Building flax fibres: more than one brick in the walls

The objective of the review is to provide fundamental knowledge on the chemical composition and structural characteristics of flax fibres. These are long and multinucleate cells without septum or partition (average length 2–5 cm) and have a secondary wall of very large thickness (5–15 μm). Fibres are gathered in bundles of one to three dozen cells that encircle the vascular cylinder. The bundle cohesion is insured by pectins, accumulating in the primary wall and cell junctions. In contrast, lignin, which is present in very low amount, does not seem to play a major role in bundle cohesion. At maturity, secondary wall is characterised by (i) a high level of cellulose with microfibrils locked into an almost axial direction and (ii) 5–15% non-cellulosic polysaccharides (NCPs). The chemical composition of NCPs depends on growth stage, indicating important cell wall remodelling, fibre position and variety. Despite the large disparity of the results reported in the literature, galactose appears to be the predominant sugar of NCPs, and β-1-4-galactan together with rhamnogalacturonan of type I (RG-I) and polygalacturonic acid (PGA) become, with fibre maturity, the most abundant tightly bound NCPs. Glycine-rich proteins (GRPs) and arabinogalactan-proteins (AGPs), also present in flax fibres, are both characterised by appreciable levels of glycine and acidic amino acid and are deficient in hydroxyproline, and may contribute to the cross-linking of pectins. (Galacto)glucomanans/glucans rather than xylans consist of cross-linking polymers in fibre secondary wall. A model is proposed where cellulose microfibrils, tethered by cross-linking (galacto)glucomanans/glucans, are embedded in a pectic matrix.     

Monitoring cell cycle distributions in MCF-7 cells using near-field photothermal microspectroscopy

Microspectroscopic techniques such as Fourier transform infrared (FTIR) have played an important role in "fingerprinting" the biochemical composition of cellular components. Based on structure and function, complex biomolecules absorb energy in the mid-infrared (lambda = 2-20 microm) yielding characteristic vibrational infrared (IR) spectra. However, optical detection FTIR microspectroscopy may not be suitable for IR-absorbing sample materials. Photothermal microspectroscopy (PTMS) permits the direct measurement of heat generated as a result of sample material absorbing radiation. This approach generates true absorption spectra and is implemented by interfacing a scanning probe microscope and an FTIR spectrometer. Detection is performed using a near-field ultra-miniaturized temperature sensor. Employing PTMS, IR spectra of MCF-7 cells were examined in spectral regions (900-2000 cm(-1)) corresponding to proteins, DNA, RNA, glycoproteins, carbohydrates, lipids, and levels of protein phosphorylation. As a cell passes through the cell cycle, its nuclear material decondenses and condenses and this has led to ambiguity as to whether the intensity of such spectral regions may be associated with the G(1)-, S- or G(2)-phases of the cell cycle. Cultured cells were tracked over a time course known to correspond to marked alterations in cell-cycle distributions, as determined using flow cytometry. Experiments were carried out in the absence or presence of lindane, a pesticide known to induce G(1)-arrest in MCF-7 cells. Significant (P < 0.05) elevations in spectral intensities were associated with exponentially growing cell populations, predominantly in S-phase or G(2)-phase, compared to more quiescent populations predominantly in G(1)-phase. Increases in the absorption band at 970 cm(-1), associated with elevated protein phosphorylation, were observed in vibrational spectra of exponentially growing cell populations compared to those exhibiting a slowing in their growth kinetics. These results seem to suggest that intracellular bulk changes, associated with transit through the cell cycle, can be tracked using PTMS.     

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.     

Structural characterization of cell wall polysaccharides from two plant species endemic to central Africa,Fleurya aestuans and Phragmenthera capitata

Fleurya aestuans (Linnaeus) Miquel and Phragmenthera capitata (Spreng) are two plants endemic to central Africa that are used in traditional medicine. However, information on their molecular constituents is lacking. In the present study and as part of our research on the structure/bioactivity relationship of plant cell wall molecules, we investigated the structure of polysaccharides isolated from leaf cell walls of both plant species. To this end, we used sequential extraction of polysaccharides, gas chromatography, matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and immuno-dot assays. Our data indicate the presence of both pectin and hemicellulosic polysaccharides in the cell walls of both plants. In particular, cell wall of F. aestuans leaves appears to contain much more pectin than those of P. capitata. Structural analysis of hemicellulosic polysaccharides revealed differences in the structure of xyloglucan isolated from both species. While only the XXXG-type was found in P. capitata, both XXXG and XXGG types were detected in F. aestuans. No arabinosylated subunits were found in any of the xyloglucan isolated from both plant species. In addition, xylan structure with non methylated-α-d-glucuronic acid on side chains was only detected in F. aestuans leaf cell walls. Finally, structural analysis of rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) shows that unlike RG-II, RG-I is qualitatively different between F. aestuans and P. capitata leaves.     

