Cell Wall In Response Research Articles

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

The Aspergillus giganteus antifungal protein AFPNN5353activates the cell wall integrity pathway and perturbs calcium homeostasis

BackgroundThe antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.ResultsWe determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells.ConclusionsThe present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.

Xylan-degrading enzymes in male and female flower nectar of Cucurbita pepo

Nectar is a very complex mixture of substances. Some components (sugars and amino acids) are considered primary alimentary rewards for animals and have been investigated and characterized in numerous species for many years. In contrast, nectar proteins have been the subject of few studies and little is known of their function. Only very recently have detailed studies and characterization of nectar proteins been undertaken, and then for only a very few species. This current work represents a first step in the identification of a protein profile for the floral nectar of Cucurbita pepo. In this regard, the species studied is of particular interest in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived from male and female flowers may differ. Manually excised spots from two-dimensional (2-D) electrophoresis were subjected to in-gel protein digestion. The resulting peptides were sequenced using nanoscale LC-ESI/MS-MS (liquid chromatography-electrospray ionization/tandem mass spectrometry). An MS/MS ions search was carried out in Swiss-Prot and NCBInr databases using MASCOT software. Two-dimensional electrophoresis revealed a total of 24 spots and a different protein profile for male and female flower nectar. Four main proteins recognized by 2-D electrophoresis most closely resemble β-d-xylosidases from Arabidopsis thaliana and have some homology to a β-d-xylosidase from Medicago varia. They were present in similar quantities in male and female flowers and had the same molecular weight, but with slightly different isoelectric points. A putative function for xylosidases in floral nectar of C. pepo is proposed, namely that they may be involved in degrading the oligosaccharides released by the nectary cell walls in response to hydrolytic enzymes produced by invading micro-organisms. Several types of oligosaccharides have been reported to increase the pathogenic potential of micro-organisms. Thus, it is possible that such a mechanism may reduce the virulence of pathogens present in nectar.

The walls have ears: the role of plant CrRLK1Ls in sensing and transducing extracellular signals

In plants, organ formation and cell elongation require the constant adjustment of the dynamic and adaptable cell wall in response to environmental cues as well as internal regulators, such as light, mechanical stresses, pathogen attacks, phytohormones, and other signaling molecules. The molecular mechanisms that perceive these cues and translate them into cellular responses to maintain integrity and remodelling of the carbohydrate-rich cell wall for the coordination of cell growth are still poorly understood. In the last 3 years, the function of six membrane-localized receptor-like kinases (RLKs) belonging to the CrRLK1L family has been linked to the control of cell elongation in vegetative and reproductive development. Moreover, the presence of putative carbohydrate-binding domains in the extracellular domains of these CrRLK1Ls makes this receptor family an excellent candidate for coordinating cell growth, cell-cell communication, and constant cell wall remodelling during the plant life cycle.

Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties

Most of the implant-associated infections are attributed to bacteria adhering to biomaterial surfaces as “biofilm” communities. Bacterial transport, first contact with the surface as well as some of the further developments can be considered and can be described using physical–chemical concepts. However, far from simple colloidal particles, bacteria have various macromolecular structures at their cell wall surface for interacting with their surroundings through specific and non-specific bindings. They are also able to modify composition and features of their cell wall in response to specific surrounding conditions. Therefore, bacteria/surface material interface is a complex topic, involving chemical and physical–chemical characteristics of both material surface and bacterial cell wall, as well as biological characteristics of bacteria. Furthermore, proteins and other biomolecules coming from surrounding medium influence the bacteria/material interface by adsorbing onto the material surface prior to any adhesion of bacteria. Finally, bacterial adhesion and biofilm formation phenomena occur at the same time as eukaryotic cell adhesion in an acute competition for adhering to and colonising the biomaterial surface. Therefore, developing biomaterials able to favour cell adhesion without promoting also bacterial adhesion appears still to be a challenge. In this article, we describe briefly the common development and particularities of biofilms before focusing on what for and whether bacterial features and material surface properties are likely to be involved in bacterial adhesion, the first step in biofilm formation. The influence of adsorbed biomolecules at the bacteria/material interface is finally addressed, as well as the current knowledge about the competition between bacteria and eukaryotic cells.

