Fatty acid metabolism and intracellular lipid trafficking in plants

前言在植物中，脂肪酸具有多种功能，既可以作为结构成分，也可以作为储存化合物和信号物质。鉴于脂肪酸的多重作用，一个复杂的调控网络显然对于匹配脂肪酸代谢的不同途径的供需是必不可少的。脂肪酸的细胞内运输和酶激活是植物脂肪酸代谢调控网络的基石，这两个方面都将在拟议的项目中得到解决。内质网和叶绿体是植物脂肪代谢的主要部位，底物在这两个亚细胞结构域之间的运输被证明是必不可少的。在拟议项目的一个部分中，我们将重点确定脂类运输机械的新组件，这些组件仍然知之甚少。在另一部分中，将分析游离脂肪酸在转录调控中的作用。脂肪酸在细胞质中的代谢可利用性强烈依赖于它们被酰辅酶A合成酶(LACS)激活。在项目的第三部分，我们将使用具有修饰脂肪酸可用性的LACS突变系来深入了解植物脂肪酸代谢的复杂网络。方法我们最近鉴定了一个拟南芥突变系，其特征是内质网和叶绿体之间的脂类运输受损，游离脂肪酸水平升高。我们将通过全转录组鸟枪测序(RNA-seq)来分析这个突变体，以确定脂类运输机制的新成分。通过相同的实验，我们将研究游离脂肪酸水平升高对转录组的影响。作为进一步表征脂质运输机制的替代方法，我们将使用免疫亲和纯化参与运输的标记版本的蛋白质，以便共纯化相关蛋白。在拟南芥中，我们观察到负责激活游离脂肪酸的活动之间的功能冗余。为了实现对该基因家族的反向遗传方法，我们生成了一个包含130多个突变系的突变集合。具有简洁表型的突变株系将通过一系列生化、遗传和细胞生物学方法进行分析，以了解特定细胞脂肪酸库的生物学作用。展望所获得的数据将揭示脂肪酸在细胞代谢中的流动，并将为未来针对植物脂肪代谢的修饰提供支持。提高对不同亚细胞室之间脂类运输的理解也将使我们能够在其他模式生物中测试类似的机制。

IntroductionIn plants, fatty acids serve multiple functions as structural components, as storage compounds, and as signaling substances. Given the multiple roles of fatty acids a complex regulatory network is obviously essential to match supply and demand of the diverse pathways of fatty acid metabolism. The regulation involves control over fatty acid biosynthesis, the activation, and the transport of fatty acids within the cell.ObjectivesIntracellular transport and enzymatic activation of fatty acids are cornerstones of the regulatory network of plant fatty acid metabolism and both will be addressed in the proposed project. The ER and the plastid are major sites of plant lipid metabolism and substrate trafficking between both subcellular domains was shown to be essential. In one part of the proposed project we will focus on identifying new components of the lipid transport machinery which is still poorly understood. In another part, the role of free fatty acids on transcriptional control will be analyzed.The metabolic availability of fatty acids in the cytoplasm is strongly dependent on their enzymatic activation by acyl-CoA synthetases (LACS). In the third part of the project we will employ LACS mutant lines with modified fatty acid availability to gain insight into the intricate network of plant fatty acid metabolism.MethodsWe identified recently an Arabidopsis mutant line characterized by impaired lipid trafficking between ER and plastids and by elevated levels of free fatty acids. We will analyze this mutant by whole transcriptome shotgun sequencing (RNA-seq) in order to identify new components of the lipid trafficking machinery. By the same experiment we will investigate the effects of elevated levels of free fatty acids on the transcriptome. As alternative approach to further characterize the lipid transport machinery we will use immunoaffinity purification of tagged versions of proteins involved in the transport in order to co-purify associated proteins.In Arabidopsis we observed functional redundancy between activities responsible for the activation of free fatty acids. To enable a reverse-genetic approach for the gene family we generated a mutant collection comprising more than 130 multiple mutant lines. Mutant lines with concise phenotypes will be analyzed by a set of biochemical, genetic, and cell biological methods in order to understand the biological roles of specific cellular fatty acid pools.OutlookThe data obtained will reveal insight into the flux of fatty acids within the cellular metabolism and will support future efforts aiming at the modification of plant lipid metabolism. Improving the understanding on lipid trafficking between different subcellular compartments will also allow us to test for similar mechanisms in other model organisms.