2012 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.
To understand the molecular mechanisms regulating calcium oxalate crystal formation in plants, we have completed genetic and molecular studies directed toward identifying new genes required for crystal accumulation in Medicago truncatula. We utilized a small DNA genomic fragment of mutant gene 1 to isolate the cDNA encoding its transcript. We have also completed oxalate measurements on the back-crossed mutants.
Studies have been initiated to produce unique transgenic crops in order to identify the relationship between minerals in the plant cell and nutrient bioavailability. Using plant mutants generated in our lab we have used Synchrotron x-ray fluorescence (SXRF) microspectroscopy to conceptualize the relationship between partitioning within plant cells and bioavailability. These approaches may foster strategies to optimize location of calcium within the plant matrix to maximize calcium absorption and utilization from dietary fruits and vegetables. These modified foods could be part of a regime undertaken in children and adults identified as at-risk for low calcium intake or absorption with the ultimate goal of decreasing the incidence and severity of inadequate bone mineralization.
We have used state of the art microscopy to collect spatially resolved elemental profiles of mineral nutrients inside of plant tissues with the aim of clarifying the influence of altered nutrient transport function on the abundance and distribution of nutrients within plant-based foods. Although not a novel technique, the application of this microscopy technique to plant molecular genetics is new. Our working hypothesis is that nutrient distribution is altered by changing the expression of nutrient transporters in plants. Our recent work has demonstrated that seeds of plants with increased expression of calcium transport expression have higher levels of calcium and other nutrients in the seed coat. These findings suggest a way to boost the calcium nutritional value of agriculturally important crops.
Several studies were conducted to understand how plants regulate the accumulation of essential minerals in edible organs. We characterized the concentrations of several minerals in diverse cultivars of broccoli, bean, rice, pea, chickpea, and lentil. For the broccoli heads, or bean and rice seeds, we have been using quantitative genetics approaches to identify which regions of the plant genome (DNA) are associated with elevated levels of various nutrients. This information can be used by breeders to make nutritional improvements in new cultivars of these crops. For the pea, chickpea, and lentil seed samples, we are working directly with newly developed breeding lines to assess seed mineral concentrations. This effort is intended to raise mineral concentrations in future cultivars of these seed crops.
Other studies were executed using mutant plants or diverse cultivars of crop plants to identify the genes or processes used by plants to increase their mineral absorption and/or mineral nutrient quality. Mutant plants (of a legume species) that exhibited altered zinc requirements were analyzed to determine the part of their DNA that was harboring the mutated gene. We narrowed in on the probable genomic region and are in the process of evaluating several candidate genes for the zinc trait. The identification of the relevant genes will contribute to our knowledge on how plants utilize zinc throughout the roots and shoots. Work also occurred with two cultivars of barley, which exhibited weaker or stronger abilities to cope with limited iron in their soil environment. When barley plants are challenged with limited amounts of iron, they grow poorly, their seed yield is diminished, and the iron concentration of their seeds is lowered. In order to help barley plants absorb more iron, we examined what happens in barley roots when the plants are presented with limited amounts of iron. In roots, we measured the activities of various enzymes that are responsible for the production of specific carbohydrate molecules. The carbohydrates we studied have been shown to help non-cereal crops cope with the stress of limited iron. The enzyme activities had not been previously studied in a cereal species like barley, at least not in relation to limited iron availability. We found the enzyme activities to be elevated in the roots of both barley cultivars when the plants were iron limited, but overall the activities were much lower than those found in iron-challenged non-cereal crops like tomato or soybean. Nonetheless, our results showed that cereal and non-cereal crop plants can activate some of the same root processes when challenged with limited amounts of iron. Future studies are needed to help us understand how to elevate these root processes even further in barley cultivars, in order to help them grow better in low iron soils.
Studies were continued to develop methods for enriching plants with non-radioactive forms of elements and to use these plants in human or animal feeding trials to understand nutrient absorption and metabolism. We grew several plants in nutrient solutions made with heavy water, a substance that contains a heavy form of hydrogen. It can be used to tag hydrogen-containing molecules in plants; these can then be measured in blood or tissue samples with special instrumentation. We tagged compounds like vitamin K in kale and beta-carotene (a precursor of vitamin A) in leaves or seeds of different crops. These specially tagged foods were fed to human or animal subjects in an effort to understand how vitamin K is absorbed, metabolized, and distributed through the body, or were used to establish how effectively beta-carotene is converted to vitamin A in foods like Golden Rice (a transgenic rice that makes beta-carotene in its grains).
Long-term trends in the mineral nutritional quality of broccoli florets. Crop breeding is often focused on yield improvement, rather than nutritional improvement. Thus, researchers asked the question whether long-term breeding efforts in broccoli had resulted in diminished concentrations of various essential minerals. Scientists at the Children's Nutrition Research Center in Houston, Texas, grew 14 broccoli cultivars released over 50 years, and harvested market-sized florets for the analysis of mineral concentrations. We demonstrated that there were significant cultivar differences in floret concentrations for several minerals, but no clear downward trends between concentration and year of cultivar release. These results confirm that broccoli mineral concentrations have not declined over the years, and provide a guide for mineral levels in broccoli that should be maintained as other characteristics are manipulated by breeders in the future.
Engineering calcium oxalate crystal formation in plants. Calcium oxalate crystals can help protect plants from chewing insects but not all plants make these crystals. Research conducted at the Children’s Nutrition Research Center in Houston, Texas, has shown that it is now possible to engineer a non-calcium oxalate crystal accumulating plant, such as Arabidopsis, to form crystals of calcium oxalate. Thus, engineering this form of plant protection in important crop plants appears possible. Such a feat would help reduce the need for chemical pesticides in producing food stuffs and other plant goods.