Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth,Sphingolipid Metabolism,Programmed Cell Death,and Mycotoxin Resistance

Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B₁ (FB₁). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB₁, whereas LOH1-overexpressing plants showed no increase in FB₁ resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB₁. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance for improved plant performance.Ceramides are central intermediates in sphingolipid biosynthesis and mediators of programmed cell death (PCD) in plants (Dunn et al., 2004; Saucedo-García et al., 2011; Ternes et al., 2011a). Ceramides are synthesized by ceramide synthase (or sphingosine N-acyltransferase; EC 2.3.1.24), which catalyzes the formation of an amide linkage between a sphingoid long-chain base (LCB) and a fatty acid using LCB and fatty acyl-CoA substrates (Mullen et al., 2012). The LCB substrate can have two or three hydroxyl groups that are referred to as dihydroxy or trihydroxy LCBs, respectively (Chen et al., 2010). The fatty acyl-CoA substrates typically have chain lengths of C16 or C22 to C26 (Dunn et al., 2004). The latter are referred to as very-long-chain fatty acids (VLCFAs). The ceramide product of ceramide synthase is used primarily as a substrate for the synthesis of either of the two major glycosphingolipids found in plants: glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC; Chen et al., 2010). These glycosphingolipids are major structural components of the plasma membrane and other endomembranes of plant cells (Verhoek et al., 1983; Sperling et al., 2005). In this role, they contribute to membrane physical properties that are important for the ability of plant cells to adjust to environmental extremes and to Golgi-mediated protein trafficking of proteins, including cell wall metabolic enzymes and auxin transporters that underlie plant growth (Borner et al., 2005; Markham et al., 2011; Mortimer et al., 2013; Yang et al., 2013). Alternatively, ceramides can be converted to ceramide-1-phosphates by ceramide kinase activity (Liang et al., 2003). The interchange of ceramides between their free and phosphorylated forms has been linked to the regulation of PCD and PCD-associated resistance to pathogens via the hypersensitive response (HR; Liang et al., 2003; Bi et al., 2014; Simanshu et al., 2014).The Arabidopsis (Arabidopsis thaliana) genome contains three ceramide synthase genes denoted LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540), LOH2 (At3g19260), and LOH3 (At1g13580; Markham et al., 2011; Ternes et al., 2011a). These studies suggest that LOH1 and LOH3 polypeptides are structurally related and catalyze primarily the amidation reaction of trihydroxy LCBs and CoA esters of VLCFAs. The LOH2 polypeptide is more distantly related to LOH1 and LOH3 and catalyzes primarily the condensation of dihydroxy LCBs and C16 fatty acyl-CoAs (Chen et al., 2008; Markham et al., 2011; Ternes et al., 2011a). The ceramide products of LOH1 and LOH3 are most prevalent in GIPC, whereas the ceramide products of LOH2 are more enriched in GlcCer (Markham and Jaworski, 2007; Chen et al., 2008; Ternes et al., 2011b). Similar to plants, the six ceramide synthase isoforms found in humans and mice have distinct specificities for their LCB and acyl-CoA substrates, and these specificities contribute to the formation of complex sphingolipids with differing structures and functions (Venkataraman et al., 2002; Riebeling et al., 2003; Mizutani et al., 2005, 2006; Laviad et al., 2008).In Arabidopsis, LOH1 and LOH3 are partially redundant, but the combined activities of the corresponding polypeptides are essential for plant cell viability, as null double mutants of these genes are lethal (Markham et al., 2011). In contrast, mutants of LOH2 are viable and display no apparent growth phenotype, which brings into question the role of LOH2 ceramide synthase in plant performance (Markham et al., 2011; Ternes et al., 2011a). Overall, these observations indicate that sphingolipids with LOH1-/LOH3-derived trihydroxy LCBs and VLCFA ceramides are essential, but LOH2-derived dihydroxy LCBs and C16 fatty acid ceramides are not required by plant cells. Related to this, LCB C-4 hydroxylase mutants that are deficient in trihydroxy LCBs accumulate elevated amounts of sphingolipids with dihydroxy LCB- and C16 fatty acid-containing ceramides via LOH2 activity (Chen et al., 2008). These mutants are severely impaired in growth and do not transition from vegetative to reproductive growth (Chen et al., 2008).Ceramide synthases are known targets for competitive inhibition by sphingosine analog mycotoxins, including fumonisin B₁ (FB₁) and AAL toxin, produced by pathogenic fungi such as various Fusarium spp. and Alternaria alternata f. sp. lycopersici (Abbas et al., 1994). Inhibition of ceramide synthase results in the accumulation of LCBs that are believed to trigger PCD and result in cytotoxicity (Abbas et al., 1994). In studies of LOH mutants, treatment of Arabidopsis seedlings with FB₁ resulted in not only increases in LCBs but also increases in C16 fatty acid-containing sphingolipids and decreases in VLCFA-containing sphingolipids (Markham et al., 2011; Ternes et al., 2011a). The interpretation of this observation was that FB₁ preferentially inhibits LOH1 and LOH3 ceramide synthases but inhibits LOH2 ceramide synthase to a lesser extent (Markham et al., 2011; Ternes et al., 2011a).Given the findings from Arabidopsis mutants that LOH1 and LOH3 ceramide synthases have distinct substrate specificities and sensitivity to FB₁ relative to LOH2, we hypothesized that the overexpression of each of these ceramide synthases would lead to the production of different sphingolipid compositions as well as different growth phenotypes. This report details experiments designed to test this hypothesis. Among the results presented is a large divergence in the effects of the overexpression of LOH1 and LOH3 versus LOH2 on the growth of Arabidopsis. LOH2 overexpression was also shown to result in sphingolipid compositional, growth, and physiological phenotypes that closely mimic those observed previously in LCB C-4 hydroxylase mutants (Chen et al., 2008).     

