2010 Annual Report
1a.Objectives (from AD-416)
Objective 1. Use genetic, molecular, and physiological approaches to define the role of specific genes and gene products in the acquisition and whole-organism partitioning of minerals (iron, zinc, calcium, and magnesium) and other factors that inhibit or promote absorption of these minerals in plant foods.
Sub-objective 1.A. Identify quantitative trait loci (QTLs) associated with elevated seed or leaf mineral concentrations or seed biomass accumulation.
Sub-objective 1.B. Identify rate-limiting and/or novel genes that contribute to tissue Fe, Zn, Ca, and Mg concentrations or seed biomass accumulation.
Sub-objective 1.C. Assess the functional role of newly identified gene products in whole-plant nutrition and nutrient partitioning.
Sub-objective 1.D. Measure the effects of modulating cation transporter functions on plant Ca, Mg, Fe and Zn content.
Sub-objective 1.E. Measure the impact of altered transport function in agriculturally important crops.
Sub-objective 1.F. Identify and isolate genes that are involved in calcium oxalate crystal formation in selected mutants.
Sub-objective 1.G. Assess the role of the identified genes in calcium oxalate crystal formation.
Objective 2. Conduct animal and human feeding studies to determine mineral bioavailability of the nutritionally enhanced crops.
Sub-objective 2.A. Use novel transgenic plants to dissect the relationship between mineral partitioning and nutrient absorption in mice feeding studies.
Sub-objective 2.B. If the rodent studies demonstrate proof of concept, initiate pilot feeding studies using young adults.
Objective 3. Develop new, cost-effective methods for the intrinsic labeling of plant foods for use in nutrient bioavailability studies.
1b.Approach (from AD-416)
The long-term objective of this project is to contribute to the development of nutritionally enhanced plant foods and to develop tools for testing nutrient bioavailability. We have chosen to work initially with plants that are tractable molecular genetic systems (Arabidopsis, Medicago, and soybean) where we can perform gene discovery quickly. We then translate these findings into agriculturally important crops that can be easily transformed and for which established protocols are in place for measuring nutrient absorption in both mice and humans. Specifically, we will work to identify and characterize genes and gene products that are involved in mineral transport throughout the plant, focusing both on whole organ accumulation and subcellular partitioning of minerals. We also will identify and characterize the molecular processes associated with calcium oxalate formation in plants. We envision this work to eventually have relevance to mineral nutrition improvement (e.g., calcium, magnesium, iron, and zinc) in several agronomic crops. In addition, we will develop new, cost-effective methods for the intrinsic labeling of plant foods, using stable isotopes, in order to facilitate nutrient bioavailability studies in humans. These efforts will expand our capabilities for assessing the absorption and metabolism of various plant-derived minerals and phytochemicals. They also will facilitate the generation of new bioavailability data for various nutrients, which will allow informed decisions when policymakers establish future dietary recommendations for humans.
We have made progress in our effort to understand how crystals of calcium oxalate form in plants. We determined that calcium oxalate mutant 1 is new and calcium oxalate mutant 2 is similar to one we previously identified. As a first step toward isolating the genes important in calcium oxalate formation, we eliminated the genes that were not involved in calcium oxalate formation from mutant 1 and 2. A library of all the genes expressed in Medicago truncatula leaves has also been prepared for use in isolating the genes required for calcium oxalate formation.
We created numerous carrots expressing higher levels of nutrient transporters. Through cooperation with the Vegetable and Fruit Improvement Center (VFIC) at Texas A&M University, the plants were grown and analyzed for nutrient concentrations. The VFIC helped in the initial seed production that is currently still ongoing. As a result, we have generated novel foods that may contain substantially higher levels of nutrients. We have completed the generation of more than 50 primary carrot transformants and greater than 200 Arabidopsis lines. We have monitored growth and fitness of these lines and done preliminary characterization of gene expression levels. To support our generation of these novel crops, we have further characterized the nutrient transporters in other biological systems. We measured transport properties and the ability of each nutrient transporter to accumulate nutrients within the cell. These data will help us dissect how the various carrot lines above accumulate more nutrients.
Genetic diversity studies using unique lines of soybean, or the model legume (Medicago truncatula), were used to identify regions of the plant's genome (DNA) that are associated with higher tissue levels of minerals. We completed the growth, mineral analyses, and statistical tests of data using a Medicago population and obtained genomic locations relevant to higher leaf levels of several minerals. Work with soybean has shown similar results for seeds. A second soybean population was grown and seeds harvested by colleagues at the Beltsville ARS laboratory; these will be analyzed next year. Backcrossing procedures were completed for several Arabidopsis and Medicago lines previously identified as maintaining good copies of genes for elevated levels of seed minerals. These new genetic lines will be analyzed to better define the mineral-enhancing genomic regions of interest. Research involving stable isotope labeling of plants, designed to provide better tools for future human nutrition studies, were carried out with Arabidopsis and soybean. Experiments with Arabidopsis, to be grown hydroponically with different concentrations of heavy water, were delayed due to problems with our hydroponic methods. The planned experiments will be completed next year. Studies with soybean were carried out to find a line with high seed concentrations of vitamin K, but no lines were identified. It was decided that other oil seed crops might need to be pursued for future studies.
The ADODR monitors activities for the project by routine site visits, and review of major purchases, use of SCA funds for foreign travel.
Enhancing yeast as a research tool. A strategy to increase the calcium concentration in fruits and vegetables is by altering the regulation of plant calcium transporters so they move increased amounts of calcium into the edible tissues of plants. A simple and quick process researchers typically use to study plant calcium transporters is by expressing these transporters in brewer's yeast where they can study their biological properties because yeast is a single celled organism that lacks the complex physiology of a plant. By using yeast to express the transporter, the researcher can study the single transporter and remove the background of the many other plant transporters that may have similar functions; however, when a particular plant transporter is inactive in yeast this creates an obstacle. In Houston, Texas, Children's Nutrition Research Center researchers have generated a process that now makes the transporter active in yeast so that their functions can be readily analyzed in a simple biological system. These findings are important since it will impact how biologists view the use of yeast as a tool to study plant transporters.
A strategy for identifying new genes for enhancing calcium bioavailability in edible plants. Calcium, when present as the calcium oxalate crystal in foods, is unavailable for nutritional absorption. Such crystals are common in edible plant foods, thereby reducing their nutritional quality. Researchers at the Children's Nutrition Research Center at Houston, Texas, have determined that using the latest integrative molecular and genetic tools (e.g., transposon-tagged mutant lines) developed in the legume model, Medicago truncatula, will expedite discovery of the genes that compose the pathways of calcium oxalate crystal formation in plants. Segregation and complementation analysis has shown that the gene containing the transposon-tag is linked to the mutant calcium oxalate phenotype. It is anticipated that the genes identified through the use of these tools will provide the molecular targets for a decreased expression strategies to enhance the nutritional value of economically important crop plants.