2005 Annual Report 4d.Progress report. This report serves to document research conducted under an agreement between ARS and both the Centro Internacional de Agricultural Tropical (CIAT) and the International Food Policy Research Institute (IFPRI), as part of the multi-institutional Biofortification Challenge Program project known as HarvestPlus. The overall scope of this project is to enhance the micronutrient content (iron, zinc, and pro-vitamin A carotenoids) of staple food crops (rice, wheat, maize, bean, cassava, and sweet potato) in order to improve the micronutrient status of nutritionally deficient populations throughout the developing world. Our research effort will be directed towards providing the HarvestPlus consortium with molecular technologies, and an evaluation of existing proof-of-concept technologies, that will assist in the conventional breeding or transformation-based generation of staple crops with enhanced micronutrient content. This research is linked to and complements research in the parent project 6250-21520-042-00D. Progress this past year included the functional analysis of iron-related transgenic plants and the identification of genes and gene products that contribute to seed iron and zinc content. Available rice transgenic lines (transformed with an Arabidopsis root iron reductase, AtFRO2, or an Arabidopsis plasmalemma iron transporter, AtIRT1) and soybean transgenic lines (transformed with an Arabidopsis root iron reductase, AtFRO2) were analyzed to ascertain the ability of these transformation strategies to improve whole-plant metal status, and the ultimate delivery of iron to seeds. AtFRO2 rice was found to lack expression of the transgene, and thus also lacked functional root iron reductase activity. The line available to us used a putative iron-deficiency inducible Arabidopsis 5' promoter for FRO2, and this apparently was not adequate to effect gene expression in rice under the various growth regimes tested. We have concluded that a constitutive promoter, such as 35S, will be needed before we can adequately test the functionality of FRO2 in a cereal. Several 35S::AtIRT1 rice lines have been screened to verify homozygosity. Lines were grown on various Fe concentrations and tissues analyzed for minerals; no elevations in leaf Fe or seed Fe levels were detected. This transgene alone does not appear to alter Fe dynamics in rice, probably due to the lack of a reductase to provide the Fe2+ substrate. Several 35S::AtFRO2 soybean events were available to us, and we found a few with high expression of AtFRO2 in all tissues, and an enhancement of iron reductase activity in roots and leaves. Detailed analysis showed activities to be roughly 3-fold higher than wild-type plants, when grown with Fe. Controlled studies have been completed in which transgenic lines were grown at elevated Fe levels. Leaf Fe concentrations increased dramatically, but seed Fe levels were not significantly increased. We are still evaluating these results, but our data appear to suggest that seed Fe levels cannot be manipulated through an enhanced source leaf Fe reductase capacity. For our gene discovery activities, we collaborated with researchers at Michigan State University to harvest developing Arabidopsis seeds at different stages of development. We isolated mRNA and performed Affymetrix-based microarray studies to assess the expression of genes potentially involved in micronutrient composition of seeds. Analysis of the expression results is currently underway. This work is important, as it will provide clues to genes involved in the storage and partitioning of metals in seeds. Other work with metal-related genes involved the creation of a cDNA macroarray using 50 rice genes with putative roles in metal transport and/or metal homeostasis. All of the genes have now been cloned as fragments, and several membranes have been spotted to generate the macroarrays. Quality control experiments have been performed to determine quantity of mRNA needed for highest achievable sensitivity, reproducibility, and cross-talk between arrayed target genes. The macroarrays appear to be functioning well, with little cross-talk, and consistent results. Several rice genotypes are now being grown for tissue collection in the next year of the project. Preliminary studies with root and leaf tissues collected from line IR68144 demonstrate that only about half of the arrayed genes are being expressed at detectable levels. This is important, as it tells us that many of the predicted metal-related genes in the rice genome may not be playing a role in metal homeostasis, and thus we can focus our attention on the subset of actively expressed genes. These macroarrays will be used extensively in the coming year. Additionally, we used our knowledge of metal transporter genes (ZIP family members) in the model legume, Medicago truncatula, and conducted a TILLING procedure (Targeting Induced Local Lesions IN Genomes) to identify gene-specific mutants in this plant. Two mutants were identified, one which demonstrated an amino acid alteration in MtZIP3 (an Fe transporter) and the other an alteration in MtZIP1 (a Zn transporter). Both mutants exhibit some level of growth retardation, and both do respond to elevated administration of Fe (MtZIP3 mutant) or Zn (MtZIP1 mutant). Plants were maintained on varying nutrient supplies to carry them through flowering, and first backcrosses have been made. F1 seeds and selfed seeds have been collected. F1 plants and homozygous mutants are now being grown to assess growth and tissue mineral concentrations in response to varying metal treatments. Cloning of the mutated genes is in progress; these will be used in the coming year along with wild-type alleles to study the kinetic properties of the proteins in a yeast expression system. These results will provide important information on the role of these proteins in whole-plant metal homeostasis.