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.     

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

Sphingolipids are a diverse group of lipids that have essential cellular roles as structural components of membranes and as potent signaling molecules. In recent years, a detailed picture has emerged of the basic biochemistry of sphingolipids—from their initial synthesis in the endoplasmic reticulum (ER), to their elaboration into complex glycosphingolipids, to their turnover and degradation. However, our understanding of how sphingolipid metabolism is regulated in response to metabolic demand and physiologic cues remains incomplete. Here I discuss new insights into the mechanisms that ensure sphingolipid homeostasis, with an emphasis on the ER as a critical regulatory site in sphingolipid metabolism. In particular, Orm family proteins have recently emerged as key ER-localized mediators of sphingolipid homeostasis. A detailed understanding of how cells sense and control sphingolipid production promises to provide key insights into membrane function in health and disease.Eukaryotic cell membranes maintain a complex and tightly regulated complement of lipids and proteins that are essential for their function. These lipids can be divided into three broad classes—sterols, glycerolipids, and sphingolipids—on the basis of their distinct chemical structures and dedicated enzymatic machineries (Fig. 1A–C). Sphingolipids typically represent ∼10%–20% of cellular lipids and have essential functions arising both from their effects on the physical properties of membranes and from their roles as signaling molecules (van Meer et al. 2008). Additionally, the activities of many transmembrane and peripheral membrane proteins are dependent on their close association with sphingolipids (Lingwood and Simons 2010). Over recent years, sphingolipids have been shown to participate in an increasingly wide range of biological processes that includes secretion, endocytosis, chemotaxis, neurotransmission, angiogenesis, and inflammation (Hannun and Obeid 2008; Lingwood and Simons 2010; Lippincott-Schwartz and Phair 2010; Blaho and Hla 2011; Lingwood 2011).Open in a separate windowFigure 1.Structures of sphingolipids and other cellular lipids. (A–C) Representative structures of (A) sphingolipids, (B) glycerolipids, and (C) sterols. (D) Formation of sphingolipids from key building blocks, long chain bases (LCBs), and coenzyme A-linked fatty acids (FA-CoAs) that often have a very long acyl chain (VLCFA-CoA). Serine palmitoyltransferase (SPT) produces the LCB intermediate 3-keto-dihydrosphingosine, which is then reduced to yield LCBs that are used by ceramide synthase (CerS) to form ceramides. Sphingolipid structural diversity arises from (a) headgroup modifications including phosphorylation, glycosylation, or phosphocholine addition, (b) LCB hydroxylation, (c) LCB desaturation, (d) variability in the length of the N-linked acyl chain, and (e) desaturation of the N-linked acyl chain.The focus of this article is the variety of regulatory mechanisms that cells use to ensure sphingolipid homeostasis. This task requires balancing sphingolipid levels in conjunction with sterols and glycerolipids and adapting sphingolipid metabolism in response to physiological cues and external stresses. A need for tight regulatory control is further highlighted by the potent signaling activities of many sphingolipid biosynthetic intermediates such as sphingosines and ceramides (Hannun and Obeid 2008; Fyrst and Saba 2010; Blaho and Hla 2011). Additionally, because most sphingolipids cannot move freely between different organelles, cells must regulate multiple intracellular pools of sphingolipids as well as lipid transport between these sites.It is noteworthy that, despite great progress in defining the enzymes that carry out sphingolipid synthesis and degradation, how cells achieve sphingolipid homeostasis remains poorly understood. In this article, I will describe recent progress in the field and highlight outstanding questions. In particular, I will discuss the emergence of the endoplasmic reticulum (ER) as a key site for sphingolipid homeostasis. Several critical enzymes in sphingolipid metabolism are found in the ER, and recent studies have identified a mechanism for matching sphingolipid production to metabolic demand that depends on the ER-localized Orm family of proteins (Breslow et al. 2010). Although many details of Orm protein function remain to be discovered, Orm proteins provide a valuable model for understanding how cells sense sphingolipids and dynamically regulate sphingolipid metabolism.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.     

