The biosynthesis of LCBs is initiated through an endoplasmic reticulum-localized reaction catalyzed by serine palmitoyltransferase (SPT) that condenses serine and palmitoyl-CoA to form the 18 carbon intermediate 3-ketosphinganine (
Chen et al. 2006;
Dietrich et al. 2008;
Teng et al. 2008). The product of this reaction is then reduced by 3-ketosphinganine reductase (KSR) to form sphinganine or d18:0, the simplest long-chain base in plants and other eukaryotes (
Chao et al. 2011).
SPT is a member of the α-oxoamine synthase subfamily and is generally regarded as the main regulated step in sphingolipid biosynthesis (
Hanada 2003). Similar to other eukaryotes, the Arabidopsis SPT functions as a heterodimer comprised of LCB1 and
Although SPT can function as a heterodimer (LCB1 and LCB2) with minimal enzymatic activity, ssSPT enhances SPT activity to levels that produce LCBs in amounts that are sufficient to support cell viability in Arabidopsis (
Kimberlin et al. 2013).
Studies of LCB1, LCB2, and ssSPT mutants have demonstrated that SPT activity is essential, and consequently, sphingolipids are required for the viability of plant cells (
Kimberlin et al. 2013;
Dietrich et al. 2008;
Teng et al. 2008).
In this regard, the fbr11-2 mutant of the single Arabidopsis LCB1 gene (At4g36480) displays male gametophytic lethality (
Teng et al. 2008). Loss of pollen viability is also observed in double mutants of the two redundant LCB2 genes LCB2a (At5g23670) and LCB2b (At3g48780) (
Dietrich et al. 2008) as well as in null mutants of ssSPTa (At1g06515), the more highly expressed of the two ssSPT genes in Arabidopsis (
Kimberlin et al. 2013). Pollen deficient in sphingolipid synthesis in lcb2a-/-lcb2b-/+ mutants lack Golgi stacks and surrounding intine layer and have vesiculated ER, all of which are consistent with the contributions of sphingolipids to the structural and functional integrity of the endomembrane system (
Dietrich et al. 2008).
In the second step of LCB synthesis, the SPT product 3-ketosphinganine is reduced by the enzyme 3-ketosphinganine reductase (KSR) to form sphinganine (d18:0), the simplest LCB found in plants. KSR is encoded by two genes in Arabidopsis thaliana, KSR-1 (At3g06060) and KSR-2 (At5g19200). Both genes are essential and contribute to the reductase activity (
Chao et al. 2011), although KSR-1 is more highly expressed throughout the plant (
Chao et al. 2011). KSR-1 and KSR-2 are functionally redundant, but KSR-1 is the primary contributor to the reductase activity (
Chao et al. 2011). Loss-of-function mutants of KSR-1 are viable but display greatly reduced reductase activity (
Chao et al. 2011). These mutants also display an altered leaf ionome that is associated with increased root suberization, altered root morphology, and altered root iron homeostasis (
Chao et al. 2011). The sphinganine (d18:0) produced from the combined activities of SPT and KSR can be used directly by ceramide synthase or modified by hydroxylation or desaturation at the C-4 position prior to use for ceramide synthesis.
The regulation of SPT is thought to occur primarily through ssSPT and a second class of interacting proteins termed orosomucoid or ORM polypeptides, rather than transcriptional mechanisms (
Markham et al. 2013).
The ssSPTs appear to be limiting and so modulation of their expression alters SPT activity. This has been shown in Arabidopsis with ssSPT over-expression leading to increased SPT activity and ssSPT RNAi suppression resulting in reduced SPT activity (
Kimberlin et al. 2013
).
Whether alteration of ssSPT levels occurs naturally in response to intracellular cues to mediate sphingolipid homeostasis is unclear. In addition, no evidence currently exists for regulation of SPT activity through post-translational modifications of ssSPT polypeptides. It is also notable that ssSPTs can dictate the acyl-CoA specificity of SPT (
Han et al. 2009
).
