Metal Uptake in Arabidopsis thaliana

铁是高等植物必不可少的营养物质，含铁化合物在光合作用和呼吸作用的电子传递系统中占有重要地位。尽管土壤中铁含量丰富，但由于其在中性ph下的溶解度低，铁的获取存在问题。作为对铁缺乏的响应，除草类外，所有植物都诱导铁（III）螯合还原酶活性、铁（II）运输活性和质子释放到根际。在这三种反应中，铁（III）还原被认为是使植物获得铁的主要因素。在目前的资助期内，发现了铁（III）螯合物还原缺陷的拟南芥突变体。在缺铁条件下，frd1不能诱导铁（III）螯合还原酶活性，而当植物生长在铁充足的条件下，frd3的铁（III）还原酶活性相对于野生型具有较高水平。frd1突变所鉴定的基因FRO2已被克隆，并显示与frd1突变互补。FRO2编码一种金属螯合还原酶，属于黄细胞色素超家族，其成员通过膜传递电子。在这项工作的过程中，还发现了另一个基因家族，ZIP基因家族，它编码能够运输Fe(II), Mn（II）和Zn（II）的金属转运蛋白。家族成员之一IRT1仅在缺铁植物的根中表达，并被认为负责从根际吸收铁（II）。该项目将以当前资助期内的发现为基础，特别侧重于使工厂获得铁的策略（即金属还原和质子释放）。本课题的具体目标是：1)通过酵母的功能表达研究和拟南芥的遗传、细胞学和生化分析，确定FRO2金属还原酶在拟南芥金属离子摄取和代谢中的作用。如果时间允许，分析FRO基因家族其他成员的作用。2)进一步鉴定frd3突变体，克隆frd3基因。作为表征FRO2基因的一部分，器官和组织特异性以及亚细胞定位将被确定。为了确定FRO2基因产物对金属离子稳态的贡献，还将鉴定携带FRO2基因插入的敲除突变体。FRO2、IRT1和AHA2的零等位基因也将被用来检验铁（III）还原确实限制了植物获取铁的假设，这是生理学研究提出的。根据迄今为止所进行的生理表征，frd3突变所鉴定的基因可能参与金属运输、金属感应或金属调节。frd3突变的精细定位将通过分析生态型间重组来完成，以促进frd3基因的克隆。了解植物对铁的吸收是极其重要的，因为世界上三分之一的土壤缺铁。此外，人类缺铁是当今世界上最普遍的营养问题，影响着发达国家和发展中国家约27亿人。对于世界上大部分地区来说，植物材料是铁的主要来源，因此确保植物具有更高的生物可利用铁储量将对解决人类营养中的一个重要问题大有帮助。

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.