Ceramide Synthase Schlank Is a Transcriptional Regulator Adapting Gene Expression to Energy Requirements

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Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma

Citrate synthase (CS), one of the key enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A to generate citrate. Increased CS has been observed in pancreatic cancer. In this study, we found higher CS expression in malignant ovarian tumors and ovarian cancer cell lines compared to benign ovarian tumors and normal human ovarian surface epithelium, respectively. CS knockdown by RNAi could result in the reduction of cell proliferation, and inhibition of invasion and migration of ovarian cancer cells in vitro. The drug resistance was also inhibited possibly through an excision repair cross complementing 1 (ERCC1)-dependent mechanism. Finally, upon CS knockdown we observed significant increase expression of multiple genes, including ISG15, IRF7, CASP7, and DDX58 in SKOV3 and A2780 cells by microarray analysis and real-time PCR. Taken together, these results suggested that CS might represent a potential therapeutic target for ovarian carcinoma.     

Photoperiod Differentially Affects the Expression of Three mPer Genes in the Mouse Heart

To investigate the mechanism that controls circadian rhythms in mammalian peripheral tissues, we housed mice in short days (6 h light: 18 h dark) or long days (18 h light: 6 h dark) and examined the rhythmic expression patterns of the mammalian clock genes mPer1 , mPer2 and mPer3 and a clock-controlled gene Dbp in the mouse heart. Northern blot analyses showed that peak levels of mPer1 mRNA expression in long days were about 50 % higher than those in short days. On the contrary the amplitude of the mPer2 mRNA peak in long days was about 25 % lower than that in short days. We could not find any effect of photoperiod on either the amplitude or waveform of the rhythms of mPer3 and Dbp mRNAs. Photoperiod differentially affected the expression of three mPer genes even in a peripheral tissue of mice.     

Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.     

Insights into Sphingolipid Miscibility: Separate Observation of Sphingomyelin and Ceramide N-Acyl Chain Melting

Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane’s physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by ²H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our ²H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids’ miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer.     

