Sphingolipid Function

Membrane Function

Sphingolipids compose an estimated ~40 % of the total lipids in plasma membrane of plants (Sperling et al. 2005), where they are enriched in the outer leaflet (van Genderen et al. 1991; Burger et al. 1996; Tjellstrom et al. 2010). Sphingolipids are also abundant lipid components of other endomembranes in the plant, including ER, Golgi, and tonoplast (Bayer et al. 2014; Mongrand et al. 2004; Sperling et al. 2005; Verhoek et al. 1983). GlcCer was first identified in the plasma membrane and tonoplast of plant cells in a number of studies conducted in the 1980s (Cahoon and Lynch 1991; Lynch and Steponkus 1987; Verhoek et al. 1983; Yoshida et al. 1986). In these membranes, GlcCer was reported to compose between 7 and 30 % of plasma membrane and tonoplast, depending on the plant species and tissue type analyzed (Cahoon and Lynch 1991; Lynch and Steponkus 1987; Uemura et al. 1995; Uemura and Steponkus 1994; Verhoek et al. 1983; Yoshida et al. 1986). More recently, it has been shown that GIPCs, rather than GlcCer, are the more abundant glycosphingolipid in plants (Markham and Jaworski 2007; Markham et al. 2006). The quantitative importance of GIPCs was largely overlooked until very recently because their highly glycosylated head groups offer challenges for extraction using typical organic solvents, such as mixtures of chloroform and methanol (Markham et al. 2006). Initially, by quantification of long-chain bases of GIPCs and GlcCer and later by LC-MS/MS analysis, GIPCs were found to be nearly twofold more abundant than GlcCer in Arabidopsis leaves, whereas amounts of GIPCs and GlcCer were nearly the same in tomato leaves (Markham and Jaworski 2007; Markham et al. 2006). GIPCs have subsequently been identified as one of the most abundant lipids of plant plasma membrane and are also enriched in detergent resistant membranes (DRMs) derived from isolated plasma membrane and in plasmodesmata (Grison et al. 2015; Cacas et al. 2012). Given their abundance in plasma membrane and tonoplast, it is likely that the content and composition of sphingolipids affect the ability of plants to respond to abiotic stress, particularly osmotic stresses such as freezing, drought, and salinity. For example, GlcCer concentrations were shown to decrease by nearly half in plasma membrane during cold acclimation of rye and Arabidopsis (Lynch and Steponkus 1987; Uemura et al. 1995; Uemura and Steponkus 1994). More recently, it was reported that GIPCs increase and GlcCer decrease in response to chilling of Arabidopsis (Nagano et al. 2014). Although this is likely an adaptive response to low temperatures, the impact of such adjustments in relative amounts of GIPCs and GlcCer on plant performance has not yet been established. In addition, the fatty acid and long-chain base composition of sphingolipids also affects plant resistance to abiotic stress. For example, Arabidopsis mutants lacking LCB Δ8 and ceramide fatty acid unsaturation display sensitivity to low temperature growth and alterations in the relative amounts of LCB cis-trans Δ8 unsaturation affects resistance of plants to aluminum (Chen et al. 2012; Chen and Thelen 2013; Ryan et al. 2007).

The unique structural components of sphingolipid hydrophobic ceramide backbones include VLCFAs and an abundance of hydroxyl groups distributed between the LCB and fatty acid moieties. Through these structural features, sphingolipids confer rigidity to membranes. In addition, the hydroxyl groups enable the formation of extensive hydrogen bonding networks that result in elevated phase transition temperatures and reduced ion permeability (Lunden et al. 1977; Pascher 1976). The rigidity and high phase transition temperatures of sphingolipid micelles is moderated by interactions with other lipids, including sterols (Curatolo 1987). Sphingolipids have been shown to cluster with sterol in membrane microdomains or lipid rafts (Cacas et al. 2012). Lipid microdomains have long been hypothesized to be present in membranes (Karnovsky et al. 1982) with sphingolipids potentially aiding in the sorting of membrane proteins, such as GPI-anchored proteins, by forming lipid domains that slow lateral protein diffusion (Simons and van Meer 1988; van Meer and Simons 1988; Brown and Rose 1992; Simons and Ikonen 1997; de Almeida et al. 2003). Indeed, pure sphingolipid membranes form a ‘solid gel’ phase with little lateral movement that is fluidized by the presence of sterols (van Meer et al. 2008; Estep et al. 1980; Roche et al. 2008; Grosjean et al. 2015). The co-localization of sterols and sphingolipids in the membrane may be due to sphingolipids complex sugar head group’s ability to shield the non-polar sterol from the bulk solvent, much like an umbrella (Huang and Feigenson 1999; Ali et al. 2006). Raft formation is also dependent on the various sphingolipid modifications, including fatty acid chain length and fatty acid and LCB hydroxylation (Klose et al. 2010).

