Stimulating Cell Wall Research Articles

Exogenous Nitric Oxide Induces Pathogenicity of Alternaria alternata on Huangguan Pear Fruit by Regulating Reactive Oxygen Species Metabolism and Cell Wall Modification.

Black spot caused by Alternaria alternata is one of the most common postharvest diseases in fruit and vegetables. A comprehensive investigation into its pathogenicity mechanism is imperative in order to propose a targeted and effective control strategy. The effect of nitric oxide (NO) on the pathogenicity of A. alternata and its underlying mechanism was studied. The results showed that treatment with 0.5 mM L-1 of sodium nitroprusside (SNP) (NO donor) increased the lesion diameter of A. alternata in vivo and in vitro, which was 22.8% and 13.2% higher than that of the control, respectively. Exogenous NO treatment also induced endogenous NO accumulation by activating nitric oxide synthase (NOS). In addition, NO triggered an increase in reactive oxygen species (ROS) levels. NO enhanced activities and gene expression levels of NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR). Moreover, NO stimulated cell wall degrading enzymes by activating the corresponding gene expression in vivo and in vitro. These results suggested that exogenous NO promoted the pathogenicity of A. alternata by inducing ROS accumulation and activating antioxidants and cell wall degrading enzymes. The present results could establish a theoretical foundation for the targeted control of the black spot disease in pear fruit.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Phyco-based synthesis of TiO2 nanoparticles and their influence on morphology, cyto-ultrastructure and metabolism of Spirulina platensis

The characterization of phyco-based synthesis of TiO2 NPs from Spirulina platensis (GSSp.TiO2 NPs) and the effect of such biosynthesized nanoparticles on morphology, growth, ultrastructure and enzymatic of the same alga have been studied. These nanoparticles have a good solubility, are stable in water and the average size was 17.3 nm. GSSp.TiO2 NPs aggregated and adsorbed on S. platensis membrane. Penetration and entrance of the nanoparticles into the Spirulina cells were also recorded which stimulated cell wall deformity, plasmolysis, and damage to both cell wall and plasma membrane, combined with the appearance of notch-like structure. A positive significant correlation was recorded between all applied concentrations of the biosynthesized nanoparticles and the antioxidant activities (CAT and APX) and LOX. More than 160 mg/l of GSSp.TiO2 NPs have a harmful impact on S. platensis, so nanoparticles have to be managed before disposal to protect our health and ecosystem.

NH4+ facilitates iron reutilization in the cell walls of rice (Oryza sativa) roots under iron-deficiency conditions

NH4+ and NO3− are two major nitrogen sources that differentially affect the uptake and transfer of other nutrients in rice (Oryza sativa), although their effects on Fe remobilization under Fe-starvation conditions are largely unknown. Here, by investigating the rice cultivar ‘Kasalath’ (Kas), which is tolerant of nutrient-deficient soil, we found that NH4+ negatively regulates the hemicellulose 1 content in root cell walls and stimulates the secretion of phenolic compounds from roots under −Fe conditions, thus facilitating the release of cell wall Fe and increasing soluble Fe levels in roots. NH4+ significantly increased the translocation of Fe from root to shoot by upregulating the expression of OsFRDL1 in roots and increasing the Fe content in the xylem. We further found that the higher putrescine content in rice roots under NH4+ conditions led to a higher nitric oxide (NO) content compared with NO3− conditions, and that NO is involved in the NH4+-stimulated cell wall Fe reutilization. The addition of the NO donor sodium nitroprusside significantly reduced the hemicellulose 1 content in cell walls and increased Fe release from cell walls, ultimately increasing the soluble Fe content in rice and alleviating chlorosis under −Fe conditions. Taken together, our findings demonstrate that NH4+ promotes Fe remobilization from the cell wall in rice.

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Stepwise and cooperative assembly of a cytokinetic core complex in Saccharomyces cerevisiae.

Actomyosin ring (AMR) contraction and the synthesis of an extracellular septum are interdependent pathways that mediate cytokinesis in the yeast Saccharomyces cerevisiae and other eukaryotes. How these interdependent pathways are physically connected is central for understanding cytokinesis. The yeast IQGAP (Iqg1p) belongs to the conserved AMR. The F-BAR-domain-containing protein Hof1p is a member of a complex that stimulates cell wall synthesis. We report here on the stepwise formation of a physical connection between both proteins. The C-terminal IQ-repeats of Iqg1p first bind to the essential myosin light chain before both proteins assemble with Hof1p into the Mlc1p-Iqg1p-Hof1p (MIH) bridge. Mutations in Iqg1p that disrupt the MIH complex alter Hof1p targeting to the AMR and impair AMR contraction. Epistasis analyses of two IQG1 alleles that are incompatible with formation of the MIH complex support the existence and functional significance of a large cytokinetic core complex. We propose that the MIH complex acts as hinge between the AMR and the proteins involved in cell wall synthesis and membrane attachment.