Hypoxia Alters Iron Homeostasis and Induces Ferritin Synthesis in Oligodendrocytes

Abstract: Both iron and the major iron-binding protein ferritin are enriched in oligodendrocytes compared with astrocytes and neurons, but their functional role remains to be determined. Progressive hypoxia dramatically induces the synthesis of ferritin in both neonatal rat oligodendrocytes and a human oligodendroglioma cell line. We now report that the release of iron from either transferrin or ferritin-bound iron, after a decrease in intracellular pH, also leads to the induction of ferritin synthesis. The hypoxic induction of ferritin synthesis can be blocked either with iron chelators (deferoxamine or phenanthroline) or by preventing intracellular acidification (which is required for the release of transferrin-bound iron) with weak base treatment (ammonium chloride and amantadine). Two sources of exogenous iron (hemin and ferric ammonium citrate) were able to stimulate ferritin synthesis in both oligodendrocytes and HOG in the absence of hypoxia. This was not additive to the hypoxic stimulation, suggesting a common mechanism. We also show that ferritin induction may require intracellular free radical formation because hypoxia-mediated ferritin synthesis can be further enhanced by cotreatment with hydrogen peroxide. This in turn was blocked by the addition of exogenous catalase to the culture medium. Our data suggest that disruption of intracellular free iron homeostasis is an early event in hypoxic oligodendrocytes and that ferritin may serve as an iron sequestrator and antioxidant to protect cells from subsequent iron-catalyzed lipid peroxidation injury.     

The Allelochemical MDCA Inhibits Lignification and Affects Auxin Homeostasis

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.Plants growing in a tight community are in continuous competition for space, light, water, and nutrients. Potential survival strategies include optimizing plant architecture and maximizing growth rate, allowing the plant to capture light and receive nutrients and water more efficiently, while placing neighboring plants in an unfavorable position (Einhellig, 1995; Weir et al., 2004). Besides developmental shifts, plants release an array of secondary metabolites (allelochemicals) into the rhizosphere to negatively affect the growth and reproduction of neighboring, competitor plants (Putnam, 1988; Bertin et al., 2003). Despite a lot of research effort having been devoted to allelopathic chemical warfare over the past decades, it remains a difficult study object due to the complexity of plant-plant interactions (Zeng, 2014). Nevertheless, the significance of allelochemicals in structuring plant communities and preserving biodiversity has been fully recognized by the scientific community. Moreover, allelochemicals show the potential to be used as an environmentally friendly alternative for weed control to improve agricultural productivity (Zeng, 2014).Strictly speaking, the term “allelochemical” refers to a compound produced and released by one organism to affect the growth and development of susceptible species (Weir et al., 2004). In practice, compounds derived from plant extracts or exudates are often cataloged as allelochemicals based on their inhibitory effect on seed germination and/or growth of other plant species in an artificial setup. Despite their importance, the molecular mode of action of a given allelochemical compound has rarely been studied in detail; however, toxicity is relatively easily demonstrated, identifying its molecular target is far more challenging. An interesting example is the phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA), which was isolated from lyophilized root tissues of Asparagus [Asparagus officinalis L.; Hartung et al. (1990)]. It was suggested to be an allelochemical based on its inhibitory effect on root and shoot growth of Lepidium sativum (Hartung et al., 1990). Independent studies revealed that MDCA acts as an efficient competitive inhibitor of 4-COUMARATE-CoA LIGASE (4CL), the enzyme converting hydroxycinnamates to their corresponding CoA-esters (Knobloch and Hahlbrock, 1977; Chakraborty et al., 2009). This conversion is an early step in the general phenylpropanoid pathway leading to a wide array of metabolites, including coumarins, stilbenes, salicylic acid, flavonoids, and monolignols (Vogt, 2010). Given that inhibition of 4CL in this metabolic pathway will have far-reaching effects on plant growth and development (Voelker et al., 2010), it is tempting to link the proposed phytotoxicity of MDCA to this metabolic block.Here, we evaluate whether the phytotoxicity of MDCA is a direct consequence of the inhibition of 4CL or if MDCA targets also other biological processes in Arabidopsis (Arabidopsis thaliana). We found that MDCA causes strong developmental defects in Arabidopsis seedlings at early developmental stages. Convincing evidence was found that MDCA affects the homeostasis of the plant signaling compound auxin. Our results provide an alternative explanation for the molecular mechanism underlying the phytotoxic properties of MDCA, and suggest that these multiple modes of action make it an attractive candidate as an environmental agrochemical or synergist.     

