Elucidating the Function in Iron Homeostasis and Transport of the VTL Family of Vacuolar Membrane Proteins in Arabidopsis

在项目的继续进行中，我们希望继续研究VTL蛋白在铁稳态中的功能，并确定VTL转运蛋白是否是增加植物铁储存能力的合适工具。将实现以下目标。1.VTL蛋白转运特性及其细胞内定位的研究我们有多条证据表明VTL蛋白存在于液泡膜上，并在铁稳态中发挥重要作用。通过VTL1、2或5的异源表达来补充ccc1突变体，进一步支持了它们在液泡运输和铁的动态平衡中的滚动。然而，有必要直接证明铁的传输，并确定垂直热释光传输的动力学特性。我们建议通过使用从过度表达VTL基因的酵母细胞中分离出的空泡进行铁转运试验来实现这一点。在异源表达的同时，还将研究VTL蛋白在细胞内的定位。2.优化植物中铁的储存。其他人也在追求的一个目标是增加工厂的铁储存能力。实现这一目标将生产出更耐寒的植物和作物，提高食品和饲料的营养价值。我们认为，有了VTL转运体，我们就有了额外的可能性来增加植物中的铁。我们已经发表了过表达VTL1、2或5的nramp3/nramp4突变种子中铁含量增加的文章(Gollhofer等人，2014年)。我们有未发表的数据表明，VTL2、4和5过表达系，现在是第三代，种子中的铁含量比Col野生型高50%到100%。因此，VTL基因的过度表达可以导致铁存储能力的增加。我们希望扩展这些发现，并将它们与增加种子含铁量的独特方法相结合，从而开发出一种模型，用于为广泛的植物物种设计增加植物铁的方法。3.筛选有效铁含量较低的土壤以促进生长。从2015年秋季开始，持续一学年，沃尔夫冈·施密特教授将从中央研究院休假到柏林洪堡大学生物研究所。在我的实验室里，我们将对拟南芥的生态型进行筛选，以了解它们在缺乏可利用的铁源(例如，pH值为7.0的FeCl3)上生长的能力。最近，铁动员策略的范围已经扩大到包括拟南芥中酚类化合物的合成和分泌(Rodríguez-Celm等人，2013年；Schmid.等人，2014年)。这种基于酚类物质的吸收策略可能对中性和碱性土壤中的铁吸收很重要，这可能是作物抵抗石灰/重碳酸盐诱导的黄化的机制。我们的目标是确定新的参与者，允许植物在贫瘠的铁质土壤上生长。

In the continuation of the project we wish to pursue the investigation of the functions of VTL proteins in iron homeostasis and to establish whether the VTL transporters are suitable tools for increasing the iron storage capacity in plants. The following goals will be pursued. 1. Investigation of the Transport Characteristics of the VTL Proteins and their intracellular localization We have multiple lines of evidence that indicate the presence of the VTL proteins on the vacuolar membrane and that they play an important role in iron homeostasis. Complementation of the ccc1 mutant by heterologous expression of VTL1, 2 or 5 lends further support for their roll in vacuolar transport and homeostasis of iron. It is necessary, however, to demonstrate iron transport directly and to determine the kinetic properties of transport by the VTLs. We propose accomplishing this by iron transport assays using vacuoles isolated from yeast cells that over-express the VTL genes. In parallel to heterologous expression, intracellular localization of the VTL proteins will be investigated. 2. Optimizing Iron Storage in Plants. A goal that is being pursued also by others is to increase the iron storage capacity of plants. Obtaining this goal would produce hardier plants and crops with improved nutritional value for food and fodder. We feel that with the VTL transporters we have additional possibilities for increasing iron in plants. We have published increased iron content in nramp3/nramp4 mutant seeds that over-express VTL1, 2 or 5 (Gollhofer et al., 2014). We have unpublished data showing that VTL2, 4 and 5 over-expression lines, now in the third generation, have from 50 to 100% higher iron in seeds than the Col wild-type. Thus, over-expression of the VTL genes can lead to an increased iron storage capacity. We wish to extend these findings and combine them with unique approaches to increase the iron content of seeds, and thereby develop a model for engineering an increase in plant iron for a wide range of plant species. 3. Screening for Improved Growth on Poorly Available Iron. Beginning in the Fall of 2015 and continuing for one academic year Prof. Wolfgang Schmidt will be on sabbatical leave from the Academia Sinica at the Institute of Biology, Humboldt-Universität zu Berlin. In my laboratory we will conduct a screening of Arabidopsis ecotypes for their ability to grow on poorly available iron sources (e.g. FeCl3 at pH 7.0). Recently, the spectrum of iron mobilization strategies has been expanded to include synthesis and secretion of phenolic compounds in Arabidopsis (Rodríguez-Celm et al., 2013; Schmid et al., 2014). This phenolic based uptake strategy is likely important for iron uptake in neutral and alkaline soils, and it may be the mechanism behind resistance to lime-/bicarbonate-induced chlorosis in crop plants. Our goal is to identify new players that permit plant growth on poorly available iron soils.