Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl

Summary Pectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pectic polysaccharides in the control of cell elongation, we have examined the distribution of polygalacturonans in the epidermal and cortical parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni²⁺) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, calcium (Ca²⁺) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calcium with potassium pyroantimonate. Our results show that, at the tissular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni²⁺ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca²⁺ distribution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methylesterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca²⁺ distribution in the epidermal wall, suggesting that this cation is more tightly bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccharides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca²⁺ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca²⁺ in this regulation.Abbreviations Cdta diamino-1,2-cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PGA polygalacturonic acid - PME pectin methylesterase - RG I rhamnogalacturonan I - SIMS secondary ion mass spectrometry - TEM transmission electron microscopy     

Drusus sharrensis sp. n. (Trichoptera,Limnephilidae), a new species from Sharr National Park in Kosovo,with molecular and ecological notes

In this paper we describe Drusus sharrensis sp. n., from the Sharr Mountains in Kosovo. Males of the new species are morphologically most similar to Drusus krusniki Malicky, 1981, Drusus kerek Oláh, 2011 and Drusus juliae Oláh, 2011 but differ mainly in exhibiting (1) a differently shaped spinose area on tergite VIII; (2) intermediate appendages anteriorly curved in lateral view with broad tips in dorsal view; (3) inferior appendages with a distinct dorsal protrusion in the proximal half. Females of the new species are morphologically most similar to Drusus krusniki, Drusus kerek, Drusus juliae, and Drusus plicatus Radovanovic, 1942 but mainly differ in (1) segment X that is longer than the supragenital plate with distinctly pointed tips; (2) supragenital plate quadrangular with a distinct round dorsal protrusion; (3) a vulvar scale with a small median lobe. Results of phylogenetic species delimitation support monophyly of Drusus sharrensis sp. n. and recover it as sister to a clade comprising (Drusus pelasgus Oláh, 2010 + Drusus juliae + Drusus arbanios Oláh, 2010 + Drusus plicatus + (Drusus dacothracus Oláh, 2010 + Drusus illyricus Oláh, 2010)). The new species is a micro-endemic of the Sharr Mountains, a main biodiversity hotspot in the Balkan Peninsula. Main threats to the aquatic ecosystems of this part of the Balkan Peninsula are discussed.     

Etiopathogenesis of adolescent idiopathic scoliosis and new molecular concepts

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis that affects a significant number of young teenagers, mainly females (0.2-6 % of the population). Historically, several hypothesis were postulated to explain the aetiology of AIS, including genetic factors, biochemical factors, mechanics, neurological, muscular factors and hormonal factors. The neuroendocrine hypothesis involving a melatonin deficiency as the source for AIS has generated great interest. This hypothesis stems from the fact that experimental pinealectomy in chicken, and more recently in rats maintained in a bipedal mode, produces a scoliosis. The biological relevance of melatonin in idiopathic scoliosis is controversial since no significant decrease in circulating melatonin level has been observed in a majority of studies. Analysis of melatonin signal transduction in musculoskeletal tissues of AIS patients demonstrated for the first time a defect occurring in a cell autonomous manner in different cell types isolated from AIS patients suffering of the most severe form of that disease. These results have led to a classification of AIS patients in three different functional groups depending on their response to melatonin, suggesting that the cause of AIS involves several genes. Molecular analysis showed that melatonin signaling dysfunction is triggered by an increased phosphorylation of Gi proteins inactivating their function. This discovery has led to development of a first scoliosis screening assay. This test, using blood sample, is currently in clinical validation process in Canada and could be used for screening children at high risk of developing AIS.     

Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco

Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.     

Tracking cell wall changes in wine and table grapes undergoing Botrytis cinerea infection using glycan microarrays

Background and AimsThe necrotrophic fungus Botrytis cinerea infects a broad range of fruit crops including domesticated grapevine Vitis vinifera cultivars. Damage caused by this pathogen is severely detrimental to the table and wine grape industries and results in substantial crop losses worldwide. The apoplast and cell wall interface is an important setting where many plant–pathogen interactions take place and where some defence-related messenger molecules are generated. Limited studies have investigated changes in grape cell wall composition upon infection with B. cinerea, with much being inferred from studies on other fruit crops.MethodsIn this study, comprehensive microarray polymer profiling in combination with monosaccharide compositional analysis was applied for the first time to investigate cell wall compositional changes in the berries of wine (Sauvignon Blanc and Cabernet Sauvignon) and table (Dauphine and Barlinka) grape cultivars during Botrytis infection and tissue maceration. This was used in conjunction with scanning electron microscopy (SEM) and X-ray computed tomography (CT) to characterize infection progression.Key ResultsGrapes infected at veraison did not develop visible infection symptoms, whereas grapes inoculated at the post-veraison and ripe stages showed evidence of significant tissue degradation. The latter was characterized by a reduction in signals for pectin epitopes in the berry cell walls, implying the degradation of pectin polymers. The table grape cultivars showed more severe infection symptoms, and corresponding pectin depolymerization, compared with wine grape cultivars. In both grape types, hemicellulose layers were largely unaffected, as was the arabinogalactan protein content, whereas in moderate to severely infected table grape cultivars, evidence of extensin epitope deposition was present.ConclusionsSpecific changes in the grape cell wall compositional profiles appear to correlate with fungal disease susceptibility. Cell wall factors important in influencing resistance may include pectin methylesterification profiles, as well as extensin reorganization.     