Phase-Specific Microstimulation Differentially Modulates Beta Oscillations and Affects Behavior

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Ablation of Neuronal Ceramide Synthase 1 in Mice Decreases Ganglioside Levels and Expression of Myelin-associated Glycoprotein in Oligodendrocytes

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.     

The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.During the transition from seed to seedling, the coordination of defense and development is critical for early survival (Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al., 2008). After emerging from the seed coat, the embryo pushes through the soil to reach the surface; at this time, it is more vulnerable to biotic and abiotic stresses (Raven et al., 2005), and underlying actors of this transition are relatively unstudied (Warpeha and Montgomery, 2016). Phe-derived compounds, the phenylpropanoids, play an important role in the first line of defense by contributing to the reinforcement of the external cuticle layer and by conferring UV light protection properties to epicuticular waxes (Steyn et al., 2002; Pollard et al., 2008); in addition, phenylpropanoids influence wax production in response to UV light exposure (Rozema et al., 2002; Pollard et al., 2008; Warpeha et al., 2008). The activity of the phenylpropanoid pathway provides an additional line of defense, as phenolic compounds take part in a nonenzymatic mechanism to efficiently scavenge reactive oxygen species (ROS), whose levels increase as a result of metabolic reactions and when plants initiate a stress response (Sharma et al., 2012; Agati et al., 2013). Moreover, by influencing the cell’s ability to balance and modulate ROS production and scavenging, phenylpropanoids allow fluctuations in ROS levels that are required to elicit stress signaling pathways for specific defense strategies (Apel and Hirt, 2004; Mittler et al., 2011).AROGENATE DEHYDRATASE3 (ADT3)/PREPHENATE DEHYDRATASE1 belongs to the arogenate dehydratase protein family, whose members catalyze the last steps of the biosynthesis of Phe (Warpeha et al., 2006; Cho et al., 2007; Tzin and Galili, 2010; Bross et al., 2011). Activation of ADT3 leads to an increase in Phe content and in the production of phenylpropanoids (Warpeha et al., 2006). Accordingly, loss of ADT3 results in an enhanced sensitivity to UV irradiation in etiolated seedlings due to the reduced synthesis of photoprotective compounds and UV light-scattering epicuticular waxes (Warpeha et al., 2008). However, the physiological and molecular bases of this phenotype and the function of ADT3 in the seed-to-seedling transition remain to be elucidated.We sought to understand the role of ADT3 postgermination, in the seed-to-seedling transition. ADT3 is expressed early in seedling growth (Warpeha et al., 2006; Hruz et al., 2008). Localization studies in Arabidopsis (Arabidopsis thaliana) using protoplasts from cell suspension and light-grown rosette leaves have placed this enzyme within the chloroplast (Rippert et al., 2009), while we have reported ADT3 activity in the cytosolic fraction in young etiolated seedlings (Warpeha et al., 2006). These reports differ likely due to the different age and growth conditions of the studied plant material. Cytosolic forms of chorismate mutase, which act at the first committed step in the Phe and Tyr biosynthesis pathway, were found in Arabidopsis and other plants (d’Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996), suggesting the possibility of extraplastidic Phe biosynthesis.Here, we report in transgenic complementation experiments that ADT3 is expressed widely in the young shoot and largely accumulates in the cytosol. Based on the role of phenylpropanoids in plant defense, we hypothesize that the ADT3 regulation of Phe supply is required to coordinate defense and development at the seed-to-seedling transition. We found that, without ADT3, the cells of the epidermis cannot buffer and restrict ROS; moreover, adt3 cotyledons enter an aberrant developmental program that results in abnormal morphology and patterning as well as several alterations at the subcellular level. Proteomic analysis of adt3 seedlings provided insights into the molecular basis of adt3 phenotypes, as it uncovered a chronic inability to buffer an excess of ROS and maintain plastid integrity. It also revealed a failed attempt to control cell rheology through up-regulation of the biochemical pathway for cuticle biosynthesis and assembly, as indicated by the increase in the permeability of adt3 epidermal cells; we propose that this also could be the cause of the defecting epidermal patterning in adt3 cotyledons. In addition, we suggest an additional role of Phe in nutrient supply in etiolated seedlings.