2009 Annual Report 1a.Objectives (from AD-416) 1) Identify and characterize plant genes whose products are involved in root iron acquisition or phloem-to-seed delivery of iron, and assess their overall contribution to seed iron content;. 2)Determine the biophysical mechanisms and molecular processes occurring within pods that regulate calcium movement into developing legume seeds;. 3)Develop cost-effective methods for the intrinsic stable isotope labeling of plants for use in bioavailability studies;. 4)Identify cod [calcium oxalate defective] mutant genes in Medicago truncatula by positional cloning;. 5)Determine whether multiple pathways of oxalate biosynthesis exist in plants;. 6)Determine the influence of light on calcium oxalate formation in plants;. 7)Demonstrate the ability to alter nutrient uptake in plants by expression of CAX2 [calcium exchanger 2] variants in Arabidopsis and tomato;. 8)Elucidate the regulation of CAX2; and. 9)Characterize plants perturbed in CAX2 expression; 10) Identify and characterize Arabidopsis CAX2 homologs. 1b.Approach (from AD-416) 1) Use diverse pea germplasm to assess the correlation between whole-plant iron partitioning, root iron reductase activity, and seed iron concentration; measure seed mineral concentrations in recombinant inbred lines of Arabidopsis thaliana and Medicago truncatula to identify quantitative trait loci associated with elevated iron content in seeds; conduct global gene expression studies with Medicago truncatula plants bearing mutations in known iron genes to identify new genes involved in iron homeostasis;. 2)Measure and characterize calcium accretion in developing seeds of chickpea and measure individual components of calcium transit from pod wall to seeds;. 3)Analyze selenate influx kinetics in wheat and broccoli roots and the timing of selenate administration, in order to optimize isotopic selenate incorporation in edible tissues; generate plants labeled with isotopic selenate, or with deuterium oxide, for collaborative bioavailability studies;. 4)Use genetic mapping technologies coupled with molecular and transgenic complementation analysis to map and identify genes required for calcium oxalate formation;. 5)Analyze metabolic precursors of oxalate, using mutants with altered oxalate levels, to determine the biosynthetic pathway of oxalate in Medicago truncatula;. 6)Measure biomass, chlorophyll, starch, and oxalate levels of plants grown under differing light intensities, and use cytochemical investigations to assess the role of oddly shaped plastids present in oxalate accumulating cells;. 7)Engineer modified versions of the Arabidopsis broad substrate specificity vacuolar antiporter CAX2 into Arabidopsis and tomato; measure mineral concentrations in tissues; measure transport capacity of variant proteins in both yeast and plant systems;. 8)Identify novel regulatory proteins that interact with the N-terminal autoinhibitory sequence of CAX2;. 9)Measure phenotypic changes in plants demonstrating an altered expression of the native CAX2 transporter; and 10) Clone and characterize CAX2 homologs in an attempt to understand functional and regulatory diversity among transporters. 3.Progress Report Knockout mutants in Arabidopsis thaliana and Medicago truncatula for several iron-related genes were obtained and Arabidopsis transgenic lines over-expressing an oligopeptide transporter (thought to be involved in iron transport) were developed and grown to maturity for seed mineral analyses; differential Fe concentrations in seeds of certain lines were informative for the role of specific gene products in whole-plant iron movement. An analysis was made of the peptide transporter clade of the oligopeptide transporter (OPT) family in rice. Expression studies and bioinformatic analyses were conducted on OPT genes; iron transport capacity of the OPT proteins was also measured. Calcium-related investigations with chickpea and Medicago pod walls were carried out, although using slightly different approaches than originally envisioned at the start of the next 5 year research project. Because genomic resources have not advanced as much as anticipated in chickpea, we directed all our efforts on Medicago pod tissues. Available on-line transcriptome databases were searched for calcium-related genes and the expression of these genes in developing pod wall tissues in order to develop hypotheses related to their role in calcium nutrition of developing legume seeds. (Project. 1)Heterologous expression of CAX2 and CAX5 in yeast indicates that both of these transporters possess an N-terminal regulatory region. Transport competition analysis was used to compare the substrate characteristics of CAX2 and CAX5 and shows that despite significant sequence similarity between these genes, there are significant differences in transport activity. (Project. 