2006 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 during the past two years 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 of one of the transgenic lines showed reductase activities to be roughly 3-fold higher than wild-type plants, when grown with Fe. The enhanced Fe reductase activity led to reduced chlorosis and increased biomass as compared to control plants grown under hydroponics that mimicked a calcareous soil environment. Iron concentration in roots and shoots were found to be up to 30% higher in transgenic plants relative to controls under higher Fe treatments. These results suggest that constitutive expression of an Fe reductase in soybean can provide a route to alleviate iron deficiency chlorosis in this commercially important crop. We also used this transgenic line to study the role that leaf iron reduction might play in the overall movement of iron to developing seeds. Controlled studies were completed in which transgenic and control lines were grown at elevated Fe levels. Leaf Fe concentrations increased dramatically in the transgenic line (up to 100% higher than controls), but seed Fe levels were increased only moderately (10%). Under the same growth conditions, Fe reductase capacity increased three-fold in leaves of the transgenic line, relative to the control. These results suggest that leaf Fe reduction may contribute to the processing of Fe, prior to its delivery to seeds, but that enhanced leaf Fe reduction alone is not adequate to yield dramatically higher levels of Fe in seeds. 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 36 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 the quantity of mRNA needed for highest achievable sensitivity, reproducibility, and cross-talk between arrayed target genes. The macroarrays have now been used to study gene expression in flag leaves and non-flag leaves of four rice genotypes, which differ in seed iron and zinc concentrations. Leaves were collected during the period of mid grain fill. Twenty-four of 36 genes exhibited low to non-detectable signals in the macroarray, while 12 genes (OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYS15, OsYS16, OsYS17, OsYS18, OsYSL18, OsNRAMP2, OsNRAMP4, and OsNRAMP7) were found to be highly expressed in both flag and non-flag leaves of all four cultivars. Of the 12 expressed genes, there were no consistent differences in expression between the flag and non-flag leaf tissues. This suggests that the products of these genes (which are all putative metal transporters) contribute to the movement of metals between and within various cellular/subcellular leaf compartments, at least during the stage of grain fill in rice. However, it appears that none of these 12 gene products plays a unique role in flag leaves, with respect to metal remobilization to seeds. Additional expression analysis using semi-quantitative or quantitative PCR provided results that were generally consistent with the macroarray, but semi-quantitative PCR confirmed that OsFER1, OsNAS1, OsZIP7, and OsZIP10 were also expressed in leaves. This specialized macroarray has provided a short list of potential candidate genes, expressed in leaves, which might contribute to the process of metal transport to distant sinks, such as seeds. These macroarrays will be used extensively in the coming year to investigate gene expression in other rice genotypes. 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 that 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. Functional studies have been completed with the wild-type MtZIP1 protein, by expressing it in a yeast strain that is defective in zinc uptake. Using this system, MtZIP1 was shown to have a Km of 1 uM for Zn and a Vmax of 8.4 pmol Zn/min/10^6 cells. Copper inhibits MtZIP1 by affecting both its affinity for zinc and the rate of zinc transport. MtZIP1 does not appear to transport iron or manganese. Expression analyses are in progress to assess mRNA levels for MtZIP1 in wild-type and mutant plants. These studies are enhancing our understanding of one of the zinc transporters in this model plant. These results will provide important information on the role of these proteins in whole-plant metal homeostasis.