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A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan 1), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In addition to establishing symbiotic relationships with arbuscular mycorrhizal fungi, legumes also enter into a nitrogen-fixing symbiosis with rhizobial bacteria that results in the formation of root nodules. Several genes involved in the development of both arbuscular mycorrhiza and legume nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONSI) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K+) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the C-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast (Saccharomyces cerevisiae) mutant toward those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant-negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants. [ABSTRACT FROM AUTHOR]

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity. [ABSTRACT FROM AUTHOR]

Examines the expression patterns of GMD1 and GMD2 Genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns. Regulation of the synthesis of fucosylated polysaccharides; Accounts on the development of Arabidopsis roots; Characterization of the L-Fuc-deficient mur1 mutant.

Examines the influence of cellulose synthase catalytic subunits in catalyzing the biosynthesis of cellulose. Accounts on the component of plant cell walls; Reduction of fiber cell wall thickness; Cause of the collapse of xylem phenotype.

Investigates the fucose destined into the xyloglucan is synthesized using one or the other or both isoforms of the GDP-D-mannose 4,6-dehydratase depending on the cell type and/or development of the cell. Recognition of fucose-containing epitope by monoclonal antibody, CCRC-MI; Usage of the CCRC-MI on the distribution of epitope throughout the mur1 mutant of Arabidopsis; Developmental regulation of the CCRC-MI epitope in the mur1 roots.