In this regard, human ssSPTa and ssSPTb polypeptides have been shown to confer different acyl-CoA specificity when bound to SPT leading to the production of either C18 LCBs using palmitoyl (16:0)-CoA substrates or C20 LCBs using stearoyl (18:0)-CoA substrates (
Han et al. 2009
). These differences in acyl-CoA substrate specificities were shown to result from a single amino acid residue that is a Met in the human ssSPTa and Val in human ssSPTb (
Han et al. 2009
). The Arabidopsis ssSPTa and ssSPTb polypeptides both contain Met at the analogous position, but mutation of Met to Val results in the aberrant production of C20 LCBs when expressed in transgenic Arabidopsis (
Kimberlin et al. 2013).
In yeast, ORM proteins have been shown to act as homeostatic negative regulators of SPT in response to intracellular sphingolipid levels (
Roelants et al. 2011
;
Han et al. 2010
;
Breslow et al. 2010
). The regulation involves TORC2-dependent phosphorylation of ORM to gradually relieve ORM suppression of SPT to enhance LCB synthesis in response to sub-optimal intracellular sphingolipid levels and reversible dephosphorylation of ORM by SAC1 phosphatase to engage ORM suppression of SPT activity in response to excess intracellular sphingolipid levels (
Muir et al. 2014
;
Roelants et al. 2011).
Arabidopsis has two homologs of the yeast ORM genes, ORM1 (At1g01230) and ORM2 (At5g42000). Although these proteins have not yet been characterized, RNAi suppression of ORM genes in rice results in temperature sensitivity and pollen abnormalities (
Chueasiri et al. 2014).
Regulation of SPT by ORM proteins in plants and other eukaryotes appears to be more complex than ORM phosphorylation/dephosphorylation in yeast.
As described above, the primary regulatory mechanism of ORM in yeast occurs through TORC2 dependent YPK1 phosphorylation of ORM that relieves inhibition of SPT that can be reversed by SAC1 phosphatase activity that restores inhibition of SPT (
Han et al. 2010
;
Muir et al. 2014
).
This mechanism is adjustable and dependent on intracellular sphingolipid levels and has been shown to be coordinated with ceramide synthase activity (
Muir et al. 2014
). An N-terminal extension of approximately 80 amino acids in yeast ORM was found to contain several Ser residues that are responsible for this phosphorylation mechanism (
Roelants et al. 2011
). This phosphorylation domain, however, is absent in plant and mammalian ORM homologs (
Roelants et al. 2011
). As a result, it is unclear if ORM phosphorylation/dephosphorylation also regulates SPT activity in plants and mammals.
Overall, homeostatic regulation of SPT in plants and mammals remains an open and active area of research.
In Plants, there are over 200 different structural variations of sphingolipids. These differences primarily manifest in the modifications of long-chain bases (LCB) and variations in fatty acids.
What is the biosynthetic and functional basis for the large amount of structural diversity found in plant sphingolipids?
What is the functional significance of LCB hydroxylation and desaturation?
The d18:0 LCB resulting from the sequential activities of SPT and KSR can undergo combinations of three modification reactions to generate trihydroxylated and unsaturated LCBs. In Arabidopsis leaves, ~90 % of the total LCBs contain three hydroxyl groups and Δ8 unsaturation. The third hydroxyl group of these LCBs occurs at the C-4 position and is introduced by a LCB C-4 hydroxylase (
Chen et al. 2008
;
Sperling et al. 2001
). This enzyme is a di-iron oxo protein with homology to desaturases and hydroxylases (
Sperling et al. 2001). The two genes that encode the LCB C-4 hydroxylase in Arabidopsis are designated SPHINGOID BASE HYDROXYLASE (SBH) 1 (At1g69640) and 2 (At1g14290). Expression of these genes in mutants of the Saccharomyces cerevisiae SUR2 gene (
Haak et al. 1997
) that encodes a related LCB C-4 hydroxylase restores trihydroxy LCB synthesis (
Chen et al. 2008
;
Sperling et al. 2001)
Although definitive evidence has yet to be reported regarding the nature of the substrate for plant LCB C-4 hydroxylases, it is likely that C-4 hydroxylation occurs primarily on free dihydroxy LCBs prior to incorporation into ceramide (
Wright et al., 2003
) and the trihydroxy LCB t18:0 is the predominate free LCB in Arabidopsis thaliana leaves (
Markham and Jaworski 2007
;
Chen et al. 2008
).
