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Involvement of sphingosine kinase in plant cell signalling
Abstract
In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.
... Phosphorylation of LCBs at their C-1 hydroxyl group is catalyzed by LCB kinases (often referred to sphingosine kinases or SPHKs). To date three LCB kinases have been identifi ed in Arabidopsis: SPHK1 (At5g23450), SPHK2 (At2g46090), and AtLCBK1 (At5g23450) (Imai and Nishiura 2005 ;Worrall et al. 2008 ;. Release of the phosphate group from LCB-P molecules is catalyzed by the enzyme LCB-P phosphatase, which are encoded by two genes in Arabidopsis (At3g58490 and At5g03080) (Nakagawa et al. 2012 ;Worrall et al. 2008 ). ...
... To date three LCB kinases have been identifi ed in Arabidopsis: SPHK1 (At5g23450), SPHK2 (At2g46090), and AtLCBK1 (At5g23450) (Imai and Nishiura 2005 ;Worrall et al. 2008 ;. Release of the phosphate group from LCB-P molecules is catalyzed by the enzyme LCB-P phosphatase, which are encoded by two genes in Arabidopsis (At3g58490 and At5g03080) (Nakagawa et al. 2012 ;Worrall et al. 2008 ). As described below, the interplay between LCB kinases and LCB-P phosphatases are believed to be important for signaling pathways in plants (Nakagawa et al. 2012 ;Worrall et al. 2008 ). ...
... Release of the phosphate group from LCB-P molecules is catalyzed by the enzyme LCB-P phosphatase, which are encoded by two genes in Arabidopsis (At3g58490 and At5g03080) (Nakagawa et al. 2012 ;Worrall et al. 2008 ). As described below, the interplay between LCB kinases and LCB-P phosphatases are believed to be important for signaling pathways in plants (Nakagawa et al. 2012 ;Worrall et al. 2008 ). ...
... To test if SLs are important in PD regulation, we used several potential inhibitors of SL metabolism (Supplemental Fig. S1; Edsall et al., 1998;Singh et al., 2000;Coursol et al., 2003;Sperling and Heinz, 2003;Wright et al., 2003;Falcone et al., 2004;Chen et al., 2006Chen et al., , 2014Delgado et al., 2006;Kang et al., 2008;Worrall et al., 2008;Melser et al., 2010;Markham et al., 2011;Su et al., 2011;Adibhatla et al., 2012;Michaelson et al., 2016). We performed PD permeability assays using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; Han et al., 2014) and Aniline Blue callose staining assays with etiolated Arabidopsis hypocotyls after treatment with SL synthetic pathway inhibitors. ...
... Second, it is known that SPHK1 is employed to produce S1P (Supplemental Fig. S1). In plants, the SL metabolite S1P is involved in guard cell signal transduction (Ng et al., 2001;Coursol et al., 2003Coursol et al., , 2005Worrall et al., 2008;Puli et al., 2016). S1P in guard cells triggers alterations in nitric oxide, cytoplasmic calcium, and cytoplasmic pH (Ng et al., 2001;Puli et al., 2016). ...
... Arabidopsis (Arabidopsis thaliana) Col-0 and the following mutants were used: SPHK1-KD (Sail_794_B01; Worrall et al., 2008;Qin et al., 2017), sphk1-2 (Salk_042166), and gcs-2 (SK31705). The following constructs for the PD reporter were previously described: p35S::SP:GFP:PdBG2 (Simpson et al., 2009;Grison et al., 2015), p35S::SP:GFP:PDCB1 (Simpson et al., 2009;Grison et al., 2015), and p35S::PDLP1:GFP (Caillaud et al., 2014). ...
Plasma membranes encapsulated in the symplasmic nano-channels of plasmodesmata (PD) contain abundant lipid rafts, which are enriched with sphingolipids (SLs) and sterols. Reduction of sterols has highlighted the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly in affecting callose enhancement. The presence of callose at PD is strongly attributed to regulation of callose accumulation and callose degradation by callose synthases and β-1,3-glucanases (BGs), respectively. Sphingolipids are implicated in signaling and membrane protein trafficking; however, the underlying processes linking sphingolipid compositions to the control of symplasmic apertures remain unknown. The wide variety of sphingolipids in plants prompted us to investigate which sphingolipid molecules are important for regulating symplasmic apertures in Arabidopsis (Arabidopsis thaliana). We introduced several potential SL pathway inhibitors and genetically modified SL contents using two independent SL pathway mutants. We were able to modulate callose deposition to control symplasmic connectivity through perturbations of sphingolipid metabolism. Alteration in glucosylhydroxyceramides or related SL composition particularly disturbed the secretory machinery for the GPI-anchored PdBG2 protein, resulting in an over-accumulation of callose. Moreover, our results revealed that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting of GPI-anchored PD proteins incorporating glucosyl SLs.
... Bei SPHK2 handelt es sich auÿerdem um ein direkt benachbartes Gen von SPHK1 mit hoher Sequenzähnlichkeit. Die Enzyme werden in unterschiedlichen Geweben der Panze exprimiert (Guo et al., 2011;Qin et al., 2017;Worrall et al., 2008). Eine knockdown-Mutante von SPHK1 weist einen reduzierten, eine Überexpressionsmutante einen erhöhten Abscisinsäure-induzierten Sto-mataschluss auf (Coursol et al., 2003(Coursol et al., , 2005Worrall et al., 2008 Die Dephosphorylierung von Sphingobasen kann durch eine LCB-P-Phosphatase erfolgen. ...
... Die Enzyme werden in unterschiedlichen Geweben der Panze exprimiert (Guo et al., 2011;Qin et al., 2017;Worrall et al., 2008). Eine knockdown-Mutante von SPHK1 weist einen reduzierten, eine Überexpressionsmutante einen erhöhten Abscisinsäure-induzierten Sto-mataschluss auf (Coursol et al., 2003(Coursol et al., , 2005Worrall et al., 2008 Die Dephosphorylierung von Sphingobasen kann durch eine LCB-P-Phosphatase erfolgen. ...
... Dagegen ist die Rolle von LCB-Ps in der Zelltodregulation bei Panzen aufgrund des Fehlens der in Tieren identizierten G-Protein-gekoppelten Rezeptoren für Sphingosin-1-Phosphat unklar. Wegen der in Arabidopsis bekannten und charakterisierten Enzyme der LCB-Kinasen (SPHK1, SPHK2, LCBK1, LCBK2) wurde in mehreren Publikationen eine Rolle von LCB-Ps an Prozessen wie Schlieÿung der Stomata und Kälteadaption beschrieben (Coursol et al., 2003(Coursol et al., , 2005Dutilleul et al., 2012;Huang et al., 2017;Worrall et al., 2008 Lachaud et al., 2010Lachaud et al., , 2011Magnin-Robert et al., 2015;Ng et al., 2001;Peer et al., 2011;Saucedo-García et al., 2011b;Takahashi et al., 2009;Testard et al., 2016) (Bi et al., 2014;Liang et al., 2003;Simanshu et al., 2014;Townley et al., 2005). ...
Sphingobasen bilden das Grundgerüst und die Ausgangsbausteine für die Biosynthese von Sphingolipiden. Während komplexere Sphingolipide einen wichtigen Bestandteil von eukaryotischen Membranen bilden, sind Sphingobasen, die auch als long-chain bases (LCBs) bezeichnet werden, als Signalmoleküle bei zellulären Prozessen in Eukaryoten bekannt. Im tierischen System wurden antagonistische Effekte von nicht-phosphorylierten Sphingobasen (LCBs) und ihren phosphorylierten Gegenstücken (LCB-Ps) bei vielen Zellfunktionen, insbesondere der Apoptose, nachgewiesen und die zugrundeliegenden Signalwege umfassend aufgeklärt. Im Gegensatz dazu sind in Pflanzen weniger Belege für einen antagonistischen Effekt und mögliche Signaltransduktionsmechanismen bekannt. Für eine regulatorische Funktion von Sphingobasen beim programmierten Zelltod (PCD) in Pflanzen existieren mehrere Hinweise: (I) Mutationen in Genen, die den Sphingobasen-Metabolismus betreffen, führen zum Teil zu spontanem PCD und veränderten Zelltodreaktionen. (II) Die Gehalte von LCBs sind bei verschiedenen Zelltod-auslösenden Bedingungen erhöht. (III) Nekrotrophe Pathogene produzieren Toxine, wie Fumonisin B1 (FB1), die mit dem Sphingolipid-Metabolismus der Wirtspflanze interferieren, was wiederum die Ursache für den dadurch ausgelösten PCD darstellt. (IV) Die Behandlung von Pflanzen mit LCBs, nicht aber mit LCB-Ps, führt zu Zelltod. In dieser Arbeit wurde die Rolle von Sphingobasen in der pflanzlichen Zelltodreaktion untersucht, wobei der Fokus auf der Überprüfung der Hypothese eines antagonistischen, Zelltod-hemmenden Effekts von LCB-Ps lag. Anhand von Leitfähigkeit-basierten Messungen bei Blattscheiben von Arabidopsis thaliana wurde der durch Behandlung mit LCBs und separater oder gleichzeitiger Zugabe von LCB-Ps auftretende Zelltod bestimmt. Mit dieser Art der Quantifizierung wurde der an anderer Stelle publizierte inhibierende Effekt von LCB-Ps auf den LCB-induzierten Zelltod nachgewiesen. Durch parallele Messung der Spiegel der applizierten Sphingobasen im Gewebe mittels HPLC-MS/MS konnte dieser Antagonismus allerdings auf eine reduzierte Aufnahme der LCB bei Anwesenheit der LCB-P zurückgeführt werden, was auch durch eine zeitlich getrennte Behandlung mit den Sphingobasen bestätigt wurde. Darüber hinaus wurde der Einfluss einer exogenen Zugabe von LCBs und LCB-Ps auf den durch Pseudomonas syringae induzierten Zelltod von A. thaliana untersucht. Für LCB-Ps wurde dabei kein Zelltod-hemmender Effekt beobachtet, ebenso wenig wie ein Einfluss von LCB-Ps auf den PCD, der durch rekombinante Expression und Erkennung eines Avirulenzproteins in Arabidopsis ausgelöst wurde. Für LCBs wurde dagegen eine direkte antibakterielle Wirkung im Zuge der Experimente mit P. syringae gezeigt, die den in einer anderen Publikation beschriebenen inhibierenden Effekt von LCBs auf den Pathogen-induzierten Zelltod in Pflanzen relativiert. In weiteren Ansätzen wurden Arabidopsis-Mutanten von Enzymen des Sphingobasen-Metabolismus (LCB-Kinase, LCB-P-Phosphatase, LCB-P-Lyase) hinsichtlich veränderter in-situ-Spiegel von LCBs/LCB-Ps funktionell charakterisiert. Der Phänotyp der Mutanten gegenüber Fumonisin B1 wurde zum einen anhand eines Wachstumstests mit Keimlingen und zum anderen anhand des Zelltods von Blattscheiben bestimmt und die dabei akkumulierenden Sphingobasen quantifiziert. Die Sensitivität der verschiedenen Linien gegenüber FB1 korrelierte eng mit den Spiegeln der LCBs, während hohe Gehalte von LCB-Ps alleine nicht in der Lage waren den Zelltod zu verringern. In einzelnen Mutanten konnte sogar eine Korrelation von stark erhöhten LCB-P-Spiegeln mit einer besonderen Sensitivität gegenüber FB1 festgestellt werden. Die Ergebnisse der vorliegenden Arbeit stellen die Hypothese eines antagonistischen Effekts von phosphorylierten Sphingobasen beim pflanzlichen Zelltod in Frage. Stattdessen konnte in detaillierten Analysen der Sphingobasen-Spiegel die positive Korrelation der Gehalte von LCBs mit dem Zelltod gezeigt werden. Die hier durchgeführten Experimente liefern damit nicht nur weitere Belege für die Zelltod-fördernde Wirkung von nicht-phosphorylierten Sphingobasen, sondern tragen zum Verständnis der Sphingobasen-Homöostase und des Sphingobasen-induzierten PCD in Pflanzen bei.
