NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Balanced callose and cellulose biosynthesis in Arabidopsis quorum-sensing signaling and pattern-triggered immunity.

The plant cell wall (CW) is one of the most important physical barriers that phytopathogens must conquer to invade their hosts. This barrier is a dynamic structure that responds to pathogen infection through a complex network of immune receptors, together with CW-synthesizing and CW-degrading enzymes. Callose deposition in the primary CW is a well-known physical response to pathogen infection. Notably, callose and cellulose biosynthesis share an initial substrate, UDP-glucose, which is the main load-bearing component of the CW. However, how these 2 critical biosynthetic processes are balanced during plant-pathogen interactions remains unclear. Here, using 2 different pathogen-derived molecules, bacterial flagellin (flg22) and the diffusible signal factor (DSF) produced by Xanthomonas campestris pv. campestris, we show a negative correlation between cellulose and callose biosynthesis in Arabidopsis (Arabidopsis thaliana). By quantifying the abundance of callose and cellulose under DSF or flg22 elicitation and characterizing the dynamics of the enzymes involved in the biosynthesis and degradation of these 2 polymers, we show that the balance of these 2 CW components is mediated by the activity of a ß-1,3-glucanase (BG2). Our data demonstrate balanced cellulose and callose biosynthesis during plant immune responses.

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian-dinoflagellate symbiosis.

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan-lectin interactions in host-symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular ß-D-galactose) residues in the establishment of the cnidarian-dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.

The state of the art in plant lipidomics.

Lipids are a group of compounds with diverse structures that perform several important functions in plants. To unravel and better understand their in vivo functions, plant biologists have been using various lipidomic technologies including liquid-chromatography (LC)-mass spectrometry (MS). However, there are still significant challenges in LC-MS based plant lipidomics, which need to be addressed. In this review, we provide an overview of the key developments in LC-MS based lipidomic approaches to detect and identify plant lipids with emphasis on areas that can be further improved. Given that the cellular lipidome is estimated to contain hundreds of thousands of lipids,1,2 many of the lipid structures remain to be discovered. Furthermore, the plant lipidome is considered to be significantly more complex compared to that of mammals. Recent technical developments in mass spectrometry have made the detection of novel lipids possible; hence, approaches that can be used for plant lipid discovery are also discussed.

Not Just a Simple Sugar: Arabinose Metabolism and Function in Plants.

Growth, development, structure as well as dynamic adaptations and remodeling processes in plants are largely controlled by properties of their cell walls. These intricate wall structures are mostly made up of different sugars connected through specific glycosidic linkages but also contain many glycosylated proteins. A key plant sugar that is present throughout the plantae, even before the divergence of the land plant lineage, but is not found in animals, is l-arabinose (l-Ara). Here, we summarize and discuss the processes and proteins involved in l-Ara de novo synthesis, l-Ara interconversion, and the assembly and recycling of l-Ara-containing cell wall polymers and proteins. We also discuss the biological function of l-Ara in a context-focused manner, mainly addressing cell wall-related functions that are conferred by the basic physical properties of arabinose-containing polymers/compounds. In this article we explore these processes with the goal of directing future research efforts to the many exciting yet unanswered questions in this research area.

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development.

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi.

A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.

Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), ß-1,6-galactotetraose (ß-1,6-Gal4) and ß-1,3-galactopentose (ß-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the ß-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.

A comprehensive comparison of four methods for extracting lipids from Arabidopsis tissues.

BACKGROUND: The plant lipidome is highly complex, and the composition of lipids in different tissues as well as their specific functions in plant development, growth and stress responses have yet to be fully elucidated. To do this, efficient lipid extraction protocols which deliver target compounds in solution at concentrations adequate for subsequent detection, quantitation and analysis through spectroscopic methods are required. To date, numerous methods are used to extract lipids from plant tissues. However, a comprehensive analysis of the efficiency and reproducibility of these methods to extract multiple lipid classes from diverse tissues of a plant has not been undertaken. RESULTS: In this study, we report the comparison of four different lipid extraction procedures in order to determine the most effective lipid extraction protocol to extract lipids from different tissues of the model plant Arabidopsis thaliana. CONCLUSION: While particular methods were best suited to extract different lipid classes from diverse Arabidopsis tissues, overall a single-step extraction method with a 24 h extraction period, which uses a mixture of chloroform, isopropanol, methanol and water, was the most efficient, reproducible and the least labor-intensive to extract a broad range of lipids for untargeted lipidomic analysis of Arabidopsis tissues. This method extracted a broad range of lipids from leaves, stems, siliques, roots, seeds, seedlings and flowers of Arabidopsis. In addition, appropriate methods for targeted lipid analysis of specific lipids from particular Arabidopsis tissues were also identified.

UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex.

Rhamnogalacturonan-II (RG-II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG-II molecules can form an RG-II-borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross-linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG-II dimer is still unclear. In this study we investigated the two homologous UDP-D-apiose/UDP-D-xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP-D-apiose (UDP-Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP-Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG-II-borate complex than wild-type Col-0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP-Api and RG-II-borate complex formation in plant growth and development.

A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia.

We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.

Profiling Cell Wall Monosaccharides and Nucleotide-Sugars from Plants.

The cell wall is an intricate mesh largely composed of polysaccharides that vary in structure and abundance. Apart from cellulose biosynthesis, the assembly of matrix polysaccharides such as pectin and hemicellulose occur in the Golgi apparatus before being transported via vesicles to the cell wall. Matrix polysaccharides are biosynthesized from activated precursors or nucleotide sugars. The composition and assembly of the cell wall is an important aspect in plant development and plant biomass utilization. The application of anion-exchange chromatography to determine the monosaccharide composition of the insoluble matrix polysaccharides enables a complete profile of all major sugars in the cell wall from a single run. While porous carbon graphite chromatography and tandem mass spectrometry delivers a sensitive and robust nucleotide sugar profile from plant extracts. Here we describe detailed methodology to quantify nucleotide sugars within the cell and profile the non-cellulosic monosaccharide composition of the cell wall. © 2019 by John Wiley & Sons, Inc.

Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3.

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.

A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids.

Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cytosolic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphorylceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids.

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.

Three UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis.

UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.

Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass.

BACKGROUND: Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. RESULTS: We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. CONCLUSION: The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.

Absolute Quantitation of In Vitro Expressed Plant Membrane Proteins by Targeted Proteomics (MRM) for the Determination of Kinetic Parameters.

The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination of the turnover number of an enzyme-substrate complex (k cat) requires that the catalytic site concentration (amount of protein) in the total reaction volume is known. As a result, we have been employing targeted proteomics (multiple reaction monitoring) with peptide standards and a triple quadrupole mass spectrometer to estimate the absolute amount of protein in a mixed protein microsomal fraction. The following method details the steps required to define the absolute quantitation of an in vitro expressed membrane protein to define complete kinetic parameters.

The elaborate route for UDP-arabinose delivery into the Golgi of plants.

In plants, L-arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDP-Araf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transported back into the lumen. This step is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Thus, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgi-localized UDP-Araf transporters in Arabidopsis The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage.

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.