Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.     

7-Dehydrobrefeldin A, a naturally occurring brefeldin A derivative, inhibits secretion and causes a cis-to-trans breakdown of Golgi stacks in plant cells.

7-Dehydrobrefeldin A (7-oxo-BFA) is a brefeldin A (BFA) analog that, like BFA, is a potent phytotoxin of Alternaria carthami, a fungal pathogen of safflower (Carthamus tinctorius L.) plants. Both BFA and 7-oxo-BFA have been shown to be causal agents of the leaf spot disease of these plants. We have investigated the effects of 7-oxo-BFA on the secretion and the structure of the Golgi stacks of sycamore maple (Acer pseudoplatanus) suspension-cultured cells to determine whether 7-oxo-BFA affects these cells in the same manner as BFA. When applied at 10 micrograms/mL for 1 h, 7-oxo-BFA inhibits secretion of proteins by approximately 80%, the same value obtained for BFA. However, electron micrographs of high-pressure frozen/freeze-substituted cells demonstrated that 7-oxo-BFA is a more potent disrupter of the Golgi stacks of sycamore maple cells than BFA. In cells treated for 1 h with 10 micrograms/mL 7-oxo-BFA, very few Golgi stacks can be discerned. Most of those that are left consist of fewer than three cisternae, all of which stain like trans-Golgi cisternae. They are surrounded by clusters of large (150-300 nm in diameter), darkly staining vesicles that are embedded in a fine-filamentous, ribosome-excluding matrix. Similarly sized and stained vesicles are seen budding from the rims of the residual trans-Golgi cisternae. Both the large vesicles and the residual Golgi stack buds stain with anti-xyloglucan polysaccharide antibodies. Recovery of Golgi stacks after removal of 7-oxo-BFA from 1-h-treated cells takes 2 to 6 h, compared with 1 to 2 h for cells treated with BFA. In contrast to 7-oxo-BFA, the BFA breakdown product BFA acid had no effect either on secretion or on the secretory apparatus. This is the first report, to our knowledge of a BFA analog inhibiting secretion in a eukaryotic cell system.     

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.     

Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules

How does a plant cell sense and respond to the status of its cell wall? Intercourse between cell wall and cytoplasm has long been supposed to involve arabinogalactan proteins, in part because many of them are anchored to the plasma membrane. Disrupting arabinogalactan proteins has recently been shown to disrupt the array of cortical microtubules present just inside the plasma membrane, implying that microtubules and arabinogalactan proteins interact. In this article, we assess possibilities for how this interaction might be mediated. First, we consider microdomains in the plasma membrane (lipid rafts), which have been alleged to link internal and external regions of the plasma membrane; however, the characteristics and even the existence of these domains remains controversial. Next, we point out that disrupting the synthesis of cellulose also can disrupt microtubules and consider whether arabinogalactan proteins are part of a network linking microtubules and nascent microfibrils. Finally, we outline several signaling cascades that could transmit information from arabinogalactan proteins to microtubules through channels of cellular communication. These diverse possibilities highlight the work that remains to be done before we can understand how plant cells communicate across their membranes.     

The reb1-1 mutation of Arabidopsis alters the morphology of trichoblasts,the expression of arabinogalactan-proteins and the organization of cortical microtubules

The root epidermal bulger 1 ( reb1) mutant of Arabidopsis thaliana (L.) Heynh. is characterized by a reduced elongation rate of the primary root and by the bulging of many, but not all, root epidermal cells. In this study, we investigated cell wall structure of root epidermal cells in reb1-1 by using serial sectioning, and light and electron microscopy in combination with immuno-cytochemistry and polysaccharide staining. We found that: (i) Cell bulging in the mutant was initiated in the zone of elongation of the root, and occurred exclusively in trichoblasts. (ii) reb1-1 and wild-type root cells stained identically with anti-pectin antibodies, such as JIM5. In contrast, the anti-arabinogalactan-protein antibodies, JIM14 and LM2, stained all epidermal cells in the wild type and trichoblasts preferentially, but in reb1-1 they stained the atrichoblasts only. (iii) Compared to the wild type, mutant trichoblasts had a thinner outer epidermal cell wall, which presented abnormal periodic acid-thio carbohydrazide silver proteinate (PATAg) staining. In addition, we investigated the organization of cortical microtubules in a reb1-1 mutant line expressing a green-fluorescent protein fused to a microtubule-binding domain from human microtubule-associated protein 4. Microtubules in the swollen trichoblasts of reb1-1 were either disordered or absent entirely. Together our findings indicate that the reb1-1 mutation results in an abnormal trichoblast cell wall, and suggest that cell surface arabinogalactan-proteins are required for anisotropic expansion and for orienting cortical microtubules.     