Comparative ultrastructure of nonwounded Mexican lime and Yuzu leaves infected with the citrus canker bacterium Xanthomonas citri pv. citri

Ultrastructural aspects of citrus canker development were investigated in nonwounded leaves of citrus species by transmission electron microscopy (TEM). A susceptible species Mexican lime and a resistant species Yuzu were spray-inoculated with a virulent strain of Xanthomonas citri pv. citri. Initial symptoms occurred on Mexican lime approximately 9 days after inoculation, whereas they appeared on Yuzu mostly 11 days after inoculation. In Mexican lime leaves, the bacterial invasion was usually accompanied by host cell wall dissolution and cellular disruption. Fibrillar materials from degenerated cell walls were usually found in intercellular spaces. Damaged host cells with necrotic cytoplasm showed the localized separation of plasma membrane from the cell wall. Bacterial multiplication and electron-transparent capsule-like structures around bacteria were commonly observed. Meanwhile, cell wall protuberances were prominent outside host cell walls in response to bacterial invasion in Yuzu leaves. Occlusion of intercellular spaces was also formed by the fusion of two or more individual cell wall protuberances originated from two adjacent host cells. Papillae-like materials accumulated locally within host cells in close proximity to bacteria. Some bacteria were found to be undergoing degeneration in xylem vessels. Also, the shrunken, inactive bacteria were surrounded by electron-translucent fibrillar materials in intercellular spaces, implying bacterial immobilization. These cellular responses are thought to be the consequences of defense responses of Yuzu leaves to invading bacteria. In both citrus species, X. citri pv. citri contained polyphosphate bodies showing electron-dense and elliptical structures in cytoplasm.

EXPRESSION PROFILING OF THE MANNURONAN C5-EPIMERASE MULTIGENIC FAMILY IN THE BROWN ALGA LAMINARIA DIGITATA (PHAEOPHYCEAE) UNDER BIOTIC STRESS CONDITIONS(1).

The aim of this study was to profile expression of mannuronan C5-epimerase (ManC5-E) genes of Laminaria digitata (Huds.) J. V. Lamour. under treatment with brown alga cell wall components known to induce defense reaction in this organism. After determining that incubation in the presence of homo-guluronates increased the accumulation of ManC5-E transcripts, we cloned a number of partial ManC5-E genes from nonelicited and elicited specimens. In addition to these fragments, previously identified genes from sporophyte and protoplast cDNA libraries were considered for a clustering analysis. Several groups of ManC5-E genes were observed, including groups containing sequences specific from nonelicited and elicited sporophytes, and sequences isolated from protoplasts. Quantitative real-time PCR experiments were then carried out using genes representative of different groups. Results of transcript accumulation during protoplast regeneration and sporophyte elicitation supported the hypothesis that specific C5-epimerase genes are recruited under pathogen attack, enabling L.digitata to modify its cell wall in response to environmental stimuli.

A Putative MAP Kinase Kinase Kinase,MCK1, Is Required for Cell Wall Integrity and Pathogenicity of the Rice Blast Fungus,Magnaporthe oryzae

Insertional mutagenesis of Magnaporthe oryzae led to the identification of MCK1, a pathogenicity gene predicted to encode mitogen-activated protein kinase kinase kinase (MAPKKK) homologous to BCK1 in Saccharomyces cerevisiae. Targeted disruption of MCK1 resulted in the fungus undergoing autolysis and showing hypersensitivity to cell-wall-degrading enzyme. The mck1 produced significantly reduced numbers of conidia and developed appressoria in a slightly retarded manner compared with the wild type. Appressorium of the mck1 mutant was unable to penetrate into plant tissues, thereby rendering the mutant nonpathogenic. Cytorrhysis assay and monitoring of lipid mobilization suggested that the appressorial wall was altered, presumably affecting the level of turgor pressure within appressorium. Furthermore, the mck1 mutant failed to grow inside plant tissue. Complementation of the mutated gene restored its ability to cause disease symptoms, demonstrating that MCK1 is required for fungal pathogenicity. Taken together, our results suggest that MCK1 is an MAPKKK involved in maintaining cell wall integrity of M. oryzae, and that remodeling of the cell wall in response to host environments is essential for fungal pathogenesis.

