Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.     

Role of salicylic acid and oxidative burst in expression of resistance to Alternaria blight

The accumulation of salicylic acid and H₂O₂ during pathogenic infection of mustard plants with Alternaria brassicae spores was investigated to understand the role of these two defense compounds in the expression of resistance. Comparisons were made between a susceptible Brassica juncea variety RH30 and a Brassica carinata variety HC1, which is known to be resistant. An oxidative burst was detected as in situ accumulation of H₂O₂, in both the Brassica spp. after pathogen application. However, H₂O₂ generation was extracellular in the resistant variety and both extra- and intracellular in the susceptible variety. Endogenous levels of SA increased over 2.5-fold in the resistant variety HC1 in response to pathogen application and this increase was observed only in conjugated SA levels. Pathogen application also led to an increase in the antioxidant enzymes, guaiacol-dependent peroxidase (GDP) and superoxide dismutase (SOD) in HC1. Exogenous SA application to leaves led to over threefold increase in the free and conjugated SA levels in both varieties. Pathogen application to the SA pretreated plants led to over 10-fold increase in endogenous SA levels in both varieties as compared to the levels in controls and this correlated with a decrease in disease symptoms in both species. SA appeared to regulate defense responses in Brassica spp. in a concentration-dependent manner. While 2.7-fold increase in endogenous SA levels (as seen in HC1) led to an induction of antioxidant enzymes, over 10-fold increases in endogenous SA levels (as seen after exogenous SA application in both varieties) brought about no induction of the antioxidant enzymes, probably because SA itself served as an antioxidant.     

A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis

A crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography GC-MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C-14 reductase acts upstream of HYD1 C-8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post-embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15-azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.     

Expression of a plant cell wall invertase in roots of Arabidopsis leads to early flowering and an increase in whole plant biomass

In order to enhance sink strength, we expressed a heterologous plant cell wall invertase (CrCIN1) under the control of a root-specific promoter (ppyk10) in Arabidopsis thaliana. Slightly elevated apoplastic invertase activity resulted in apparent phenotypic changes. Transgenic plants developed more secondary roots and subsequently, possibly because of a higher capacity to acquire nutrients, a higher shoot and whole plant biomass. Furthermore, an early flowering phenotype was detected. The data presented here demonstrate that it is possible to modulate carbohydrate metabolism by ectopic expression of cell wall invertases and thereby influence sink organ size and whole plant development.     

The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation

Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type. Additionally, xylans from irx10 stem tissues have an almost complete loss of the GlcUA side chain, whereas the level of 4- O -Me-GlcUA was similar to that in wild type. Deletion of the predicted signal peptide from the N terminus of IRX10 or IRX10-L results in an inability to rescue the irx10 irx10-L double mutant phenotype. These findings demonstrate that IRX10 and IRX10-L perform a critical function in the synthesis of glucuronoxylan during secondary cell-wall formation, and that this activity is associated with the formation of the xylan backbone structure. This contrasts with the proposed function of the tobacco NpGUT1, which is closely related to the Arabidopsis IRX10 and IRX10-L proteins, in rhamnogalacturonan II biosynthesis.     

Glucose alleviates cadmium toxicity by increasing cadmium fixation in root cell wall and sequestration into vacuole in Arabidopsis

Glucose(Glu) is involved in not only plant physiological and developmental events but also plant responses to abiotic stresses. Here, we found that the exogenous Glu improved root and shoot growth, reduced shoot cadmium(Cd) concentration, and rescued Cdinduced chlorosis in Arabidopsis thaliana(Columbia ecotype,Col-0) under Cd stressed conditions. Glucose increased Cd retained in the roots, thus reducing its translocation from root to shoot signi fi cantly. The most Cd retained in the roots was found in the hemicellulose 1. Glucose combined with Cd(Glu t Cd) treatment did not affect the content of pectin and its binding capacity of Cd while it increased the content of hemicelluloses 1 and the amount of Cd retained in it signi fi cantly. Furthermore, Leadmium Green staining indicated that more Cd was compartmented into vacuoles in Glu t Cd treatment compared with Cd treatment alone, which was in accordance with the R e ssigni fi cant upregulation of the expression of tonoplastlocalized metal transporter genes, suggesting that compartmentation of Cd into vacuoles also contributes to the Glu-alleviated Cd toxicity. Taken together, we demonstrated that Glu-alleviated Cd toxicity is mediated through increasing Cd fi xation in the root cell wall and sequestration into the vacuoles.     

