Eric Nguema-Ona

Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.

The reb1-1 Mutation of Arabidopsis. Effect on the Structure and Localization of Galactose-Containing Cell Wall Polysaccharides

The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-d-Glucose 4-epimerases involved in the synthesis of d-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for α-l-Fucp-(1→2)-β-d-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.

Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation.

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as ‘pectic plasticizers’. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.

Arabinogalactan proteins in root and pollen-tube cells: distribution and functional aspects

BACKGROUND Arabinogalactan proteins (AGPs) are complex proteoglycans of the cell wall found in the entire plant kingdom and in almost all plant organs. AGPs encompass a large group of heavily glycosylated cell-wall proteins which share common features, including the presence of glycan chains especially enriched in arabinose and galactose and a protein backbone particularly rich in hydroxyproline residues. However, AGPs also exhibit strong heterogeneities among their members in various plant species. AGP ubiquity in plants suggests these proteoglycans are fundamental players for plant survival and development. SCOPE In this review, we first present an overview of current knowledge and specific features of AGPs. A section devoted to major tools used to study AGPs is also presented. We then discuss the distribution of AGPs as well as various aspects of their functional properties in root tissues and pollen tubes. This review also suggests novel directions of research on the role of AGPs in the biology of roots and pollen tubes.

Arabinogalactan proteins in root–microbe interactions

Arabinogalactan proteins (AGPs) are among the most intriguing sets of macromolecules, specific to plants, structurally complex, and found abundantly in all plant organs including roots, as well as in root exudates. AGPs have been implicated in several fundamental plant processes such as development and reproduction. Recently, they have emerged as interesting actors of root-microbe interactions in the rhizosphere. Indeed, recent findings indicate that AGPs play key roles at various levels of interaction between roots and soil-borne microbes, either beneficial or pathogenic. Therefore, the focus of this review is the role of AGPs in the interactions between root cells and microbes. Understanding this facet of AGP function will undoubtedly improve plant health and crop protection.

Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

BackgroundPolygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection.ResultsGlobal gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity.ConclusionsThis evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs.

Overexpression of the grapevine PGIP1 in tobacco results in compositional changes in the leaf arabinoxyloglucan network in the absence of fungal infection

BackgroundConstitutive expression of Vitis vinifera polygalacturonase-inhibiting protein 1 (Vvpgip1) has been shown to protect tobacco plants against Botrytis cinerea. Evidence points to additional roles for VvPGIP1, beyond the classical endopolygalacturonase (ePG) inhibition mechanism, in providing protection against fungal infection. Gene expression and biochemical datasets previously obtained, in the absence of infection, point to the cell wall, and particularly the xyloglucan component of transgenic VvPGIP1 lines as playing a role in fungal resistance.ResultsTo elucidate the role of wall-associated processes in PGIP-derived resistance pre-infection, a wall profiling analysis, using high-throughput and fractionation techniques, was performed on healthy leaves from wild-type and previously characterized transgenic lines. The cell wall structure profile during development was found to be altered in the transgenic lines assessed versus the wild-type plants. Immunoprofiling revealed subtle changes in pectin and cellulose components and marked changes in the hemicellulose matrix, which showed reduced binding in transgenic leaves of VvPGIP1 expressing plants. Using an enzymatic xyloglucan oligosaccharide fingerprinting technique optimized for tobacco arabinoxyloglucans, we showed that polysaccharides of the XEG-soluble domain were modified in relative abundance for certain oligosaccharide components, although no differences in ion profiles were evident between wild-type and transgenic plants. These changes did not significantly influence plant morphology or normal growth processes compared to wild-type lines.ConclusionsVvPGIP1 overexpression therefore results in cell wall remodeling and reorganization of the cellulose-xyloglucan network in tobacco in advance of potential infection.

Profiling the main cell wall polysaccharides of grapevine leaves using high-throughput and fractionation methods.

Vitis species include Vitis vinifera, the domesticated grapevine, used for wine and grape agricultural production and considered the worlds most important fruit crop. A cell wall preparation, isolated from fully expanded photosynthetically active leaves, was fractionated via chemical and enzymatic reagents; and the various extracts obtained were assayed using high-throughput cell wall profiling tools according to a previously optimized and validated workflow. The bulk of the homogalacturonan-rich pectin present was efficiently extracted using CDTA treatment, whereas over half of the grapevine leaf cell wall consisted of vascular veins, comprised of xylans and cellulose. The main hemicellulose component was found to be xyloglucan and an enzymatic oligosaccharide fingerprinting approach was used to analyze the grapevine leaf xyloglucan fraction. When Paenibacillus sp. xyloglucanase was applied the main subunits released were XXFG and XLFG; whereas the less-specific Trichoderma reesei EGII was also able to release the XXXG motif as well as other oligomers likely of mannan and xylan origin. This latter enzyme would thus be useful to screen for xyloglucan, xylan and mannan-linked cell wall alterations in laboratory and field grapevine populations. This methodology is well-suited for high-throughput cell wall profiling of grapevine mutant and transgenic plants for investigating the range of biological processes, specifically plant disease studies and plant-pathogen interactions, where the cell wall plays a crucial role.

The Developmental Regulator SEEDSTICK Controls Structural and Mechanical Properties of the Arabidopsis Seed Coat

A molecular network controlled by STK regulates cell wall properties of the seed coat, showing that, in addition to specifying organ identity, the role of STK in seed development includes modification of the cell wall structure. Although many transcription factors involved in cell wall morphogenesis have been identified and studied, it is still unknown how genetic and molecular regulation of cell wall biosynthesis is integrated into developmental programs. We demonstrate by molecular genetic studies that SEEDSTICK (STK), a transcription factor controlling ovule and seed integument identity, directly regulates PMEI6 and other genes involved in the biogenesis of the cellulose-pectin matrix of the cell wall. Based on atomic force microscopy, immunocytochemistry, and chemical analyses, we propose that structural modifications of the cell wall matrix in the stk mutant contribute to defects in mucilage release and seed germination under water-stress conditions. Our studies reveal a molecular network controlled by STK that regulates cell wall properties of the seed coat, demonstrating that developmental regulators controlling organ identity also coordinate specific aspects of cell wall characteristics.