Analysis of the Golgi Apparatus in Arabidopsis Seed Coat Cells during Polarized Secretion of Pectin-Rich Mucilage

Department of Botany, University of British Columbia, Vancouver, Canada V6T 1Z4.
The Plant Cell (Impact Factor: 9.34). 06/2008; 20(6):1623-38. DOI: 10.1105/tpc.108.058842
Source: PubMed


Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.

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Available from: George W Haughn, Sep 29, 2015
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    • "High resolution imaging with the aid of specific monoclonal antibodies (mAbs) is very helpful to place the structural complexity of cell wall polysaccharides including pectin in cell biological and developmental contexts (Willats et al. 2001a). A large set of mAbs against pectin motifs is already available (e.g.: through Plant Probes and CCRC websites) amongst which several mAbs are specifically directed against RGI motifs (Clausen et al. 2003; Jones et al. 1997; Ralet et al. 2010; Verhertbruggen et al. 2009a; Willats et al. 1998; Young et al. 2008). However, antibodies directed to highly-branched pectic domains or to the junction between the side chains and the RGI backbone have not yet been isolated. "
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    ABSTRACT: 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 assembling of pectin structural domains and their integration and remodelling in cell walls.
    Plant and Cell Physiology 09/2015; DOI:10.1093/pcp/pcv128 · 4.93 Impact Factor
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    • "Both layers are composed primarily of the pectin RGI with the adherent layer also containing other pectins and minor amounts of hemicellulose and cellulose (Western et al., 2000; Penfield et al., 2001; Willats et al., 2001; Macquet et al., 2007; Young et al., 2008). A number of previous studies provide data supporting a role for cellulose in seed coat mucilage: i) Calcofluor, Congo red and Pontamine fast scarlet S4B stains of seed mucilage are consistent with presence of cellulose in the set of rays deposited across the inner layer of seed coat mucilage (Windsor et al., 2000; Willats et al., 2001; Macquet et al., 2007; Harpaz-Saad et al., 2011; Mendu et al., 2011b); ii) Fluorescently labeled cellulose-binding modules identify the presence of crystalline and amorphous cellulose (Blake et al., 2006; Young et al., 2008; Dagel et al., 2011; Sullivan et al., 2011); iii) pectolytic enzymes could not degrade the set of rays radiating from the seed unless combined with cellulase treatment (Macquet et al., 2007); and iv) Most recently, genetic studies confirmed the cellulosic composition of the rays of seed coat mucilage, identifying CELLULOSE SYNTHASE 5 (CESA5) as an essential player in cellulose deposition in this context (Harpaz-Saad et al., 2011; Mendu et al., 2011b; Sullivan et al., 2011). Altogether, this data demonstrates the essential structural role of cellulose in anchoring the pectic component of seed coat mucilage to the seed surface. "
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    ABSTRACT: Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells (MSCs) is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE 2 (COBL2), a member of the GPI-anchored COBRA-LIKE gene family, as coexpressed with other genes involved in cellulose deposition in MSCs. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic-rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised as demonstrated by scanning electron microscopy and in-situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole seed crystalline cellulose content compared to wild type. The data establishes that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. Copyright © 2015, American Society of Plant Biologists.
    Plant physiology 01/2015; 167(3). DOI:10.1104/pp.114.240671 · 6.84 Impact Factor
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    • "Developing seeds at 4, 7, and 10 DPA were high-pressure frozen, resin embedded, and sectioned according to a previously published method (Rensing et al., 2002; Young et al., 2008). Developing seeds at 4, 7, and 10 DPA were dissected and pierced with an insect pin before being frozen in the presence of hexadecene using a Leica EM HPM 100 High-Pressure Freezer (Leica). "
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    ABSTRACT: Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially de-methyl esterified by pectin methyl esterase (PME) to strengthening or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage we identified 7 PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 Days Post Anthesis (DPA) both in the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PMEI5 over-expression line (OE) suggest that HMS acts independently from other cell wall modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves, and that its role in the seed coat is masked by redundancy. Copyright © 2015, American Society of Plant Biologists.
    Plant physiology 01/2015; 167(3). DOI:10.1104/pp.114.255604 · 6.84 Impact Factor
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