... The LCBs and their phosphorylated derivatives (LCB-Ps) are critical regulators of many physiological processes and stress responses (Ng et al., 2001;Coursol et al., 2003Coursol et al., , 2005Coursol et al., , 2015Shi et al., 2007;Chen et al., 2008;Worrall et al., 2008;Teng et al., 2008;Islam et al., 2012;Dutilleul et al., 2012Dutilleul et al., , 2015Markham et al., 2013;Wu et al., 2014). Phosphorylation of LCBs at their C-1 hydroxyl group is catalysed by LCB kinases (LCBKs), often referred to as sphingosine kinases (SPHKs), and four genes (LCBK1, LCBK2, SPHK1, and SPHK2) encode these enzymes in Arabidopsis . ...
... Phosphorylation of LCBs at their C-1 hydroxyl group is catalysed by LCB kinases (LCBKs), often referred to as sphingosine kinases (SPHKs), and four genes (LCBK1, LCBK2, SPHK1, and SPHK2) encode these enzymes in Arabidopsis . SPHKs function as important signal transducers in stress responses (Imai and Nishiura, 2005;Worrall et al., 2008;Dutilleul et al., 2012;Guo et al., 2012;Saucedo-García et al., 2011). In Arabidopsis, abscisic acid (ABA) stimulates the enzymatic activity of SPHK1 and SPHK2 during stomatal closure (Ng et al., 2001;Coursol et al., 2003Coursol et al., , 2005, LCBK1 functions as a positive regulator of freezing tolerance (Huang et al., 2017), and recent findings indicate a role of SPHK1 in modulating fumonisin B1-triggered cell death via salicylic acid (SA) and jasmonic acid (JA) pathway interactions (Qin et al., 2017). ...
... Although the availability of Arabidopsis mutants has led to discoveries of the involvement of sphingolipids in reproduction and embryogenesis, endomembrane trafficking, abiotic stress responses and pathogen defence Teng et al., 2008;Worrall et al., 2008;Guillas et al., 2011;Markham et al., 2011;Berkey et al., 2012;Dutilleul et al., 2012;Wu et al., 2015a;Tartaglio et al., 2017), little is known about sphingolipids in relation to early fruit growth and developmental processes in fleshy-fruited species. ...
... However, later observations indicated that the regulation of stomatal pore size by S1P in ABA signaling pathway actually involved heterotrimeric G protein α subunit (GPA1) (Coursol et al. 2003). The sphingosine phosphorylating enzyme, sphingosine kinase (SPHK), also inhibited seed germination (Worrall et al. 2008). Other enzymes in the sphingolipid pathway that participate in ABA response are long-chain base phosphate lyase (LCBPL), long-chain base phosphate phosphatases (LCBPPs) and LCB Δ4 desaturase (Worrall et al. 2008;Michaelson et al. 2009). ...
... The sphingosine phosphorylating enzyme, sphingosine kinase (SPHK), also inhibited seed germination (Worrall et al. 2008). Other enzymes in the sphingolipid pathway that participate in ABA response are long-chain base phosphate lyase (LCBPL), long-chain base phosphate phosphatases (LCBPPs) and LCB Δ4 desaturase (Worrall et al. 2008;Michaelson et al. 2009). In the perspectives of these observations, Guo et al. (2012) questioned the involvement of other LCBPs in ABA-induced stomatal closure. ...
Carotenoids are vital pigments for higher plants and play a crucial function in photosynthesis and photoprotection. Carotenoids are precursors of vitamin A synthesis and contribute to human nutrition and health. However, cereal grain endosperm contains a minor carotenoid measure and a scarce supply of provitamin A content. Therefore, improving the carotenoids in cereal grain is of major importance. Carotenoid content is governed by multiple candidate genes with their additive effects. Studies on genes related to carotenoid metabolism in cereals would increase the knowledge of potential metabolic steps of carotenoids and enhance the quality of crop plants. Recognizing the metabolism and carotenoid accumulation in various staple cereal crops over the last few decades has broadened our perspective on the interdisciplinary regulation of carotenogenesis. Meanwhile, the amelioration in metabolic engineering approaches has been exploited to step up the level of carotenoid and valuable industrial metabolites in many crops, but wheat is still considerable in this matter. In this study, we present a comprehensive overview of the consequences of biosynthetic and catabolic genes on carotenoid biosynthesis, current improvements in regulatory disciplines of carotenogenesis, and metabolic engineering of carotenoids. A panoptic and deeper understanding of the regulatory mechanisms of carotenoid metabolism and genetic manipulation (genome selection and gene editing) will be useful in improving the carotenoid content of cereals.
... In addition, sphingolipids and LCBs are bioactive molecules involved in signal transduction processes in plants. Levels of specific LCBs have been functionally linked with processes such as stomatal closure (Ng and Hetherington, 2001;Coursol et al., 2003Coursol et al., , 2005Worrall et al., 2008;Dutilleul et al., 2012) and programmed cell death (Shi et al., 2007;Wang et al., 2008;Lachaud et al., 2010;Alden et al., 2011;Ternes et al., 2011;Magnin-Robert et al., 2015;Yanagawa et al., 2017;Glenz et al., 2019). High levels of LCBs are detected during programmed cell death reactions (Peer et al., 2010), and are also induced by the toxin Fumonisin B 1 , which serves as a virulence factor for necrotrophic pathogenic strains of the Fusarium clade (Abbas et al., 1994;Shi et al., 2007;Williams et al., 2007). ...
... LCBs can also be phosphorylated by specific kinases (SPHK1/2 and LCBK1 and 2), which is required for their degradation by dihydrosphingosine-1-phosphate lyase (DPL1) to phosphoethanolamine and hexadecanal (Tsegaye et al., 2007;Nishikawa et al., 2008). In addition to these reactions, LCB levels are influenced by the degradation of ceramides (ceramidase activity), or by dephosphorylation of LCB-Ps by phosphatases (SPP1; Imai and Nishiura, 2005;Worrall et al., 2008;Nakagawa et al., 2012). ...
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.
... SBH and Δ4-DES use d18:0 as substrates; therefore, C-4 hydroxylation of d18:1 precludes Δ4 desaturation, and conversely, Δ4 desaturation of d18:1 prevents C-4 hydroxylation . Several LCB kinases (LCBK1, LCBK2, SPHK1, and SPHK2) phosphorylate LCBs at the C-1 OH position to form LCB-1-phosphates (LCB-Ps) (Imai and Nishiura 2005;Worrall et al. 2008). These LCB-Ps can be degraded by PHYTO-S1P PHOSPHATASE (SPP1) or PHYTO-S1P LYASE (DPL1) (Nishikawa et al. 2008;Worrall et al. 2008;Nakagawa et al. 2012). ...
... Several LCB kinases (LCBK1, LCBK2, SPHK1, and SPHK2) phosphorylate LCBs at the C-1 OH position to form LCB-1-phosphates (LCB-Ps) (Imai and Nishiura 2005;Worrall et al. 2008). These LCB-Ps can be degraded by PHYTO-S1P PHOSPHATASE (SPP1) or PHYTO-S1P LYASE (DPL1) (Nishikawa et al. 2008;Worrall et al. 2008;Nakagawa et al. 2012). ...
Sphingolipids (lipids with a sphingoid base backbone) are important components of eukaryotic membrane systems and key signaling molecules that are essential for controlling cellular homeostasis, acclimating to stress, and regulating plant immunity. Studies using sphingolipid treatments, measuring sphingolipids in infected plants, and functionally studying sphingolipid biosynthetic mutants demonstrated that sphingolipids participate in plant cell death and defense responses. In this review, we present an updated map of sphingolipid signaling and review recent progress in understanding the functions of sphingolipids in plant immunity as structural components of biological membranes, and as mediators of defense signaling. Moreover, several pressing questions, such as how sphingolipids in the plasma membrane, particularly microdomains, act to perceive pathogens and transduce signals during plant–pathogen interactions, remain to be further explored in future research.
... This important physiological response certainly requires further investigation, particularly the identification of additional genetic components associated with PCD signalling. The kinases responsible for phosphorylating LCBs, SPHINGOSINE KINASE 1 and SPHINGOSINE KINASE 2, have primarily been studied in A. thaliana (Coursol et al., 2005;Worrall et al., 2008;Alden et al., 2011;Guo et al., 2011). LCB-Ps can be depleted either by dephosphorylation by LONG-CHAIN BASE PHOSPHATE PHOSPHATASEs (LCB-PP1 and LCB-PP2) (Worrall et al., 2008) or by cleavage of the LCB backbone to produce hexadecanal and phosphoethanolamine by LONG-CHAIN BASE PHOSPHATE LYASE (DPL) (Tsegaye et al., 2007). ...
... The kinases responsible for phosphorylating LCBs, SPHINGOSINE KINASE 1 and SPHINGOSINE KINASE 2, have primarily been studied in A. thaliana (Coursol et al., 2005;Worrall et al., 2008;Alden et al., 2011;Guo et al., 2011). LCB-Ps can be depleted either by dephosphorylation by LONG-CHAIN BASE PHOSPHATE PHOSPHATASEs (LCB-PP1 and LCB-PP2) (Worrall et al., 2008) or by cleavage of the LCB backbone to produce hexadecanal and phosphoethanolamine by LONG-CHAIN BASE PHOSPHATE LYASE (DPL) (Tsegaye et al., 2007). ...
