2005 Annual Report
The unit contains three individual projects:.
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 This research project is evaluating 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, and 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. Our lab will employ a combination of molecular and cellular approaches in these systems to characterize the expression and physiological function of vacuolar ion transporters and will use these ion transporters as "bait" in yeast and plant systems to identify the molecules that interact with these transporters and, thus, regulate ion homeostasis. Upon characterizing and identifying the ensemble of ion transporters and their regulatory molecules, our lab will begin to manipulate ion storage, signal transduction events, and the environmental constraints of traditional agricultural practices.
We believe 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, and it binds calcium to a state that renders the calcium unavailable for human absorption. It is important to note that direct absorption of free oxalate from plant foods has been shown to be a problem for many prone to urinary stone formation, with over 75% of all urinary stones containing calcium oxalate as their primary component. 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 aid scientists in determining the pathway(s) of oxalate biosynthesis and where this biosynthesis occurs within a cell. The linkage of oxalate biosynthesis to other metabolic processes is 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 scientists the ability to alter the production of oxalate in plants, thereby permitting alterations in a plant's mineral bioavailability and making the plant parts more nutritious. It is important to know if a plant that normally has oxalate and calcium oxalate crystals will be impacted 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 contribute to the scientific community’s knowledge on manipulating these items.
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 CAX 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
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
Project 2: Genetic engineering of vacuolar H+/metal antiport activity CALCIUM LEVELS ALTERED IN POTATO The capacity to alter calcium levels in potato tubers through genetic manipulations has not been previously addressed, and this potential alteration is important since increased calcium in potatoes may increase the production rate by enhancing tuber quality and storability. Additionally, increased calcium levels in this and other important agricultural crops may help reduce the incidence of osteoporosis. Scientists at the Children's Nutrition Research Center at Houston, TX, have demonstrated that potato tubers expressing the Arabidopsis H+/Ca2+ transporter sCAX1 contain up to three times more calcium (throughout the tuber) than wild-type tubers. Furthermore, increased calcium levels in sCAX1-expressing tubers do not appear to alter tuber morphology or yield. Given the preponderance of potato consumption worldwide, these transgenic plants may be a means of marginally increasing calcium intake levels and to our knowledge this study represents the first attempts to use biotechnology to increase the potato calcium content.
Project 3: Investigations into calcium oxalate formation in plants MANIPULATING PLANT CALCIUM OXALATE LEVELS Information is needed to determine if altering oxalate levels in Medicago truncatula affects the partitioning of carbon into other nutrients. Researchers at the Children's Nutrition Research Center at Houston, TX, have analyzed the starch, chlorophyll, and ascorbate levels in plants with altered calcium oxalate crystal formation. Our lab has found that increasing oxalate accumulation in plants resulted in a decrease in the accumulation of ascorbate, starch and chlorophyll. Yet, decreasing the amount of oxalate had no effect on the levels of these same nutrients. Therefore reducing oxalate levels appears to be a feasible approach to improving the nutritional quality of plant foods without impacting key nutrients.
UNDERSTANDING CALCIUM OXALATE ABSORPITON A system to evaluate the absorbability of calcium and soluble oxalate from plants is needed to aid our research on oxalate bioavailability. Scientists at the Children's Nutrition Research Center in Houston, TX, have established a simple in vitro system to quickly assess the absorbability of calcium and soluble oxalate from plant foods. Our lab showed the utility of this system using the edible cactus, which is enriched in calcium but is mostly in the form of calcium oxalate crystals; in oxalate form the calcium is not readily absorbed. This system will allow CNRC researchers to quickly and inexpensively test whether the plants we have isolated, with altered calcium oxalate content, have the desired changes in calcium and oxalate bioavailability.
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 now underway to identify candidate genes within the loci that play a role in seed iron levels. This information will ultimately lead to new molecular markers for use by plant breeders.
