinant (RI) populations of Arabidopsis thaliana were grown to maturity in order to harvest seeds and to analyze these for mineral concentrations. Quantitative trait locus (QTL) analysis was performed to identify genetic loci with relevance to various essential minerals, but especially iron. Seed iron concentrations varied 3-fold among the RI lines, and 6 QTLs were identified for seed iron concentration between the two populations. Efforts are underway to identify candidate genes within the loci that play a role in seed iron levels. Several candidate genes are currently being assessed through the use of knock-out mutants for these genes. This information will ultimately lead to new molecular markers for use by plant breeders to help elevate seed iron concentrations in several crops. We pursued the analysis of an RI population from the model legume, Medicago truncatula, as we believed this plant would allow us to identify more genes relevant to seed metal concentration. At the start of the project we had planned to generate and map a new RI population, which would have required two years to complete. Fortunately, a pre-existing RI population was made available to us by collaborators in France, and we opted to use this population. Seed and leaf mineral analyses have been completed and several QTLs identified for seed iron and zinc concentrations. Additional molecular markers are being tested for this population and we have begun to generate Near Introgressed Lines (NILs) to fine map the loci. This information will enable breeders to increase the micronutrient concentration of important agronomic legumes. Our lab worked with colleagues from the University of California-Davis to use the TILLING procedure to identify Medicago truncatula mutants defective in metal-related genes. Three putative mutants were identified with altered sequences in the gene MtZIP1, which encodes for a zinc transport protein. One of the mutants was confirmed as a gene knockout, and ongoing studies show it to have altered zinc physiology, and possibly altered copper physiology. Efforts are underway to develop a growth protocol for this mutant, and to backcross it with its wild-type parent. Analyses of growth, seed production, and zinc uptake are being conducted with mutant and wild-type plants. These studies will contribute to our overall understanding of how zinc is handled within plants, and which genes regulate its distribution to edible tissues. The contribution of leaf iron reductase activity to seed iron accumulation in soybean was investigated. Iron is transported to soybean leaves as ferric citrate, but we don't know if a reduction step is required before the iron is remobilized from leaves to seeds in the phloem transport pathway. We studied a transgenic line of soybean in which a gene (AtFRO2) from Arabidopsis thaliana conferred enhanced iron reductase activity throughout the plant. Protoplasts isolated from leaf cells of the transgenic and wild-type plants showed that AtFRO2 expression increased leaf iron reduction capacity up to 3-fold. Iron analysis of different shoot tissues revealed increased iron concentrations in the transgenic plants, including up to a 100% increase in leaves, a 60% increase in pod walls, but only a 10% increase in seeds. It appears that enhanced leaf iron reductase capacity has a minimal impact on seed iron levels, but that it does contribute to the overall process. These results will help molecular biologists design transgenic strategies in various crops to help elevate the iron concentration of harvested seeds. To understand how calcium is delivered to legume seeds, we characterized calcium concentration and content in developing seeds of chickpea and pea. The two legumes have different pod morphologies, which helps to provide clues about the pathway of calcium movement into seeds. Chickpea seeds are suspended within the interior of an inflated pod during early seed development, and are connected to the pod wall only through their funiculus, while pea seeds remain in lateral and funicular contact with the pod wall throughout seed growth. Both legumes demonstrated calcium accretion throughout seed development, suggesting that calcium diffusion along the funiculus is an important pathway for calcium accretion in legume seeds. Because this calcium comes from adjacent pod wall tissues, studies are underway to assess the pool of calcium in pod wall tissues throughout seed development. These studies will help provide information