The functional significance of LCB C-4 hydroxylation in Arabidopsis thaliana was recently examined by the generation of double mutants and RNAi suppression lines for the two hydroxylase genes (
Chen et al. 2008
). Based on the lack of a growth phenotype in the Saccharomyces cerevisiae sur2 mutant (
Haak et al. 1997
;
Grilley et al., 1998
), it was anticipated that the Arabidopsis thaliana mutants would have no obvious phenotypes. Instead, double mutants were found to be severely dwarfed and did not progress from vegetative to reproductive growth (
Chen et al. 2008
).
In addition, the degree of growth reduction in RNAi lines was found to be more severe as the relative content of trihydroxy LCBs decreased. Unexpectedly, the sphingolipid content in double mutants was 2.5- to three-fold higher than in wild type plants, and the accumulation of sphingolipids was primarily the result of increased amounts of molecular species with C16 fatty acids, rather than the more typical VLCFAs, in all sphingolipid classes (
Chen et al. 2008
).
As described above, the increased levels of ceramide backbones with C16 fatty acids and dihydroxy LCBs is likely reflective of the substrate specificities of ceramide synthases in Arabidopsis thaliana. Overall, these results indicate that LCB C-4 hydroxylation is a critical sphingolipid structural modification for growth and for the regulation of sphingolipid content and composition in Arabidopsis thaliana. These findings also suggest that the synthesis of trihydroxy LCBs is important for mediating flux into the sphingolipid biosynthetic pathway, perhaps through regulation of SPT activity, to meet the demands for growth.
The LCB Δ8 desaturase is absent in Saccharo-myces cerevisiae and mammalian cells, but does occur in plants and many fungi. The LCB Δ8 desaturase was first identified in plants as a novel peptide consisting of an N-terminal cytochrome b5 domain fused to a desaturase-like polypeptide (
Sperling et al. 1998
). Heterologous expression of the Arabidopsis thaliana and Brassica napus cDNAs in Saccharomyces cerevisiae allows the biosynthesis of cis and trans isomers of t18:1Δ8 (
Sperling et al. 1998
). These results demonstrate that the plant LCB Δ8 desaturase is bifunctional with regard to the stereospecificity of double bond insertion. In most plants, the trans isomers of Δ8 unsaturated LCBs predominate in the total sphingolipid extract. However, the cis isomer of t18:1Δ8 is much more enriched in GlcCers relative to GIPCs in plants, such as Arabidopsis thaliana (
Sperling et al. 2005
;
Markham et al. 2006
;
Markham and Jaworski 2007
). It is not yet clear if this difference in stereoisomer composition results from distinct LCB Δ8 desaturases that may be associated with GlcCers and GIPCs. Recently, a plant LCB Δ8 desaturase from the legume Sty-losanthes hamata was shown to produce more of the cis isomer of t18:1Δ8 when expressed in Sac-charomyces cerevisiae (
Ryan et al. 2007
). This is in contrast to findings with the Arabidopsis thal-iana and Brassica napus enzymes, which generated mostly the trans isomer upon expression in Saccharomyces cerevisiae (
Sperling et al. 1998). These studies demonstrate that LCB Δ8 desatu-rase with different stereoselective properties have evolved in plants. Mechanistic studies with the sunflower LCB Δ8 desaturase indicate that the bifunctionality of this enzyme arises from two different conformations that the LCB substrate can assume in the active site (
Beckmann et al. 2002
). Interestingly, expression of the Stylosan-thes hamata desaturase in Arabidopsis thaliana was shown to not only increase the content of t18:1Δ8cis but also confer increased tolerance to aluminum toxicity (
Ryan et al. 2007
). This finding strongly indicates that the relative amounts of cis—trans isomers of unsaturated LCBs in sphin-golipids impact the plant's ability to adapt to at least some abiotic stresses. However, it has yet to be established if LCB Δ8 unsaturation is essential in plants. A number of metabolic questions regarding the LCB Δ8 desaturase also remain unanswered. For example, it is not known if this enzyme uses a free LCB, ceramide, or complex sphingolipid as a substrate. In addition, it has not been established if distinct LCB Δ8 desaturases are involved in the synthesis of monounsaturated and diunsaturated LCBs. This is particularly relevant given the occurrence of two LCB Δ8 desaturase genes in Arabidopsis thaliana (At3g61580, SLD1; At2g46210, SLD2).
