Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.     

Mutations in Multiple XXT Genes of Arabidopsis Reveal the Complexity of Xyloglucan Biosynthesis

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.     

Arabidopsis GT34 family contains five xyloglucan α-1,6-xylosyltransferases

The Arabidopsis genome includes seven family 34 glycosyltransferase (GT34) encoding genes. XXT1 and XXT2 have previously been shown to encode XyG α-1,6-xylosyltransferases, while knockout mutants of a third, XXT5, exhibit decreased XyG content, suggesting a similar activity. Here, we extend the study to the rest of the Arabidopsis GT34 genes in terms of biochemical activity and their roles in XyG biosynthesis. The enzyme activity of XXTs was investigated using recombinant protein expressed in E. coli. XyG analysis of single and double T-DNA insertion knockouts, together with overexpression of GT34s in selected mutant lines, provided detailed function of each gene. We reveal the activity of the third member of the GT34 gene family (XXT4) that exhibits xylosyltransferase activity. Double mutants for either xxt2 or xxt5 had a large impact on XyG content, structure and size distribution. Overexpression of the remaining member, XXT3, was able to restore XyG epitopes in xxt2, xxt5 and xxt2 xxt5 double knockouts, suggesting that it also encodes a protein with XXT activity. Our work demonstrates that five of the seven Arabidopsis GT34 genes encode XXT enzymes.     

Changes in cell wall biomechanical properties in the xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis

The main load-bearing network in the primary cell wall of most land plants is commonly depicted as a scaffold of cellulose microfibrils tethered by xyloglucans. However, a xyloglucan-deficient mutant (xylosyltransferase1/xylosyltransferase2 [xxt1/xxt2]) was recently developed that was smaller than the wild type but otherwise nearly normal in its development, casting doubt on xyloglucan's role in wall structure. To assess xyloglucan function in the Arabidopsis (Arabidopsis thaliana) wall, we compared the behavior of petiole cell walls from xxt1/xxt2 and wild-type plants using creep, stress relaxation, and stress/strain assays, in combination with reagents that cut or solubilize specific components of the wall matrix. Stress/strain assays showed xxt1/xxt2 walls to be more extensible than wild-type walls (supporting a reinforcing role for xyloglucan) but less extensible in creep and stress relaxation processes mediated by α-expansin. Fusicoccin-induced "acid growth" was likewise reduced in xxt1/xxt2 petioles. The results show that xyloglucan is important for wall loosening by α-expansin, and the smaller size of the xxt1/xxt2 mutant may stem from the reduced effectiveness of α-expansins in the absence of xyloglucan. Loosening agents that act on xylans and pectins elicited greater extension in creep assays of xxt1/xxt2 cell walls compared with wild-type walls, consistent with a larger mechanical role for these matrix polymers in the absence of xyloglucan. Our results illustrate the need for multiple biomechanical assays to evaluate wall properties and indicate that the common depiction of a cellulose-xyloglucan network as the major load-bearing structure is in need of revision.     

Xyloglucan Xylosyltransferases XXT1, XXT2, and XXT5 and the Glucan Synthase CSLC4 Form Golgi-Localized Multiprotein Complexes

Xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants and has important structural and physiological functions in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the 1,4-β-glucan synthase, Cellulose Synthase-Like C4 (CSLC4), and three xylosyltransferases, XXT1, XXT2, and XXT5, act in the Golgi to form the xylosylated glucan backbone during xyloglucan biosynthesis. However, the functional organization of these enzymes in the Golgi membrane is currently unknown. In this study, we used bimolecular fluorescence complementation and in vitro pull-down assays to investigate the supramolecular organization of the CSLC4, XXT1, XXT2, and XXT5 proteins in Arabidopsis protoplasts. Quantification of bimolecular fluorescence complementation fluorescence by flow cytometry allowed us to perform competition assays that demonstrated the high probability of protein-protein complex formation in vivo and revealed differences in the abilities of these proteins to form multiprotein complexes. Results of in vitro pull-down assays using recombinant proteins confirmed that the physical interactions among XXTs occur through their catalytic domains. Additionally, coimmunoprecipitation of XXT2YFP and XXT5HA proteins from Arabidopsis protoplasts indicated that while the formation of the XXT2-XXT2 homocomplex involves disulfide bonds, the formation of the XXT2-XXT5 heterocomplex does not involve covalent interactions. The combined data allow us to propose that the proteins involved in xyloglucan biosynthesis function in a multiprotein complex composed of at least two homocomplexes, CSLC4-CSLC4 and XXT2-XXT2, and three heterocomplexes, XXT2-XXT5, XXT1-XXT2, and XXT5-CSLC4.     

