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

• Applicant: Privatdozent Dr. Frank Waller
• Subject Area: Plant Physiology ; Plant Breeding and Plant Pathology ; Plant Cell and Developmental Biology
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 290163576

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.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Agnes Fekete