Proteomic analysis of DRMs prepared from plant plasma membrane has revealed an enrichment of proteins in lipid rafts related to signaling, responses to biotic and abiotic stress, cellular trafficking, auxin transport, and cell wall synthesis and degradation (Brodersen et al. 2002; Lefebvre et al. 2007; Lin et al. 2008; Morel et al. 2006), suggesting that raft regions contribute to these cellular functions. Similar to DRMs, plasmodesmata have also recently been shown to be enriched in sphingolipids and sterols and contain specific GPI-anchored proteins (Bayer et al. 2014; Grison et al. 2015).

Endomembrane Trafficking

Given their abundance in the endomembrane system, sphingolipids are presumed to play a major role in ER export and Golgi-mediated trafficking of proteins through the secretory system. Consistent with this, Arabidopsis pollen deficient in sphingolipids have been shown to have vesiculated ER and lack Golgi stacks (Dietrich et al. 2008). Consistent with defects in Golgi-trafficking to the plasma membrane, the sphingolipid-deficient pollen lacked a surrounding intine layer (Dietrich et al. 2008). Similarly, chemical inhibition of GlcCer synthesis has been shown to alter Golgi morphology and impair Golgi-mediated trafficking of secretory proteins to the plasma membrane (Melser et al. 2010, 2011). Recent studies using an Arabidiopsis KSR mutant and GlcCer synthase and ceramide synthase inhibitors demonstrated the importance of sphingolipids in trafficking of ATP-binding cassette B19 (ABCB19) auxin transporter to the Golgi, trans-Golgi network, and plasma membrane (Yang et al. 2012). Similar studies targeting sterols indicated that sterols have greater significance for post-Golgi transport of ABCB19 from trans-Golgi to the plasma membrane (Yang et al. 2012). Of particular importance for trafficking of proteins through the secretory system is the presence of the very long-fatty acid (VLCFA) component of sphingolipids, which are enriched in GIPCs. Mutants defective in VLCFA synthesis or that have reduced activity of Class II ceramide synthases that incorporate VLCFAs in ceramides have impaired trafficking of secretory proteins, including PIN1 and AUX1 that are required for auxin transport and plant growth (Bach et al. 2008, 2011; Markham et al. 2011; Zheng et al. 2005).

ABA-Dependent Guard Cell Closure

In addition to the contributions of the glycosphingolipids GIPCs and GlcCer to membrane structure and function, the less abundant sphingolipid biosynthetic intermediates LCBs, LCBPs, ceramides, and ceramide-1-phosphates have been linked to the mediation of numerous, diverse physiological processes in plant cells. An important contributor to the formation of these physiological mediators are kinase and phosphatase reactions that convert LCBs and ceramides between their phosphorylated and free forms, as described above. The phosphorylation status of LCBs and ceramides are key to the particular physiological process that they regulate.