Sphingolipids are essential metabolites found in all plant species. They are required for plasma membrane integrity, tolerance of and responses to biotic and abiotic stresses, and intracellular signalling. There is extensive diversity in the sphingolipid content of different plant species, and in the identities and roles of enzymes required for their processing. In this review, we survey results obtained from investigations of the classical genetic model Arabidopsis thaliana, from assorted dicots with less extensive genetic toolkits, from the model monocot Oryza sativa, and finally from the model bryophyte Physcomitrium patens. For each species or group, we first broadly summarize what is known about sphingolipid content. We then discuss the most insightful and puzzling features of modifications to the hydrophobic ceramides, and to the polar headgroups of complex sphingolipids. Altogether, this data can serve as a framework for our knowledge of sphingolipid metabolism across the plant kingdom. This chemical and metabolic heterogeneity underpins equally diverse functions. With greater availability of different tools for analytical measurements and genetic manipulation, our field is entering an exciting phase of expanding our knowledge of the biological functions of this persistently cryptic class of lipids.
... Both sphingolipids and LCBs are involved in important functions including developmental processes and responses to abiotic and biotic stress [reviewed in (Markham et al. 2013, Michaelson et al. 2016]. LCBs, and their phosphorylated forms (LCB-Ps) have been characterized as bioactive molecules which regulate processes such as stomatal closure (Ng and Hetherington 2001, Coursol et al. 2003, Coursol et al. 2005, Worrall et al. 2008) and programmed cell death (PCD) (Shi et al. 2007, Wang et al. 2008, Lachaud et al. 2010, Alden et al. 2011, Peer et al. 2011, Berkey et al. 2012. Several lines of evidence link LCB levels with plant cell death: (i) Mutations in Arabidopsis thaliana disrupting specific steps of sphingolipid metabolism show cell death-related phenotypes, e.g. ...
... All mutants of A. thaliana were in the Col-0 background. The T-DNA insertion mutants dpl1-1 and dpl1-2 (SALK_020151 and SALK_093662; Tsegaye et al. 2007), spp1-1 (SALK_035202; Nakagawa et al. 2012), SPP1-OE (GK-126D07), SPHK1-KD and SPHK1-OE (SAIL_794_B01 and GK-288D07; Worrall et al. 2008) were created by SALK (Alonso et al. 2003), SAIL (McElver et al. 2001) and GABI-Kat (Kleinboelting et al. 2012), respectively, and obtained from the Nottingham Arabidopsis Stock Centre [NASC, University of Nottingham, Loughborough, UK (Scholl et al. 2000)]. The Line Dex:AvrRpm1-HA for the dexamethasone inducible expression of the effector protein AVRRPM1 was kindly provided by Geng and Mackey (2011). ...
Long-chain bases (LCBs), also termed sphingobases, are building blocks of sphingolipids, which make up a significant proportion of the cellular membrane system. They are also bioactive molecules regulating intracellular processes. Elevated levels of LCBs like phytosphingosine and dihydrosphingosine can induce cell death in plants and correlate with programmed cell death (PCD) reactions after pathogen recognition. We investigated the previously hypothesized antagonism between phosphorylated and non-phosphorylated LCBs with respect to cell death in Arabidopsis thaliana. Using HPLC-MS/MS, we determined levels of phosphorylated and non-phosphorylated LCBs after cell death induction by LCB application or by Fumonisin B1 treatment. We show that previously reported antagonistic effects of phosphorylated LCBs after simultaneous application with non-phosphorylated LCBs are linked to reduced uptake of non-phosphorylated LCBs into the tissue. Furthermore, phosphorylated LCBs did not antagonize PCD induced by avirulence protein recognition. In a functional approach, we used Arabidopsis lines with perturbed levels of phosphorylated LCBs. In these plants, the degree of Fumonisin B1-induced cell death did not consistently correlate negatively with levels of phosphorylated LCBs, but positively with levels of major non-phosphorylated LCBs phytosphingosine and dihydrosphingosine. As treatment with phosphorylated LCBs did not antagonize cell death, and elevated in vivo levels of these LCB species did not reduce Fumonisin B1-induced cell death, we conclude that the hypothesized general cell death-antagonizing effect of phosphorylated LCBs in plant cell death reactions should be rejected. Instead, our time-course analysis of LCB levels during cell death reactions showed a positive correlation between levels of non-phosphorylated LCBs and cell death.
... In the GO term phosphatidic acid binding (GO: 0070300), the gene AT4G21534 encodes sphingosine kinase (SPHK2). Six SphKs genes were identified in the Arabidopsis genome (Worrall et al. 2008;Guo et al. 2011), and SPHK1, SPHK2/phyto-S1P, and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis (Coursol et al. 2005;Worrall et al. 2008;Michaelson et al. 2009;Guo et al. 2011). Gene AT2G44640 encodes TriGalactosylDiacylglycerol protein (TGD4). ...
... In the GO term phosphatidic acid binding (GO: 0070300), the gene AT4G21534 encodes sphingosine kinase (SPHK2). Six SphKs genes were identified in the Arabidopsis genome (Worrall et al. 2008;Guo et al. 2011), and SPHK1, SPHK2/phyto-S1P, and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis (Coursol et al. 2005;Worrall et al. 2008;Michaelson et al. 2009;Guo et al. 2011). Gene AT2G44640 encodes TriGalactosylDiacylglycerol protein (TGD4). ...
Main conclusion
Alternative splicing EVENTS were genome-wide identified for four legume species, and nitrogen fixation-related gene families and evolutionary analysis was also performed.
Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. Investigation of the genome-wide conserved AS events across different species will help with the understanding of the evolution of the functional diversity in legumes, allowing for genetic improvement. Genome-wide identification and characterization of AS were performed using the publically available mRNA, EST, and RNA-Seq data for four important legume species. A total of 15,165 AS genes in Glycine max, 6077 in Cicer arietinum, 7240 in Medicago truncatula, and 7358 in Lotus japonicus were identified. Intron retention (IntronR) was the dominant AS type among the identified events, with IntronR occurring from 53.76% in M. truncatula to 43.91% in C. arietinum. We identified 1159 AS genes that were conserved among four species. Furthermore, nine nitrogen fixation-related gene families with 237 genes were identified, and 80 of them were AS, accounting for the 43.48% in G. max and 27.78% in C. arietinum. An evolutionary analysis showed that these AS genes tended to be located adjacent to each other in the evolutionary tree and are unbalanced in the distribution in the sub-family. This study provides a foundation for future studies on transcription complexity, evolution, and the role of AS on plant functional regulation.
... Coursol et al. (2003) provided further complexity to this mechanism under drought stress when they reported the involvement of the a subunit (GPA1) of the heterotrimeric G protein in the S1P-promoted regulation of the stomatal pore size in the ABA signaling pathway (Coursol et al., 2003). Worrall et al. (2008) reported that, apart from the role of sphingosine kinase (SPHK) in ABA-promoted stomatal response, the enzyme also inhibited seed germination. Long-chain base phosphate lyase (LCBPL) and long-chain base phosphate phosphatases (LCBPPs) also have been shown to participate in ABA and drought responses reported in Arabidopsis RNAi lines (Worrall et al., 2008). ...
... Worrall et al. (2008) reported that, apart from the role of sphingosine kinase (SPHK) in ABA-promoted stomatal response, the enzyme also inhibited seed germination. Long-chain base phosphate lyase (LCBPL) and long-chain base phosphate phosphatases (LCBPPs) also have been shown to participate in ABA and drought responses reported in Arabidopsis RNAi lines (Worrall et al., 2008). In subsequent studies, the functional characterization of the Arabidopsis LCB D4 desaturase mutants revealed that these mutants responded in a similar manner as the wild-type to water deficits and stomatal closure during ABA treatment (Michaelson et al., 2009). ...
Plant sphingolipids are not only structural components of the plasma membrane and other endomembrane systems but also act as signaling molecules during biotic and abiotic stresses. However, the roles of sphingolipids in plant signal transduction in response to environmental cues are yet to be investigated in detail. In this review, we discuss the signaling roles of sphingolipid metabolites with a focus on plant sphingolipids. We also mention some microbial sphingolipids that initiate signals during their interaction with plants, because of the limited literatures on their plant analogs. The equilibrium of nonphosphorylated and phosphorylated sphingolipid species determine the destiny of plant cells, whereas molecular connections among the enzymes responsible for this equilibrium in a coordinated signaling network are poorly understood. A mechanistic link between the phytohormone–sphingolipid interplay has also not yet been fully understood and many key participants involved in this complex interaction operating under stress conditions await to be identified. Future research is needed to fill these gaps and to better understand the signal pathways of plant sphingolipids and their interplay with other signals in response to environmental stresses. Sphingolipids are the least explored signaling molecules in plants. Receptors, targets, and the mediators of the sphingolipid-mediated signaling pathway are barely identified in plants, which provides a great opportunity to investigate plant sphingolipids and their signaling roles in response to environmental stresses.
... Previous work has shown that Arabidopsis plants with a partial loss of SPHINGOSINE 217 KINASE1 (SPHK1) were less sensitive to application of exogenous ABA (Worrall et al., 2008). ...
... Previously, it has been reported that plants with disrupted sphingolipid biosynthesis have altered 296 responses to ABA, including reduced sensitivity to ABA-dependent stomatal closure and 297 suppression of germination (Ng et al., 2001;Worrall et al., 2008;Wu et al., 298 2015). Therefore, analogous to their role in mammalian systems, sphingolipid molecules such as 299 sphingosine-1-phosphate and ceramide may act as signaling molecules. ...
Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structure, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis thaliana, and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain N-acetylglucosamine (GlcNAc) and glucosamine (GlcN; collectively GlcN(Ac)). In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, “rice-like” GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.
... Previous work has shown that Arabidopsis plants with a partial loss of SPHINGOSINE 217 KINASE1 (SPHK1) were less sensitive to application of exogenous ABA (Worrall et al., 2008). ...
... Previously, it has been reported that plants with disrupted sphingolipid biosynthesis have altered 296 responses to ABA, including reduced sensitivity to ABA-dependent stomatal closure and 297 suppression of germination (Ng et al., 2001;Worrall et al., 2008;Wu et al., 298 2015). Therefore, analogous to their role in mammalian systems, sphingolipid molecules such as 299 sphingosine-1-phosphate and ceramide may act as signaling molecules. ...
Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structure, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis thaliana, and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain N-acetylglucosamine (GlcNAc) and glucosamine (GlcN; collectively GlcN(Ac)). In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.
... Likewise, LPPα3 and LPPα4 are not predicted to localize to chloroplasts, and LPPα2 has been detected at the plasma membrane and chloroplast OEM (Katagiri et al. 2005;Nguyen and Nakamura 2023). LPPβ is present in the ER, where it is likely involved in oil synthesis (Cotton 2015), while LPPδ was determined to be a sphingoid phosphate phosphatase and renamed SPHINGOID PHOSPHATE PHOSPHATASE 1 (SPP1) (Worrall et al. 2008;Nakagawa et al. 2012). It is therefore unlikely that the plastid pathway PAP is a known LPP, and it may be necessary to search for another enzyme class associated with PAP activity. ...