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

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.Higher plants rely on their roots to acquire water and other nutrients in the soil to grow and develop (Esau, 1977). At the tip of every growing root is a conical covering consisting of several layers of cells called the root cap that plays a major role in root protection and its interaction with the rhizosphere (Rougier, 1981; Baluška et al., 1996; Barlow, 2003).Root tips of most plant species produce a large number of cells programmed to separate from the root cap and to be released into the external environment (Hawes et al., 2003). This process occurs through the action of cell wall-degrading enzymes that solubilize the interconnections between root cap peripheral cells, causing the cells to separate from each other and from the root as populations of single cells (Hawes et al., 2003). Because of their specific position at the interface between root and soil, these living cells are defined as root border cells. It has been shown that the number of these cells per root varies between plant families: from about 100 (e.g. the Solanaceae family) to several thousands (e.g. 10,000 or more for the Pinaceae; Hawes et al., 2003). It has also been suggested that species of the Brassicaceae family including Arabidopsis (Arabidopsis thaliana) do not produce border cells (Hawes et al., 2003). Indeed, the Arabidopsis root tip does not produce isolated border cells per se, but it does produce and release cells that remain attached to each other, forming a block of several cell layers called border-like cells (Vicré et al., 2005; Fig. 1). This also occurs in other Brassicaceae species, including rapeseed (Brassica napus), mustard (Brassica juncea), and Brussels sprout (Brassica oleracea gemmifera), indicating that such an organization might be specific to this family (Driouich et al., 2007).Open in a separate windowFigure 1.Morphological phenotypes of root tips showing border-like cells (BLC) of the wild type and cell wall mutants of Arabidopsis. Wild-type Columbia (Col O; A), wild-type Wassilewskija (Ws; B), mur3 (C), mur2-1 (D), kor1 (E), rsw1 (F), epc1-1 (G), arad1-1 (H), qua1-1 (I), and qua2-1 (J) are shown. Border-like cells are released from the root tip in organized cell layers (arrows) in the wild type and in all mutants examined with the exception of qua1-1 and qua2-1. Note also that border-like cell organization is similar between Columbia and Wassilewskija. M, Mucilage. Bars = 20 μm (A, B, D–H, and J) or 50 μm (C and I).The unique organization pattern of Arabidopsis border-like cells (e.g. they do not disperse individually) suggests that they might have a specific cell wall composition and/or structure that makes them resistant to cell wall-hydrolyzing enzymes or that the enzymes are not present or not functional (Driouich et al., 2007). The only information on cell wall composition of Arabidopsis border-like cells was obtained from immunocytochemical studies, in which it has been shown that the cell wall of border-like cells is rich in pectic homogalacturonan and arabinogalactan-proteins, two wall polymers believed to be involved in cell adhesion in plants (Vicré et al., 2005). Based on this observation, we postulated that pectic polysaccharides of the cell wall may serve as a glue to cement border-like cells together, leading to that particular organization (Vicré et al., 2005).The cell wall of higher plants comprises mainly polysaccharides and proteoglycans. Cell wall polysaccharides are assembled into complex macromolecules, including cellulose, hemicellulose, and pectin. Cellulose forms microfibrils, which constitute an ordered, fibrous phase, whereas pectin and hemicellulose form an amorphous matrix phase surrounding the microfibrils (Cosgrove, 1997). Pectins constitute a highly complex family of cell wall polysaccharides, including homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Homogalacturonan domains consist of α-d-(1→4)-GalUA residues, which can be methyl esterified, acetylated, and/or substituted with β-(1→3)-Xyl residues to form xylogalacturonan (Schols et al., 1995; Willats et al., 2001; Vincken et al., 2003). Deesterified blocks of homogalacturonan can be cross-linked by calcium, leading to the formation of a gel that is believed to be involved in cell adhesion (Jarvis et al., 2003). Rhamnogalacturonan I consists of a backbone of up to 100 repeats of the disaccharide α-(1→4)-GalUA-(1→2)-rhamnose, which carries complex and variable side chains. The rhamnose residues are commonly substituted with polymeric β-(1→4)-linked d-galactosyl residues and/or α-(1→5)-linked l-arabinosyl residues (Ridley et al., 2001). Rhamnogalacturonan II is a highly complex but conserved molecule consisting of a homogalacturonan-like backbone substituted with four different side chains containing specific sugars (O''Neill et al., 2004).Xyloglucan is the major hemicellulosic polysaccharide of the primary wall of dicotyledonous plants, and it consists of a β-d-(1→4)-glucan backbone to which are attached side chains containing xylosyl, galactosyl-xylosyl, or fucosyl-galactosyl-xylosyl residues. Xyloglucan is the principal polysaccharide that cross-links the cellulose microfibrils. The xyloglucan-cellulose network forms a major load-bearing structure that contributes to the control of cell expansion (Hayashi, 1989; Cosgrove, 1999).Glycoproteins, such as arabinogalactan-proteins, are also present in the cell wall matrix (Showalter, 1993; Seifert and Roberts, 2007). Arabinogalactan-proteins are highly glycosylated members of the Hyp-rich glycoprotein superfamily. Many of these glycoproteins, the so-called classical arabinogalactan-proteins, are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor and have the potential to bind both cell wall components (Immerzeel et al., 2006) and cytosolic cortical microtubules (Schultz et al., 2002; Sardar et al., 2006; Nguema-Ona et al., 2007). These proteoglycans have been implicated in many aspects of plant life, including cell expansion, cell signaling and communication, embryogenesis, and wound response (Johnson et al., 2003; Seifert and Roberts, 2007; Driouich and Baskin, 2008).Although cell-to-cell interaction is a fundamental feature of plant growth and development, the molecular bases of intercellular adhesion and its loss are not fully understood (Roberts et al., 2002; Jarvis et al., 2003; Willats et al., 2004). This study aims at investigating the role of cell wall polysaccharides in cell attachment and the organization of border-like cells in Arabidopsis. To this end, we took advantage of the recent characterization of several Arabidopsis mutants affected in the biosynthesis of different classes of cell wall polysaccharides, including pectin, xyloglucan, and cellulose. We thus examined the pattern of border-like cells released by the root tip of selected Arabidopsis mutants using microscopy and immunocytochemistry. These mutants are (1) quasimodo1-1 (qua1-1) and qua2-1 (Bouton et al., 2002; Mouille et al., 2007), ectopically parting cells1-1 (epc1-1; Singh et al., 2005), and arabinan deficient1-1 (arad 1-1; Harholt et al., 2006), which all have been reported to be possibly affected in pectin biosynthesis; (2) murus2-1 (mur2-1) and mur3, which make altered xyloglucan (Vanzin et al., 2002; Madson et al., 2003); and (3) radially swollen1 (rsw1) and korrigan1 (kor1), which are affected in cellulose biosynthesis (Arioli et al., 1998; Nicol et al., 1998).Our data show that the organization of border-like cells had a wild-type phenotype in all of the mutants examined except in qua1-1 and qua2-1. In both of these mutants, border-like cells had lost the wild-type phenotype, as they were released as single cells separated from each other. This phenotype was far more pronounced in qua1-1 than in qua2-1. Further analysis of qua1-1 using immunocytochemistry and Fourier transform infrared microspectrometry showed a substantial loss of homogalacturonan content in border-like cells. In addition, border-like cells in the qua1-1 mutant secreted an abundant mucilage enriched in xylogalacturonan and arabinogalactan-protein epitopes.     