CD36 antisense expression in 3T3-F442A preadipocytes

An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretinsensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF3 8 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation.     

A role for pectin-associated arabinans in maintaining the flexibility of the plant cell wall during water deficit stress

One of the main components of pectin, a primary constituent of higher plant cell walls, is rhamnogalacturonan I. This polymer comprised of linked alternating rhamnose and galacturonic acid residues is decorated with side chains composed of arabinose and galactose residues. At present, the function of these side chains is not fully understood. Our research on Southern African resurrection plants, plants that are capable of surviving severe dehydration (desiccation), has revealed that their cell walls are capable of extreme flexibility in response to water loss. One species, Myrothamnus flabellifolia, has evolved a constitutively protected leaf cell wall, composed of an abundance of arabinose polymer side chains, suggested to be arabinans and/or arabinogalactans, associated with the pectin matrix. In this article, we propose a hypothetical model that explains how the arabinan rich pectin found in the leaves of this desiccation-tolerant plant permits almost complete water loss without deleterious consequences, such as irreversible polymer adhesion, from occurring. Recent evidence suggesting a role for pectin-associated arabinose polymers in relation to water dependent processes in other plant species is also discussed.Key words: arabinans, cell wall, desiccation, resurrection, rehydration, rhamnogalacturonan IThe flowering plant cell wall is a composite structure consisting of a skeletal framework of cellulose and hemicellulose embedded within a matrix of pectin polysaccharides and cell wall glycoproteins.^1,2 The pectin matrix, in turn, is composed of three primary types of polysaccharides, these being rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII) and homogalacturonan (HG).¹ RGII is a complex polysaccharide, consisting of many unusual sugar moieties, and is not present in large amounts in the wall.³ HG is effectively a linear homopolymer of galacturonic acid and is believed to facilitate the formation of tight junctions, ‘egg boxes’, by complexing with calcium ions present in the cell wall.¹ RGI is a polymer composed of a backbone of alternating glycosidically linked rhamnose and galacturonic acid residues.¹ Side chains, consisting of either arabinogalactan polymers or linear chains of arabinans and/or galactans, are then attached to the rhamnose residues of the RGI backbone.¹ The manner with which these polymers are attached or become entangled with each other and cellulosic polymers to form the pectin matrix has been a matter of debate. The classical theory is that the RGI and HG polymers alternate with each other as block polymers and that the side chains interact with neighbouring polysaccharide chains. Recently, this standard theory has been questioned and an argument whereby the HG polymers are actually side chains of a RGI backbone polymer has been advanced.⁴ Nevertheless, the complexity of pectin polysaccharides is such that ascribing definitive functions to this matrix of polysaccharides has proven quite difficult. The physical properties of the pectin matrix suggest a number of possible functions. The water binding properties of the galacturonic acid residues indicate that polymers containing these groups have the capacity to hydrate and swell and so possibly help maintain polymer separation in the wall.⁵ The side chains of RGI include arabinan and galactan polymers which have been shown to be highly mobile^6,7,8 with the potential to interact with each other forming a temporally entangled matrix.⁹ It is also believed that arabinan chains, which have been shown to contain ferulate residues attached to terminal arabinose groups, are able to oxidatively cross-link via the formation of diferulate bridges between arabinan chains that originate on separate RGI polysaccharides.¹⁰ The pectin matrix is now believed to contain sub-domains of RGI, HG and RGII which may interact with different polysaccharide components of the cell wall such as cellulose or xyloglucan.^11,12 Hence, it is possible that the pectin matrix may form these associations with other polysaccharides via covalent⁹ and/or non-covalent¹¹ (e.g., H-bonding) interactions and in so doing ensure the integrity of the wall and its polymer organisation. Although a number of general functions, such as hydration and ion binding, have been proposed for the pectin matrix, in particular the RGI polymer and its neutral side chains, there has been difficulty in elucidating specific functions for these polysaccharides. A number of molecular genetic studies have been performed with the aim of establishing specific functions for the RGI side chains. A recent study showed that genetic removal of the arabinan side chains in the cell walls of Nicotiana plumbaginfolia results in the formation of a non-organogenic callus culture with loosely attached cells.¹³ Furthermore, it has been shown that ‘in muro’ fragmentation of the RG1 backbone in Solanum tuberosum results in abnormal development of the periderm.¹⁴ This suggests that these side chains may play at least some role in normal cell attachment and cell development. However, the real problem is that no obvious phenotypic differences between wild type and mutant plants (in which neutral side chains have been modified) have been observed.