Galactolipids comprise the majority of chloroplast membranes in plants, and their biosynthesis requires dephosphorylation of phosphatidic acid at the chloroplast envelope membranes. In Arabidopsis (Arabidopsis thaliana), the lipid phosphate phosphatases LPPγ, LPPε1, and LPPε2 have been previously implicated in chloroplast lipid assembly, with LPPγ being essential, as null mutants were reported to exhibit embryo lethality. Here, we show that lppγ mutants are in fact viable and that LPPγ, LPPε1, and LPPε2 do not appear to have central roles in the plastid pathway of membrane lipid biosynthesis. Redundant LPPγ and LPPε1 activity at the outer envelope membrane is important for plant development, and the respective lppγ lppε1 double mutant exhibits reduced flux through the ER pathway of galactolipid synthesis. While LPPε2 is imported and associated with interior chloroplast membranes, its role remains elusive and does not include basal nor phosphate limitation-induced biosynthesis of glycolipids. The specific physiological roles of LPPγ, LPPε1, and LPPε2 are yet to be uncovered, as does the identity of the phosphatidic acid phosphatase required for plastid galactolipid biosynthesis.
... Two earlier studies (Imai & Nishiura, 2005;Worrall et al., 2008) have explored this interaction inconclusively, suggesting a potential coordinated regulation between LCBK and its reversible counterpart. This underscores the need for more comprehensive research to thoroughly understand the interactions between these enzymes and their collective role in maintaining cellular homeostasis. ...
Sphingolipids are pivotal for plant development and stress responses. Growing interest has been directed towards fully comprehending the regulatory mechanisms of the sphingolipid pathway. We explore its de novo biosynthesis and homeostasis in Arabidopsis thaliana cell cultures, shedding light on fundamental metabolic mechanisms. Employing 15N isotope labeling and quantitative dynamic modeling approach, we developed a regularized and constraint-based Dynamic Metabolic Flux Analysis (r-DMFA) framework to predict metabolic shifts due to enzymatic changes. Our analysis revealed key enzymes such as sphingoid-base hydroxylase (SBH) and long-chain-base kinase (LCBK) to be critical for maintaining sphingolipid homeostasis. Disruptions in these enzymes were found to affect cellular viability and increase the potential for programmed cell death (PCD). Thus, this work enhances our understanding of sphingolipid metabolism and demonstrates the utility of dynamic modeling in analyzing complex metabolic pathways.
... The accumulation of LCBs stimulates CPK3, a calcium-dependent kinase, which then phosphorylates its 14-3-3 protein binding partners and triggers PCD (Lachaud et al., 2013). Moreover, LCB-Ps are involved in ABA, cold, and drought stress responses (Worrall et al., 2008;Guillas et al., 2013). While the role of sphingolipids in animals is well understood, the sphingolipid signaling system in plants is still largely unknown. ...
Lipids are a principal component of plasma membrane, acting as a protective barrier between the cell and its surroundings. Abiotic stresses such as drought and temperature induce various lipid-dependent signaling responses, and the membrane lipids respond differently to environmental challenges. Recent studies have revealed that lipids serve as signal mediators forreducing stress responses in plant cells and activating defense systems. Signaling lipids, such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, and N-acylethanolamines, are generated in response to stress. Membrane lipids are essential for maintaining the lamellar stack of chloroplasts and stabilizing chloroplast membranes under stress. However, the effects of lipid signaling targets in plants are not fully understood. This review focuses on the synthesis of various signaling lipids and their roles in abiotic stress tolerance responses, providing an essential perspective for further investigation into the interactions between plant lipids and abiotic stress.
... In addition, ceramide [mainly Cer (d18:1/26:0)] closely interacted with soil microbial communities in comparison with the naturally fermented soybean fertilization in enzymatic fermented soybean fertilization. Sphingolipids were not only bio-active components of cells with signal transduction function (Worrall et al., 2008), but also the second messenger of plant defense mechanisms participating in various plant stress responses (Markham et al., 2013;Michaelson et al., 2016). Ceramide was the key intermediate of the sphingolipid metabolism pathway, and its synthesis was the starting point of biosynthesis in various complex sphingolipid compounds (Hou et al., 2016). ...
Compared with traditional organic fertilizer, fermented soybean is a better fertilizer resource in tea plantations. The application of organic fertilizer is a feasible practice to mitigate the soil degradation caused by the overuse of chemical fertilizers, which can effectively regulate soil microbial communities in tea plantations. However, the effects of fermented soybean on soil microbial communities, soil metabolites and metabolites in tea new shoots have not been systematically demonstrated, and their interactions have never been studied. Here, we investigated the responses of the soil microbial community, soil metabolites and metabolites of tea new shoots to urea fertilization (UF), naturally fermented soybean fertilization (NFS) and enzymatic fermented soybean fertilization (EFS), and analyzed the relationships between soil microbes, soil metabolites and metabolites in tea new shoots. The results showed that soil bacterial communities were dominated by Pseudomonas, Romboutsia, Candidatus_Nitrosotalea and Helicobacter , and soil fungal communities were dominated by Peziza, Fusarium, Candida and Cheilymenia at the genus level. In EFS, bacterial genera ( Glutamicibacter and Streptomyces ) and fungal genera ( Candida and Actinomucor ) presented high abundances, which were correlated with soil carbohydrate and lipid including D-Mannitol, D-Sorbitol, 9,12-Octadecadienoic acid and (Z)-13-Docosenoic acid. Enzymatic fermented soybean fertilization also affected the lipid metabolites in tea new shoots. Glycerolipids and glycerophospholipids significantly increased in EFS, which positively correlated with some soil microbial communities. Besides, the application of fermented soybean fertilizer could increase the contents of TP, AP and AK, which were also important environmental factors affecting the structure of soil microbial community in tea plantation. It was concluded that fermented soybean fertilization could improve soil nutrition, regulate associated microbial communities, and positively affect lipid metabolites in tea new shoots. This study not only explores the relationships between soil microbes and metabolites in tea plants, but also provides feasible technical guidance to cultivate high-quality tea using soybean as high-grade fertilizer.
... Besides being the common backbone of all sphingolipids, free LCBs also act as second messengers in multiple processes, such as regulation of stomatal closure (Worrall et al., 2008;Guo et al., 2012), freezing tolerance (Huang et al., 2017), autophagy (Zheng et al., 2018), programmed cell death (PCD; Shi et al., 2007), and immunity (Magnin-Robert et al., 2015;Liu et al., 2019). PCD is essential for normal growth and development as well as responses to a variety of biotic and abiotic stresses in plants (Locato and De Gara, 2018). ...
Ceramide synthases (CSs) produce ceramides from long-chain bases (LCBs). However, how CSs regulate immunity and cell death in Arabidopsis thaliana remains unclear. Here, we decipher the roles of two classes of CS, CSI (LAG1 HOMOLOG 2, LOH2) and CSII (LOH1/3), in these processes. The loh1-2 and loh1-1 loh3-1 mutants were resistant to the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) DG3 and exhibited programmed cell death (PCD), along with increased LCBs and ceramides, at later stages. In loh1-2, the Psm resistance, PCD, and sphingolipid accumulation were mostly suppressed by inactivation of the lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4), and partly suppressed by loss of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2). The LOH1 inhibitor fumonisin B1 (FB1) triggered EDS1/PAD4-independent LCB accumulation, and EDS1/PAD4-dependent cell death, resistance to Psm, and C16 Cer accumulation. Loss of LOH2 enhances FB1-, and sphinganine-induced PCD, indicating that CSI negatively regulates the signaling triggered by CSII inhibition. Like Cer, LCBs mediate cell death and immunity signaling, partly through the EDS1/PAD4 pathway. Our results show that the two classes of ceramide synthases differentially regulate EDS1/PAD4-dependent PCD and immunity via subtle control of LCBs and Cers in Arabidopsis.
... In yeast, phytosphingosine, thought to be a putative sphingolipid second messenger, acts as a mediator in regulation of heat stress signaling and activation of ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways (Chung et al., 2000). Based on the accumulated findings, sphingolipids function as bioactive signals and play critical roles in a variety of physiological processes and environmental responses, including programmed cell death (Liang et al., 2003;Shi et al., 2007;Simanshu et al., 2014), pathogen-induced hypersensitive response (HR; Liang et al., 2003;Wang et al., 2008;Gan et al., 2009), ABA-dependent guard cell closure (Ng et al., 2001;Coursol et al., 2003;Worrall et al., 2008), host-pathogen interactions (Takahashi et al., 2009;FIGURE 7 | Heat map of the significantly differential lysophospholipids in leaves of B. grandis in response to heat stress. Fully extended leaves detached from 173-d-old plants of B. grandis were at control condition (22°C for 2 h) or challenged with heat stress (45°C for 2 h). ...
Characterization of the alterations in leaf lipidome in Begonia (Begonia grandis Dry subsp. sinensis) under heat stress will aid in understanding the mechanisms of stress adaptation to high-temperature stress often occurring during hot seasons at southern areas in China. The comparative lipidomic analysis was performed using leaves taken from Begonia plants exposed to ambient temperature or heat stress. The amounts of total lipids and major lipid classes, including monoacylglycerol (MG), diacylglycerol (DG), triacylglycerols (TG), and ethanolamine-, choline-, serine-, inositol glycerophospholipids (PE, PC, PS, PI) and the variations in the content of lipid molecular species, were analyzed and identified by tandem high-resolution mass spectrometry. Upon exposure to heat stress, a substantial increase in three different types of TG, including 18:0/16:0/16:0, 16:0/16:0/18:1, and 18:3/18:3/18:3, was detected, which marked the first stage of adaptation processes. Notably, the reduced accumulation of some phospholipids, including PI, PC, and phosphatidylglycerol (PG) was accompanied by an increased accumulation of PS, PE, and phosphatidic acid (PA) under heat stress. In contrast to the significant increase in the abundance of TG, all of the detected lysophospholipids and sphingolipids were dramatically reduced in the Begonia leaves exposed to heat stress, suggesting that a very dynamic and specified lipid remodeling process is highly coordinated and synchronized in adaptation to heat stress in Begonia plants.
... Sphingolipids are not only a crucial component of biomembranes but also are important bioactive molecules that mediates a variety of cell processes [43], such as programmed cell death [71][72][73][74], low temperature signal transduction [75][76][77], pathogen-induced hypersensitivity [72], host-pathogen crosstalk [78], the closure of stomatal guard cells regulated by ABA signaling [79][80][81][82], and the regulation of membrane stability [83]. Recent studies indicated that sphingolipids also participate in cotton ovule growth and fiber cell development [84,85]. ...
