pectins and 2-keto-3-deoxyoctonate

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.

Topics: Arabidopsis; Azides; Cell Wall; Cells, Cultured; Nicotiana; Nucleotidyltransferases; Pectins; Plant Roots; Seedlings; Staining and Labeling; Sugar Acids

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation.. Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy.. Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary.. This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Wall; Gene Expression Regulation, Plant; Genes, Reporter; Mutation; Organ Specificity; Pectins; Phenotype; Pollen; Pollen Tube; Polymers; Sialyltransferases; Sugar Acids

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. It contains roughly 30 monosaccharide units (MW approximately 5 kDa) including very unusual residues such as Kdo, Dha, aceric acid and apiose. Previous studies have demonstrated that these monomers are arranged into four structurally well-defined oligosaccharide side chains (A-D), linked to a homogalacturonan mainchain, but the specific attachment sites of these branches on the pectic backbone have not yet been elucidated. In the present work, fairly complete assignments of the 750 MHz 1H NMR spectra and partial assignments of the 13C NMR spectra of the sodium-borohydride-reduced RG-II monomer were obtained for a 5 mM sample isolated from red wine. On the whole, these data corroborate the primary structures of the sidechains previously established by methylation analysis, partial hydrolysis and FAB-MS spectrometry but some heterogeneity has been demonstrated (partial substitution at B5, B6, and A5). The preferred orientations of the majority of the sidechain glycosidic linkages in the RG-II monomer have been determined from the sequential nOe data and the solution structure is generally in good agreement with the stable conformers previously obtained by molecular modeling (MM3) of the disaccharide and sidechain oligosaccharide building blocks. All of a two-residue, a three-residue, and a four-residue segment of the backbone have been tentatively identified from long range interactions between sidechain protons as well as in the mainchain. Taking into account the length of the 9-mer galacturonan mainchain described in prior work, these building blocks constitute almost the complete structure of RG-II (Scheme 2).

Topics: Borohydrides; Carbohydrate Sequence; Carbon Isotopes; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Pectins; Pentoses; Protons; Solutions; Spectrometry, Mass, Fast Atom Bombardment; Sugar Acids; Wine