Functional characterization of sphingobase metabolism and the role of sphingobases in plant programmed cell death

鞘氨醇碱代谢的功能表征以及鞘氨醇碱在植物程序性细胞死亡中的作用

• 批准号: 290163576
• 负责人: Privatdozent Dr. Frank Waller
• 金额: --
• 依托单位: Julius-von-Sachs-Institut für Biowissenschaften
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/290163576?language=en
• 关键词: Functional; characterization; sphingobase; metabolism; role

Sphingolipids are essential components of eukaryotic cell membranes. They are composed of a sphingobase and a fatty acid and may contain a headgroup, e.g. a phosphate or sugar residue. Beside their structural role, specific sphingobases and -lipids fulfil important signaling functions in yeast and in vertebrates by regulating cellular development, stress responses, and programmed cell death.In plants, high levels of sphingobases trigger cell death, and sphingobase-induced cell death displays typical features of programmed cell death (PCD). Some signal transduction elements which are required for sphingobase-induced PCD have already been identified in plants, and mutant screens identified sphingolipid metabolism genes as being responsible for different cell death phenotypes.In previous work we observed sustained high levels of the free sphingobase phytosphingosine (t18:0) in Arabidopsis leaves reacting with PCD to inoculation with an avirulent (but not to a virulent) strain of the bacterial pathogen Pseudomonas syringae. Elevated t18:0 levels preceded cell death symptoms. We therefore hypothesized that free sphingobases like t18:0 could be signaling molecules responsible for triggering cell death in plants.To establish such a signaling role of free sphingobases in plants, the project uses two approaches: First, to analyze mechanisms which regulate the levels of free sphingobases in the plant, and second, the identification and functional characterization of transcripts regulated during sphingobase-induced cell death.The regulation of sphingobase levels will be addressed by manipulating sphingobase levels in vivo using Arabidopsis sphingobase metabolism mutants, as well as inducible overexpression and knock-down lines which have already been prepared for this project. Plants will be assessed in physiological tests, e.g. cell death assays, by quantification of selected sphingobases and sphingolipids using high-resolution mass-spectrometry coupled with high resolution lipid chromatography, and also by identifying the metabolic fate of isotope-labelled sphingobases.As signal transduction pathways for the induction of programmed cell death are still fragmentary, a transcriptome analysis of wild type plants and of non-responding mutant plants following sphingobase treatment will be performed. Functional tests of selected gene candidates identified in the transcriptome analysis will identify further signaling elements and mechanisms which execute cell death processes in plants.The two functional approaches will utilize specifically prepared Arabidopsis mutants and transgenic lines and cover both transcriptional regulation and regulation of sphingobases and -lipids to identify regulatory and signal transduction mechanisms. The project is expected to lead to a better understanding of the regulation of plant cell death, as well as the metabolism and possible signaling role of free sphingobases in plants.

鞘磷脂是真核细胞膜的重要组成部分。它们由狮身人面像碱基和脂肪酸组成，并可能含有头基，例如磷酸盐或糖残基。除了它们的结构作用外，特定的狮身人面像碱和脂类通过调节细胞发育、应激反应和程序性细胞死亡在酵母和脊椎动物中发挥重要的信号功能。在植物中，高水平的狮身人面像触发细胞死亡，而狮身人面像诱导的细胞死亡表现出典型的程序性细胞死亡(PCD)的特征。在植物中已经鉴定了一些狮身人面像诱导PCD所需的信号转导元件，突变筛选发现鞘磷脂代谢基因与不同的细胞死亡表型有关。在以前的工作中，我们观察到在PCD与PCD反应的拟南芥叶片中持续高水平的游离鞘氨醇(t18：0)，接种一种无毒(但不是致病)的细菌病原菌紫草假单胞菌。升高的t18：0水平先于细胞死亡症状。因此，我们假设像t18：0这样的游离狮身人面像碱基可能是触发植物细胞死亡的信号分子。为了在植物中建立这种信号转导作用，该项目使用了两种方法：第一，分析调节植物中游离斯芬戈碱基水平的机制，第二，鉴定和功能鉴定在斯芬戈碱基诱导细胞死亡过程中调节的转录物。斯芬戈碱基水平的调节将通过使用拟南芥斯芬戈碱基代谢突变体在体内操纵斯芬戈碱基水平，以及已经为该项目准备的可诱导的过度表达和敲除品系来解决。植物将在生理测试中进行评估，例如细胞死亡分析，通过使用高分辨率质谱仪和高分辨率脂层析对选定的狮身人面像碱基和鞘磷脂进行量化，以及通过确定同位素标记的狮身人面像碱基的代谢命运来评估植物。由于诱导程序性细胞死亡的信号转导途径仍然不完整，因此将对野生型植物和经过狮身人面像碱基处理后无反应的突变植物进行转录组分析。对转录组分析中确定的候选基因的功能测试将进一步确定在植物中执行细胞死亡过程的信号元件和机制。这两种功能方法将利用专门准备的拟南芥突变体和转基因株系，涵盖转录调控和狮身人面像碱基和脂类的调控，以确定调控和信号转导机制。该项目有望更好地了解植物细胞死亡的调控，以及植物体内游离狮身人面像碱基的新陈代谢和可能的信号作用。