to breeders to help them design strategies to enhance seed calcium levels in crop legumes. Selenate uptake studies were initiated in wheat and broccoli in order to investigate concentration-dependent kinetics and to assess competitive interactions with sulfate. Experiments and data analysis are ongoing. The results of these studies will be used to develop more effective strategies for stable isotope labeling of these crop plants for use in human studies. Heavy water labeling procedures were designed and tested in order to generate labeled carotenoids in maize, golden rice, sweet potato, peppers, and the blue-green algae Spirulina. Labeled material has been sent to collaborators at Tufts University for use in clinical trials that will assess carotenoid absorption and the conversion of selected carotenoids to vitamin A. The techniques to label these plants provide a useful tool for human nutritionists to study nutrient absorption and metabolism. The ultimate results of these studies will be used by nutritionists and policy makers to revise dietary recommendations for carotenoids and vitamin A. Project 2: Genetic engineering of vacuolar H+/metal antiport activity We have previously shown that expression of N-terminally truncated CAX2 (sCAX2, lacking amino acids 1-42) in yeast can suppress the Ca2+ and Mn2+ hypersensitivity of a yeast mutant (K667), indicating the ability to transport these cations. Full-length CAX2 cannot suppress the Ca2+ and Mn2+ sensitivity of the K667 strain when grown on high metal concentrations but can weakly suppress both Ca2+ and Mn2+ sensitivity at lower concentrations, indicating that an N-terminal regulatory mechanism regulates both Ca2+ and Mn2+ transport activity. By comparison with the N-terminal regulatory mechanism for CAX1, it is assumed that cation transport in full-length CAX2 is inhibited by an autoinihibitory mechanism and that regulatory proteins that interact with the CAX2 N-terminus activate cation transport. Yeast screens have been performed by co-expressing an Arabidopsis cDNA library into CAX2-expressing K667 to identify these interacting proteins which may activate either Ca2+ or Mn2+ transport activity of CAX2. Ca and Mn concentrations were chosen that do not allow growth of full-length CAX2 when expressed alone in K667. Yeast colonies that were able to grow on Ca or Mn media were screened and cDNA clones isolated by plasmid rescue. Currently 9 cDNA clones have been isolated and sequenced, the majority of which encode proteins with no known function, while none are of known function or have been previously characterized. None of the clones are identical to any of the cDNAs identified in the previous CAX1 activator screen. So far all of the identified cDNAs that have been re-tested allow suppression of both Ca2+ and Mn2+ sensitivity of CAX2-expressing yeast, regardless of whether they were identified in the Ca2+ or Mn2+ screen. In addition, they do not activate either CAX1 or CAX5 transport activity. Further experiments are validating the interactions and further candidate clones are being isolated for sequencing. The most closely related CAX-like genes to CAX2 are CAX5 and CAX6. CAX5 and CAX6 knockout lines have been identified and generation of CAX2 CAX5 and CAX2 CAX6 double knockout lines has been initiated. Project 3: Genetic investigation of calcium oxalate formation Increasing the amount of available calcium in plant foods will provide more calcium intake, thus a healthier diet may be achieved. Researchers at the Children's Nutrition Research Center have determined that genetically reducing plant oxalate content is possible and can be used to increase calcium availability in plant foods. Biologists will benefit from the basic knowledge gained regarding the impact of high and low oxalate levels on plant growth and development. The consumer demanding a more healthful food supply will benefit through the future production of nutritionally enhanced plant foods, as well as will the farmers and breeders involved in production of the modified crop. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability Stable-isotopically labeled foods have been transferred to human nutrition colleagues to support clinical investigations. Current scientific results have been transferred to other research scientists through meeting presentations and scientific publications. Project 2: Genetic engineering of vacuolar H+/metal antiport activity We have been working with a biotech company to try to develop the technology to make lettuce contain more calcium. Project 3: Genetic investigation of calcium oxalate formation The information gained from this research is primarily destined for use by other scientists. Research results have been reported at national and international meetings, and detailed findings have been published in international scientific journals. