Metal Uptake in Arabidopsis thaliana

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Iron is an essential nutrient for higher plants, with iron-containing compounds figuring prominently in the electron transport systems of photosynthesis and respiration. Although iron is abundant in the soil, the acquisition of iron is problematic due to its low solubility at neutral pH. In response to iron deficiency, all plants except the grasses induce Fe(III) chelate reductase activity, Fe(II) transport activity and proton release into the rhizosphere. Of the three responses, Fe(III) reduction is thought to be the primary factor in making iron available to plants. During the current funding period, Arabidopsis mutants that have defects in Fe(III) chelate reduction were identified. frd1 cannot induce Fe(III) chelate reductase activity in response to iron deficiency and frd3 has high levels of Fe(III) reductase activity relative to wild type when plants are grown under iron sufficient conditions. The gene identified by the frd1 mutation, FRO2, has been cloned and shown to complement the frd1 mutant. FRO2 encodes a metal chelate reductase belonging to a superfamily of flavocytochromes whose members pass electrons across membranes. During the course of this work, another gene family, the ZIP gene family, was also identified that encodes metal transporters capable of transporting Fe(II), Mn(II) and Zn(II). One of the family members, IRT1, is only expressed in the roots of iron deficient plants and is suggested to be responsible for uptake of Fe(II) from the rhizosphere. This project will build on the discoveries made during the current funding period, specifically focusing on strategies to make Fe available to the plant (i.e., metal reduction and proton release). The specific objectives of this proposal are to: 1) determine the role of the FRO2 metalloreductase in Arabidopsis metal ion uptake and metabolism using functional expression studies in yeast and genetic, cytological and biochemical analyses in Arabidopsis. As time permits, analyze the role of other members of the FRO gene family.2) further characterize the frd3 mutant and clone the FRD3 gene.As part of the characterization of the FRO2 gene, organ and tissue specificity as well as subcellular localization will be determined. A knockout mutant that carries an insertion in FRO2 gene will also be identified in order to determine the contribution of the FRO2 gene product to metal ion homeostasis. Null alleles of FRO2, IRT1 and AHA2 will also be used to test the hypothesis that Fe(III) reduction is indeed limiting iron acquisition by plants, as has been suggested from physiological studies. The gene identified by the frd3 mutation may be involved in metal transport, metal sensing or metal regulation, based on the physiological characterization carried out to date. Fine mapping of the frd3 mutation will be done by analyzing inter-ecotype recombinants to facilitate the cloning of the FRD3 gene. Understanding iron uptake in plants is extremely important as one-third of the world's soils are iron deficient. Furthermore, iron deficiency in humans is the most prevalent nutritional problem in the world today, affecting an estimated 2.7 billion people in developed and developing countries. For most of the world, plant material is the major source of iron so ensuring that plants have higher stores of bio-available iron will go a long way towards solving an important problem in human nutrition.