Analysis of an RI population from the model legume, Medicago truncatula, for seed mineral concentrations is needed, 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 have now grown this population and harvested seeds and vegetative tissues. The mineral and QTL analyses for Medicago have now been moved up to year 2 of the project. This accomplishment enabled CNRC researchers to speed up the research process and build collaboration with international researchers.
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 preliminary studies show it to have altered zinc physiology. Efforts are underway to develop a growth protocol for this mutant, and to backcross it with its wild type parent.
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.
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.
Project 2: Genetic engineering of vacuolar H+/metal antiport activity Our lab has demonstrated that potato tubers expressing the Arabidopsis H+/Ca2+ transporter sCAX1 contain up to three times more calcium (throughout the tuber) than wild-type tubers. The increased calcium levels in sCAX1-expressing tubers do not appear to alter tuber morphology or yield. Given the preponderance of potato consumption worldwide, these transgenic plants may be a means of marginally increasing calcium intake levels.
Our lab has demonstrated that fruit from tomato plants expressing Arabidopsis H+/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to the control tomatoes. The sCAX1-expressing tomato lines demonstrate increased vacuolar H+/Ca2+ transport, when measured in root tissue, and elevated fruit Ca2+ content and prolonged shelf-life, but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life, but no deleterious effects on plant growth. Such findings suggest that CAX expression may fortify plants with Ca2+, and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities and the capability to harvest vine-ripened tomatoes and ship great distances without deleterious effects.
Project 3: Investigations into calcium oxalate formation in plants We have determined that manipulating the amount of oxalate in plants is possible. 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 the farmers and breeders involved in production of the modified crop.
Project 2: Genetic engineering of vacuolar H+/metal antiport activity 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. New information on mineral content in crop varieties has been specifically presented to ARS, academia, and others. Adoption of these cultivars by farmers and the consumer will depend on market interest in utilizing food and food products of improved nutritional quality.
Project 3: Investigations into calcium oxalate formation in plants 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.
Grusak, M.A. 2004. HarvestPlus biofortification project: Using plant nutritional genomics to alleviate human micronutrient deficiencies. Department of Biology Seminar Series, University of North Texas, October 7, 2004, Denton, Texas. (invited)
Grusak, M.A. 2004. Nutritional genomics: Zinc absorption and retranslocation. HarvestPlus Wheat Biofortification Meeting, Sabanci University, September 14, 2004, Istanbul, Turkey.
Grusak, M.A. 2004. Strategies to enhance the iron and zinc concentrations of staple food crops. CNRC Seminar Series, October 28, 2004, Houston, Texas.
Grusak, M.A. 2004. The role of nutritional genomics in the effort to improve crop micronutrient levels. Agronomy Society of America Annual Meetings, November 1, 2004, Seattle, Washington. (invited)
Grusak, M.A. 2004. Nutritional, anti-nutritional, allergenic, and other health-related aspects of food and forage legumes. Cross-Legume Advances through Genomics (CATG) Conference, December 14, 2004, Santa Fe, New Mexico. (invited)
Grusak, M.A. 2005. The application of transgenic methods. 2005 American Association for the Advancement of Science Annual Meeting, Symposium on Biofortification: Science for Better Health, February 21, 2005, Washington, DC. (invited)
Grusak, M.A. 2005. Understanding plant metal homeostasis to improve the nutritional quality of plant foods. University of Massachusetts-Amherst, Plant Biology Graduate Program, February 17, 2005, Amherst, Massachusetts. (invited)
Grusak, M.A. 2005. Understanding plant metal homeostasis to improve the nutritional quality of plant foods. University of Texas-El Paso, Department of Chemistry, February 25, 2005, El Paso, Texas. (invited)
Project 2: Genetic engineering of vacuolar H+/metal antiport activity Dr. Kendal Hirschi was an invited speaker at the Texas Produce Convention, August 11, 2005. He communicated his lab research to the growers of Texas.
