Glycosylinositol phosphorylceramides (GIPCs) constitute an important class of plasma-membrane lipids in plants. The complex glycan headgroups of GIPCs vary between plant species and tissues. Recent studies have shown that the structure of the glycan headgroup is important for plant development, abiotic stress tolerance, and interactions with pathogenic and symbiotic microorganisms

Mortimer, Jenny C

Scheller, Henrik Vibe

English

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UC BerkeleyUC Berkeley Previously Published WorksTitleSynthesis and Function of Complex Sphingolipid GlycosylationPermalinkhttps://escholarship.org/uc/item/6xn8z69sJournalTrends in Plant Science, 25(6)ISSN1360-1385AuthorsMortimer, Jenny CScheller, Henrik VibePublication Date2020-06-01DOI10.1016/j.tplants.2020.03.007 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of California 1 Article submission number: PLANTS-D-20-00012 1 Synthesis and function of complex sphingolipid glycosylation  2 Jenny C. Mortimer1,2 and Henrik Vibe Scheller1,2,3 3 1Joint BioEnergy Institute, Emeryville, California 94608, United States 4 2Environmental Genomics and Systems Biology Division, Lawrence Berkeley National 5 Laboratory, Berkeley, California 94720, United States 6 3Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, 7 California 94720 8  9 *Correspondence: jcmortimer@lbl.gov (J.C. Mortimer) or hscheller@lbl.gov (H.V. 10 Scheller) 11 Keywords 12 GIPC, glycosylinositol phosphorylceramide, plasma membrane, phytoglycolipid 13 Abstract 14 Glycosylinositol phosphorylceramides (GIPCs) constitute an important class of plasma 15 membrane lipids in plants. The complex glycan headgroup of GIPCs varies between plant 16 species and tissues. Recent studies have shown that the structure of the glycan 17 headgroup is important for development, abiotic stress tolerance, and interactions with 18 pathogenic and symbiotic microorganisms.   19   20  2 Main text 1 Glycosylinositol phosphorylceramides (GIPCs) are the major class of sphingolipids in 2 plants. They are thought to be found primarily in the outer leaflet of the plasma membrane, 3 where they are estimated to make up more than half of the total lipid. They consist of a 4 complex polar glycan headgroup, which protrudes into the apoplast, linked to a ceramide 5 (Figure 1, 2). Sphingolipids were first identified in mammals in 1884 by Johann 6 Thudichum, and it is claimed that he named them after the riddle of the Sphinx, due to 7 their enigmatic nature. GIPCs were first identified in plants in the 1950s 8 (“phytoglycolipids”). However, research on them essentially ceased in the 1970s, in part 9 due to their insolubility in standard lipid-extraction protocols. New analytical techniques 10 developed in 2006 allowed them to be “rediscovered” [1]. GIPCs are unique to plants, 11 fungi and some protozoans. While animal sphingolipids also have a ceramide group, the 12 polar headgroup is different from the glycosylinositol group in plants.   13 A recent review described important roles of GIPCs in programmed cell death and 14 response to both abiotic and biotic stress in plants [2]. The review by Huby et al. focused 15 on mutants affected in the biosynthesis of the ceramide part of GIPCs, which are generally 16 severely impacted in their ability to synthesize GIPCs. However, several recent studies 17 have shown that modification to the glycan headgroup of GIPCs can cause severe growth 18 defects or lethality, depending on which sugars are lost, even if ceramides are 19 synthesized and accumulate normally. Following synthesis of the ceramide in the ER, it 20 moves to the Golgi where the inositol phosphate ceramide synthases (IPCSs) add the 21 inositol phosphate group [3]. We and our collaborators then identified the proteins 22 responsible for three enzymatic activities in the subsequent glycosylation pathway (and 23  3 showed their importance in arabidopsis (Arabidopsis thaliana], rice (Oryza sativa) and 1 Medicago truncatula) [4–7]. We also identified three Golgi-localized transporters required 2 for delivering substrates to the GIPC glycosyltransferases [8–10]. Knock-out mutations in 3 the IPUT1 gene encoding the glucuronosyltransferase that adds the first sugar in the 4 glycan headgroup cause lethality, even though the precursor inositol phosphorylceramide 5 accumulates in the plants [6]. Mutant pollen tubes grow at normal rates but are severely 6 impacted in their ability to reach the ovule and cause fertilization [5]. However, rescue of 7 the pollen function results in plants that are severely affected in vegetative development, 8 showing that GIPC glycosylation is also important for other stages of development. While 9 all plant GIPCs have glucuronic acid as the first sugar, the subsequent sugars vary 10 between tissues and species [11]. In arabidopsis, mannose is the predominant second 11 sugar residue, and plants deficient in adding mannose to glucuronic acid are strongly 12 inhibited in growth [4,9,10]. The first mutants found to be