The Novel Cyst Nematode Effector Protein 19C07 Interacts with the Arabidopsis Auxin Influx Transporter LAX3 to Control Feeding Site Development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

Differential Histone Modification and Protein Expression Associated with Cell Wall Removal and Regeneration in Rice (Oryza sativa)

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Electric Field Mediated Fusion of Erugrostis Tef and Sorghum Bicolor Protoplasts and their Electroporation Conditions

An extraction solution that is optimal for protoplast release in Eragrostis tef (Zucc.) Trotter and Sorghum bicolor (L.) Moench consists of 1% Cellulysin, 0.5 % Macerozyme and 2 % Cellulase Onozuka, 1 mM CaCl2, 5 mM 2, (n-morpholino) ethane sulfonic acid, and 0.4 M mannitol. A culture medium consisting of two parts of Gamborg et al. medium, three parts of Murashige and Skoog medium, 1 % Schenk and Hildebrandt powder vitamin, 1 mg/l matin, 10 mM glutamine, 10 mg/l of 2,4-dichlorophenoxyacetic acid, and 10 mg/l naphthaleneacetic acid, was optimal for the survival and division of a small fraction of cultured protoplasts and protoplast fusion products. Electroporation and heat shock treatments stimulated cell wall formation and cell division in both sorghum and tef. Electrical parameters and conditions for fusing tef and sorgum were determined. Field pulse conditions for eventual electrogene transfer in tef and sorghum consisted of 700 and 400 V/cm, respectively, at 20°C and 40μs field pulse duration.

Regulation of cell wall glucanase activities by non-enzymic proteins in maize coleoptiles

Auxin stimulates cell wall glucanase activities in isolated segments of maize coleoptiles with no detectable change in the amounts of the enzymic peptides. To disclose the regulatory mechanism for this enhanced growth, we examined the possibility that protein interactions account for stimulating cell wall enzyme activities. One putative regulatory peptide, an acidic wall protein (AW), was separated from the wall protein fractionation profile, and this non enzymic protein was able to stimulate both exo- and endoglucanase activities in vitro. The results are consistent with the hypothesis that a pertinent glucanase/glucan interaction might be facilitated by AW mobilization within the wall in response to auxin-treatment.

Leaf growth of hybrid poplar following exposure to elevated CO2

summaryLeaf extension was stimulated following exposure of three interamerican hybrid poplar clones (Populus trichocarpa P. deltoides); ‘Unal’, ‘Boelare’, and ‘Beaupre’ and a euramerican clone ‘Primo’ (Populus nigra×P. deltoides) to elevated CO2, in controlled environment chambers. For all three interamerican clones the evidence suggests that this was the result of increased leaf cell expansion associated with enhanced cell wall extensibility (WEx), measured as tensiomerric increases in cell wall plasticity. For the interameriean clone ‘Boelare’, there was also a significant increase in cell wall elasticity following exposure to elevated CO2 (P⩽ 0.001). The effect of elevated CO2 in stimulating cell wall extensibility was confirmed in a detailed spatial analysis of extensibility made across the lamina of expanding leaves of the clone ‘Boelare’. For two of the interamerican hybrids, ‘Unal’ and ‘Beaupre’, both leaf cell water potential Ψ and turgor pressure (P) were lower in elevated than in ambient CO2. By contrast, no significant effects on the cell wall properties or leaf water relations for the euramerican hybrid ‘Primo’ were observed following exposure to elevated CO2. suggesting that the mechanism for increased leaf extension in elevated CO2, differed, depending on clone. The cumulative total length of leaves of ‘Boelare’ grown in elevated CO2, was significantly increased (P≤ 0.05) and since leaf number was not significantly increased in any inter‐american clone it is hypothesized that final leaf size was stimulated in elevated CO2 for these clones. By contrast, there was no significant effect of CO2 on cumulative total leaf length for the euramerican clone ‘Primo’, but leaf number was significantly increased by elevated CO2. The measurements suggest that total tree leaf area was stimulated for a range of poplar hybrids exposed to elevated CO2. Given the short rotation of a coppiced crop, it is likely that increased leaf areas will result in enhanced stemwood production when hybrid poplars are grown in the CO, concentrations predicted for the next century.

Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases

The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.

Characterization of theBacillus subtilisCwbA protein which stimulates cell wall lytic amidases

Characterization of the<i>Bacillus subtilis</i>CwbA protein which stimulates cell wall lytic amidases

Effect of gibberellic acid on epicotyl growth and carbohydrate distribution in derooted Pisum sativum cuttings with or without cotyledons

The effect of gibberellic acid (GA) on subhook growth in derooted cuttings of pea (Pisum sativum L. cv. Alaska) grown in the dark was studied in relation to the distribution of sugar‐related compounds in the epicotyl and cotyledons. GA stimulated subhook growth of cuttings with or without cotyledons. In cuttings with cotyledons, the net inflow of sugar‐related compounds (soluble sugars, starch, cell wall polysaccharides and sugars consumed by respiration) to the epicoiyl balanced with the net outflow from the cotyledons. GA stimulated the net inflow of sugar‐related compounds to the epicotyl and the net outflow from cotyledons. Among these compounds, GA substantially increased the amount of soluble sugars, starch and cell wall polysaccharides in the subhook. In cuttings without cotyledons, on the other hand, the net inflow of sugar‐related compounds to the subhook almost balanced with the net outflow from the epicotyl below the subhook. GA stimulated the net inflow of sugar‐related compounds to the subhook and the net outflow from the epicotyl below the subhook. Among these compounds, GA substantially increased the amount of soluble sugars and cell wall polysaccharides in the subhook. These results suggest that GA stimulates an increase in the net inflow of sugar‐related compounds to the subhook, thereby preventing an increase in osmotic potential and stimulating cell wall polysaccharide synthesis, when pea subhook growth is stimulated.