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability MA Grusak presented the seminar "Nutritional Enhancement and Evaluation of Plant Foods," USDA-ARS Children's Nutrition Research Center, Faculty Seminar Series, Houston, TX. October 2005. MA Grusak presented the invited Symposium Presentation "Health-Promotive and Nutritional Attributes of Pulse Crops: Benefits to Humans and Other Animals," International Annual Meetings of the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Salt Lake City, UT. November 2005. MA Grusak presented the seminar "Nutritional Evaluation of Plant Foods Using Stable Isotope Technologies: Research at the Interface of Plant and Human Nutrition Sciences," Department of Food and Nutrition Seminar Series, Purdue University, West Lafayette, IN. December 2005. MA Grusak presented the invited Talk "Applications of Food Biotechnology to Human Nutrition: Golden Rice and Selenium in Wheat," American Baker's Association - USDA/ARS Technical Issues Conference, Houston, TX. January 2006. MA Grusak presented the seminar "Nutritional Evaluation of Plant Foods Using Stable Isotope Methodologies," Food Science Seminar Series, Cornell University, Ithaca, NY. February 2006. MA Grusak presented the invited Symposium Presentation "Golden Rice: Recent Developments in its Biotechnology, Safety, and Nutritional Value," 31st Rice Technical Working Group Meeting, The Woodlands, TX. February 2006. MA Grusak presented the seminar "Strategies to Improve the Nutritional Value of Rice – Efforts to Alleviate Micronutrient Malnutrition in the Developing World," USDA/ARS Dale Bumpers National Rice Research Center, Stuttgart, AR. May 2006. MA Grusak presented the invited Oral Presentation "Nutritional Diversity in Germplasm Collections: Valuable Resources to Enhance the Nutritional Quality of Food Crops," USDA/ARS Plant Genetic Resources Conference, Ames, IA. June 2006. MA Grusak presented the invited Oral Presentation "Plant Sources of Dietary Iron: Diversity in Tissue Iron Concentration," 13th International Symposium on Iron Nutrition and Interaction in Plants, Montpellier, France. July 2006. Cockrell J (2006) Researcher in the News - Dr. Michael Grusak at the USDA-ARS Children's Nutrition Research Center. Texas Rice 6:5-7. Tiefert R (2006) The Bean Scene: Green beans provide the right nutrition to turn kids into lean, "mean," healthy machines. School Foodservice & Nutrition 60:59-68. Project 2: Genetic engineering of vacuolar H+/metal antiport activity KD Hirschi Invited Speaker: "Molecular Biology and Food Biotechnology," American Bakers Association (ABA)/ARS Technical Issues Conference. Houston, TX. January 2006. Project 3: Genetic investigation of calcium oxalate formation PA Nakata Invited Speaker: "Developing strategies to improve the nutritional quality and production of plant foods through the manipulation of calcium oxalate formation," International Symposium in Memory of Vincent R. Fransceshi, Washington State University, Pullman, WA. June 2006. "The secret life of plant crystals," by Ivan Amato. Chemical Engineering News. 2006, 84:26-27. "Calcium oxalate crystals defend again chewing insects" by Peter Minorsky. On the inside Plant Physiology, 2006:141:1-2. Review Publications Park, S., Kang, T.S., Kim, C.K., Han, J.S., Kim, S., Smith, R.H., Pike, L.M., Hirschi, K.D. 2005. Genetic manipulation for enhancing calcium content in potato tuber. Journal of Agricultural and Food Chemistry. 53(14):5598-5603. Shigake, T., Barkla, B.J., Miranda-Vergara, M.C., Zhao, J., Pantoja, O., Hirschi, K. 2005. Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/h+ exchanger cax1. Journal of Biological Chemistry. 280(34):30136-30142. Shigake, T., Kole, M., Ward, J.M., Sze, H., Hirschi, K. 2005. Cre-loxp recombination vectors for the expression of Riken Arabidopsis full-length cDNAs in plants. Biotechniques. 39(3):301-303. Park, S., Cheng, N., Pittman, J.K., Yoo, K.S., Park, J., Smith, R.H., Hirschi, K. 2005. Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis h+/ca2+ transporters. Plant Physiology. 