deficient in mannosylated GIPCs 13 were mutated in a gene encoding the Golgi-localized GDP-mannose transporter, 14 GONST1 [9]. The only known function of this transporter and its homolog GONST2 [10] 15 is to provide GDP-mannose for the GIPC mannosyltransferase GMT1. This transferase 16 was identified in a mutant with a phenotype similar to gonst1 (including a cell wall with 17 reduced cellulose content, and a constitutive defense response), and was shown to 18 belong to glycosyltransferase family GT64 [4]. Another member of this family, GINT1, was 19 shown to add N-acetylglucosamine (GlcNAc) rather than mannose to glucuronic acid of 20 GIPCs [7]. In rice the second sugar is always GlcN[Ac] and Osgint1 mutants exhibit 21 seedling lethality. Arabidopsis has GlcN[Ac] GIPCs as a minor form in seeds, but this form 22 is not essential for seed development. In Medicago truncatula roots, the mannose form is 23  4 the major form present, while the GlcN[Ac] form is a minor component. However, the 1 GINT1 GlcNAc transferase gene is highly upregulated in root nodules and in roots 2 infected with arbuscular mycorrhizal fungi. Downregulation of GINT1 has no apparent 3 impact on root growth in the absence of symbionts but strongly inhibits the development 4 of functional root nodules and arbuscules (Moore, Mortimer and Scheller, in review). 5 Notably, arabidopsis has a third member of the GT64 family with so far unknown function. 6 It seems likely that it is involved in GIPC biosynthesis, perhaps in a specific molecular 7 form that has not been clearly identified. 8 More recently, GIPC glycosylation has been identified as a receptor for a toxin from the 9 plant pathogen Phytophthora infestans [12] and a salt sensor for the plasma membrane 10 Ca2+ influx channel [13]. Many plant pathogens produce necrosis and ethylene-inducing 11 peptide 1-like (NLP)-proteins, which can aid infection. NLPs from oomycetes were shown 12 to bind to GIPCs (but not other plant lipids), as well as to oligosaccharides that correspond 13 to GIPC headgroups, although with lower affinity than to the GIPCs [12]. Only eudicots 14 are sensitive to NLPs, whereas monocots are resistant, with the exception of the 15 monocots Phalaenopsis amabilis. Monocot GIPC pools tend to be dominated by 16 headgroups with three sugar residues on the inositol phosphorylceramide (IPC) core, 17 whereas dicots tend to have two-sugar headgroups. Interestingly, P. amabilis also has a 18 mixture of 2- and 3-sugar GIPCs, implying that the number of sugar units could be an 19 important determinant of pathogenicity. Testing of glycan headgroup mutants for NLP 20 sensitivity could help to confirm this. In another recent study, a new iput1 allele was 21 identified in a forward genetic screen for arabidopsis mutants defective in salt-dependent 22 calcium influxes, moca1 [13]. moca1 has a 4 amino acid deletion in IPUT1, but still retains 23  5 ~10% of wild-type GIPCs, and it grows normally under standard conditions.  Previously, 1 the gint1 mutant had been shown to be less sensitive to the effects of salinity on 2 germination rates [7], supporting a role for GIPC glycosylation in salt sensing. Jiang et al. 3 used isothermal titration calorimetry to demonstrate that GIPCs can bind Na+, and then 4 proposed a model in which this depolarizes the cell surface potential and gates the Ca2+ 5 channel [13].  Multiple other processes that center around plasma membrane-spanning 6 proteins are also disrupted in GIPC-glycan headgroup mutants, including cellulose 7 synthesis [4] and signal transduction [4,5]. Taken together, these studies show that subtle 8 changes to the molecular structure of the GIPC headgroup can have dramatic effects on 9 function.  10 Future work will require better tools for GIPC analysis. Given the condition/cell-specific 11 nature of the phenotypes described above, tools that support live cell imaging such as 12 GIPC-specific dyes, and pharmacological inhibitors which can specifically inhibit 13 synthesis, are particularly critical. Identification of a gene encoding a GIPC-specific 14 phospholipase (such as that reported from white cabbage (Brassica oleracea) in [14]) 15 would enable facile release of the glycan headgroup, and support detailed analysis of 16 sugar identity and linkage. Finally, the discovery of additional glycosyltransferases 17 responsible for the headgroup synthesis could be combined with synthetic biology tools, 18 and potentially deliver crops with altered plant-microbe interactions, such as resistance 19 to pathogens or enhanced arbuscular mycorrhizal symbiosis. 20  21 Acknowledgements 22  6 JCM and HVS are funded as part of the DOE Joint BioEnergy Institute 1 (http://www.jbei.org) supported by the US Department of Energy, Office of Science, 2 through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory 3 and the US Department of Energy.  