Project 3: Investigations into calcium oxalate formation in plants Nakata, P. 2005. Investigations into calcium oxalate crystal formation in Medicago truncatula. Model Legume Congress, June 2005, Pacific Grove, California. (invited)
Nakata, P. 2005. Insights into oxalate biosynthesis: Developing strategies to improve the nutritional quality and production of plant foods. Summer FASEB Research Conference, July 2005, Tucson, Arizona. (invited)
Pomper, K.W., Grusak, M.A. 2004. Calcium uptake and whole-plant water use influence pod calcium concentration in snap bean plants. Journal of the American Society for Horticultural Science. 129(6):890-895.
Park, S., Kim, C., Pike, L., Smith, R., and Hirschi, K. 2004. Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter. Molecular Breeding. 14:275-282.
Pittman, J.K., Cheng, N., Shigaki, T., Kunta, M., Hirschi, K. 2004. Functional dependence on calcineurin by variants of the saccharomyces cerevisiae vacuolar ca2+/h+ exchanger vcx1p. Molecular Microbiology. 54(4):1104-1116.
Hirschi, K. 2004. The calcium conundrum. Both versatile nutrient and specific signal. Plant Physiology. 136:2338-2342.
Sze, H., Padmanaban, S., Cellier, F., Honys, D., Cheng, N., Bock, K.W., Conejero, G., Li, X., Twell, D., Ward, J.M., Hirschi, K. 2004. Expression patterns of a novel atchx gene family highlight potential roles in osmotic adjustment and k+ homeostasis in pollen development1[w]. Plant Physiology. 136:2532-2547.
Grusak, M.A. 2005. Golden rice gets a boost from maize. Nature Biotechnology. 23(4):429-430.
Yang, S., Carter, S.A., Cole, A.B., Cheng, N., Nelson, R. 2004. A natural variant of a host rna-dependent rna polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proceedings of the National Academy of Sciences. 101(16):6297-6302.
Grusak, M.A., Cakmak, I. 2005. Methods to improve the crop-delivery of minerals to humans and livestock. In: Broadley, M.R., White, P.J., editors. Plant Nutritional Genomics. Oxford:Blackwell Publishing. p. 265-286.
Kim, K.M., Park, Y.H., Kim, C.K., Hirschi, K., Sohn, J.K. 2005. Development of transgenic rice plants overexpressing the arabidopsis h(+)/ca(2+) antiporter cax1 gene. Plant Cell Reports. 23(10-11):678-682.
Vasconcelos, M., Musetti, V., Li, C., Datta, S.K., Grusak, M.A. 2004. Functional analysis of transgenic rice (Oryza sativa L.) transformed with an Arabidopsis thaliana ferric reductase (atfr02). Soil Science and Plant Nutrition. 50:151-1157.
Han, J.S., Kim, C.K., Park, S.H., Hirschi, K., Mok, I.G. 2005. Agrobacterium-mediated transformation of bottel gourd (Lagenaria siceraria Standl.). Plant Cell Reports. 23(10-11):692-698.
Lopez-Millan, A., Ellis, D.R., Grusak, M.A. 2005. Effect of zinc and manganese supply on the activities of superoxide dismutase and carbonic anhydrase in Medicago truncatula wild type and raz mutant plants. Plant Science. 168:1015-1022.
Abbo, S., Molina, C., Jungmann, R., Grusak, M.A., Berkovitch, Z., Reifen, R., Kahl, G., Winter, P., Reifen, R. 2005. Qtl governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.). Theoretical and Applied Genetics. 111(2):185-195.
Pittman, J.K., Shigaki, T., Marshall, J.L., Morris, J.L., Cheng, N., Hirschi, K. 2004. Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter. Plant Molecular Biology Reporter. 56(6):959-971.
Grusak, M.A., Li, C., Moffet, M., Weeden, N.F. 2004. Map position of the FRO1 locus in Pisum sativum. Pisum Genetics. 36:6-8.