Cotton Fiber Cell Walls of Gossypium hirsutum and Gossypium barbadense Have Differences Related to Loosely-Bound Xyloglucan

Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.     

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.     

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H₂O₂) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H₂O₂ attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H₂O₂ generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys¹⁸⁹) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.     

Role of catalase,H2O2 and phenolics in resistance of pigeonpea towards Helicoverpa armigera (Hubner)

Rapid generation of superoxide radicals and accumulation of H₂O₂ is a characteristic early response of plants following perception of insect herbivory signals. Induction of oxidative burst on account of herbivory triggers various defense mechanisms in plants. Response of superoxide and H₂O₂-metabolizing enzymes and secondary metabolites in nine pigeonpea genotypes to Helicoverpa armigera feeding was investigated. Out of nine, four genotypes were found to be moderately resistant, three were intermediate and two were moderately susceptible. In general, H. armigera infestation resulted in increase in superoxide dismutase activity, H₂O₂ and phenolics content and decrease in catalase (CAT) activity in leaves, developing seeds and pod wall of pigeonpea genotypes. Peroxidase activity was found only in leaves. Among genotypes, the increase in phenolic constituents was found greater in moderately resistant genotypes than in moderately susceptible genotypes; this might determine their contribution in providing resistance to genotypes against H. armigera infestation. The capability of moderately resistant genotypes to maintain relatively lower H₂O₂ content and higher CAT activity in pod wall and developing seeds also appeared to determine resistance of genotypes towards H. armigera. Expression of resistance to H. armigera was found to be associated with a negative correlation of H₂O₂-metabolizing enzymes and phenolics with pod damage as well as with negative association between CAT activity and H₂O₂ content. A positive correlation found between H₂O₂ content and pod damage suggested the accumulation of H₂O₂ in response to pod borer attack. In addition, correlation analysis also revealed a positive association between CAT, phenolic compounds and DPPH radical scavenging activity following pod borer attack; this indicated their contribution in resistance mechanisms against H. armigera herbivory.     

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Abstract

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H₂O₂ accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.     

GhTZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic Arabidopsis

Redox homeostasis is important for plants to be able to maintain cellular metabolism, and disrupting cellular redox homeostasis will cause oxidative damage to cells and adversely affect plant growth. In this study, a cotton CCCH-type tandem zinc finger gene defined as GhTZF1, which was isolated from a cotton cell wall regeneration SSH library in our previous research, was characterized. GhTZF1 was predominantly expressed during early cell wall regeneration, and it was expressed in various vegetative and reproductive tissues. The expression of GhTZF1 was substantially up-regulated by a variety of abiotic stresses, such as PEG and salt. GhTZF1 also responds to methyl jasmonate (MeJA) and H₂O₂ treatment. Overexpression of GhTZF1 enhanced drought tolerance and delayed drought-induced leaf senescence in transgenic Arabidopsis. Subsequent experiments indicated that dark- and MeJA-induced leaf senescence was also attenuated in transgenic plants. The amount of H₂O₂ in transgenic plants was attenuated under both drought conditions and with MeJA-treatment. The activity of superoxide dismutase and peroxidase was higher in transgenic plants than in wild type plants under drought conditions. Quantitative real-time PCR analysis revealed that overexpression of GhTZF1 reduced the expression of oxidative-related senescence-associated genes (SAGs) under drought conditions. Overexpression of GhTZF1 also enhanced oxidative stress tolerance, which was determined by measuring the expression of a set of antioxidant genes and SAGs that were altered in transgenic plants during H₂O₂ treatment. Hence, we conclude that GhTZF1 may serve as a regulator in mediating drought stress tolerance and subsequent leaf senescence by modulating the reactive oxygen species homeostasis.     

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers

In animals, the small GTP-binding proteins, Rac and Rho, of theras superfamily participate in the signal rransduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.     

Does polyamine catabolism influence root development and xylem differentiation under stress conditions?

Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H₂O₂). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H₂O₂ production. Here, we suggest that H₂O₂ derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H₂O₂ balance.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

In Vitro Prenylation of the Small GTPase Rac13 of Cotton

Previous work (D.P. Delmer, J. Pear, A. Andrawis, D. Stalker [1995] Mol Gen Genet 248: 43-51) has identified a gene in cotton (Gossypium hirsutum), Rac13, that encodes a small, signal-transducing GTPase and shows high expression in the fiber at the time of transition from primary to secondary wall synthesis. Since Rac13 may be important in signal transduction pathway(s), regulating the onset of fiber secondary wall synthesis, we continue to characterize Rac13 by determining its ability to undergo posttranslational modification. In animals Rac proteins contain the C-terminal consensus sequence CaaL (where "a" can be any aliphatic residue), which is a site for geranylgeranylation (B.T. Kinsella, R.A. Erdman, W.A. Maltese [1994] J Biol Chem 266: 9786-9794). We have identified activities in developing cotton fibers that resemble in specificity the geranylgeranyl- and farnesyltransferases of animals and yeast. In addition, using prenyltransferases from rabbit reticulocytes, we show that Rac13, having a C-terminal sequence of CAFL, can serve as an in vitro substrate for geranylgeranylation but not farnesylation. However, the presence of the uncommon penultimate F residue appears to slow the rate of prenylation considerably compared with other acceptors.     

Hydrogen peroxide induces hyphal differentiation in Candida albicans

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H₂O₂ in Candida albicans. This finding is confirmed by the changing intracellular concentration of H₂O₂. In order to induce the same level of differentiation, low concentrations of exogenous H₂O₂ are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Interaction between HY1 and H2O2 in auxin-induced lateral root formation in Arabidopsis

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H₂O₂) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H₂O₂ in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H₂O₂ generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H₂O₂. This conclusion was supported by analyzing the removal of H₂O₂ with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H₂O₂-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H₂O₂ generation, since Znpp inhibition of HY1 blocked the NAA-induced H₂O₂ production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H₂O₂ production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H₂O₂ production and LR formation, both of which were decreased by DMTU. Moreover, H₂O₂ and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H₂O₂ and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H₂O₂ existing in auxin-induced LR formation in Arabidopsis.