2008 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.
Our lab analyzed unique genetic lines of Medicago truncatula, Lotus japonicus, and Arabidopsis thaliana to identify genomic locations associated with elevated mineral concentrations in seeds. Genetic mapping studies and bioinformatic analyses occurred to focus on these genomic locations and to identify candidate genes that might be responsible for the seed mineral traits. Studies with Medicago truncatula zinc and iron mutants were continued to demonstrate the role of unique mutations on the zinc and iron nutrition of this model legume. Studies in chickpea and Medicago, designed to understand the physiology of calcium movement from pods to developing seeds, were initiated and are still in progress. Heavy water labeling of carrots, tomato, and Golden Rice were completed, with materials sent to a collaborator in Boston for use in human nutritional studies [NP 107, Component 2](Project 1). The progress in our lab has been directed at characterizing the functions of Arabidopsis transporters. Every transporters capable of moving different nutrients, and we are making substantial progress toward functional characterization of numerous transport proteins. This should aid both plant and human health [NP 107, Component 2](Project 2). Our lab has progressed in our understanding of how plant calcium oxalate crystals form, how researchers can manipulate crystal accumulation in edible tissues, and how such manipulations impact the nutritional quality of plant foods. Our genetic, molecular, and biochemical studies have given us new insight into the number and types of genes important in calcium oxalate crystal formation and how the gene products function in the process of calcium oxalate crystal formation. Using isolated plant mutants that differ in the amount of calcium they tie-up in the calcium oxalate crystal, coupled with an in vitro system that simulates human digestion and absorption, we have been able to show that decreasing and increasing the amount of calcium tied-up in the crystalline form positively and negatively impacts calcium bioavailability, respectively [NP 107, Component 2](Project 3).
Enhancing Bioavailable Calcium in Vegetables:
Calcium is an important and essential nutrient for humans. Children's Nutrition
Research Center researchers have increased the levels of bioavailable calcium in
vegetables. Our lab directly evaluated the nutritional consequence of transgenic
foods in both animal and human feeding studies. Our team demonstrated that modifyinga single plant calcium transporter leads to more calcium in the food and more calcium absorbed from the transgenic food. This finding is important since the approach in this work can serve as a paradigm about the role of other plant alterations to bioavailability of nutrients contained within the plant matrix. [NP 107, Component 5] (CNRC Project 2)
Identifying Targets for Improving Calcium Bioavailability in Plant Foods:
Oxalate is an antinutrient that when present in foods can tie-up the available
calcium in the nutritionally unavailable form of the calcium oxalate crystal. Such crystals are quite common in edible plants, reducing their nutritional quality. Researchers at the Children's Nutrition Research Center at Houston, TX, discovered, using genetic plant mutants and an integration of molecular, llular, and biochemical methodologies, that knocking out the expression of a particular molecular component dramatically reduces the amount of calcium that gets tied-up in the calcium oxalate crystal. Such a molecular component appears to be a viable universal target that can be utilized in efforts to improve calcium bioavailability in a broad range of edible crop plants. [NP 107,Component 2] (CNRC Project 3)
Understanding Potassium Transport for Improved Plant Productivity:
Potassium plays an essential role in plant growth and development. The Arabidopsis genome contains genes for numerous cation:proton antiporters (named CHX), whose encoded proteins may mediate potassium transport; however, no work has been done looking at the function of CHX transporters. Children's Nutrition Research Center researchers focused on the functional analysis of AtCHX13. We expressed this transporter in various yeast strains in order to compare and contrast transport properties to previously characterized potassium transporters. We monitored where AtCHX13 was expressed in yeast cells and in plants, and we perturbed AtCHX13 expression in plants to examine the effect on potassium transport and plant growth in the transgenic plants. Through this work we have biochemically characterized the first CHX transporter from plants, which should aid in the engineering of CHX transporters for possible improvements in plant productivity. [NP 107, Component 5] (CNRC Project 2)
Identifying Genomic Sites that Influence Seed Mineral Concentration:
Biofortification of foods, achieved by increasing the concentrations of minerals such as iron and zinc, is a goal of plant scientists. Understanding genes that influence seed mineral concentration in a model plant such as Arabidopsis thaliana could help in the development of nutritionally enhanced crop cultivars. Quantitative trait locus mapping for seed concentrations of calcium, copper, iron, potassium, magnesium, manganese, phosphorus, sulfur, and zinc was performed by Children's Nutrition Research Center researchers using two recombinant inbred populations of Arabidopsis, grown on multiple occasions. Over 100 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 indicate that Arabidopsis thaliana is a suitable and convenient model for the discovery of genes that can affectseed mineral levels, and that this plant could be exploited to improveagronomically important food crops. [NP 107, Component 5](CNRC Project 1)
Kriseman, M.L., Grusak, M.A. 2007. Developmental expansion of the hilum in chickpea seed coats. Journal of Semi-Arid Tropical Agricultural Research [serial online]. 5(1). Available: http://www.icrisat.org/Journal/volume5/ChickPea_PigeonPea/cp2.pdf.
