Reprogramming sphingolipid glycosylation is required for endosymbiont persistence in Medicago truncatula.

Plant endosymbiosis relies on the development of specialized membranes that encapsulate the endosymbiont and facilitate nutrient exchange. However, the identity and function of lipids within these membrane interfaces is largely unknown. Here, we identify GLUCOSAMINE INOSITOL PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a sphingolipid glycosyltransferase highly expressed in Medicago truncatula root nodules and roots colonized by arbuscular mycorrhizal (AM) fungi and further demonstrate that this enzyme functions in the synthesis of N-acetyl-glucosamine-decorated glycosyl inositol phosphoryl ceramides (GIPCs) in planta. MtGINT1 expression was developmentally regulated in symbiotic tissues associated with the development of symbiosome and periarbuscular membranes. RNAi silencing of MtGINT1 did not affect overall root growth but strongly impaired nodulation and AM symbiosis, resulting in the senescence of symbiosomes and arbuscules. Our results indicate that, although M. truncatula root sphingolipidome predominantly consists of hexose-decorated GIPCs, local reprogramming of GIPC glycosylation by MtGINT1 is required for the persistence of endosymbionts within the plant cell.

The Three Members of the Arabidopsis Glycosyltransferase Family 92 are Functional ß-1,4-Galactan Synthases.

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched ß-1, 4-galactan. Plants in which all three genes were inactivated had no detectable ß-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) Is a GlcNAc-Containing Glycosylinositol Phosphorylceramide Glycosyltransferase.

Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis.

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis.

Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.

Identification of a sphingolipid α-glucuronosyltransferase that is essential for pollen function in Arabidopsis.

Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants.

Xylan biosynthesis.

Plant cells are surrounded by a rigid wall made up of cellulose microfibrils, pectins, hemicelluloses, and lignin. This cell wall provides structure and protection for plant cells. In grasses and in dicot secondary cell walls, the major hemicellulose is a polymer of ß-(1,4)-linked xylose units called xylan. Unlike cellulose--which is synthesized by large complexes at the plasma membrane--xylan is synthesized by enzymes in the Golgi apparatus. Xylan synthesis thus requires the coordinated action and regulation of these synthetic enzymes as well as others that synthesize and transport substrates into the Golgi. Recent research has identified several genes involved in xylan synthesis, some of which have already been used in engineering efforts to create plants that are better suited for biofuel production.

Pectin biosynthesis: GALS1 in Arabidopsis thaliana is a ß-1,4-galactan ß-1,4-galactosyltransferase.

ß-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, ß-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but ß-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased ß-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher ß-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto ß-1,4-galactopentaose. GALS1 specifically formed ß-1,4-galactosyl linkages and could add successive ß-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as ß-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

Three members of the Arabidopsis glycosyltransferase family 8 are xylan glucuronosyltransferases.

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of ß(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 µm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development.

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.

A comprehensive picture of phloem loading strategies.

Mechanisms of phloem loading in the minor veins of leaves are known for only a few species. We propose that there are a limited number of loading strategies for the primary photoassimilates, sucrose and sugar alcohols. These strategies can be predicted based on thermodynamic and anatomical considerations and identified by autoradiography of veins following uptake of (14)C-labeled compounds, analysis of leaf solute composition and concentrations, and plasmodesmatal counting. Experiments on 45 dicotyledonous species identified the predicted loading patterns. Over 50-fold differences in concentrations of sucrose and sugar alcohols in leaves were measured. The cumulative concentrations of transport compounds in leaves correlated with loading mechanisms, a previously unrecognized association. Comparisons of solute concentrations and osmotic potentials of whole leaves suggest that sucrose and sugar alcohols are more concentrated in the cytosol than in the vacuoles of mesophyll cells, thus increasing the driving force for passive loading in species that employ this strategy. Passive loading is more widespread than previously thought, especially in trees. The results indicate that plants have exploited all thermodynamically feasible and structurally compatible loading strategies and that these strategies can be identified with straightforward protocols.

Phloem loading strategies in three plant species that transport sugar alcohols.

Many plants translocate sugar alcohols in the phloem. However, the mechanism(s) of sugar alcohol loading in the minor veins of leaves are debated. We characterized the loading strategies of two species that transport sorbitol (Plantago major and apple [Malus domestica]), and one that transports mannitol (Asarina scandens). Plasmodesmata are abundant at all interfaces in the minor vein phloem of apple, and in one of two types of phloem in the minor veins of A. scandens. Few plasmodesmata are present in the minor veins of P. major. Apple differs from the other two species in that sugar alcohol and sucrose (Suc) are present in much higher concentrations in leaves. Apple leaf tissue exposed to exogenous [(14)C]sorbitol, [(14)C]Suc, or (14)CO(2) did not accumulate radiolabel in the minor veins, as determined by macroautoradiography. P. major minor veins accumulated radiolabel from [(14)C]Suc, [(14)C]sorbitol, and (14)CO(2). A. scandens minor veins accumulated (14)C from [(14)C]Suc and (14)CO(2), but not from [(14)C]mannitol. We conclude that the movement of sugar alcohol from the mesophyll into the phloem in apple and A. scandens is symplastic and passive, but in P. major it involves an apoplastic step and is energized. We also suggest that apple leaves transport sorbitol in high concentrations to avoid the feedback limitation of photosynthesis that would result from driving passive movement of solute into the phloem with high levels of Suc alone. The loading pathways and the mechanisms by which hydrostatic pressure is maintained in the minor vein phloem of these species are discussed.