Cereal root exudates contain highly structurally complex polysaccharides with soil‐binding properties

Rhizosheaths function in plant?soil interactions, and are proposed to form due to a mix of soil particle entanglement in root hairs and the action of adhesive root exudates. The soil‐binding factors released into rhizospheres to form rhizosheaths have not been characterised. Analysis of the high‐molecular‐weight (HMW) root exudates of both wheat and maize plants indicate the presence of complex, highly branched polysaccharide components with a wide range of galactosyl, glucosyl and mannosyl linkages that do not directly reflect cereal root cell wall polysaccharide structures. Periodate oxidation indicates that it is the carbohydrate components of the HMW exudates that have soil‐binding properties. The root exudates contain xyloglucan (LM25), heteroxylan (LM11/LM27) and arabinogalactan‐protein (LM2) epitopes, and sandwich‐ELISA evidence indicates that, in wheat particularly, these can be interlinked in multi‐polysaccharide complexes. Using wheat as a model, exudate‐binding monoclonal antibodies have enabled the tracking of polysaccharide release along root axes of young seedlings, and their presence at root hair surfaces and in rhizosheaths. The observations indicate that specific root exudate polysaccharides, distinct from cell wall polysaccharides, are adhesive factors secreted by root axes, and that they contribute to the formation and stabilisation of cereal rhizosheaths.     

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN‐LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d ‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d ‐glucan.     

A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.     

Secretion of cell wall polysaccharides in Vicia root hairs

Root hairs of hairy winter vetch ( Vicia villosa Roth) synthesize and secrete abundant cell wall matrix polysaccharides, making this an excellent system for the study of secretion during tip growth. Roots with newly formed hairs were preserved by cryofixation and freeze substitution. Cryofixed root hairs showed excellent structural and antigenic preservation. Ultrastructural analyses showed numerous vesicles near the tip and a concentration of Golgi bodies in the subapical region of the hair. The distribution of polygalacturonic acid and xyloglucan in the endomembrane system and cell wall were revealed by immunolabeling, using previously characterized monoclonal antibodies. De-esterified polygalacturonic acid was present on the external surface of the cell wall, but was not detected within the cell, although chemical de-esterification revealed abundant antigen in Golgi bodies and secretory vesicles. Methyl-esterified polygalacturonic acid epitopes were detected within the medial and trans cisternae of Golgi bodies, in secretory vesicles, and throughout the wall, indicating that pectin is secreted in a neutral form and may then be de-esterified in muro . Xyloglucan was also detected within the trans cisternae of Golgi bodies, secretory vesicles and throughout the cell wall. Double labeling experiments demonstrated that both polysaccharides occur simultaneously in the same Golgi bodies, and that secretory vesicles containing both polygalacturonic acid and xyloglucan deliver the polysaccharides to the cell wall at the growing tip.     

Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.     

A Barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3;1,4)-beta-D-glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.     

Cell wall polysaccharide distribution in Sandersonia aurantiaca flowers using immuno-detection

The localization of cell wall polysaccharides of the fused petals of monocotyledonous Sandersonia aurantiaca flowers has been identified using antibodies directed to pectin and xyloglucan epitopes and detection by fluorescence microscopy. Cross sections of the petal tissue were taken from cut flowers in bud and at various stages of maturity and senescence. Patterns of esterification in pectin backbones were identified by JIM5 and 2F4 labelling. Pectic galactan and arabinan side branches were detected by LM5 and LM6, respectively, while fucosylated xyloglucan was identified by CCRC-M1. The labelling patterns highlighted compositional differences between walls of the outer/inner epidermis compared to the spongy parenchyma cells of the interior mesophyll for fucosylated xyloglucan and arabinan. Partially esterified homogalacturonan was present in the junction zones of the outer epidermis and points of contact between cells of the mesophyll, and persisted throughout senescence. Pectic galactans were ubiquitous in the outer and inner epidermal cell walls and walls of the interior mesophyll at flower opening, whereas pectic arabinan was found predominantly in the epidermal cells. Galactan was lost from walls of all cells as flowers began to senesce, while fucosylated xyloglucan appeared to increase over this time. Such differences in the location of polysaccharides and the timing of changes suggest distinct combinations of certain polysaccharides offer mechanical and rheological advantages that may assist with flower opening and senescence.     