^15,16,17 It may be that the conditions under which phenotypic differences between wild type and mutant plants would arise have not yet been investigated. We believe the water binding and attachment properties of the pectin matrix are particularly important. This is especially so given the role pectin plays in the middle lamella ensuring attachment of cells to each other and in the formation of the apoplast where water mediated transport of solutes occurs.¹ Our research has focused on a group of Southern African plants termed ‘Resurrection plants’ because of their unique ability to survive severe dehydration (desiccation) to an almost air-dry state.¹⁸ We have been interested in how the cell walls of angiosperm resurrection plants such as Craterostigma wilmsii^19,20 and Myrothamnus flabellifolia^21,22 may have become adapted to survive this extreme water deficit stress (desiccation). We have shown that in the case of the Myrothamnus flabellifolia leaf cell wall, which becomes considerably folded when dried, does not undergo dramatic changes in composition or polymer location in response to desiccation.²¹ Rather we propose that this plant has evolved a constitutively protected cell wall which is able to undergo repeated cycles of desiccation and rehydration.^21,22 We have observed that the pectin component of the leaf cell wall in this species was unusually rich in arabinose polymers, most likely arabinan and arabinogalactan in nature, which we advanced was the reason that the cell wall of this species was able to tolerate desiccation.²¹ Here we provide a simple model (Fig. 1) whereby the arabinan side chains of the pectin polysaccharides are responsible for possibly buffering/replacing the lost water during desiccation and in so doing prevent the formation of tight junctions (e.g., egg boxes) or strong H-bonding interactions between the normally separate ‘skeletal’ polysaccharides (e.g., cellulose microfibrils and xyloglucan tethers) embedded in the pectin matrix. Our model is supported by the observation that cell wall arabinans play a crucial role in the response of guard cells to turgor pressure.²³ It was shown that removal of arabinans by enzymatic digestion of leaf strips of Commelina communis resulted in locking of the guard cell walls in either the open or closed position.²³ Additional roles for arabinan polymers in cell walls have recently been implied with respect to the salt tolerance of Mesembryanthemum crystallinum,²⁴ ensuring hydration of the seed endosperm of Gleditsia triacanthos during germination²⁵ and the tolerance of tropical legume seeds to dehydration.²⁶ We believe that the arabinan side chains of RGI play a critical role in the ability of cell walls to remain flexible during plant growth and may have important functions in relation to the water content of the cell. Further studies aimed at determining the relationship between wall water content, RGI side chains and cell wall flexibility may reveal hitherto unsuspected functions for these polysaccharides in the life of the plant.Open in a separate windowFigure 1A model proposing the role of arabinose rich pectin polymers in stabilising the cell wall against water loss. (A) Pectin consisting of short arabinan chains in the hydrated state, (B) pectin consisting of short arabinan chains in the dehydrated state; (C) pectin consisting of long arabinan chains in the hydrated state; (D) pectin consisting of long arabinan chains in the dehydrated state. The likelihood of irreversible tight junctions (e.g., egg boxes) forming in arabinan poor cell walls during dehydration is demonstrated in (B) while the reversible buffering effect of arabinan rich cell walls is proposed in (D) as would possibly occur in Myrothamnus flabellifolia. For simplicity arabinan chains not participating in the buffering interactions between the RGI backbone chains have been shortened to two arabinose residues in length. Note in (A) and (B) all arabinan chains are two arabinose residues in length.     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56–66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei—the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

Root extracellular traps versus neutrophil extracellular traps in host defence,a case of functional convergence?

The root cap releases cells that produce massive amounts of mucilage containing polysaccharides, proteoglycans, extracellular DNA (exDNA) and a variety of antimicrobial compounds. The released cells – known as border cells or border‐like cells – and mucilage secretions form networks that are defined as root extracellular traps (RETs). RETs are important players in root immunity. In animals, phagocytes are some of the most abundant white blood cells in circulation and are very important for immunity. These cells combat pathogens through multiple defence mechanisms, including the release of exDNA‐containing extracellular traps (ETs). Traps of neutrophil origin are abbreviated herein as NETs. Similar to phagocytes, plant root cap‐originating cells actively contribute to frontline defence against pathogens. RETs and NETs are thus components of the plant and animal immune systems, respectively, that exhibit similar compositional and functional properties. Herein, we describe and discuss the formation, molecular composition and functional similarities of these similar but different extracellular traps.