Cotton fiber is a seed trichome that protrudes from the outer epidermis of cotton ovule on the day of anthesis (0 day past anthesis, 0 DPA). The initial number and timing of fiber cells are closely related to fiber yield and quality. However, the mechanism underlying fiber initiation is still unclear. Here, we detected and compared the contents and compositions of sphingolipids and sterols in 0 DPA ovules of Xuzhou142 lintless-fuzzless mutants (Xufl) and Xinxiangxiaoji lintless-fuzzless mutants (Xinfl) and upland cotton wild-type Xuzhou142 (XuFL). Nine classes of sphingolipids and sixty-six sphingolipid molecular species were detected in wild-type and mutants. Compared with the wild type, the contents of Sphingosine-1-phosphate (S1P), Sphingosine (Sph), Glucosylceramide (GluCer), and Glycosyl-inositol-phospho-ceramides (GIPC) were decreased in the mutants, while the contents of Ceramide (Cer) were increased. Detail, the contents of two Cer molecular species, d18:1/22:0 and d18:1/24:0, and two Phyto-Cer molecular species, t18:0/22:0 and t18:0/h22:1 were significantly increased, while the contents of all GluCer and GIPC molecular species were decreased. Consistent with this result, the expression levels of seven genes involved in GluCer and GIPC synthesis were decreased in the mutants. Furthermore, exogenous application of a specific inhibitor of GluCer synthase, PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol), in ovule culture system, significantly inhibited the initiation of cotton fiber cells. In addition, five sterols and four sterol esters were detected in wild-type and mutant ovules. Compared with the wild type, the contents of total sterol were not significantly changed. While the contents of stigmasterol and campesterol were significantly increased, the contents of cholesterol were significantly decreased, and the contents of total sterol esters were significantly increased. In particular, the contents of campesterol esters and stigmasterol esters increased significantly in the two mutants. Consistently, the expression levels of some sterol synthase genes and sterol ester synthase genes were also changed in the two mutants. These results suggested that sphingolipids and sterols might have some roles in the initiation of fiber cells. Our results provided a novel insight into the regulatory mechanism of fiber cell initiation.
... Sphingolipids are major structural components of the plasma membrane, vacuole membrane, and inner membrane and are enriched in the lipid rafts of cell membranes [13][14][15]. Sphingolipids are not only a crucial component of biomembranes but are also an important bioactive molecule that mediates a variety of cell processes, such as programmed cell death [16][17][18][19], pathogen-induced hypersensitivity [18,20,21], the closure of stomatal guard cells regulated by ABA signaling [20][21][22][23], host-pathogen crosstalk [24], low temperature signal transduction [25][26][27], and the regulation of membrane stability [28]. ...
Cotton fiber is a single-celled seed trichrome that arises from the epidermis of the ovule’s outer integument. The fiber cell displays high polar expansion and thickens but not is disrupted by cell division. Therefore, it is an ideal model for studying the growth and development of plant cells. Sphingolipids are important components of membranes and are also active molecules in cells. However, the sphingolipid profile during fiber growth and the differences in sphingolipid metabolism at different developmental stages are still unclear. In this study, we detected that there were 6 classes and 95 molecular species of sphingolipids in cotton fibers by ultrahigh performance liquid chromatography-MS/MS (UHPLC-MS/MS). Among these, the phytoceramides (PhytoCer) contained the most molecular species, and the PhytoCer content was highest, while that of sphingosine-1-phosphate (S1P) was the lowest. The content of PhytoCer, phytoceramides with hydroxylated fatty acyls (PhytoCer-OHFA), phyto-glucosylceramides (Phyto-GluCer), and glycosyl-inositol-phospho-ceramides (GIPC) was higher than that of other classes in fiber cells. With the development of fiber cells, phytosphingosine-1-phosphate (t-S1P) and PhytoCer changed greatly. The sphingolipid molecular species Ceramide (Cer) d18:1/26:1, PhytoCer t18:1/26:0, PhytoCer t18:0/26:0, PhytoCer t18:1/h20:0, PhytoCer t18:1/h26:0, PhytoCer t18:0/h26:0, and GIPC t18:0/h16:0 were significantly enriched in 10-DPA fiber cells while Cer d18:1/20:0, Cer d18:1/22:0, and GIPC t18:0/h18:0 were significantly enriched in 20-DPA fiber cells, indicating that unsaturated PhytoCer containing hydroxylated and saturated very long chain fatty acids (VLCFA) play some role in fiber cell elongation. Consistent with the content analysis results, the related genes involved in long chain base (LCB) hydroxylation and unsaturation as well as VLCFA synthesis and hydroxylation were highly expressed in rapidly elongating fiber cells. Furthermore, the exogenous application of a potent inhibitor of serine palmitoyltransferase, myriocin, severely blocked fiber cell elongation, and the exogenous application of sphingosine antagonized the inhibition of myriocin for fiber elongation. Taking these points together, we concluded that sphingolipids play crucial roles in fiber cell elongation and SCW deposition. This provides a new perspective for further studies on the regulatory mechanism of the growth and development of cotton fiber cells.
... Sphingolipids are considered as the major structural components (~40%) of the plasma membrane but they are also abundant in other endomembranes. Analysis of genes involved in sphingolipid metabolism showed that they have an essential role in optimal plant growth and development, as well as in stress responses such as during cold stress [20][21][22][23][24][25][26]. Plant sphingolipids can be divided into four major classes: free long-chain bases [LCBs; including sphingosine (Sph), dihydro-sphingosine (dh-Sph) and Phyto-shingosine (Phyto-Sph)], ceramides, glycosylceramides (GlcCers) and inositol-phosphoceramides (IPCs) [27,28]. ...
Fumonisin B1 (FB1) is the most harmful mycotoxin which prevails in several crops and affects the growth and yield as well. Hence, keeping the alarming consequences of FB1 under consideration, there is still a need to seek other more reliable approaches and scientific knowledge for FB1-induced cell death and a comprehensive understanding of the mechanisms of plant defence strategies. FB1-induced disturbance in sphingolipid metabolism initiates programmed cell death (PCD) through various modes such as the elevated generation of reactive oxygen species, lipid peroxidation, cytochrome c release from the mitochondria, and activation of specific proteases and nucleases causing DNA fragmentation. There is a close interaction between sphingolipids and defence phytohormones in response to FB1 exposure regulating PCD and defence. In this review, the model plant Arabidopsis and various crops have been presented with different levels of susceptibility and resistivity exposed to various concentration of FB1. In addition to this, regulation of PCD and defence mechanisms have been also demonstrated at the physiological, biochemical and molecular levels to help the understanding of the role and function of FB1-inducible molecules and genes and their expressions in plants against pathogen attacks which could provide molecular and biochemical markers for the detection of toxin exposure.
... In these allocations, complex sphingolipids function as structural components of the membrane matrix. However, small amounts of complex sphingolipid precursors, which are transiently produced, have been involved as second messengers in a variety of physiological plant processes (Worrall et al., 2008;Peer et al., 2010;Guo et al., 2012;Markham et al., 2011;Saucedo-Garcia et al., 2011;Chen et al., 2012;Dutilleul et al., 2012;Luttgeharm et al., 2016). ...
Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. 'Picual'). Here, we investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate that the concentration of LCB [t18:1(8Z), t18:1(8E), t18:0, d18:2(4E/8Z), d18:2(4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.
... Sphingolipids are widely found in eukaryotes and in a few prokaryotic membranes [9]. Having complex and diverse structures [10] not only they are the main structural components of membranes, but they are also important bioactive molecules, and are involved in various signal transduction pathways, including programmed cell death (PCD) [11][12][13], hypersensitivity induced by pathogens [11,14,15], ABA-dependent guard cell closure [16][17][18], host-pathogen interaction [19] and low temperature signal transduction [20,21]. ...
Sphingolipids are essential biomolecules and membrane components, but their regulatory role in cotton fiber development is poorly understood. Here, we found that fumonisin B1 (FB1)—a sphingolipid synthesis inhibitor—could block fiber elongation severely. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we detected 95 sphingolipids that were altered by FB1 treatment; of these, 29 (mainly simple sphingolipids) were significantly increased, while 33 (mostly complex sphingolipids) were significantly decreased. A quantitative analysis of the global proteome, using an integrated quantitative approach with tandem mass tag (TMT) labeling and LC-MS/MS, indicated the upregulation of 633 and the downregulation of 672 proteins after FB1 treatment. Most differentially expressed proteins (DEPs) were involved in processes related to phenylpropanoid and flavonoid biosynthesis. In addition, up to 20 peroxidases (POD) were found to be upregulated, and POD activity was also increased by the inhibitor. To our knowledge, this is the first report on the effects of FB1 treatment on cotton fiber and ovule sphingolipidomics and proteomics. Our findings provide target metabolites and biological pathways for cotton fiber improvement.
... However, at present only sphk1 and sphk2 single mutants have been characterized, both of which have normal vegetative growth. 48,49 Thus, additional genetic studies are needed to test the relative importance of each subsequent stage in GlcCer catabolism. ...
The Aminophospholipid ATPase (ALA) family of plant lipid flippases is involved in the selective transport of lipids across membrane bilayers. Recently, we demonstrated that double mutants lacking both ALA4 and −5 are severely dwarfed. Dwarfism in ala4/5 mutants was accompanied by cellular elongation defects and various lipidomic perturbations, including a 1.4-fold increase in the accumulation of glucosylceramides (GlcCers) relative to total sphingolipid content. Here, we present a potential model for flippase-facilitated GlcCer catabolism in plants, where a combination of ALA flippases transport GlcCers to cytosolic membrane surfaces where they are degraded by Glucosylceramidases (GCDs). GCDs remove the glucose headgroup from GlcCers to produce a ceramide (Cer) backbone, which can be further degraded to sphingoid bases (Sphs, e.g, phytosphingosine) and fatty acids (FAs). In the absence of GlcCer-transporting flippases, GlcCers are proposed to accumulate on extracytoplasmic (i.e., apoplastic) or lumenal membrane surfaces. As GlcCers are potential precursors for Sph production, impaired GlcCer catabolism might also result in the decreased production of the secondary messenger Sph-1-phosphate (Sph-1-P, e.g., phytosphingosine-1-P), a regulator of cell turgor. Importantly, we postulate that either GlcCer accumulation or reduced Sph-1-P signaling might contribute to the growth reductions observed in ala4/5 mutants. Similar catabolic pathways have been proposed for humans and yeast, suggesting flippase-facilitated GlcCer catabolism is conserved across eukaryotes.
... 这些结果进一步支持 S1P 通过诱 导保卫细胞胞质碱化参与黑暗诱导气孔关闭的结论. 谢物 S1P 参与调节许多细胞反应, 包括增殖、存活、 骨架组成与机能、迁移、分化、神经突起退缩和缠 绕 [55,56] . 目前, 已经知道 S1P 和鞘氨醇激酶(SphK, 一种 LCBK)参与 ABA 调控的气孔运动 [5,14,15] . 然而, 拟南芥 ∆4-去饱和酶基因插入突变体既不含 S1P, 也未 干扰 ABA 诱导气孔关闭, 显示 S1P 不参与 ABA 诱导 气孔关闭 [8] . ...
... SPHK1 is an enzyme that phosphorylates d18:1 and t18:0. Stomata of SPHK1-OE and of Atspp1 mutant (which accumulates d18:1-P) displayed a higher sensitivity than WT to ABA (Worrall et al., 2008;Nakagawa et al., 2012). Therefore, LCB-P content regulated by LCB kinases and phosphatases plays a key role in the ABA signalling pathway. ...