Microscopic studies on mature flax fibers embedded in LR white: immunogold localization of cell wall matrix polysaccharides.

Flax fibers have been the subject of many biochemical studies, which revealed that cellulose and pectins are the major constituents of their walls. In contrast, little is known about the location of these polymers within the walls of mature fibers by microscopic methods. This has been technically hampered by the very thick secondary wall of fibers, resulting in inadequate tissue preservation unsuitable for immunogold microscopy. In this study, we adapted the basic chemical fixation, dehydration and infiltration methods to achieve a good preservation of the cell structures of mature fibers and reduced damage to antigens. We were able to apply postembedding immunocytochemical techniques to map the location of various pectic epitopes within the walls of mature fibers. Our immunolabeling data show that homogalacturonans were exclusively found in the middle lamellae and the cell junctions but were not detectable in the secondary wall. In contrast, rhamnogalacturonan I (RG I)-associated epitopes, as well as galactan and arabinan epitopes, were abundantly distributed over the secondary wall of mature fibers.     

Composition and desiccation-induced alterations of the cell wall in the resurrection plant Craterostigma wilmsii

Resurrection plants have the unique capacity to revive from an air-dried state. In order to tolerate desiccation they have to overcome a number of stresses, mechanical stress being one. In leaves of the Craterostigma species, an extensive shrinkage occurs during drying as well as a considerable cell wall folding. Our previous microscopically analysis using immunocytochemistry on the resurrection plant Craterostigma wilmsii , has shown an increase in labelling of xyloglucan and unesterified pectins in the cell wall during drying. In this study, we have undertaken a biochemical approach to separate, quantify and characterize major cell wall polysaccharides in fully hydrated and dry leaves of C. wilmsii . Our results show that the overall cell wall composition of C. wilmsii leaves was similar to that of other dicotyledonous plants with respect to the pectin content. However, the structure of the hemicellulosic polysaccharide xyloglucan was characterized to be XXGG-type. The data also demonstrate marked changes in the hemicellulosic wall fraction from dry plants compared to hydrated ones. The most conspicuous change was a decrease in glucose content in the hemicellulosic fraction of dry plants. In addition, xyloglucan from the cell wall of dry leaves was relatively more substituted with galactose than in hydrated walls. Together these findings show that dehydration induces significant alteration of polysaccharide content and structure in the cell wall of C. wilmsii , which in turn might be involved in the modulation of the mechanical properties of the wall during dehydration.     

Immunocytochemical characterization of early-developing flax fiber cell walls

Summary The deposition and formation of a thick secondary wall is a major event in the differentiation of flax (Linum usitatissimum) fibers. This wall is cellulose-rich; but it also contains significant amounts of other matrix polymers which are noncellulosic such as pectins. We have used immunocytochemical techniques with antibodies specific for various epitopes associated with either pectins or arabinogalactan proteins (AGPs) to investigate the distribution of these polymers within the walls of differentiating young fibers of 1- and 2-week-old plants. Our results show that different epitopes exhibit distinct distribution patterns within fiber walls. Unesterified pectins recognized by polygalacturonic acid-rhamnogalacturonan I (PGA/RG-I) antibodies and rhamnogalacturonan II recognized by anti-RG-II-borate complex antibodies are localized all over the secondary wall of fibers. PGA/RG-I epitopes, but not RG-II epitopes, are also present in the middle lamellae and cell junctions. In marked contrast, -(14) galactans recognized by the LM5 monoclonal antibody and AGP epitopes recognized by anti--(16) galactan and LM2 antibodies are primarily located in the half of the secondary wall nearest the plasma membrane. LM2 epitopes, present in 1-week-old fibers, are undetectable later in development, suggesting a regulation of the expression of certain AGP epitopes. In addition, localization of cellulose with the cellobiohydrolase I-gold probe reveals distinct subdomains within the secondary walls of young fibers. These findings indicate that, in addition to cellulose, early-developing flax fibers synthesize and secrete different pectin and AGP molecules.     

Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum,revealed a variability in PME proteolytic maturation

Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.¹ Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca²⁺ ions during their intracellular transport.² The highly methylesterified pectins are then secreted into the apoplasm³ and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca²⁺ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.⁴ PMEs belong to a large multigene family. Sixty­six PME-related genes are predicted in the Arabidopsis genome.¹ Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.⁵ In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.