Destructive Influence

Certain bacterial pathogens inject their effector proteins into the target cell to wreak havoc. Nomura et al. now show what a Pseudomonas protein does once it is inside an Arabidopsis plant cell. The virulence protein, HopM1, targets a plant defense protein, AtMIN7, by escorting that protein to its destruction by the plant's own proteasome. AtMIN7 normally functions in the vesicle trafficking that builds up a cell-wall response to pathogen invasion. K. Nomura, S. DebRoy, Y. H. Lee, N. Pumplin, J. Jones, S. Y. He, A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313 , 220-223 (2006). [Abstract] [Full Text]

‘Defence lignin’ and hydroxycinnamyl alcohol dehydrogenase activities in wounded Eucalyptus gunnii

‘Defence lignin’ and hydroxycinnamyl alcohol dehydrogenase activities in wounded <i>Eucalyptus gunnii</i>

The expression of a germin‐like protein with superoxide dismutase activity in the halophyte Atriplex lentiformis is differentially regulated by wounding and abscisic acid

The cDNA clone encoding a putative germin‐like protein (GLP) was isolated from the halophyte Atriplex lentiformis (Torr.) Wats. The deduced amino acid sequence of the protein showed high homology to those of various plant germins or germin‐like proteins. The protein was designated as AlGLP (A. lentiformis germin‐like protein). It was found that AlGLP has superoxide dismutase (SOD; EC 1.15.1.1) activity by in‐gel SOD assays after immunoprecipitation, and that it is a glycosylated oligomer in native form. The AlGLP was strongly expressed in calli and weakly in roots, but not in stems or leaves. Interestingly, the transcript level in roots was decreased by salt or abscisic acid (ABA) treatment. We present evidence that the accumulation level of AlGLP mRNA in leaves is increased by methyl jasmonate treatment or wounding, and that the induction is suppressed by ABA treatment. These results suggest that AlGLP expression is increased to fulfil H2O2 supply to modify the structure of cell wall in response to wounding.

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protein in yeast.

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein synthesis, indicating that preexisting cytosolic enzyme is incorporated into the cell wall, (ii) nor the participation of the ubiquitin yeast stress response system, as no differences were observed between wild-type and polyubiquitin-depleted (Deltaubi4) strains.

Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs.

Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked beta-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036-1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S i) and the swelling coefficient (K cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pHi = f (S i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S t cat) and the anion exchange (S t an) groups were determined in the root cell walls. The number of the functional group of each type (ΔS j) was estimated, and the corresponding values of pK a j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK a j = f (C K+)] were determined. It was found that the swelling of root cell walls changes with pH, C K+ and strongly depends on plant species. Within the experimental pH and C K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (ΔS j, pK a j, K cw), the equation {pK a j = f (CK+)} and the dependencies {K cw = f (CK+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

Two antigenically related glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell wall in response to water deficit in Phaseolus vulgaris. In this report, we show that p33 and p36 are able to adhere to leaf protoplasts, and that they bind to plasma membrane (PM) vesicles in a divalent cation-dependent manner. Data from the partial amino acid sequence of the p33 and p36 proteins indicate that they contain repeats of the decapeptide POVYKPOVEK; therefore, they are related to proline-rich proteins. Binding assays demonstrate that both proteins specifically bind to an 80 kDa PM protein. This binding is competed with a peptide that contains the RGD motif, as well as with fibronectin, which also includes this sequence, suggesting that the 80 kDa PM protein has an integrin-like function whose natural ligands are p33 and p36. This is the first case where a PM ligand for a higher plant cell wall protein has been identified.

Influence of Ripening and Turgor on the Tensile Properties of Pears: A Microscopic Study of Cellular and Tissue Changes

The tensile properties of european pear (Pyrus communis L. `Beurre Bosc') and asian pear (Pyrus pyrifolia Nakai `Choguro') were examined using a microscope-mounted apparatus that allowed direct observation and recording of cell and tissue changes during testing. To manipulate turgor potential, tissue slices from fruit of different firmness (ripeness) were incubated in sucrose solutions of differing water potential. Solution water potentials were adjusted for individual fruit, and varied between -2.5 and 1 MPa from the water potential of the expressed juice. Fruit firmness declined from 100 to 20 N and from 60 to 25 N during ripening of european and asian pears, respectively. For both european and asian pears the relationship between fruit firmness and tensile strength of tissue soaked in isotonic solutions was sigmoidal, with the major mechanism of tissue failure being cell wall failure and cell fracture at high firmness and intercellular debonding at low firmness. In the intermediate zone, where fruit firmness and tissue tensile strength decreased simultaneously, a mixture of cell wall rupture and intercellular debonding could be observed. Tissue and cell extension at maximum force both declined similarly as fruit softened. Tensile strength of tissue from firm pears (&gt;50 N firmness, &gt;0.8 N tensile strength) decreased by as much as 0.6 N during incubation in solutions that were more concentrated than the cell sap (hypertonic solutions). When similar tissue slices were incubated in solutions that were less concentrated than the cell sap (hypotonic solutions), the tensile strength increased by up to 0.4 N. This is interpreted as stress-hardening of the cell wall in response to an increase in cell turgor. Tensile strength of tissue from soft pears was not affected by osmotic changes, as the mechanism of tissue failure is cell-to-cell debonding rather than cell wall failure.

