Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion

Abstract

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.

The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan

“…Consequently, this would lead to limited retention of xylogalacturonan and arabinogalactan-proteins in the cell wall and would facilitate their diffusion and release into the extracellular mucilage. Such a hypothesis has been proposed previously to explain the increase in the amount of extracellular polymers released in the medium of qua1-1 suspension-cultured cells compared with wild-type cells (Leboeuf et al, 2005;Rondeau-Mouro et al, 2008). An alternative explanation is that the qua1-1 mutant border-like cells compensate for the lack of homogalacturonan in their wall by an increase in de novo synthesis and secretion of xylogalacturonan/arabinogalactan-proteins abundant in mucilage in order to retain nonattached border-like cells in close proximity to the root tip.…”

“…Immersion immunofluorescence staining, which allows detection of epitopes at the surface of the tissue only, did not detect any differences in terms of intensity of fluorescence between the qua1-1 mutant and the wild type. However, as reduction in homogalacturonan in the cell walls of qua1-1 was reported to be particularly associated with the tricellular junction in suspension-cultured cells (Leboeuf et al, 2005), we performed the same labeling experiment with JIM5 on cryofixed and resin-embedded sections of root tips of the mutant and the wild type (Fig. 4).…”

Pectin Metabolism and Assembly in the Cell Wall of the Charophyte Green Alga Penium margaritaceum

“…three lines of evidence (Leboeuf et al, 2005): (1) unesterified HG is cross-linked by Ca 2+ in vitro to form gel networks; (2) low DE HG and Ca 2+ have been reported to colocalize in the middle lamella, cell junction zones adjacent to intracellular spaces, where cell adhesion is presumed to be particularly important; and (3) treatment of plant tissues with pectinases or Ca 2+ -chelating agents can induce cell separation. However, there is little direct evidence for this pectin assembly model occurring in vivo.…”

GALACTURONOSYLTRANSFERASE-LIKE5 Is Involved in the Production of Arabidopsis Seed Coat Mucilage

“…AtGATL5 belongs to Carbohydrate-Active Enzymes family GT8 (Yin et al, 2010), several of whose members have been implicated in the synthesis of HG (Bouton et al, 2002;Leboeuf et al, 2005;Orfila et al, 2005;Sterling et al, 2006;Atmodjo et al, 2011), including one functionally characterized GalAT, GAUT1 (Sterling et al, 2006). Thus, we tested whether AtGATL5 functions as a HG:GalAT.…”