Location: Children's Nutrition Research Center2011 Annual Report
1a. Objectives (from AD-416)
The overall goal of our research is to develop nutritionally enhanced plant foods that provide increased nutrient bioavailability and absorption in children. Ultimately, this plant food research in combination with mineral nutrition research in children will allow researchers to provide guidance regarding food intake and fortification, specifically related to iron, zinc, Vitamin C and calcium. Specific objectives of this research include: 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, Vitamin C, calcium, and magnesium) and other factors that inhibit or promote absorption of these minerals in plant foods; 2) Conduct animal and human feeding studies to determine mineral bioavailability of the nutritionally enhanced crops; 3) develop new, cost-effective methods for the intrinsic labeling of plant foods for use in nutrient bioavailability studies; 4) determine the absorption of dietary calcium, magnesium, iron, and zinc in children and the influence of other nutrients and dietary factors on the absorption; 5) (deleted due to resignation of investigator); 6) determine the effect of dietary components on the upregulation of intestinal iron transporter genes in human models; 7) characterize dynamic indices of bone formation by quantitative histomorphometry and micro computed tomography in 7 mouse models; 8) quantitate specific gene expression in calvarial osteoblasts derived from mouse models; and 9) determine the effects of hormone ablation, iron loading, ASC feeding and plant derived antioxidants on bone parameters in vivo. These efforts will expand our capabilities for assessing the absorption and metabolism of various plant-derived minerals and phytochemicals and will provide novel information directly useful to government, industry and the consumer related to dietary requirements. The generation of new bioavailability data for various plant-derived nutrients will be established and such data will have global application and provide a strong basis for evidence-based nutritional recommendations to be developed.
1b. Approach (from AD-416)
These research studies will utilize diverse plant species, human cell culture systems, or human subjects. CNRC scientists will focus on characterizing plant genes and gene products that are involved with mineral transport in the plant, with a focus on iron, zinc, calcium, and magnesium. We will use specifically manipulated transgenic lines, various plant mutants, or unique plant genotypes to assess the impact of altered genes on mineral transport and storage throughout various plant tissues. In order to facilitate studies of bioavailability of plant-based nutrients, we will develop new, cost-effective methods for the intrinsic, stable-isotopic labeling of plant foods, by testing different hydroponic strategies and altered timings of isotope application to the plants. Food-based factors associated with the dietary delivery of the essential minerals calcium, iron, and zinc will be investigated using human in vitro cell culture and human subject-based experiments. We will conduct a controlled trial of vitamin D supplementation to assess the effects of vitamin D status on calcium absorption in small children. We will evaluate different types of whole diets (lacto-ovo vegetarian) on iron status and the effects of differing intakes of zinc on zinc and copper absorption. We will determine if benefits previously seen for prebiotic fibers in enhancing calcium absorption also occur for iron absorption. Low abundance stable isotopes of each element will be used to track absorption in each of these human studies. In vitro cell culture models will seek to identify the genetic basis for iron and zinc absorption in intestinal cells, by monitoring mineral absorption in combination with the differential expression of various metal transporter genes. We will explore the roles of aldose reductase and aldehyde reductase in modulating oxidative stress in cells, as well as their separate role in providing the starting substrates for the ascorbate synthesis pathway. Ultimately we will have a better understanding of the role of vitamin C in our diet.
