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

鞘脂是真核细胞膜的重要组成部分。它们由鞘氨醇碱基和脂肪酸组成，并且可以含有头基，例如磷酸盐或糖残基。在酵母和脊椎动物中，除了它们的结构作用外，特定的鞘氨醇和鞘氨醇脂还通过调节细胞发育、应激反应和程序性细胞死亡来实现重要的信号功能。在植物中，高水平的鞘氨醇酶触发细胞死亡，并且鞘氨醇酶诱导的细胞死亡显示出程序性细胞死亡（PCD）的典型特征。鞘氨醇诱导的PCD所需的一些信号转导元件已被确定在植物中，和突变体筛选确定鞘脂代谢基因是负责不同的细胞死亡phenotypes.In以前的工作中，我们观察到持续高水平的游离鞘氨醇植物鞘氨醇（t18：0）在拟南芥叶片与PCD反应接种与无毒（但不是有毒）菌株的细菌病原体假单胞菌。t18：0水平升高先于细胞死亡症状。因此，我们假设像t18：0这样的游离鞘氨醇碱可能是负责触发植物细胞死亡的信号分子。为了确定植物中游离鞘氨醇碱的这种信号作用，该项目使用了两种方法：首先，分析调节植物中游离鞘氨醇水平的机制，其次，在鞘氨醇基酶过程中调控的转录物的鉴定和功能表征，诱导细胞死亡。鞘氨醇水平的调节将通过使用拟南芥鞘氨醇代谢突变体在体内操纵鞘氨醇水平来解决，以及已经为本项目制备的诱导型过表达和敲低系。植物将在生理学试验中进行评估，例如细胞死亡测定，通过使用高分辨率质谱联用高分辨率脂质色谱法对选定的鞘氨醇碱和鞘脂进行定量，以及通过鉴定同位素标记的鞘氨醇碱的代谢命运来进行评估。进行野生型植物和鞘氨醇碱处理后无应答突变植物的转录组分析。在转录组分析中确定的候选基因的功能测试将进一步确定执行植物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制备的拟南芥突变体和转基因株系，并涵盖转录调控和鞘氨醇和-脂质的调控，以确定调控和信号转导机制。该项目预计将导致更好地了解植物细胞死亡的调节，以及植物中游离鞘氨醇的代谢和可能的信号作用。

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.