Infrared spectroscopy with multivariate analysis potentially facilitates the segregation of different types of prostate cell

The prostate gland is conventionally divided into zones or regions. This morphology is of clinical significance as prostate cancer (CaP) occurs mainly in the peripheral zone (PZ). We obtained tissue sets consisting of paraffin-embedded blocks of cancer-free transition zone (TZ) and PZ and adjacent CaP from patients (n = 6) who had undergone radical retropubic prostatectomy; a seventh tissue set of snap-frozen PZ and TZ was obtained from a CaP-free gland removed after radical cystoprostatectomy. Paraffin-embedded tissue slices were sectioned (10-mum thick) and mounted on suitable windows to facilitate infrared (IR) spectra acquisition before being dewaxed and air dried; cryosections were dessicated on BaF(2) windows. Spectra were collected employing synchrotron Fourier-transform infrared (FTIR) microspectroscopy in transmission mode or attenuated total reflection-FTIR (ATR) spectroscopy. Epithelial cell and stromal IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences between cells in situ in different tissue regions. After spectroscopic analysis, plotted clusters and their loadings curves highlighted marked variation in the spectral region containing DNA/RNA bands ( approximately 1490-1000 cm(-1)). By interrogating the intrinsic dimensionality of IR spectra in this small cohort sample, we found that TZ epithelial cells appeared to align more closely with those of CaP while exhibiting marked structural differences compared to PZ epithelium. IR spectra of PZ stroma also suggested that these cells are structurally more different to CaP than those located in the TZ. Because the PZ exhibits a higher occurrence of CaP, other factors (e.g., hormone exposure) may modulate the growth kinetics of initiated epithelial cells in this region. The results of this pilot study surprisingly indicate that TZ epithelial cells are more likely to exhibit what may be a susceptibility-to-adenocarcinoma spectral signature. Thus, IR spectroscopy on its own may not be sufficient to identify premalignant prostate epithelial cells most likely to progress to CaP.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

New Findings in a Global Approach to Dissect the Whole Phenotype of PLA2G6 Gene Mutations

Mutations in PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients’ muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.