Corrigendum: Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

This corrects the article DOI: 10.1038/nature21725.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

Transport of UDP-rhamnose by URGT2, URGT4, and URGT6 modulates rhamnogalacturonan-I length.

Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.

RUBY, a Putative Galactose Oxidase, Influences Pectin Properties and Promotes Cell-To-Cell Adhesion in the Seed Coat Epidermis of Arabidopsis.

Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis (Arabidopsis thaliana) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE (RUBY), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUBY is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 (mum2) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains.

Natural Variation Reveals a Key Role for Rhamnogalacturonan I in Seed Outer Mucilage and Underlying Genes.

On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat.

Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer.

Local evolution of seed flotation in Arabidopsis.

Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 ß-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells.

Imbibed seeds of the Arabidopsis thaliana accession Djarly are affected in mucilage release from seed coat epidermal cells. The impaired locus was identified as a pectin methylesterase inhibitor gene, PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), specifically expressed in seed coat epidermal cells at the time when mucilage polysaccharides are accumulated. This spatio-temporal regulation appears to be modulated by GLABRA2 and LEUNIG HOMOLOG/MUCILAGE MODIFIED1, as expression of PMEI6 is reduced in mutants of these transcription regulators. In pmei6, mucilage release was delayed and outer cell walls of epidermal cells did not fragment. Pectin methylesterases (PMEs) demethylate homogalacturonan (HG), and the majority of HG found in wild-type mucilage was in fact derived from outer cell wall fragments. This correlated with the absence of methylesterified HG labeling in pmei6, whereas transgenic plants expressing the PMEI6 coding sequence under the control of the 35S promoter had increased labeling of cell wall fragments. Activity tests on seeds from pmei6 and 35S:PMEI6 transgenic plants showed that PMEI6 inhibits endogenous PME activities, in agreement with reduced overall methylesterification of mucilage fractions and demucilaged seeds. Another regulator of PME activity in seed coat epidermal cells, the subtilisin-like Ser protease SBT1.7, acts on different PMEs, as a pmei6 sbt1.7 mutant showed an additive phenotype.

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter-connections of plant cell-wall matrix glycans.

Plant cell walls are complex, multi-macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell-wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell-wall matrix glycan sub-populations and inter-connections. The method combines chromatographic separations with use of glycan-directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell-wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub-populations and their modulation in A. thaliana organs.

The Deconstruction of Pectic Rhamnogalacturonan I Unmasks the Occurrence of a Novel Arabinogalactan Oligosaccharide Epitope.

Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls.

MAIN CONCLUSION: The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.

Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls.

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG-I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG-I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG-I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.

Plant and algal cell walls: diversity and functionality.

BACKGROUND: Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. SCOPE: The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.

Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants.

BACKGROUND: The epidermal cells of the seed coat of certain species accumulate polysaccharides during seed development for cell wall reinforcement or release on imbibition to form mucilage. Seed-coat epidermal cells show natural variation in their structure and mucilage production, which could explain the diverse ecophysiological roles proposed for the latter. Arabidopsis mucilage mutants have proved to be an important tool for the identification of genes involved in the production of seed-coat polysaccharides. SCOPE: This review documents genes that have been characterized as playing a role in the differentiation of the epidermal cells of the arabidopsis seed coat, the natural variability in polysaccharide features of these cells and the physiological roles attributed to seed mucilage. CONCLUSIONS: Seed-coat epidermal cells are an excellent model for the study of polysaccharide metabolism and properties. Intra- and interspecies natural variation in the differentiation of these epidermal cells is an under-exploited resource for such studies and promises to play an important part in improving our knowledge of polysaccharide production and ecophysiological function.

Characterization of citrus pectin samples extracted under different conditions: influence of acid type and pH of extraction.

BACKGROUND AND AIMS: Pectin is a complex macromolecule, the fine structure of which is influenced by many factors. It is used as a gelling, thickening and emulsifying agent in a wide range of applications, from food to pharmaceutical products. Current industrial pectin extraction processes are based on fruit peel, a waste product from the juicing industry, in which thousands of tons of citrus are processed worldwide every year. This study examines how pectin components vary in relation to the plant source (orange, lemon, lime, grapefruit) and considers the influence of extraction conditions on the chemical and macromolecular characteristics of pectin samples. METHODS: Citrus peel (orange, lemon, lime and grapefruit) from a commercial supplier was used as raw material. Pectin samples were obtained on a bulk plant scale (kilograms; harsh nitric acid, mild nitric acid and harsh oxalic acid extraction) and on a laboratory scale (grams; mild oxalic acid extraction). Pectin composition (acidic and neutral sugars) and physicochemical properties (molar mass and intrinsic viscosity) were determined. KEY RESULTS: Oxalic acid extraction allowed the recovery of pectin samples of high molecular weight. Mild oxalic acid-extracted pectins were rich in long homogalacturonan stretches and contained rhamnogalacturonan I stretches with conserved side chains. Nitric acid-extracted pectins exhibited lower molecular weights and contained rhamnogalacturonan I stretches encompassing few and/or short side chains. Grapefruit pectin was found to have short side chains compared with orange, lime and lemon. Orange and grapefruit pectin samples were both particularly rich in rhamnogalacturonan I backbones. CONCLUSIONS: Structural, and hence macromolecular, variations within the different citrus pectin samples were mainly related to their rhamnogalacturonan I contents and integrity, and, to a lesser extent, to the length of their homogalacturonan domains.

Pectin-modifying enzymes and pectin-derived materials: applications and impacts.

Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Biochemical data documenting variations in mucilage polysaccharides in a range of glycosyltransferase mutants.

During Arabidopsis seed coat development, copious amounts of mucilage polysaccharides are produced in the epidermal cells. When hydrated on imbibition, these polysaccharides expand and are released to encapsulate the seed as a two-layered hydrogel. Polysaccharides are synthesized from UDP-sugars by glycosyltransferases (GTs) and several GTs, with differing activities, have been identified that contribute to mucilage polysaccharide synthesis. How these GTs orchestrate production of the complex polysaccharides found in mucilage remains to be determined. In this study, we generated a range of multiple GT mutants using either CRISPR/Cas9 targeted mutation or genetic crosses of existing T-DNA insertion mutants. Four traits for mucilage amounts or macromolecular properties were examined for four replicate seed lots from 31 different GT mutant combinations. This data provides a valuable resource for future genetic, biochemical, structural, and functional studies of the roles and properties of polysaccharides present in Arabidopsis mucilage and the relative contributions of different GTs to mucilage production.