3. Progress Report
Project 1. We completed recruitment for the vitamin D supplementation trial in 4-8 yr old children and the absorption of calcium, zinc and magnesium in these subjects. All samples have been analyzed on the mass spectrometer and data entered. We completed IRB submission for the last phase of the study and began the recruitment process. We also conducted experiments in which we treated the mammalian cell line, Caco-2 cells, with different concentrations of iron for different durations of exposure at 6, 24, 48, 72 and 96 hrs. We measured DMT1 expression under different iron concentrations/exposures using a qPCR. We will measure the expression of two proteins located on the brush border of the cell, also involved in iron uptake called Dcytb1 and Dcytb2 under the same conditions when able. We also began to define the structures and mode of interactions of Osterix (which is a novel transcription factor that is essential for bone formation) with N066. N066 is an ascorbate dependent enzyme that acts as a repressor of Osterix. N066 is also a histone demethylase, a reaction which requires an ascorbate-dependent hydroxylation. Ascorbate deficiency leads to the accumulation of inactive Osterix in immature cells that are responsible for bone formation. Thus we hypothesize that N066-Osterix interaction is the key to cell differentiation for bone formation. We have now succeeded in expressing N066 and Osterix. Project 2. We have completed genetic and molecular studies directed toward identifying new genes required for crystal accumulation in Medicago truncatula. We have completed three back-crossings of two calcium oxalate crystal insertion mutants to allow easier identification of the defective genes. We have been able to isolate a small fragment of genomic DNA that contains an insertion in mutant gene 1. We are currently using this small fragment of DNA to screen a gene library to isolate mutant gene 1. We also completed mineral analysis on both back-crossed mutants and determined that their mineral composition is similar to control plants. We continued our work to understand how plant calcium transporters have evolved. We have used biochemical and genetic tools to show that a particular group of transporters work in tandem to perform specific functions. The concept of these transporters working in teams is novel. We used unique populations of soybean, bean, or other model plant species to study seed mineral concentrations and root processes that help the plant absorb nutrients, such as iron. We have initiated the seed mineral analysis of a large population of soybeans, grown in replicated field plots in Beltsville, MD. We have grown several plant species to assess root traits, leaf mineral, or seed mineral characteristics. Data have been analyzed to identify regions of the plant genome that are linked to specific seed or leaf mineral concentrations, or enhanced ability of the root system to absorb iron. Parent lines have now been crossed with unique genetic lines, to produce progeny with an altered genetic makeup. These lines will be used to test and confirm which regions of the DNA are most associated with the measured nutritional traits.
1. 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 in Houston, TX, have identified a small region of DNA within the genome of the model legume Medicago truncatula that contains a previously unidentified gene required for calcium oxalate crystal production. When this gene is "turned off" there is a dramatic reduction in the plants ability to produce these crystals. It is anticipated that the isolation of such a key gene will provide the molecular target that, when inactivated, will enhance the nutritional value of economically important crop plants.
2. Improved yield and nutritional content in melons. Grafting is a method of plant propagation where the tissues of one plant are encouraged to fuse with those of another. It is commonly used for the propagation of many melons grown commercially. Plant scientists at the Children's Nutrition Research Center have successfully used genetically modified rootstocks for grafting of melons. These modified rootstocks produced larger, more robust melons when compared with typical plants. Using genetically engineered rootstocks could be a means of boosting plant productivity for many commercial crops that use grafting techniques. This technique would be particularly compelling for the general public in that the genetic modifications do not enter the food supply.
3. Plant root responds to differing iron deficiency conditions. Plants acquire iron from soils via processes functioning in their roots, but the availability of iron in different soils can sometimes make it difficult for those roots to absorb enough iron to meet their needs. Soils that are alkaline (high pH) and contain high levels of calcium carbonate (also known as calcareous soils) are poor sources of iron, but some plants have found ways to acquire iron in these very challenging soils. Plant scientists at the Children's Nutrition Research Center have found that the roots of a certain legume plant could synthesize and release compounds that increased the levels of available iron in the soil. Moreover these plant roots could also change their internal biochemical properties to help them function more effectively with less iron. The identification of these changes and the identification of some of the genes responsible for them are providing tools and insights to help us develop new crops with improved abilities to acquire iron.
Sperotto, R.A., Boff, T., Duarte, G.L., Santos, L.S., Grusak, M.A., Fett, J.P. 2010. Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains. Journal of Plant Physiology. 167(17):1500-1506.