Metabolic routing of sphingolipid intermediates

Apart from their physiological function in plants, structural modifications on the ceramide backbone may also have a role in channelling ceramide substrates into downstream complex sphingolipid synthesis. As mentioned, GlcCer and GIPC formation display two alternative pathways in sphingolipid metabolism (Fig. 2). Especially the hydroxylation and desaturation state of the LCB moiety is considered to be responsible for dictating the metabolic fate of precursor compounds. Previous studies on A. thaliana showed that the trihydroxy LCB, mostly t18:1 with the double bond in Δ8 position, is the most abundant moiety in GIPCs while LCB Δ4-desaturation likely plays a key role in channelling substrates into GlcCer formation (Chen et al., 2008; Michaelson et al., 2009). The t18:1 LCB moiety of GIPCs is mainly found in association with α-hydroxylated VLCFAs, while the d18:2 LCB moiety of A. thaliana pollen GlcCers and of species such as tomato (Solanum lycopersicum) and soybean (Glycine max) is mostly connected with the α-hydroxylated LCFA C16. The channelling function of the C-4 hydroxylation and the LCB Δ4-desaturation is supported by the fact that both reactions happen on the C-4 position of the LCB moiety which makes them mutually exclusive.

17 The prevalence of certain ceramide modifications in GlcCers and GIPCs appears to have direct effects on the physiological functions of the two complex sphingolipid classes.

The typical ceramide backbone found in GlcCer and GIPC species is usually highly hydroxylated, both on the LCB as well as on the fatty acid side. The hydroxylation status of sphingolipids is considered essential for building up an extensive hydrogen bond network with other membrane components (Slotte, 1999; Mombelli et al., 2003; Slotte, 2016). This is especially important for the interaction with phytosterols during lipid raft formation (Mamode Cassim et al., 2019). A. thaliana has two functionally redundant LCB C-4 hydroxylases, SBH1 and SBH2. The combined activities of both enzymes account for all trihydroxy LCB formation in the plant. Both genes were able to complement the S. cerevisae LCB C-4 hydroxylase knockout sur2Δ (Sperling et al., 2001). Knockout of both hydroxylase encoding genes led to severely dwarfed plants that were likely disturbed in cell elongation and division and that did not reach reproductive maturity (Chen et al., 2008). Additionally, knockout plants showed necrotic cotyledon lesions which were accompanied by the up-regulation of defence-related marker genes. Knockout of both genes caused serious alterations in all sphingolipid classes (Chen et al., 2008). The most prominent observation was a drastic accumulation of sphingolipids containing dihydroxy LCB moieties and C16 fatty acid moieties. Furthermore, the most abundant LCB moiety in all sphingolipid classes switched from trihydroxy LCBs to dihydroxy LCBs (Chen et al., 2008). Since sphingolipid content and composition were both affected in the mutant, the phenotype was speculated to derive either from the unusual accumulation of sphingolipids with dihydroxy LCBs and C16 fatty acids or from a global change in the most abundant LCB moiety from trihydroxy to dihydroxy LCBs. Similar to the LCB C-4 hydroxylases, A. thaliana harbours two redundant fatty acid hydroxylases, FAH1 and FAH2 (König et al., 2012). The fah1 fah2 mutant has reduced levels of α-hydroxylated sphingolipids and instead showed elevated levels of sphingolipids with unhydroxylated fatty acid moieties. Furthermore, trihydroxy LCBs and ceramides were enriched five- and ten-fold, respectively, and total GlcCer levels were reduced by 25 % compared to the wild type. These alterations in the sphingolipidome were accompanied by reduced plant size, elevated SA levels, constitutive PR gene expression and an associated increased resistance against the obligate biotrophic pathogen Golovinomyces cichoracearum (König et al., 2012).

Another important feature is the prevalence of VLCFAs in GlcCers and GIPCs.

Longer acyl chains increase the hydrophobicity and the membrane transition from fluid to gel phase state (Pinto et al., 2014; Mamode Cassim et al., 2019). Additionally, the chain length of the fatty acid moiety is considered a crucial feature in the interdigitation and therefore in the connection of the inner and outer PM monolayers (Mamode Cassim et al., 2019). Sphingolipids with VLCFAs are reported to have a crucial role in development

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(Markham et al., 2011; Molino et al., 2014). Inhibition of ceramide synthases that are specific for VLCFA substrates caused defects in root-outgrowth. On a subcellular level, defects in VLCFA-containing sphingolipids resulted in impaired membrane trafficking of auxin proteins to the PM (Markham et al., 2011). Molino et al. (2014) also demonstrated altered cell plate formation in plants whose VLCFA incorporating ceramide synthase LOH1 was blocked by the mycotoxin FB1 (Molino et al., 2014). The authors propose a function for VLCFA-containing sphingolipids in lipid bilayer fusion and therefore in vesicle dynamics during development.