Waters, B.M., Grusak, M.A. 2008. Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3. New Phytologist. 177(2):389-405.
Waters, B.M., Grusak, M.A. 2008. Quantitative trait locus mapping for seed mineral concentrations in two Arabidopsis thaliana recombinant inbred populations. New Phytologist. 179:1033-1047.
Grusak, M.A. 2008. Genetic diversity for seed mineral composition in the wild legume Teramnus labialis. Plant Foods for Human Nutrition. 63(3):105-109.
Morris, J., Hirschi, K. 2008. Calcium transporters: From fields to the table. In: Jaiwal, P.K., Singh, R.P., Dhankher, O.P., editors. Plant Membrane and Vacuolar Transporters. Oxfordshire, United Kingdom: CABI Head Office. p. 51-82.
Barkla, B.J., Hirschi, K.D., Pittman, J.K. 2008. Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca(2+) exchangers. Plant Signaling & Behavior. 3(5):354-356.
Mei, H., Zhao, J., Pittman, J.K., Lachmansingh, J., Park, S., Hirschi, K.D. 2007. "In planta" regulation of the "Arabidopsis" Ca(2+)/H(+) antiporter CAX1. Journal of Experimental Botany. 58(12):3419-3427.
Korenkov, V., Hirschi, K., Crutchfield, J.D., Wagner, G.J. 2007. Enhancing tonoplast Cd/H antiport activity increases Cd, Zn, and Mn tolerance, and impacts root/shoot Cd partitioning in Nicotiana tabacum L. Planta. 226(6):1379-1387.
Zhao, J., Barkla, B.J., Marshall, J., Pittman, J.K., Hirschi, K.D. 2008. The "Arabidopsis cax3" mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H(+)-ATPase activity. Planta. 227(3):659-669.
Morris, J., Hawthorne, K.M., Hotze, T., Abrams, S.A., Hirschi, K.D. 2008. Nutritional impact of elevated calcium transport activity in carrots. Proceedings of the National Academy of Sciences. 105(5):1431-1435.
Coyne, C.J., Mcclendon, M.T., Walling, J.G., Timmerman-Vaughn, G.M., Murray, S., Meksem, K., Lightfoot, D.A., Shultz, J.L., Keller, K.E., Martin, R.R., Inglis, D.A., Rajesh, P.N., Mcphee, K.E., Weeden, N.F., Grusak, M.A., Storlie, E.W. 2007. Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes. Genome 50:871-875.
O'Rourke, J.A., Charlson, D.V., Gonzalez, D.O., Vodkin, L.O., Graham, M.A., Cianzio, S.R., Grusak, M.A., Shoemaker, R.C. 2007. Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines. Biomed Central (BMC) Genomics. 8:476.
Wang, J., Wang, Y., Wang, Z., Li, L., Qin, J., Lai, W., Fu, Y., Suter, P., Russell, R., Grusak, M.A., Tang, G., Yin, S. 2008. Vitamin A equivalence of spirulina beta-carotene in Chinese adults assessed by stable isotope dilution and reference techniques. American Journal of Clinical Nutrition. 87:1730-1737.