139:1194-1206. Coyne, C.J., Brown, A., Timmerman-Vaughan, G.M., Mcphee, K.E., Grusak, M.A. 2005. Refined usda-ars pea core collection based on 26 quantitative traits. Pisum Genetics. 37:3-6. Coyne, C.J., Grusak, M.A., Razai, L. 2005. Variation for pea seed protein concentration in the usda pisum core collection. Pisum Genetics. 37:7-11. Mcphee, K.E., Grusak, M.A. 2006. Identification of qtl controlling seed mineral content in pea [abstract]. International Conference on Legume Genomics and Genetics. p. 109. Hodnett, G.L., Burson, B.L., Rooney, W.L., Dillon, S.L., Price, H.J. 2005. Pollen-pistil interactions result in reproductive isolation between Sorghum bicolor and divergent Sorghum species. Crop Science. 45:1403-1409. Korth, K.L., Doege, S.J., Park S.H., Goggin, F.L., Wang, Q., Gomez, S.K., Liu, G., Jia, L., Nakata, P.A. 2006. Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing insects. Plant Physiology. 141(1):188-195. Cheng, N., Pittman, J.K., Shigaki, T., Lachmansingh, J., Leclere, S., Lahner, B., Salt, D.E., Hirschi, K.D. 2005. Fuctional association of arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis. Plant Physiology. 138:2048-2060. Vasconcelos M., Grusak M.A. 2006. Status and future developments involving plant iron in animal and human nutrition. In: Barton, L.L., Abadia, J., editors. Iron Nutrition in Plants and Rhizospheric Microorganisms. New York: Springer-Verlag. p. 1-22. Grusak, M.A. 2005. Plant foods as sources of vitamin A: Application of a stable isotope approach to determine vitamin A activity. Trees for Life Journal. 1:4. Grusak, M.A., Tang, G., Russell, R.M. 2006. Golden rice: developments in its biotechnology, safety, and nutritional evaluation. In: Proceedings of the 31st Rice Technical Working Group Meeting, February 26-March 1, 2006, The Woodlands, Texas. Abstract. p. 3. Grusak, M.A. 2006. Plant sources of dietary iron: Diversity in tissue iron concentration. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. 2006. Abstract p. 56. Waters, B.M., Li, C.-L., Blair, M., Beebe, S., Grusak, M.A. 2006. Influence of rhizosphere pH on whole-root ferric reductase activity in diverse accessions of Phaseolus vulgaris. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 89. Narayanan, N.N., Vasconcelos, M.W., Grusak, M.A. 2006. Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using cDNA macroarrays. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 90. Waters, B.M., Grusak, M.A. 2006. Using natural variation for gene discovery to improve seed iron nutritional value. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 128. Sankaran, R., Grusak, M.A. 2006. Identification of quantitative trait loci contributing to seed iron concentration in Medicago truncatula. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 127. Alcaniz, S., Cerdan, M., Sanchez-Sanchez, A., Jorda, J.D., Grusak, M.A. 2006. Iron uptake from FeEDDHA isomers by pea plants at different pH values. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 115. Narayanan, N.N., Grusak, M.A. 2006. Assessing shoot-root communication in the regulation of iron homeostasis in the fefe melon mutant. In: Proceedings of the Thirteenth International Symposium on Iron Nutrition and Interactions in Plants, July 3-7, 2006, Montpellier, France. p. 91. Stephens, B.W., Cook, D.R., Grusak, M.A. 2006. Functional characterization and expression analysis of the ZIP1 metal transporter in Medicago truncatula. In: Plant Biology 2006 Final Program, August 5-9, 2006, Boston, Massachusetts. p. 160. Nakata, P.A., McConn, M.M. 2005. Isolation and characterization of medicago truncatula mutants with increased calcium oxalate accumulation [abstract]. 2005 Model Legume Congress. Paper No. 20. Park, S., Wang, Q., Nakata, P.A., Korth, K. 2005. The role of calcium oxalate crystals in Medicago truncatula defense against chewing insects [abstract]. American Society of Plant Biologists Annual Meeting. p. 157. Nakata, P.A., Mcconn, M.M. 2005. Investigations into calcium oxalate crystal formation in medicago truncatula[abstract]. 2005 Model Legume Congress. p. 20. Nakata, P.A., Mc Conn, M. 2006. A genetic mutation that reduces calcium oxalate content increases calcium availability in medicago truncatula [abstract]. Plant Biology. p.303. Morris, J., Park, S., Kim, J., Nakata, P.A., Hirschi, K. 2006. Expression of an arabidopsis ca2+/h+ transporter increases bioavailable ca2+ in edible carrot roots [abstract]. Plant Biology. p. 161. Naryanan, N.N., Vasconcelos, M.W., Grusak, M.A. 2006. 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