Plants exist in an environment of changing abiotic and biotic stresses. They have developed a complex set of strategies to respond to these stresses and over recent years it has become clear that sphingolipids are a key player in these responses. Sphingolipids are not universally present in all three domains of life. Many bacteria and archaea do not produce sphingolipids but they are ubiquitous in eukaryotes and have been intensively studied in yeast and mammals. During the last decade there has been a steadily increasing interest in plant sphingolipids. Plant sphingolipids exhibit structural differences when compared with their mammalian counterparts and it is now clear that they perform some unique functions. Sphingolipids are recognised as critical components of the plant plasma membrane and endomembrane system. Besides being important structural elements of plant membranes, their particular structure contributes to the fluidity and biophysical order. Sphingolipids are also involved in multiple cellular and regulatory processes including vesicle trafficking, plant development and defence. This review will focus on our current knowledge as to the function of sphingolipids during plant stress responses, not only as structural components of biological membranes, but also as signalling mediators.
... The protein product of this gene phosphorylates sphingosine (to S1P) and phytosphingosine (to phytoS1P) in plants [33], and increased levels of S1P and abscisic acid dependent stomatal closure have been reported in response to drought [34]. Knock-down of sphingosine kinase expression significantly decreased sensitivity to abscisic acid induced stomatal closure compared to Col0 [35], indicating that At2++ with reduced sphingosine kinase may be more sensitive to drought. One gene showed a negative correlation under GO term GO:009819 (drought recovery), a serine/theronine kinase (AT1G78290), a member of the SNF1-related protein kinase (SnRK2) family whose activity is activated by osmotic stress and dehydration [36]. ...
This research was undertaken to investigate the global role of the plant inositol phosphorylceramide synthase (IPCS), a non-mammalian enzyme previously shown to be associated with the pathogen response. RNA-Seq analyses demonstrated that over-expression of inositol phosphorylceramide synthase isoforms AtIPCS1, 2 or 3 in Arabidopsis thaliana resulted in the down-regulation of genes involved in plant response to pathogens. In addition, genes associated with the abiotic stress response to salinity, cold and drought were found to be similarly down-regulated. Detailed analyses of transgenic lines over-expressing AtIPCS1-3 at various levels revealed that the degree of down-regulation is specifically correlated with the level of IPCS expression. Singular enrichment analysis of these down-regulated genes showed that AtIPCS1-3 expression affects biological signaling pathways involved in plant response to biotic and abiotic stress. The up-regulation of genes involved in photosynthesis and lipid localization was also observed in the over-expressing lines.
... ABA-promoted stomatal response, the enzyme also inhibited seed germination. Long-chain base phosphate lyase (LCBPL) and long-chain base phosphate phosphatases (LCBPPs) also have been shown to participate in ABA and drought responses reported in Arabidopsis RNAi lines (Worrall et al., 2008). In subsequent studies, the functional characterization of the Arabidopsis LCB ∆4 desaturase mutants revealed that these mutants responded in a similar manner as the wild-type to water deficits and stomatal closure during ABA treatment (Michaelson et al., 2009) In another study conducted by Nakagawa et al. (2012), an enhanced level of dihydrosphingosine-1-phosphates was observed in Arabidopsis lcbpp mutants compared to wild-type (Nakagawa et al., 2012). ...
Roles of sphingolipids in biotic and abiotic stresses
... In contrast, two transcripts coding for a sphingosine kinase and a GNS1/SUR4 Both proteins are known to be involved in stress signalling (Guo & Wang, 2012;Quist et al., 2009). The Arabidopsis orthologue of the sphingosine kinase, which was also increased under heat plate conditions, was reported to control abscisic acid (ABA) -induced stomatal closure (Worrall et al., 2008), and AtGNS1/SUR4 regulates very long chain fatty acid composition, which are essential precursors of sphingolipids (Quist et al., 2009). This suggests that lipid signalling was activated in tubers at elevated temperatures which may influence the photosynthetic performance through an indirect effect on the stomatal aperture. ...
Potato is an important staple food with increasing popularity worldwide. Elevated temperatures significantly impair tuber yield and quality. Breeding heat‐tolerant cultivars is therefore an urgent need to ensure sustainable potato production in the future. An integrated approach combining physiology, biochemistry and molecular biology was undertaken to contribute to a better understanding of heat effects on source‐ (leaves) and sink‐organs (tubers) in a heat‐susceptible cultivar. An experimental set up was designed allowing tissue‐specific heat application. Elevated day and night (29°/27°C) temperatures impaired photosynthesis and assimilate production. Biomass allocation shifted away from tubers towards leaves indicating reduced sink strength of developing tubers. Reduced sink strength of tubers was paralleled by decreased sucrose synthase activity and expression under elevated temperatures. Heat‐mediated inhibition of tuber growth coincided with a decreased expression of the phloem‐mobile tuberisation signal SP6A in leaves. SP6A expression and photosynthesis were also affected, when only the belowground space was heated, while leaves were kept under control conditions. By contrast, the negative effects on tuber metabolism were attenuated, when only the shoot was subjected to elevated temperatures. This, together with transcriptional changes discussed, indicated a bidirectional communication between leaves and tubers to adjust the source capacity and/or sink strength to environmental conditions.
... Two LCB-1-phosphate phosphatases, encoded by the LCB3 and YSR3 genes and localized in the ER have been identified in yeast [66,67]. In Arabidopsis, three LCB kinases SPHK1 (At5g23450), SPHK2 (At2g46090), and AtLCBK1 (At5g23450) [68][69][70] and a LCB-1-phosphate phosphatase, AtSPP1 (At3g58490) [71] have been identified. In mammalian cells, formation of sphingosine-1-phosphate from sphingosine and DHS-1-P from DHS is catalyzed by two sphingosine kinases (SK1 and SK2), which are cytosolic enzymes that associate peripherally with the plasma membrane and can also move into the nucleus [26,72,73]. ...
This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature, with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as they can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants.
... Plant sphingolipids mostly consist of glycosylated inositol phosphoryl ceramides or glucosylceramides (Markham et al. 2006;Berkey et al. 2012). These derivatives are involved in drought, cold, dehydration, heat, and ABA responses (Ng et al. 2001;Worrall et al. 2008;Guillas et al. 2013;Alden et al. 2011). PA binding activates sphingolipid formation stimulating stomatal closure in an ABA-dependent manner (Guo et al. 2011). ...
Plants differ from animals by lacking the ability to escape from their environmental conditions. Plants adapt to the seasonal as well as nonseasonal perturbations by means of stress-responsive genes. Manipulation of such genes has been shown to provide abiotic stress tolerance in plants. Since abiotic stress is a polygenic trait, overexpression of single stress-responsive gene would not serve the purpose of getting stress-tolerant plants. So, the focus needs to be shifted towards the “master regulators” which are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery. In plants, there are various second messengers including calcium, ROS, phosphoinositides, cyclic nucleotides, etc., which are known to initiate the downstream signaling cascade leading to response against different, multiple, and simultaneous ambient cues. A better understanding of these elements will allow us to engineer a particular stress-responsive pathway, to achieve better stress-tolerant plants.
... In these allocations, complex sphingolipids function as structural components of the membrane matrix. However, small amounts of complex sphingolipid precursors, which are transiently produced, have been involved as second messengers in a variety of physiological plant processes, such as programmed cell death (Saucedo-García et al., 2011), guardcell closure (Worrall et al., 2008;Guo et al., 2012), cell polarity , and responses to stress such as low temperatures (Chen et al., 2012;Dutilleul et al., 2012) and pathogens (Peer et al., 2010). ...
Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.
... (2018) Ó 2018 The Authors New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com Research New Phytologist opening, but not in closure (Wang et al., 2001), a sphingosine-1-phosphate phosphatase, long-chain base phosphate lyase double mutant (sppasedpl1), which displays WT behavior during ABA-induced closure, but is slightly impaired in the ABA inhibition of stomatal opening response (Worrall et al., 2008), PI-phospholipase C, which is involved in the ABAinhibition of opening, but not closure (Mills et al., 2004), and the observation that some members of the PYR/PYL ABA receptor family involved in stomatal opening inhibition are different from those involved in stomatal closure induction (Yin et al., 2013). The second striking result to emerge from these experiments is that BIG is not involved in ABA-induced reductions in stomatal aperture (Fig. 4c,d). ...
We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2.
This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2insensitive 1 (cis1).
BIG mutants are compromised in elevated CO2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA).
Our results indicate that BIG is a signaling component involved in the elevated CO2‐mediated control of stomatal development. In the control of stomatal aperture by CO2, BIG is only required in elevated CO2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2‐mediated responses.
... In plants, sphingolipids represent more than 10% of total lipid and are present mainly in non-photosynthetic tissues . Although their role is still not well defined, recent studies indicate that they play critical functions, such as structural components of membranes, bioactive molecules involved in signal transduction and metabolic mediators of cellular processes such as programmed cell death (Coursol et al. 2003;Worrall et al. 2008;Chao et al. 2011;Michaelson et al. 2016). Further, they were related to plant responses to stresses as pathogens, hypoxia, cooling and freezing (Steponkus and Lynch 1989;Uemura et al. 1995;Saucedo-García et al. 2011;Xie et al. 2015). ...
AimsNitrogen deficiency is one of the most critical abiotic stresses for maize (Zea mays) cultivation worldwide. For a productive and sustainable scenario, developing genotypes more efficient in nitrogen use is essential. This study aimed to identify single nucleotide polymorphism (SNP) markers associated with nitrogen use efficiency (NUE) traits under field conditions and candidate genes related to these markers by genome-wide association study (GWAS). Methods
Sixty-four tropical maize inbred lines were evaluated in ideal and low nitrogen conditions for total root length (TRL) and low nitrogen tolerance index (LNTI). GWAS was performed using a fixed and random model circulating probability unification method, with marker-based principal components to correct for population stratification. Genotypic values were predicted using mixed model equations. ResultsSeven significant markers were identified. Among the primary biological processes, candidate genes are related to transcription control and regulation, detected to all evaluated traits, and the synthesis of Guanosine Monophosphate Synthetase, enzyme directly involved in the provision and recycling of nitrogen. ConclusionsGWAS analysis revealed genomic regions in tropical maize associated with NUE under field conditions. The main biological process identified as related to these markers/regions evidence cellular processes and functions associated with different process of nitrogen synthesis and recycling.
... The balance between LCBs and LCBPs has been suggested as the rheostat that controls the steady state of sphingolipid metabolism (Olivera and Spiegel, 2001;Stunff et al., 2004). The involvement of Phyto-S1P in Arabidopsis stomatal movement and plant adaptation to various environmental stresses has been well documented (Coursol et al., 2003;Worrall et al., 2008;Nakagawa et al., 2012;Dutilleul et al., 2012;Puli et al., 2016). Drought rapidly increases Phyto-S1P concentration which in turn elevates the guard cells cytosolic free calcium (Ca 2þ ) concentrations ([Ca 2þ ] cyt ) in Commelina communis (Ng et al., 2001). ...