Mechanisms for the generation of reactive oxygen species in plant defence response

Recent data indicate that plants, in a manner similar to the situation found in mammalian phagocytotic cells, produce reactive oxygen species (ROS) in response to pathogen infection. This reaction could be very quick when using pre-existing, usually exocellular, components and/or, when biochemical machinery of the cell is activated, relatively late and long-lasting. The oxidative burst is defined as a rapid, transient production of high levels of ROS in response to external stimuli. Two major models depicting the origin of ROS in the oxidative burst are described, namely: the NADPH oxidase system and the pH-dependent generation of hydrogen peroxide by exocellular peroxidases. Additionally, the participation of exocellular ROS-generating enzymes, like germin-like oxalate oxidases and amine oxidases, in plant defence response is demonstrated. The involvement of protoplasmic ROS-generating systems is also indicated.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

Arabidopsis G‐protein interactome reveals connections to cell wall carbohydrates and morphogenesis

The heterotrimeric G‐protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G‐protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G‐protein associates with heptahelical G‐protein‐coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G‐protein effectors and scaffold proteins, we screened a set of proteins from the G‐protein complex using two‐hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G‐protein interactome. Within this core, over half of the interactions comprising two‐thirds of the nodes were retested and validated as genuine in planta. Co‐expression analysis in combination with phenotyping of loss‐of‐function mutations in a set of core interactome genes revealed a novel role for G‐proteins in regulating cell wall modification.     

Production of reactive oxygen species,impairment of photosynthetic function and dynamic changes in mitochondria are early events in cadmium‐induced cell death in Arabidopsis thaliana

Background information. Cadmium (Cd) is a highly toxic heavy metal that causes changes in plant metabolism through inhibiting photosynthesis and respiration. The effects of Cd on the morphology and function of the chloroplast and mitochondria, as well as on the production and localization of ROS (reactive oxygen species), were studied at the single‐cell level in Arabidopsis. Results. The present study showed that the morphology of chloroplasts changed after Cd treatment, and the photochemical efficiency dramatically declined prior to obvious morphological distortion in the chloroplasts. A quick burst of ROS was detected after Cd treatment. The ROS appeared first in the mitochondria and subsequently in the chloroplast. Simultaneously, the mitochondria clumped irregularly around the chloroplasts or aggregated in the cytoplasm, and the movement of mitochondria was concomitantly blocked. Furthermore, the production of ROS was decreased after pre‐treatment with ascorbic acid or catalase, which prevented inhibition of photosynthesis, organelle changes and subsequent protoplast death. Our results suggest that the distribution and mobility of mitochondria, the morphology of chloroplasts and the accumulation of ROS play important roles in Cd‐induced cell death. The results are in good agreement with previous reports of many types of apoptotic‐like cell death. Conclusion. The changes in the distribution and mobility of mitochondria, and morphology of chloroplasts, as well as the accumulation of ROS, play important roles in Cd‐induced cell death.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species

The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased susceptibility to AAL-toxin due to the knockout of a gene involved in sphingolipid metabolism. Genetic screening of mutagenized loh2 seeds resulted in the isolation of AAL-toxin-resistant mutant atr1.Atr1 displays a wild type phenotype when grown on soil but it develops less biomass than loh2 on media supplemented with 2% and 3% sucrose. Atr1 was also more tolerant to the reactive oxygen species-generating herbicides aminotriazole (AT) and paraquat. Microarray analyses of atr1 and loh2 under AT-treatment conditions that trigger cell death in loh2 and no visible damage in atr1 revealed genes specifically regulated in atr1 or loh2. In addition, most of the genes strongly downregulated in both mutants were related to cell wall extension and cell growth, consistent with the apparent and similar AT-induced cessation of growth in both mutants. This indicates that two different pathways, a first controlling growth inhibition and a second triggering cell death, are associated with AT-induced oxidative stress.