EAGER: Iron sensing and signaling in Arabidopsis thaliana

生物体保持着高度活性金属铁的水平，小心地控制以避免细胞损伤。在植物中，这种控制的一部分是一种机制，允许最需要铁的茎部向发生初级铁吸收的根发出铁状态的信号。这种从茎到根的信号传导机制的分子细节尚不清楚，但可能对植物在可食用部分积累额外的生物可利用铁的成功工程至关重要。该项目探索了一种假设，即黄色条纹1样蛋白负责在植物的茎和根之间传递铁状态的信号。我们目前对植物铁稳态机制的无知是设计铁生物强化主食方法的主要障碍。生物强化是指对主粮作物进行基因工程，在可食用部分积累额外的生物可利用铁；它被广泛认为是改善全球20 - 30亿人铁营养的可持续手段，这些人的饮食不足导致缺铁性贫血。如果我们希望改善边缘土壤中作物的生长，提高我们对植物铁稳态机制的理解也至关重要，因为缺铁经常限制作物生长。拟南芥黄条纹样1和黄条纹样3双突变体（ysl1ysl3）具有与铁有关的严重多效性生长缺陷。ysl是铁和其他过渡金属（Zn, Cu, Mn, Ni）与植物特异性金属螯合化合物烟胺（NA）配合的转运体。值得注意的是，组织定位（主要在叶脉中）和调节（因缺铁而下调）都反对这些蛋白质参与从土壤中吸收初级金属的观点。ysl1ysl3植物的表型包括缺铁萎黄、组织中铁含量低、衰老过程中叶片中铜和锌的重新转运失败、花粉失败和胚胎发育不完整，这些最初都被认为是由AtYSL1和AtYSL3的金属na运输失败造成的。但ysl1ysl3植物也表现出铁信号缺陷，这很难解释为叶脉中金属运输缺陷的简单结果。新的证据表明，AtYSL1和AtYSL3植物普遍对缺铁缺乏反应迟钝，并提出了一种新的假设，即AtYSL1和AtYSL3作为“受体”的功能：转运体也作为植物铁状态的受体。在这个项目中，我们将研究AtYSL1和AtYSL3除了作为金属na转运体的作用外，还在芽铁状态的信号传导中起作用的假设。这一想法的一个关键测试是使YSL1和YSL3突变影响受体功能而不影响转运体功能，反之亦然。在这个项目中，将使用定点诱变技术制造YSL1和YSL3的转运缺陷版本，并将在酵母中测试其功能。然后将三个适当定位的运输缺陷版本在各自的启动子下引入ysl1ysl3双突变体，并对产生的植株进行ysl1ysl3表型测试。在第二个更有限的实验中，将测试YSL1和YSL3的酸性n端结构域(s)。这些结构域可能与铁结合，因此可能与铁感应有关。我们将测试去除这些结构域是否会影响酵母的运输功能，如果没有，我们将在植物中测试这些蛋白质是否会与ysl1ysl3表型互补。这些实验将阐明YSL1和YSL3是否直接参与铁信号传导。该项目将整合到本科生的实验课程中，并提供生物信息学和基因组学方法的培训，以及假设驱动的研究过程。

Living organisms keep the level of the highly reactive metal, iron, carefully controlled to avoid cellular damage. In plants, part of this control is a mechanism to allow the shoot, where iron is most heavily needed, to signal iron status to roots where primary iron uptake occurs. The molecular details of this shoot to root signaling mechanism are unknown, but are likely to be essential for successful engineering of plants to accumulate additional bioavailable iron in edible parts. This project explores the hypothesis that Yellow Stripe1-Like proteins are responsible for signaling the iron status between the shoot and the root of plants. Our current state of ignorance about many of the mechanisms involved in plant iron homeostasis is a major obstacle to devising approaches for biofortification of staple foods with iron. Biofortification refers to the genetic engineering of staple crops to accumulate additional bioavailable iron in edible parts; it is widely regarded as a sustainable means of improving the iron nutrition of the 2-3 billion people worldwide whose inadequate diet causes iron deficiency anemia. Improving our understanding of plant iron homeostatic mechanisms is also critical if we wish to improve growth of crops in marginal soils, where iron deficiency frequently limits crop growth.A double mutant with null mutations in the Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 (ysl1ysl3) has severe pleiotropic growth defects related to iron. YSLs are transporters of iron and other transition metals (Zn, Cu, Mn, Ni) complexed with the plant-specific metal chelating compound nicotianamine (NA). It is important to note that both tissue localization (mainly in the leaf veins) and regulation (down-regulated by iron deficiency) argue against the idea that these proteins participate in primary metal uptake from the soil. The phenotype of ysl1ysl3 plants, which includes iron deficiency chlorosis, low Fe levels in tissues, failure to re-translocate Cu and Zn from leaves during senescence, pollen failure, and incomplete embryo development, were at first all thought to be consequences of failed metal-NA transport by AtYSL1 and AtYSL3. But ysl1ysl3 plants also exhibit defects in Fe signaling, which are difficult to explain as a simple consequence of metal transport defects in leaf veins. New evidence has indicated that ysl1ysl3 plants have a pervasive inability to respond vigorously to iron deficiency, and a new hypothesis is put forward that AtYSL1 and AtYSL3 function as "transceptors": transporters that also function as receptors for the iron status of the plant. In this project, we will investigate the hypothesis that, in addition to their roles as metal-NA transporters, AtYSL1 and AtYSL3 function in signaling the iron status of shoots. A key test of this idea is to make mutations in YSL1 and YSL3 that affect receptor function without affecting transporter function, and vice versa. In this project, transport defective versions of YSL1 and YSL3 will be made using site directed mutagenesis and will be tested for functionality in yeast. Three properly localized transport defective versions will then be introduced into the ysl1ysl3 double mutant under their own promoters, and the resulting plants will be tested for ysl1ysl3 phenotypes. In a second, more limited experiment, the acidic N-terminal domain(s) of YSL1 and YSL3 will be tested. These domains may be iron binding, and thus could be involved in iron sensing. We will test whether removing these domains affects transport function in yeast, and if it does not, the proteins will be tested in planta for complementation of ysl1ysl3 phenotypes. These experiments will elucidate whether YSL1 and YSL3 are directly involved in iron signaling. This project will be integrated into a laboratory course for undergraduates and provide training in bioinformatics and genomics methodologies, as well as the process of hypothesis-driven research.