Although GlcCer and GIPC architectures share some structural features, the A. thaliana GlcCer pool differs in certain molecular species from the GIPC pool.

A. thaliana GlcCers are enriched in the Δ4,8-diunsaturated, d18:2, LCB moiety compared to GIPCs (Markham et al., 2006). Plants lacking the two LCB Δ8-desaturases, SLD1 and SLD2, have GlcCer levels that are 50 % reduced compared to the wild type and the mutants are more sensitive to cold stress (Chen et al., 2012). It might be that the configuration state of the Δ8 double bond plays a role in shunting t18:1 species into GlcCer or GIPC formation. In contrast to that, knockout mutants of the A. thaliana LCB Δ4-desaturase did not show any obvious phenotypes (Michaelson et al., 2009). In A. thaliana the d18:2 LCB moiety is enriched in pollen and floral tissue (Michaelson et al., 2009). GlcCer levels were also significantly reduced in the LCB Δ4-desaturase knockout plant, indicating that LCB Δ4-desaturation has indeed a channelling function for GlcCer formation (Michaelson et al., 2009). However, pollen and general plant viability was not affected in the A. thaliana mutant plants. While Δ8-desaturation is one of the most abundant LCB modifications found in A. thaliana sphingolipids, LCB Δ4-desaturation appears to not have a significant physiological role in Brassicaceae (Markham et al., 2006). A lipidomics screen covering 21 plants from different lineages identified the prevalence of the LCB double bond position in d18:1 LCB moieties (Islam et al., 2012). They revealed that LCB Δ4-desaturation is most common to non-vascular plants and to the Poales family whereas LCB Δ8-desaturation is most abundant in plants like Brassicaceae. The authors speculated that LCB Δ4-desaturation appears to be more ancient than LCB Δ8-desaturation. Interestingly, in plants like tomato and soybean ceramides with a Δ4,8-diunsaturated LCB moiety and C16 fatty acids are most abundant (Markham et al., 2006). This suggests that the LCB desaturation state was subject to divergent evolution and that LCB Δ4-desaturation likely has a more important physiological role in plants outside the Brassicaceae family. In the filamentous fungus Pichia pastoris loss of LCB Δ4-desaturation has more pronounced metabolic effects resulting in complete abolishment of GlcCers (Michaelson et al., 2009).

Plants that lack all GlcCers are seedling lethal and plants with a disturbed GlcCer formation show defects in cell differentiation and organogenesis, likely due to an impaired intracellular

19 2017). The head group composition is strongly specific for certain plant species and tissue types (Buré et al., 2011; Cacas et al., 2013; Luttgeharm et al., 2015b). Alterations of the glycan head group composition is reported to have severe effects on plant viability.

Knockout of one of the three inositolphosphoceramide synthases, ERH1, resulted in GIPC reduction and accumulation of the ceramide precursor, which was accompanied by the onset of cell death symptoms (Wang et al., 2008). Knockout of subsequent enzymes that catalyse conjugation of different sugar residues resulted in mutants that were either lethal or had severe growth defects (Mortimer et al., 2013; Rennie et al., 2014; Tartaglio et al., 2017).

Taken together, studies on distinct A. thaliana sphingolipid mutants indicate that LCB modifications have a strong influence on the metabolic flux of sphingolipid compounds, and that distinct structural features of GlcCers and GIPCs have important effects on their physiological function. Especially LCB C-4 hydroxylation and LCB Δ4-desaturation appear to be of great importance for the downstream metabolic fate of sphingolipids. In A. thaliana, the channelling of sphingolipid metabolites seems to also be partially controlled by the ratio of cis and trans Δ8 double bonds (Markham et al., 2006; Markham & Jaworski, 2007).

However, the exact channelling process of sphingolipid intermediates in plants is not yet fully elucidated, in part because of the large complexity of the A. thaliana sphingolipidome.

Furthermore, although sphingolipid biosynthesis is broadly conserved among plants of different taxonomic groups, sphingolipid composition differs between plant species and even between different tissues of the same plant (Sperling et al., 2005; Markham et al., 2006; Markham & Jaworski, 2007; Luttgeharm et al., 2015b). This underlines that distinct sphingolipid species have different physiological roles which may be more or less important in certain plants and plant tissues. This also includes a potential divergent evolution of different pathogen interaction systems in plants of different taxonomic groups. A recent study gave a great example for this and showed that GIPCs can act as necrosis and ethylene-inducing peptide 1–like (NLP) toxin receptors in eudicots but not in monocots (Lenarčič et al., 2017). The authors concluded that this host selectivity may be due to the glycan head group composition which is known to be plant species- and tissue-dependent (Buré et al., 2011; Cacas et al., 2013). These observations indicate that sphingolipid metabolism diverged during land plant evolution. However, the functional relevance for this diversification is largely unknown.

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