The LCB Δ4 desaturase introduces the trans-Δ4 unsaturation that is found in GlcCers of many plant species, but is typically absent in GIPCs (
Markham et al. 2006).
In contrast to mammals, Δ4 monounsaturated LCBs are present in low abundance in plant sphingolipids (
Sperling et al. 2005
;
Markham et al. 2006). Instead, Δ4 double bonds occur together with cis- or trans- Δ8 double bonds in the diunsaturated LCB d18:2Δ4,8 (sph-ingadiene). The plant LCB Δ4 desaturase is most related to animal LCB Δ4 desaturases, including the Drosophila melanogaster Δ4 desaturase encoded by the DEGENERATIVE SPERMATO-CYTE-1 gene (DES-1) (
Ternes et al. 2002
). In addition, the mouse LCB Δ4 desaturase DES2 has been shown to act as a bifunctional enzyme that can also catalyze C-4 hydroxylation of dihy-droxy LCBs (
Omae et al. 2004
). This gene family also includes LCB Δ4 desaturases from a number of fungal species (
Ternes et al. 2002
).
Functional identification of plant LCB Δ4 desaturases has only recently been reported. The Arabidopsis thaliana LCB Δ4 desaturase (At4g04930) was shown to restore the synthesis of d18:2 to a Pichia pastoris Δ4 desaturase null mutant (
Michaelson et al. 2009
). It is notable that functional expression of plant LCB Δ4 desaturases has not been achievable in Saccharo-myces cerevisiae (
Michaelson et al. 2009
). This may be reflective of the substrate specificity of the plant Δ4 LCB desaturase. Because Δ4 unsaturation is found almost entirely in GlcCers, one possibility is that the LCB Δ4 desaturase uses GlcCers as substrates. However, GlcCers do not occur in Saccharomyces cerevisiae. Another possibility is that the Δ4 desaturase activity requires the presence of a Δ8 double bond in LCB substrates, given that Δ4 monounsaturated LCBs are of low abundance in plants. Like the previous metabolic scenario, Saccharomyces cerevisiae does not have a Δ8 desaturase, and to our knowledge, the co-expression of plant LCB Δ4 and Δ8 desaturases in Saccharomyces cerevisiae has not been reported.
As with the LCB C-4 hydroxylase and Δ8 desaturase, the substrate for the plant LCB Δ4 desaturase has yet to be defined. In addition to the possibility that this enzyme uses a GlcCer substrate, it cannot be ruled out that the Δ4 unsaturation is introduced into ceramides, which are then selectively used for GlcCer synthesis. The latter hypothesis is supported by the observation that in Pichia pastoris knock-out of the LCB Δ4 desaturase results in a complete loss of GlcCers (
Michaelson et al. 2009
). This observation supports the idea that Δ4 unsaturation is introduced prior to the attachment of the glucose head group and as such, is necessary for channeling of ceramides into GlcCers in fungi.
The functional significance of the LCB Δ4 desaturase in plants is also not clear. Some plants are enriched in Δ4 unsaturated LCBs in GlcCers (e.g., tomato), whereas other species (e.g., Arabidopsis thaliana) contain very low levels of these LCBs in GlcCers (
Markham et al. 2006).
The near absence of Δ4 unsaturated sphingolipids in Arabidopsis thaliana suggests that the LCB Δ4 desaturase may be of little importance in this plant. Indeed, a T-DNA knock-out of the corresponding gene (At4g04930) did not affect the growth and development of Arabidopsis thaliana (
Michaelson et al. 2009
).