Nonlinear active force generation by cochlear outer hair cell.

We analyze the nonlinear behavior of the longitudinal and circumferential components of the active force generated by the outer hair cell wall in response to changes of its transmembrane potential. We treat the material of the wall as electroelastic, linear orthotropic in terms of strains and as nonlinear in terms of the transmembrane potential. To describe the nonlinear behavior of the active force versus the transmembrane potential, we use two (Boltzmann and simple exponential) types of approximation. We estimate free parameters of these approximations by combining the previously reported passive stiffnesses with the active strains measured in the microchamber experiment. We analyze the sensitivity of the estimated parameters corresponding to changes of the cell axial stiffness, a characteristic independently measured by several groups. We also study the effect of combining the active strains measured in the microchamber experiment with those measured in the whole cell recording experiment. We show agreement between our prediction of the active force and measurements in the whole cochlea and in isolated cells.

Distribution within the plant or compartmentation does not contribute substantially to the detoxification of excess boron in sunflower (Helianthus annuus)

Distribution and compartmentation of boron as possible mechanisms for the notable tolerance of sunflower (Helianthus annuus L.) to excess B supply have been studied. Following a short- term treatment (6 h) with 1000 µМ B, the distribution of B between different shoot organs was preferentially directed to the readily transpiring ones. Thus, B distribution in sunflower plants seems to be mainly influenced by transpiration and does not act as a B tolerance mechanism in sunflower. As a second possible mechanism for B tolerance, the compartmentation of B between water insoluble residue (i.e. cell wall) and cell sap (i.e. symplasm) was determined. Additional binding of B in the cell wall in response to excess B supply for 6 h was very low in leaves and negligible in roots. Thus, a substantial contribution of the cell wall in detoxification of excess B can be ruled out. In an additional experiment with a B supply of 1 µМ (marginal) to 1000 µМ (moderately toxic), the compartmentation of B between the apoplasmic fluid and the cell sap was investigated. The B concentration in the apoplasmic fluid was always lower than that in the symplasm. Thus, B was not excluded from the symplasm, but accumulated within it. The apoplasmic fluid did not contribute substantially to the detoxification of excess B in the leaf.

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of β(1–3),(1–4)-D-glucan and acid pH-induced extension of isolated Zea mays cell walls has been investigated using a constant-load extension technique. Acidic buffer (4.5) was able to induce an additional extension (Ea) on cell walls already extended at pH 6.8 buffer under a 20 g-mass load, indicating that the additional extension (Ea) was the parameter that better represented the effect of the different treatments on the mechanical properties of maize coleoptile cell walls. The additional extension in response to acidic pH was higher when cell walls had been previously autolysed for 24 h at pH 5.5. Furthermore, the acid-pH effect was dependent on the presence during the constant load extension of some thermo-labile factors, suggesting the participation of expansins. Acid pH increased Ea of native cell walls through an increase in the plastic extension (Ep) in agreement with a one step mechanism leading directly to irreversible (plastic) wall extension as suggested by Cosgrove (1977). The autolytic degradation of β(1–3), (1–4)-D-glucan was also able to modify the mechanical properties of maize coleoptile cell walls increasing its elastic extension (Ee) in response to pH 4.5 buffer but that modification only leads to an increase in wall extension when expansins are active, suggesting a cooperation between β-glucan turnover and expansin action.

Microbial growth and polysaccharidase activity against straw cell walls in response to the nature of added carbohydrates

Microbial growth and polysaccharidase activity against straw cell walls in response to the nature of added carbohydrates