One of the first links between sphingolipids and control of cellular processes was the observation that the LCB-P sphingosine-1-phosphate or S1P participates in the ABA-mediated signaling pathway that controls stomatal aperture by elevating cytosolic Ca2+ levels which, in turn, activates ion channels in guard cell membranes, with the resulting K+ efflux causing loss of guard cell turgor pressure and stomatal closing (Kim et al. 2010). After drought treatment of the plant Commelina communis, S1P levels were found to increase in leaves, and when applied exogenously, S1P resulted in a Ca2+ spike followed by stomatal closure (Ng et al. 2001). Phytosphingosine-1-phosphate elicits the same response, although sphinganine-1-phosphate does not, indicating some level of LCB specificity in the mediation of guard cell closure (Coursol et al. 2003). Treatment of Arabidopsis plants with ABA was found to activate LCB kinase, and this activity was sensitive to the mammalian LCB kinase inhibitor, N,N-dimethylsphingosine (Coursol et al. 2003). As in mammals, the target of S1P in plants is presumed to be a G-protein coupled receptor, and Arabidopsis mutants lacking the G-protein α-subunit (GPA1) did not respond upon exogenous S1P application (Coursol et al. 2005 ; Strub et al. 2010). More recently, a connection between phospholipids and sphingolipids in the signaling pathway for ABA-dependent guard cell closure has been proposed. In this regard, ABA and phosphatidic acid (PA) produced by phospholipase Dα1 (PLDα1) have been shown to activate sphingosine kinase (SPK) to promote production of LCB-P (Guo et al. 2012; Guo and Wang 2012; Worrall et al. 2008). PA enhancement of SPK activity was found to occur by direct interaction of PA with this enzyme (Guo et al. 2012; Guo and Wang 2012). Given that LCB-P induction of guard cell closure requires a functional PLDα1, it was proposed that LCB-P functions upstream of PLDα1 in this signaling pathway (Guo et al. 2012; Guo and Wang 2012).

Programmed Cell Death

Sphingolipids, primarily in the form of ceramides and LCBs, have also been strongly implicated in mediation of programmed cell death (PCD ) in plants. As described above, an initial indication of the role of ceramides as PCD triggers was obtained from the Arabidopsis acd5 mutant that is defective in a proposed ceramide kinase (Greenberg et al. 2000; Liang et al. 2003). This mutant accumulates enhanced levels of free ceramides and displays early onset of PCD relative to wild-type controls (Greenberg et al. 2000; Liang et al. 2003), resulting in part to the enhanced release of mitochondrial reactive oxygen species (Bi et al. 2014). PCD induction in the acd11 mutant has also been linked to ceramide accumulation associated with defects in ceramide-1-phosphate transport in this mutant (Simanshu et al. 2014). Similar findings have been obtained by treatment of Arabidopsis cell cultures with C2 ceramide at a concentration of 50 μM (Townley et al. 2005). This treatment induces a transient increase in cytosolic Ca2+and hydrogen peroxide production, followed by cell death, which was reversed by inhibition of Ca2+ release (Townley et al. 2005). These findings implicate Ca2+ as an essential component of ceramide induction of PCD. Notably, C2 ceramides containing 2- or α-hydroxylated fatty acids were not effective in PCD induction in Arabidopsis cell cultures (Townley et al. 2005). Consistent with this observation, the ability of Bax inhibitor-1 (BI-1) to suppress cell death in Arabidopsis is dependent on 2-hydroxylation of ceramide VLCFAs (Nagano et al. 2012).

Similar to results with ceramides, application of the free LCBs d18:1, d18:0, and t18:0 to Arabidopsis leaves also induces PCD, albeit at concentrations lower than that observed with ceramides (Shi et al. 2007). This induction of PCD was also dependent on ROS generation, but was suppressed by application of LCB-P along with free LCBs (Alden et al. 2011; Shi et al. 2007). These findings suggest that the ratio of free LCB to LCB-P, mediated by LCB kinases and LCB-P phosphatases, is an important “rheostat” for regulation of PCD (Alden et al. 2011; Shi et al. 2007). This is analogous to the dependence of PCD induction on relative levels of ceramides and ceramide-1-phosphates (Greenberg et al. 2000; Liang et al. 2003). The transduction pathway for elicitation of PCD by free LCBs has been shown to be dependent in Arabidopsis on mitogen-activated protein kinase 6 (MPK6) (Saucedo-García et al. 2011) as well as 14-3-3 protein phosphorylation by calcium-dependent kinase 3 (CPK3) that is activated by LCB-triggered release of cytosolic Ca2+ (Lachaud et al. 2013).