4  5 References 6  7 1  Markham, J.E. et al. (2006) Separation and identification of major plant sphingolipid 8 classes from leaves. J. Biol. Chem. 281, 22684–22694 9 2  Huby, E. et al. (2020) Sphingolipids: towards an integrated view of metabolism 10 during the plant stress response. New Phytol. 225, 659–670 11 3  Wang, W. et al. (2008) An inositolphosphorylceramide synthase is involved in 12 regulation of plant programmed cell death associated with defense in Arabidopsis. 13 Plant Cell 20, 3163–3179 14 4  Fang, L. et al. (2016) Loss of inositol phosphorylceramide sphingolipid 15 mannosylation induces plant immune responses and reduces cellulose content in 16 arabidopsis. Plant Cell 28, 2991–3004 17 5  Tartaglio, V. et al. (2017) Glycosylation of inositol phosphorylceramide sphingolipids 18 is required for normal growth and reproduction in Arabidopsis. Plant J. 89, 278–290 19 6  Rennie, E.A. et al. (2014) Identification of a sphingolipid α-glucuronosyltransferase 20 that is essential for pollen function in Arabidopsis. Plant Cell 26, 3314–3325 21 7  Ishikawa, T. et al. (2018) GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE 22 TRANSFERASE1 (GINT1) Is a GlcNAc-Containing Glycosylinositol 23  7 Phosphorylceramide Glycosyltransferase. Plant Physiol. 177, 938–952 1 8  Ebert, B. et al. (2018) A Golgi UDP-GlcNAc transporter delivers substrates for N-2 linked glycans and sphingolipids. Nat. Plants 4, 792–801 3 9  Mortimer, J.C. et al. (2013) Abnormal glycosphingolipid mannosylation triggers 4 salicylic acid-mediated responses in Arabidopsis. Plant Cell 25, 1881–1894 5 10  Jing, B. et al. (2018) GONST2 transports GDP-Mannose for sphingolipid 6 glycosylation in the Golgi apparatus of Arabidopsis. BioRxiv DOI: 10.1101/346775 7 11  Buré, C. et al. (2014) Characterization of glycosyl inositol phosphoryl ceramides 8 from plants and fungi by mass spectrometry. Anal. Bioanal. Chem. 406, 995–1010 9 12  Lenarčič, T. et al. (2017) Eudicot plant-specific sphingolipids determine host 10 selectivity of microbial NLP cytolysins. Science 358, 1431–1434 11 13  Jiang, Z. et al. (2019) Plant cell-surface GIPC sphingolipids sense salt to trigger 12 Ca2+ influx. Nature 572, 341–346 13 14  Tanaka, T. et al. (2013) Identification of a sphingolipid-specific phospholipase D 14 activity associated with the generation of phytoceramide-1-phosphate in cabbage 15 leaves. FEBS J. 280, 3797–3809 16   17 Figures legends 18 Figure 1: Structure of GIPCs. (A) Example structure of a GIPC showing the ceramide 19 (black), inositol phosphate and glucuronic acid (GlcA) (green), and mannose (purple). 20 The inositol phosphorylceramide (IPC) linked to a GlcA is conserved in higher plants. 21 The additional sugars vary based on species and tissue type. (B) Common GIPC 22 structures discussed in this review. The shown structures have only two or three sugar 23  8 residues, but GIPCs with longer glycan groups also exist. The Symbol Nomenclature for 1 Glycans (SNFG) is used to represent the structures. This figure was created using 2 BioRender (https://biorender.com/). 3  4 Figure 2: A schematic of our current view of the plant plasma membrane. The figure 5 highlights the asymmetry between the inner and outer leaflet, and how the glycan 6 headgroups can be tightly packed on the surface. Examples of classes of membrane 7 proteins are shown (not to scale), the functions of which have been proposed to be 8 affected by GIPC headgroup structure. The Cellulose Synthase Complex is shown with 9 examples of proteins which affect cellulose synthesis, including GPI-anchored proteins 10 (e.g. COBRA) and transmembrane proteins (e.g. KORRIGAN). This figure was created 11 using BioRender (https://biorender.com/). 12  13  14 (B)IPCIPCIPCIPCFigure 1: (A) Example structure of a GIPC showing the ceramide(black), inositol phosphate and glucuronic acid (GlcA) (green), andmannose (purple). The inositol phosphorylceramide (IPC) linked to aGlcA is conserved in higher plants. The additional sugars vary basedon species and tissue type. (B) Common GIPC structures discussed inthis review. The shown structures have only two or three sugarresidues, but GIPCs with longer glycan groups also exist. The SymbolNomenclature for Glycans (SNFG) is used to represesent thestructures. GlcAHex GlcNAcManIPCIPCGlcN(A)Celluose synthasecomplex (CSC) Iontransporter ReceptorsCytosolApoplastFigure 2: A schematic of our current view of the plantplasma membrane. It highlights the assymetery betweenthe inner and outer leaflet, and how the glycan headgroupscan be tightly packed on the surface. Examples of classesof membrane proteins are shown (not to scale), thefunctions of which have been proposed to be affected byGIPC headgroup structure. The CSC is shown withexamples of proteins which affect cellulose synthesis,including GPI anchored proteins (e.g. COBRA) andtransmembrane proteins (e.g. KORRIGAN).GIPCSterolGlucosylceramide PhospholipidGPI-anchored protein

Synthesis and Function of Complex Sphingolipid Glycosylation

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Synthesis and Function of Complex Sphingolipid Glycosylation.

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