2) During the past year we have made progress in our effort to understand how crystals of calcium oxalate form in plants. Complementation studies verified that the gene identified from our mapping efforts has an essential function in calcium oxalate formation. Our biochemical studies have given us insight into how this component functions in the process of calcium oxalate crystal formation and how researchers might use this information to nutritionally improve plant foods. (Project 3) 4.Accomplishments 1. Enhancing Seed Mineral Concentrations in Legumes: The nutritional improvement of plant foods, especially seed crops, is a goal of plant scientists. Understanding the regions of the genome and the underlying genes that influence seed mineral concentration could help in the development of nutritionally enhanced crop cultivars. Children's Nutrition Research Center scientists measured seed concentrations of calcium, copper, iron, potassium, magnesium, manganese, phosphorus, sulfur, and zinc in genetically defined lines of the model legumes Lotus japonicus and Medicago truncatula. Over 150 genomic locations harboring genes that affected seed mineral concentration were identified; a number of candidate genes within these genomic regions were also described. These results can be used by plant scientists to identify comparable genomic regions in agronomic legumes, thereby helping plant breeders develop tools and strategies for improving the nutritional value of various crop legumes. (CNRC Project 1: Understanding plant nutrient transport to improve food crop nutritional quality and to assess phytonutrient bioavailability) 2. New Genes Identified for Enhancing Calcium Bioavailability in Edible Plants: Calcium, when present as calcium oxalate crystal in foods, is unavailable for nutritional absorption in humans. These crystals are common in edible plant foods, thereby reducing their nutritional quality and potential health benefit. Researchers at the Children's Nutrition Research Center in Houston, TX, have used an integrative molecular, genetic, and biochemical approach with the legume model Medicago truncatula to show that decreasing the expression of certain genes can dramatically reduce the amount of calcium that gets sequestered in the calcium oxalate crystal. Our researchers have used bioinformatic methodologies to show that similar genes are present in a number of different economically important crop plants, such as dry beans, soybeans, and leafy green plants like spinach. Thus, the translation of this discovery toward the improvement of edible plants appears to be in hand. (CNRC Project 3: Investigations into calcium oxalate formation in plants) 3. Identifying Transporters for Increased Calcium in Plant Foods: Improving nutrient levels in popular vegetables is of importance to research nutritionists to improve the food supply for the general public and for developing countries. Children's Nutrition Research Center researchers have successfully characterized a particular group of nutrient transporters (CAX2, CAX5, and CAX6) and have studied how these transporters could be engineered to improve the movement of nutrients from the soil to edible portions of the plant. The transporters move particular minerals, specifically calcium, into storage depots inside of plant cells. Our researchers have characterized the biochemical and genetic properties of these transporters to determine which nutrients they move with high efficiency. The impact of this accomplishment will be the application of this technology into the development of crops with improved calcium content in the edible tissues of the plants. (Project 2: Genetic engineering of vacuolar H+/metal antiport activity) 6.Technology Transfer Number of Active CRADAs 1 Review Publications Klein, M.A., Grusak, M.A. 2009. Identification of nutrient and physical seed trait QTLs in the model legume, Lotus japonicus. Genome. 52:677-691. Morris, J., Tian, H., Park, S., Sreevidya, C.S., Ward, J.M., Hirschi, K.D. 2008. AtCCX3 is an Arabidopsis endomembrane H(+)-dependent K(+) transporter. Plant Physiology. 148(3):1474-1486. Zhao, J., Cheng, N-H., Motes, C.M., Blancaflor, E.B., Moore, M., Gonzales, N., Padmanaban, S., Sze, H., Ward, J.M., Hirschi, K.D. 2008. AtCHX13 is a plasma membrane K(+) transporter. Plant Physiology. 148(2):796-807. Park, S., Elless, M.P., Park, J., Jenkins, A., Lim, W., Chambers IV, E., Hirschi, K.D. 2008. Sensory analysis of calcium-biofortified lettuce. Plant Biotechnology Journal. 7(1):106-117. Fu, X., Peterson, J.W., Hdeib, M., Booth, S.L., Grusak, M.A., Lichtenstein, A.H., Dolnikowski, G. 2009. Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry (LC/MS). Analytical Chemistry. 81:5421-5425. Vasconcelos, M.W., Li, G.W., Lubkowitz, M.A., Grusak, M.A. 2008. Characterization of the PT clade of oligopeptide transporters in rice. The Plant Genome. 1(2):77-88. Tang, G., Qin, J., Dolnikowski, G.G., Russell, R.M., Grusak, M.A. 2009. Golden Rice is an effective source for vitamin A. American Journal of Clinical Nutrition. 89:1776-1783. Park, S., Doege, S.J., Nakata, P.A., Korth, K.L. 2009. Medicago truncatula-derived calcium oxalate crystals have a negative impact on chewing insect performance via their physical properties. Entomologia Experimentalis et Applicata. 131:208-215. Sankaran, R.P., Huguet, T., Grusak, M.A. 2009. Identification of QTL affecting seed mineral concentrations and content in the model legume Medicago truncatula. Theoretical and Applied Genetics. 119:241-253. Blair, M.W., Astudillo, C., Grusak, M.A., Graham, R., Beebe, S.E. 2009. Inheritance of seed iron and zinc concentrations in common bean (Phaseolus vulgaris L.). Molecular Breeding. 23:197-207. Grusak, M.A. 2009. The Lentil: Botany, Production and Uses. Preston, UK: CABI. pp. 368-390.