The fungal toxin Fumonisin B1 (FB1) is a strong inducer to trigger plant hypersensitive responses (HR) along with increased long chain bases (LCB) and long chain base phosphates (LCBP) contents, though the regulatory mechanism of FB1 action and how the LCB/LCBP signalling cassette functions during the process is still not fully understood. Here, we report sphingosine kinase 1 (SPHK1) as a key factor in FB1-induced HR by modulating the salicylic acid (SA) pathway and reactive oxygen species (ROS) accumulation in Arabidopsis thaliana. Overexpression of SPHK1 increases the FB1-induced accumulations of ROS and SA. The double mutant that simultaneously overexpresses SPHK1 and suppresses the SPPASE or DPL1, two enzymes are mainly responsible for Phyto-sphingosine-1-phosphate (Phyto-S1P) removal, showed enhanced susceptibility to FB1 killing and FB1-induced SA activation than the plants overexpress SPHK1 alone. Exogenous sphingosine-1-phosphate (S1P) can modulate the transcription of the SA-responsive marker gene PR1 in a concentration-dependent biphasic manner. Suppression of SPHK1 decreases SA production whereas promotes jasmonic acid (JA) biosynthesis in response to FB1 applications. Our findings indicate a role of SPHK1 in modulating FB1-triggered cell death via SA and JA pathway interactions.
Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there...
Cold temperatures often severely restrict the growth, distribution, and productivity of plants. The freezing tolerance of plants from temperate climates can be improved by undergoing periods of cold acclimation. Lipids play an important role in the mechanism of frost or cold tolerance in plants. Lipids are widely distributed in the plant kingdom. They are found in abundance in reproductive tissues (e.g., seeds and fruits) of some higher plants where they form important reserve food material such cotyledon of sunflower, rape, peanuts, and almond; endosperm of castor beans, coconut plants, etc.; and mesocarp of avocado pears. Plant membrane lipids have the tendency to change from gel to liquid crystalline phase in response to low-temperature stress. This process is due to the increased level of lipid desaturation. The responsible components of this process are, among others, the fatty acid desaturases. Controlling the activity of these enzymes affects the amount of polyunsaturated fatty acids on the glycerol backbone and eventually controls the plant sensitivity to low-temperature stress. These metabolic processes trigger a series of changes at the transcriptional level, causing differential expression in genes. Numerous approaches toward this process from chemical to the advanced mass spectrometry were taken during the past decades. Metabolomics and transcriptomics seem to be the keys toward describing these complex mechanisms and providing the necessary understanding of lipid metabolic response to low-temperature stress.KeywordsRole of lipids in plantEffect of lipid metabolism to cold stressEffects of cold stress on different plantsRole of lipid signaling in stress responseRole of free ftty acid in signalingRole of lipid in mitigating cold stressMechanism of temperature adaptationCold stress symptomsSensing and strategiesRole of triglycerides to cold responseGlycerolipid response to freezing temperatureLipid hydrolysis under low-temperature stress
Sphingolipids are major structural components of membrane and active signaling molecules and play an important role in plant developmental processes and stress responses. As land salinization has increased globally, salinity has compromised the growth and productivity of crops such as cotton. Understanding the mechanisms of plant adaptation to salt stress is essential for breeding salt-tolerant crops. In this study, we explored the comprehensive metabolic profile of sphingolipids in cotton root under salt stress using lipidomics. 118 sphingolipid molecular species were identified, of which PhytoSph, PhytoCer, PhytoCer-OHFA, IPC, and GIPC were relatively high in content, and PhytoSph, PhytoCer, PhytoCer-OHFA, Phyto-GluCer, and IPC showed significant changes after salt stress, especially inositol phosphatidyl ceramide (IPC), which was significantly upregulated after salt treatment. Subsequently, we identified the genes encoding IPC synthase (IPCS), and ectopic expression of GhIPCS1 enhanced salt sensitivity in Arabidopsis, which might result from the disruption on the balance between various sphingolipid classes and/or molecular species. Overall, this study reveals key lipids and genes response to salt stress in cotton and provides a theoretical basis for the use of genetic engineering to improve cotton stress resistance.
Sphingolipids and their intermediates play multiple roles in biological processes. The sphingoid long-chain base component of sphingolipids has emerged as a participant in the regulation of plant biotic and abiotic stress responses. The phytohormone abscisic acid (ABA) regulates many stress responses in plants for environmental adaptation. However, the relationship between the sphingoid bases and ABA is undetermined. In this study, mhp1-1 (the yeast Mpo1 homolog in plants) was isolated through a sodium chloride (NaCl)-sensitivity screen of Arabidopsis transfer DNA (T-DNA) insertion mutants. mhp1-1 was hypersensitivity to salt/osmotic stress and ABA. MHP1 encodes a protein with a domain of unknown function 962 (DUF962). Endoplasmic reticulum-localized MHP1 was found to interact with ABI1. MHP1, a homolog of yeast dioxygenase Mpo1, rescued the growth arrest of mpo1Δ cells caused by ER stress, suggesting functional homology of MHP1 to Mpo1. Overall, MHP1 plays important roles in response to ABA.
Sphingolipids are fundamental lipids involved in various cellular, developmental and stress-response processes. As such, they orchestrate not only vital molecular mechanisms of living cells but also act in diseases, thus qualifying as potential pharmaceutical targets. Sphingolipids are universal to eukaryotes and are also present in some prokaryotes. Some sphingolipid structures are conserved between animals, plants and fungi, whereas others are found only in plants and fungi. In plants, the structural diversity of sphingolipids, as well as their downstream effectors and molecular and cellular mechanisms of action, are of tremendous interest to both basic and applied researchers, as about half of all small molecules in clinical use originate from plants. Here, we review recent advances towards a better understanding of the biosynthesis of sphingolipids, the diversity in their structures as well as their functional roles in membrane architecture, cellular processes such as membrane trafficking and cell polarity, and cell responses to environmental or developmental signals.
Plants employ multiple mechanisms to cope with a constantly changing and challenging environment including utilizing the ubiquitin proteasome system (UPS) to alter their proteome to assist in initiating, modulating and terminating responses to stress. We previously reported that the ubiquitin ligase XBAT35.2 mediates the proteasome‐dependent degradation of Accelerated Cell Death 11 (ACD11) to promote pathogen defense. Here, we demonstrate roles for XBAT35.2 and ACD11 in abiotic stress tolerance. Similar to pathogen infection, abiotic stress stabilizes XBAT35.2 and protein abundance rose consistently with increasing concentrations of abscisic acid (ABA) and salt. Surprisingly, exposure to ABA and salt increased ACD11 stability and overexpression of ACD11 improves survival of salt and drought stress, suggesting a role for ACD11 in promoting tolerance. However, prolonged exposure to high concentrations of ABA/salt resulted in ubiquitination and proteasome‐dependent degradation of ACD11. The stress‐induced turnover of ACD11 requires XBAT35.2 as degradation is slowed in the absence of the E3 ubiquitin ligase. Consistent with XBAT35.2 mediating the proteasome‐dependent degradation of ACD11, loss of E3 ubiquitin ligase function enhances tolerance of salt and drought stress, while overexpression increases sensitivity. A model is presented where upon abiotic stress, ACD11 abundance increases to promote tolerance. Meanwhile, XBAT35.2 accumulates and in turn promotes the degradation of ACD11 to attenuate the stress response. The results characterize XBAT35.2 as an E3 ubiquitin ligase with opposing roles in abiotic and biotic stress. Supporting Information
The plasma membranes encapsulated in the plasmodesmata (PDs) with symplasmic nano-channels contain abundant lipid rafts, which are enriched by sphingolipids and sterols. The attenuation of sterol compositions has demonstrated the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly affecting in the callose enhancement. The presence of callose at PD is tightly attributed to the callose metabolic enzymes, callose synthases (CalSs) and β-1,3-glucanases (BGs) in regulating callose accumulation and callose degradation, respectively. Sphingolipids have been implicated in signaling and membrane protein trafficking, however the underlying processes linking sphingolipid compositions to the control of symplasmic apertures remain unknown. A wide variety of sphingolipids in plants prompts us to investigate which sphingolipid molecules are important in regulating symplasmic apertures. Here, we demonstrate that perturbations of sphingolipid metabolism by introducing several potential sphingolipid (SL) pathway inhibitors and genetically modifying SL contents from two independent SL pathway mutants are able to modulate callose deposition to control symplasmic connectivity. Our data from pharmacological and genetic approaches show that the alteration in glucosylhydroxyceramides (GlcHCers) particularly disturb the secretory machinery for GPI-anchored PdBG2 protein, resulting in an over accumulated callose. Moreover, our results reveal that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting to specific destination of GPI-anchored PD proteins incorporated with GlcHCers contents.
A new review covering up to 2018
Sphingolipids are essential molecules that, despite their long history, are still stimulating interest today. The reasons for this are that, as well as playing structural roles within cell membranes, they have also been shown to perform a myriad of cell signalling functions vital to the correct function of eukaryotic and prokaryotic organisms. Indeed, sphingolipid disregulation that alters the tightly-controlled balance of these key lipids has been closely linked to a number of diseases such as diabetes, asthma and various neuropathologies. Sphingolipid biogenesis, metabolism and regulation is mediated by a large number of enzymes, proteins and second messengers. There appears to be a core pathway common to all sphingolipid-producing organisms but recent studies have begun to dissect out important, species-specific differences. Many of these have only recently been discovered and in most cases the molecular and biochemical details are only beginning to emerge. Where there is a direct link from classic biochemistry to clinical symptoms, a number a drug companies have undertaken a medicinal chemistry campaign to try to deliver a therapeutic intervention to alleviate a number of diseases. Where appropriate, we highlight targets where natural products have been exploited as useful tools. Taking all these aspects into account this review covers the structural, mechanistic and regulatory features of sphingolipid biosynthetic and metabolic enzymes.
Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network’s domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 10²⁴ system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca²⁺ (Ca²⁺c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca²⁺c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.
We show that guard cells from Arabidopsis thaliana plants carrying the abscisic acid-insensitive mutations abi1 and abi2 fail to respond to CO2 and extracellular calcium. This demonstrates that the signal transduction pathways for all three stimuli converge on, or close to, the ABI1 and ABI2 gene products.
Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.
A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.
Stomata, the small pores on the surfaces of leaves and stalks, regulate the flow of gases in and out of leaves and thus plants as a whole. They adapt to local and global changes on all timescales from minutes to millennia. Recent data from diverse fields are establishing their central importance to plant physiology, evolution and global ecology. Stomatal morphology, distribution and behaviour respond to a spectrum of signals, from intracellular signalling to global climatic change. Such concerted adaptation results from a web of control systems, reminiscent of a 'scale-free' network, whose untangling requires integrated approaches beyond those currently used.