In addition, changes in stomatal aperture in response to ABA treatment was also not affected in the Arabidopsis thaliana LCB Δ4 knockout mutant relative to the wild-type control (
Michaelson et al. 2009
). This finding brings into question the purported role of Δ4 unsaturated LCB-1-phosphates in ABA-dependent stomatal closure. Still, it cannot be ruled out that the Δ4 desaturase has some important physiological functions in plants, such as tomato, that contain high levels of these LCBs in GlcCers.
Ceramide Synthesis
Ceramides are synthesized by the condensation of a long-chain base and fatty acyl-CoA through an acyltransferase-type reaction catalyzed by ceramide synthase. Three ceramide synthases have been identified in Arabidopsis through homology with the yeast ceramide synthase encoded by LAG1 (LONGEVITY ASSURANCE GENE1). These enzymes are designated Lag One Homolog (LOH)-1, -2, and -3 and correspond to genes encoded by LOH1, At3g25440; LOH2, At3g19260; and LOH3, At1g13580, respectively (
Ternes et al. 2011
;
Markham et al. 2011). Homologs of these three enzymes are found throughout the plant kingdom and appear to form two distinct evolutionary branches, LOH1/LOH3-related isoforms and LOH2-related isoforms (
Ternes et al. 2011
;
Markham et al. 2011). Arabidopsis LOH1 and LOH3 share approximately 90 % amino acid sequence identity, while LOH2 shares approximately 60 % identity with LOH1 and LOH3 (
Ternes et al. 2011
;
Markham et al. 2011). In other mammals, multiple ceramide synthases occur that have distinct specificity for fatty acyl-CoAs and/or long-chain bases (
Venkataraman et al. 2002;
Laviad et al. 2008;
Mizutani et al. 2005,
2006;
Riebeling et al. 2003).
Studies of Arabidopsis LCB C-4 hydroxylase mutants initially pointed to the likelihood that two functional classes of ceramide synthases occur in plants (
Chen et al. 2008
). Loss of or reduced LCB C-4 hydroxylation has been shown to result in the aberrant accumulation of high levels of sphingolipids with ceramides containing C16 fatty acids bound to dihydroxy LCBs (
Chen et al. 2008
). Based on this observation, it was proposed that Arabidopsis has one class of ceramide synthase that links C16 fatty acyl-CoAs with dihydoxy LCBs (termed “Class I”), and a second class (“Class II”) that primarily links very long-chain fatty acyl CoAs with trihydroxy LCBs (
Chen et al. 2008
).
Model of ceramide synthase mediated long-chain base (LCB) and fatty acid routing. The Arabidopsis gene names are shown as reference. As indicated, Class I ceramide synthase (CSI) encoded by LOH2 displays strict substrate specificity of C16 fatty acid acyl-CoAs and dihydroxy LCBs, and Class II ceramide synthase (CSII) encoded by LOH1 or LOH3 display strict substrate specificity for very long-chain fatty acyl-CoAs and trihydroxy LCBs. One or more products of the CSII pathway appear to negatively regulate serine palmitoyltransferase (SPT) activity. In addition, sphingolipids with ceramides from the CSI pathway do not support growth, while those from the CSII pathway are essential for plant growth. The mycotoxin fumonisin B1 (FB 1) appears to preferentially inhibit CSII enzymes. KSR, 3-ketosphinganine reductase; SBH, LCB C-4 hydroxylase
This prediction was supported by the identification, biochemical and genetic characterization of LOH1, LOH2, and LOH3 in Arabidopsis. Studies using yeast complementation showed that LOH2 prefers C16 acyl-CoAs, similar to the predicted Class I ceramide synthase (
Ternes et al. 2011). Similarly, Arabidopsis LOH2 mutants were found to be deficient in sphingolipids with ceramide backbones containing C16 fatty acids and dihydroxy fatty acids (
Markham et al. 2011). Consistent with the substrate properties of Class II ceramide synthase, partial knock-out mutants of LOH1 and LOH3 contained reduced amounts of ceramides with very long-chain fatty acids and trihydroxy LCBs (
Markham et al. 2011). It is notable that under ideal growth conditions, null mutants of LOH2 are viable, suggesting that the Class I ceramide synthase and hence ceramides with C16 fatty acids and dihydroxy LCBs are not essential in Arabidopsis (
Markham et al. 2011). Conversely, double null mutants of LOH1 and LOH3 were not recoverable, indicating that the Class II ceramide synthase and ceramides with very long-chain fatty acids and trihydroxy LCBs are essential (
Markham et al. 2011).