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots

Arabidopsis root hair formation is determined by the patterning genes CAPRICE ( CPC ), GLABRA3 ( GL3 ), WEREWOLF ( WER ) and GLABRA2 ( GL2 ), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy–principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase ( CESA1 – 10 ) and 4 of 33 xyloglucan endotransglucosylase ( XTH1 – 33 ) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.     

Characterization of cell wall polysaccharides from the medicinal plant Panax notoginseng

Panax notoginseng is a commonly used medicinal plant in south-western China. Recent studies indicate that wall polysaccharides are responsible for some of the immunostimulatory activity. Fractionation of the P. notoginseng root powder alcohol insoluble residue (AIR) and its compositional analysis enabled us to deduce the polysaccharide and protein composition of the root cell walls. P. notoginseng walls are composed primarily of polysaccharide (approximately 97% w/w) and some protein. The polysaccharides include pectic polysaccharides (neutral Type I 4-galactan (21%), arabinan (5%), acidic rhamnogalacturonan I (RG I, 2%) and homogalacturonan (HGA, 24%), non-cellulosic polysaccharides (heteroxylan, 3%), xyloglucan (XG, 3%) and heteromannan (1%)) and cellulose (24%). The root AIR also contains Type II AG/AGPs (5% w/w) typically associated with the plasma membrane and extracellular matrix. Thus, P. notoginseng roots contain polysaccharides typical of Type I primary cell walls but are distinguished by their very high levels of Type I 4-galactans and low levels of XGs. The major amino acids in the AIR were Leu (14 mol%), Asx (16 mol%), Glx (10 mol%), Ala (9 mol%), Thr (9 mol%) and Val (9 mol%).     

Hetero‐trans‐β‐glucanase,an enzyme unique to Equisetum plants,functionalizes cellulose

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell‐wall polysaccharides (xyloglucan, mannans, mixed‐linkage β‐glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero‐trans‐β‐glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early‐diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero‐transglycosylation: its preferred donor substrates (cellulose or mixed‐linkage β‐glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose–xyloglucan and mixed‐linkage β‐glucan–xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter‐linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better‐known xyloglucan‐acting homo‐transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable ‘green’ technology for industrially manipulating biomass.     

The FRIABLE1 Gene Product Affects Cell Adhesion in Arabidopsis

Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.     

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.     

Cell wall alterations in the arabidopsis emb30 mutant

The Arabidopsis EMB30 gene is essential for controlling the polarity of cell growth and for normal cell adhesion during seedling development. In this article, we show that emb30 mutations also affect the growth of undifferentiated plant cells and adult tissues. EMB30 possesses a Sec7 domain and, based on similarities to other proteins, presumably functions in the secretory pathway. The plant cell wall depends on the secretory pathway to deliver its complex polysaccharides. We show that emb30 mutants have a cell wall defect that sometimes allows material to be deposited into the interstitial space between cells instead of being restricted to cell corners. In addition, pectin, a complex polysaccharide important for cell adhesion, appears to be abnormally localized in emb30 plants. In contrast, localization of epitopes associated with xyloglucan or arabinogalactan was similar in wild-type and emb30 tissues, and the localization of a marker molecule to vacuoles appeared normal. Therefore, emb30 mutations do not cause a general defect in the secretory pathway. Together, these results suggest that emb30 mutations result in an abnormal cell wall, which in turn may account for the defects in cell adhesion and polar cell growth control observed in the mutants.     

In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana

Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis. Received: 13 July 2000 / Accepted: 15 September 2000     

Quantitative trait loci analysis of primary cell wall composition in Arabidopsis

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 x Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.