TY - JOUR A1 - Lambour, Benjamin A1 - Glenz, René A1 - Forner, Carmen A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Fekete, Agnes A1 - Waller, Frank T1 - Sphingolipid long-chain base phosphate degradation can be a rate-limiting step in long-chain base homeostasis JF - Frontiers in Plant Science N2 - Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels. KW - sphingolipid KW - long-chain base KW - plant sphingolipid metabolism KW - cell death KW - metabolic flux analysis KW - dihydrosphingosine-1-phosphate lyase KW - LC–MS/MS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-277679 SN - 1664-462X VL - 13 ER - TY - THES A1 - Lambour, Benjamin T1 - Regulation of sphingolipid long-chain bases during cell death reactions and abiotic stress in \(Arabidopsis\) \(thaliana\) T1 - Regulation von Sphingobasen während der Zelltodreaktion und abiotischem Stress in \(Arabidopsis\) \(thaliana\) N2 - Sphingobasen (LCBs) sind die Bausteine der Biosynthese von Sphingolipiden. Sie werden als Strukturelemente der pflanzlichen Zellmembran definiert und spielen eine wichtige Rolle für das Schicksal der Zellen. Komplexe Ceramide machen einen wesentlichen Teil der gesamten Sphingolipide aus, die einen großen Teil der eukaryotischen Membranen bilden. Gleichzeitig sind LCBs bekannte Signalmoleküle für zelluläre Prozesse in Eukaryonten und sind an Signalübertragungswegen in Pflanzen beteiligt. Es hat sich gezeigt, dass hohe LCB-Konzentrationen mit der Induktion des programmierten Zelltods sowie mit dem durch Pathogene ausgelösten Zelltod in Verbindung stehen. Mehrere Studien haben die regulierende Funktion der Sphingobasen beim programmierten Zelltod (PCD) in Pflanzen bestätigt: (i) Spontaner PCD und veränderte Zelltodreaktionen, die durch mutierte verwandte Gene des Sphingobasen-Stoffwechsels verursacht werden. (ii) Zelltodbedingungen erhöhen den Gehalt an LCBs. (iii) PCD aufgrund eines gestörten Sphingolipid-Stoffwechsels, der durch von nekrotrophen Krankheitserregern produzierte Toxine wie Fumonisin B1 (FB1) hervorgerufen wird. Um den Zelltod zu verhindern und die Zelltodreaktion zu kontrollieren, kann daher die Regulierung des Gehalts an freien LCBs entscheidend sein. Die Ergebnisse der vorliegenden Studie stellten das Verständnis der Sphingobasen und Sphingolipidspiegel während der PCD in Frage. Wir lieferten eine detaillierte Analyse der Sphingolipidspiegel, die Zusammenhänge zwischen bestimmten Sphingolipidarten und dem Zelltod aufzeigte. Darüber hinaus ermöglichte uns die Untersuchung der Sphingolipid-Biosynthese ein Verständnis des Fluxes nach Akkumulation hoher LCB-Konzentrationen. Weitere Analysen von Abbauprodukten oder Sphingolipid-Mutantenlinien wären jedoch erforderlich, um vollständig zu verstehen, wie die Pflanze mit hohen Mengen an Sphingobasen umgeht. N2 - Sphingolipid long-chain bases (LCBs) are the building blocks of the biosynthesis of sphingolipids. They are defined as structural elements of the plant cell membrane and play an important role determining the fate of the cells. Complex ceramides represent a substantial fraction of total sphingolipids which form a major part of eukaryotic membranes. At the same time, LCBs are well known signaling molecules of cellular processes in eukaryotes and are involved in signal transduction pathways in plants. High levels of LCBS have been shown to be associated with the induction of programmed cell death as well as pathogen-derived toxin-induced cell death. Indeed, several studies confirmed the regulatory function of sphingobases in plant programmed cell death (PCD): (i) Spontaneous PCD and altered cell death reaction caused by mutated related genes of sphingobase metabolism. (ii) Cell death conditions increases levels of LCBs. (iii) PCD due to interfered sphingolipid metabolism provoked by toxins produced from necrotrophic pathogens, such as Fumonisin B1 (FB1). Therefore, to prevent cell death and control cell death reaction, the regulation of levels of free LCBs can be crucial. The results of the present study challenged the comprehension of sphingobases and sphingolipid levels during PCD. We provided detailed analysis of sphingolipids levels that revealed correlations of certain sphingolipid species with cell death. Moreover, the investigation of sphingolipid biosynthesis allowed us to understand the flux after the accumulation of high LCB levels. However, further analysis of degradation products or sphingolipid mutant lines, would be required to fully understand how high levels of sphingobases are being treated by the plant. KW - PCD KW - Sphingolipids KW - LCB KW - Ackerschmalwand KW - programmed cell death KW - arabidopsis thaliana KW - abiotic stress Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325916 ER -