Ceramide synthases are known targets for competitive inhibition by sphinganine analog mycotoxins (SAMs) such as fumonisin B1 or FB1 produced by a variety of Fusarium species and AAL toxin produced by Alternaria alternata f. sp. lycopersici (
Abbas et al. 1994). These compounds, particularly FB1, have been widely used as tools for induction of programmed cell death (PCD) in plants, presumably due to the accumulation of cytotoxic LCBs from their inhibition of ceramide synthases (
Stone et al. 2000). Recent evidence using FB1 treatment of Arabidopsis ceramide synthase mutants has suggested that FB1 is a more potent inhibitor of Class II ceramide synthases (i.e. LOH1 and LOH3 ceramide synthases) (
Markham et al. 2011). Interestingly, in addition to accumulation of free LCBs, elevated levels of ceramides with C16 fatty acids and dihydroxy LCBs formed by Class I ceramide synthases (i.e. LOH2 ceramide synthase) are detectable following treatment of Arabidopsis with FB1 (
Markham et al. 2011). These results suggest that FB1 cytotoxicity and PCD induction may be triggered by accumulated ceramides rather than or in addition to accumulated LCBs. FB1 has also been used as a tool to study sphingolipid homeostasis in plants based on the observation that down-regulation of serine palmitoyltransferase (SPT) activity reduces FB1 cytotoxicity and up-regulation of SPT activity enhances sensitivity of plants to FB1 (
Kimberlin et al. 2013;
Shi et al. 2007).
Glucosylceramide Synthesis
Following its synthesis by Class I or Class II ceramide synthases, the ceramide backbone can be glycosylated at its C-1 OH to form either of two classes of glycosphingolipids: glucosylceramides (GlcCer) or glycosyl inositolphosphoceramides (GIPCs). GlcCer are the simplest glycosphingolipid and occur broadly in eukaryotes, with the notable exception of Saccharomyces cerevisiae (
Lynch and Dunn 2004). GlcCer consist of a glucose bound to the ceramide backbone by a 1,4-glycosidic linkage and are formed by the condensation of a ceramide substrate with UDP-glucose (
Leipelt et al. 2001). This reaction is catalyzed by GlcCer synthase, an ER-localized enzyme in Arabidopsis that is encoded by At2g19980 (
Melser et al. 2010). Compared to GIPCs, GlcCer are more enriched in ceramides with C16 fatty acids and dihydroxy LCBs (
Markham et al. 2006;
Sperling et al. 2005). In plants such as tomato and soybean, ceramides with C16 fatty acids and the LCB d18:2 predominate (
Markham et al. 2006;
Sperling et al. 2005). Based on this composition, it appears that a large portion of the GlcCer ceramide backbone is channeled from Class I-type ceramide synthases that have substrate preference for C16 fatty acids and dihydroxy LCBs (
Markham et al. 2011). Although it is an abundant glycosphingolipid in plants, null mutants of the LCB Δ4 deasaturase in Arabidopsis have 30 % reductions in GlcCer levels in flowers (
Michaelson et al. 2009) and 50 % reduction in GlcCer levels in pollen (
Luttgeharm et al. 2015) without any apparent effect on flower and pollen physiology and function (
Michaelson et al. 2009). GlcCer synthase is potently inhibited by d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (Melser et al. 2010). Treatment of Arabidopsis roots with PDMP results in altered Golgi morphology, including reduced numbers of Golgi stacks, and defects in endomembrane trafficking (
Melser et al. 2010,
2011). PDMP application to Arabidopsis root cells has also been shown to result in rapid vacuolar fusion and altered vacuole morphology including the appearance of vacuolar invaginations (
Kruger et al. 2013). Arabidopsis GlcCer synthase mutants devoid of GlcCer have yet to be described. Such mutants will clarify whether GlcCer are essential in plants, which is an open question because viable fungal cells can be recovered that lack GlcCer (
Michaelson et al. 2009;
Rittenour et al. 2011).