Changes in osmotic pressure and cell wall properties during auxin‐ and ethylene‐induced growth of intact coleoptiles of rice

Indole‐3‐acetic acid and 1‐aminocyclopropane‐1‐carboxylic acid, the precursor of ethylene, stimulated elongation of coleoptiles of seedlings of intact rice (Oryza sativa L. cv. Sasanishiki) submerged in buffer solution with constant air‐bubbling. The osmotic pressure of the cell sap decreased during elongation of coleoptiles. In the presence of 30 μM aminooxyacetic acid, an inhibitor of ethylene biosynthesis, in‐dole‐3‐acetic acid at 30 μM accelerated the decrease in the osmotic pressure in the early stage of growth. 1‐Aminocyclopropane‐1‐carboxylic acid at 30 μM did not influence the decrease in the osmotic pressure.Both indole‐3‐acetic acid and 1‐aminocyclopropane‐1‐carboxyIic acid decreased the minimum stress‐relaxation time and the relaxation rate of the cell wall, suggesting that both auxin and ethylene induce elongation of rice coleoptiles by stimulating cell wall loosening. These growth regulators caused an increase in the level of glucose in hemicelluloses in the early stage of growth and a decrease in the level in the subsequent last growth phase. Indole‐3‐acetic acid decreased the hydroxyproline and glucosamine levels per unit dry weight of the cell wall. These changes in the level of cell wall components may be associated with the changes in the mechanical properties of the cell walls caused by auxin and ethylene.

Factors that Influence the Yield, Stability in Culture and Cell Wall Regeneration of Spinach Mesophyll Protoplasts

A study has been made of factors that influence the yield, stability in culture, and ability to regenerate cell walls of isolated spinach (Spinacia olevacea L.) mesophyll protoplasts. The presence of 7 mM CaZ + or 7 mM Mg2+ in the isolation medium, which also included 1.5 % (w/v) Driselase, 0.25 % (w/v) pectinase and 0.8 M sorbitol, increased the yield and stability in culture of the protoplasts in a liquid nutrient medium. This latter medium has been used previously in the culture of spinach leaf discs, and does not support the division of spinach mesophyll cells. Protoplasts isolated in the presence of CaZ+ showed a greater capacity for cell wall regeneration compared with protoplasts isolated in the absence of ions or in the presence of Mg2+. Though darkness during culture improved the initial protoplast stability, it inhibited wall regeneration. The initial stability of protoplasts appeared to be dependent on plasma membrane stability, but after a few days in culture the most effective treatments were those which stimulated cell wall regeneration. In many cases, associated with cell wall regeneration, there was a budding off of small vesicles which sometimes contained chloroplasts. Protoplasts isolated in the presence of Ca2+ and incubated in low light had a 50% survival rate after 8 days culture and most had regenerated cell walls. Such culture of spinach protoplasts has not been shown previously and should be useful in developmental studies of spinach chloroplasts.

Regulation of Glucose Metabolism and Cell Wall Synthesis in Avena Stem Segments by Gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent beta-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only beta-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only beta-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.

Nutritional Control of Differentiation (Sclerotization) of the Myxomycete Physarum flavicomum

During differentiation (sclerotization) of the Myxomycete Physarum flavicomum, the acellular phasmodium converts into numerous dormant cells surrounded by cell walls. This work establishes that a condition of nutrient imbalance triggers the differentiation process. Specifically, the unavailability of an adequate spectrum of amino acids in the medium initiates the metabolic and morphological alterations characteristic of the sclerotizing plasmodium. In the absence of extracellular amino acids, the cellular pool of amino acids and cellular protein were catabolized as differentiation proceeded. The pattern of amino acids in the cellular pool also changed during differentiation, as the content of pool amino acids was reduced at least 75 percent. The decrease in protein content was negligible after 12 h incubation but was about 40 percent at 48 h when differentiation was complete. However, in the presence of extracellular amino acids, protein degradation, amino acid pool depletion, and differentiation were all inhibited. Ammonium ions (12.4 mM) similarly delayed differentiation. Differentiation, amino acid pool depletion, and the degradation of cellular protein readily occurred in the presence of an extracellular supply of dextrose, which stimulated cell wall formation. The effect of dimethyl sulfoxide, cyclic 3'-5'-adenosine monophosphate, glutathione, diamide, and other compounds on the differentiation process are reported also.