Fungal-derived sphingosine-analog mycotoxins (SAMs) including fumonisin B1 produced by Fusarium species and AAL toxin produced by Alternaria alternata lycopersici are also potent triggers of PCD in plants. These molecules competitively inhibit ceramide synthases leading to the accumulation of free LCBs that, in turn, elicit PCD (Abbas et al. 1994). Consistent with this, reduction of LCB synthesis by chemical inhibition of serine palmitoyltransferase (SPT) activity enhances resistance of plants to SAMs (Spassieva et al. 2002). Increased resistance to FB1-triggered PCD induction has also been observed in an Arabidopsis LCB1 mutant and in small subunit of SPT RNAi suppression lines that have reduced SPT activity ( Kimberlin et al. 2013; Shi et al. 2007). Recent evidence has also emerged that FB1 not only increases levels of free LCBs in plant cells but also elevates levels of ceramides containing C16 fatty acids (Markham et al. 2011; Ternes et al. 2011). This finding suggests that FB1 most effectively inhibits Class II ceramide synthases (i.e., LOH1, LOH3) that produce ceramides with VLCFAs and are less effective inhibitors of Class I ceramide synthase (i.e., LOH2) that produces ceramides with C16 fatty acids (Markham et al. 2011; Ternes et al. 2011). These findings suggest that the potency of SAMs for PCD-induction is due to their ability to enhance accumulation of LCBs and ceramides.

Pathogen Resistance

The hypersensitive response (HR) is an important process for resistance to bacterial and fungal pathogens that is characterized by localized induction of PCD that reduces or prevents the spread of pathogens in plants. Given the importance of LCBs and ceramides to PCD induction, a considerable body of research has emerged linking sphingolipids to bacterial and fungal pathogen resistance as described in a recent review (Berkey et al. 2012). Notably, ceramide accumulation in acd5 and acd11 mutants has been shown to be associated with salicylic acid (SA)-dependent upregulation of HR-type PCD and pathogen-resistance genes, including genes for PR1, ERD11, and chitinase (Brodersen et al. 2002; Greenberg et al. 2000). More recently, Arabidopsis mutants defective in 2-hydroxylation of ceramide fatty acids were found to have elevated LCB and ceramide levels, as well as, increased levels of free and glycosylated SA and constitutive induction of PR1 and PR2 genes (Konig et al. 2012). These mutants also displayed enhanced resistance to the biotrophic fungal pathogen Golovinomyces cichoracearum (Konig et al. 2012). In addition, infection of Arabidopsis with the bacterial pathogen Pseudomonas syringae was accompanied by transient increases in the LCB phytosphingosine (t18:0) and induction of ROS and cell death (Peer et al. 2010; Bach et al. 2011). Furthermore, resistance to the bacterial pathogen Pseudomonas cichorii was compromised in tobacco upon chemical inhibition of SPT and an accompanying reduction in LCB synthesis (Takahashi et al. 2009). This resistance appears to be mediated by MPK6, as FB1-elicited Arabidopsis mpk6 mutants displayed reduced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato avrRpm1 due to compromised induction of PCD in this mutant (Saucedo-García et al. 2011).

Cold Stress Signaling

Sphingolipids as abundant components of plasma membrane and tonoplast contribute to the ability of plants to resist chilling and freezing stresses. As evidence of this, Arabidopsis mutants lacking LCB Δ8 unsaturation have increased sensitivity to prolonged exposure to low, non-freezing temperatures (Chen et al. 2012). In addition to their roles as membrane components, recent studies have implicated sphingolipids in cold stress signaling pathways (Cantrel et al. 2011; Guillas et al. 2011, 2013). Exposure of Arabidopsis plants to 4 °C resulted in accumulation of PA and nitrous oxide (NO). In addition, within 5 min of this cold treatment amounts of the LCB-P phytosphingosine phosphate and ceramide-1-phosphate increased by ~50 % (Cantrel et al. 2011). This increase was negatively regulated by NO, as chemical inhibition of NO production enhanced the accumulation of these molecules but chemically-induced enhancement of NO levels reduced accumulation of the phosphorylated LCB and ceramides (Cantrel et al. 2011). From these findings, it was suggested that NO may regulate the relative levels of phosphorylated and dephosphorylated LCBs and ceramides as part of a rapid signaling response pathway to low, non-freezing temperatures (Cantrel et al. 2011; Guillas et al. 2011, 2013). The mechanistic details of this potential signaling pathway remain uncharacterized.