Inositolphosphoceramide Synthesis
As an alternative fate to GlcCer synthesis, ceramides can be used for the production of GIPCs (
Glycosyl-
Inositol
Phospho
Ceramide). GIPCs, which are approximately twofold more abundant in Arabidopsis leaves than GlcCer, are typically enriched in ceramides with VLCFAs and trihydroxy LCBs that arise from Class II ceramide synthases
Markham et al. 2006). The first step in GIPC synthesis occurs by the transfer of the inositolphosphoryl head group of phosphatidylinositol (PI) onto ceramide to form inositolphosphoceramides (IPCs) (
Mina et al. 2010;
Wang et al. 2008). This activity is catalyzed by IPC synthase, a phosphatidic acid phosphatase-2 (PAP2)-related enzyme, that is encoded by three genes in Arabidopsis: IPCS1 (or ERH1), At2g37940; IPCS2, At2g37940; IPCS3, At2g29525 (
Mina et al. 2010;
Wang et al. 2008).
In contrast to the ER localization of GlcCer synthase, IPC synthases predominantly occur in Golgi bodies of Arabidopsis. Plant IPC synthases are most closely related to analogous enzymes in the protozoa Leishmania major and Trypanosoma brucei than to the Saccharomyces cerevisiae IPC synthase (encoded by the AUR1 gene) (
Mina et al. 2010;
Wang et al. 2008). Despite this, the three Arabidopsis IPC synthase genes are able to rescue lethality associated with the loss of IPC production in the Saccharomyces cerevisiae AUR1 mutant (
Mina et al. 2010;
Wang et al. 2008). Although triple mutants of the three Arabidopsis IPC synthase genes have not been reported, it is presumed that IPC biosynthesis is essential, as the three genes are likely partially redundant.
Abbreviated plant sphingolipid biosynthetic pathway. Abbreviations: LCB long-chain base, Glc glucose, PI phosphatidylinositol, DAG diacylglycerol, IP inositolphosphate, GIPCase glycosyl inositolphosphoceramidase, IPUT1 inositol phosphorylceramide glucuronosyltransferase 1
Following the synthesis of IPC, up to seven additional sugar residues can be added to the inositolphosphoryl head group to form an array of different GIPCs (
Bure et al. 2011;
Cacas et al. 2013). The first residue added to the inositophosphoryl head group is a glucuronic acid moiety (
Rennie et al. 2014). This reaction, which uses a UDP-glucuronic acid substrate, was recently shown to be catalyzed by a glycosyltransferase encoded by IPUT1 (At5g18480) in Arabidopsis (
Rennie et al. 2014). T-DNA null mutants of IPUT1 are not transmitted through pollen, indicating that this gene is essential in Arabidopsis (
Rennie et al. 2014). The remaining glycosyltransferases associated with GIPC synthesis have yet to be identified. Interestingly, a Golgi lumen-localized GDP-mannose transporter encoded by GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER 1 (GONST1, At2g13650) in Arabidopsis was found to be deficient in GIPCs with glycosylation beyond the glucuronic acid introduced by the IPUT1-encoded glycosyltransferase (
Mortimer et al. 2013). This suggests that like Saccharomyces cerevisiae, Arabidopsis GIPCs contain mannose, presumably bound to the glucuronic acid moiety. Interestingly, GONST1 mutants display a dwarfed phenotype, constitutive induction of the hypersensitive response, and elevated salicylic acid levels, consistent with a connection between GIPC synthesis and plant pathogen defense (
Mortimer et al. 2013).