Sphingolipids are a major component of membrane lipids and their metabolite sphingosine-1-phosphate (S1P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S1P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Delta4-unsaturated long-chain sphingoid bases sphingosine (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydrosphingosine and phytosphingosine (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK1 efficiently phosphorylated Sph, dihydrosphingosine, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-100 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phytosphingosine-1-phosphate, similar to S1P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein alpha-subunit gene, GPA1.
Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis
of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization
and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural
determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1)
and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred
the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1
is mediated largely by specific lipid-protein interactions.
Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.
Terrestrial plants lose water primarily through stomata, pores on the leaves. The hormone abscisic acid (ABA) decreases water loss by regulating opening and closing of stomata. Here, we show that phospholipase Dalpha1 (PLDalpha1) mediates the ABA effects on stomata through interaction with a protein phosphatase 2C (PP2C) and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis. PLDalpha1-produced phosphatidic acid (PA) binds to the ABI1 PP2C to signal ABA-promoted stomatal closure, whereas PLDalpha1 and PA interact with the Galpha subunit of heterotrimeric G protein to mediate ABA inhibition of stomatal opening. The results reveal a bifurcating signaling pathway that regulates plant water loss.
In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA1 (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.
The sphingoid long chain bases (LCBs) and their phosphorylated derivatives (LCB-Ps) are important signaling molecules in eukaryotic organisms. The cellular levels of LCB-Ps are tightly controlled by the coordinated action of the LCB kinase activity responsible for their synthesis and the LCB-P phosphatase and lyase activities responsible for their catabolism. Although recent studies have implicated LCB-Ps as regulatory molecules in plants, in comparison with yeast and mammals, much less is known about their metabolism and function in plants. To investigate the functions of LCB-Ps in plants, we have undertaken the identification and characterization of Arabidopsis genes that encode the enzymes of LCB-P metabolism. In this study the Arabidopsis At1g27980 gene was shown to encode the only detectable LCB-P lyase activity in Arabidopsis. The LCB-P lyase activity was characterized, and mutant plant lines lacking the lyase were generated and analyzed. Whereas in other organisms loss of LCB-P lyase activity is associated with accumulation of high levels of LCB/LCB-Ps and developmental abnormalities, the sphingolipid profiles of the mutant plants were remarkably similar to those of wild-type plants, and no developmental abnormalities were observed. Thus, these studies indicate that the lyase plays a minor role in maintenance of sphingolipid metabolism during normal plant development and growth. However, a clear role for the lyase was revealed upon perturbation of sphingolipid synthesis by treatment with the inhibitor of ceramide synthase, fumonisin B(1).
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Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM.
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.
TheAgrobacteriumvacuum infiltration method has made it possible to transformArabidopsis thalianawithout plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containingAgrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, andAgrobacteriumcould be applied to plants at a range of cell densities. Repeated application ofAgrobacteriumimproved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation ofArabidopsisfor efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Abscisic acid (ABA) insensitive mutants of Arabidopsis thaliana (L.) Heynh. were isolated by selecting plants which grew well on a medium containing 10 μM ABA. From the progeny of approximately 3500 mutagen-treated seeds, five mutants of at least three different loci were isolated. Three mutants were characterized, moreover, by a reduced seed dormancy and by symptoms of withering, indicating disturbed water relations and, therefore, resembled phenotypically the ABA-deficient mutants we described earlier in this species. Two mutants showed in addition only a reduction of seed dormancy. Compared to wild type, all mutants showed similar or increased levels of endogenous ABA in developing seeds and fruits (siliquae). The role of the different genes involved is discussed in relation to the mechanism of ABA action.
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
The phosphorylation of long chain sphingoid bases on the primary alcohol group occurs in cells by the action of sphingosine
kinase (1–3). Sphingosine kinase is present in the cytosolic fraction of most cells (4–7) and in the membrane fraction of certain tissues and organisms (8,9). The reaction product, sphingosine-1-phosphate (SPP), was considered for more than 20 yr to be merely an intermediate in
the catabolism of long-chain sphingoid bases to palmitaldehyde and phosphoethanolamine (3,10). Studies in our lab stimulated new interest in the potential roles of SPP as a second messenger. Initially, we found that
exogenous SPP initiated cell division of quiescent Swiss 3T3 fibroblasts (11) and induced inositol trisphosphate-independent release of calcium from intracellular stores (11,12). SPP also has been shown to affect several signal transduction pathways including phospholipase D activation (13), stimulation of the Raf/MEK/ERK signaling pathway (14,15), and inhibition of ceramide-induced activation of stress-activated protein kinase (SAPK/JNK) that leads to apoptotic responses
(15). Additional effects of SPP include stimulation of tyrosine phosphorylation of focal adhesion kinase (FAK) and the cytoskeleton-associated
protein paxillin (16). These tyrosine phosphorylations are mediated through the activation of the small G protein rho, which also mediates stress
fiber formation induced by SPP (16).
Sphingolipid long-chain base (LCB) kinase catalyses the phosphorylation of sphingolipid LCB to form LCB 1-phosphate. Based on sequence identity to a murine sphingosine kinase (murine SPHK1a), we isolated and characterized a LCB kinase-like cDNA in Arabidopsis thaliana. The deduced amino acid sequence of the homologous cDNA shows several regions that are highly conserved in LCB kinases from mouse, yeast, human and Caenorhabditis elegans. These regions are not similar to those of other known kinase families. For a functional identification, the homologous cDNA from A. thaliana was expressed in Escherichia coli, and LCB kinase activity was measured. The recombinant AtLcbk1 protein was found to utilize ATP and sphinganine. These results indicate the first identification of a gene coding for a LCB kinase in plants.
Stomata form pores on leaf surfaces that regulate the uptake of CO2 for photosynthesis and the loss of water vapour during transpiration. An increase in the cytosolic concentration of free calcium ions ([Ca2+]cyt) is a common intermediate in many of the pathways leading to either opening or closure of the stomatal pore. This observation has prompted investigations into how specificity is controlled in calcium-based signalling systems in plants. One possible explanation is that each stimulus generates a unique increase in [Ca2+]cyt, or 'calcium signature', that dictates the outcome of the final response. It has been suggested that the key to generating a calcium signature, and hence to understanding how specificity is controlled, is the ability to access differentially the cellular machinery controlling calcium influx and release from internal stores. Here we report that sphingosine-1-phosphate is a new calcium-mobilizing molecule in plants. We show that after drought treatment sphingosine-1-phosphate levels increase, and we present evidence that this molecule is involved in the signal-transduction pathway linking the perception of abscisic acid to reductions in guard cell turgor.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-1 (EDG-1) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S1P are low and tightly regulated in a spatial-temporal manner not only by sphingosine kinase (SPHK) but also by degradation catalyzed by S1P lyase, specific S1P phosphohydrolases, and by general lipid phosphate phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S1P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S1P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S1P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S1P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing sphingosine and ceramide, bioactive sphingolipids that often have opposing biological actions to S1P. By regulating the cellular ratio between ceramide/sphingosine and S1P, S1P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S1P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S1P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S1P and how turnover of S1P can regulate sphingolipid metabolites signaling.
The evolutionarily conserved actions of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), in yeast, plants and mammals have shown that it has important functions. In higher eukaryotes, S1P is the ligand for a family of five G-protein-coupled receptors. These S1P receptors are differentially expressed, coupled to various G proteins, and regulate angiogenesis, vascular maturation, cardiac development and immunity, and are important for directed cell movement.
The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.
Biologically active sphingolipids have key roles in the regulation of several fundamental biological processes that are integral to cancer pathogenesis. Recent significant progress in understanding biologically active sphingolipid synthesis, specifically within ceramide and sphingosine-1-phosphate (S1P)-mediated pathways, has identified crucial roles for these molecules both in cancer development and progression. Ceramide — a central molecule in sphingolipid metabolism — in effect functions as a tumour-suppressor lipid, inducing antiproliferative and apoptotic responses in various cancer cells. Conversely, S1P induces responses that, on aggregate, render S1P a tumour-promoting lipid. These discoveries are paving the way for the advancement of anticancer therapies.
Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177-->Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1-->108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.
Sphingoid long-chain base (LCB) kinase catalyzes the phosphorylation of LCBs to form LCB 1-phosphates. Based on sequence identity to murine sphingosine kinase (mSPHK1), we cloned and characterized the first plant LCB kinase gene from Arabidopsis (AtLCBK1). Using recombinant AtLCBK1 protein from Escherichia coli cells, we confirmed that the enzyme specifically phosphorylated D-erythro-dihydrosphingosine (DHS), but not N-acetyl-DHS or D-threo-DHS. AtLCBK1 also phosphorylated D-erythro-sphingosine, trans-4, trans-8-sphingadienine and phytosphingosine. We found that AtLCBK1 mRNA is highly expressed in flowers. AtLCBK1 transcripts were slightly increased by low humidity or abscisic acid treatments, suggesting that AtLCBK1 is constitutively expressed under these treatments.
New plant genes are being discovered at a rapid pace. Yet, in most cases, their precise function remains elusive. The recent advent of recombinational cloning techniques has significantly improved our ability to investigate gene functions systematically. For example, proteins fused with diverse fluorescent tags can be expressed at will using versatile cloning cassettes. In addition, novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering.
The phosphorylated sphingolipid metabolites sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) have emerged as potent bioactive agents. Recent studies have begun to define new biological functions for these lipids. Generated by sphingosine kinases and ceramide kinase, they control numerous aspects of cell physiology, including cell survival and mammalian inflammatory responses. Interestingly, S1P is involved in cyclooxygenase-2 induction and C1P is required for the activation and translocation of cPLA2. This suggests that these two sphingolipid metabolites may act in concert to regulate production of eicosanoids, important inflammatory mediators. Whereas S1P functions mainly via G-protein-coupled receptors, C1P appears to bind directly to targets such as cPLA2 and protein phosphatase 1/2A. S1P probably also has intracellular targets, and in plants it appears to directly regulate the G protein alpha subunit GPA1.
The balance between the bioactive sphingolipid ceramide and its phosphorylated derivative has been proposed to modulate the amount of programmed cell death (PCD) in eukaryotes. We characterized the first ceramide kinase (CERK) mutant in any organism. The Arabidopsis CERK mutant, called accelerated cell death 5, accumulates CERK substrates and shows enhanced disease symptoms during pathogen attack and apoptotic-like cell death dependent on defense signaling late in development. ACD5 protein shows high specificity for ceramides in vitro. Strikingly, C2 ceramide induces, whereas its phosphorylated derivative partially blocks, plant PCD, supporting a role for ceramide phosphorylation in modulating cell death in plants.
The role of stomata in sensing and driving environmental change
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Assay and product analysis
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Characterization of sphingolipid long-chain base kinase in Arabidopsis thaliana
- Nishiura
Sphingosine-1-phosphate: an enigmatic signalling lipid
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The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana
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