Regulation of Plant NADPH Oxidase

The production of Reactive Oxygen Species (ROS) is one of the key events occurring during the response of plants to environmental changes, and contributing to establish adaptive signaling pathways. A plasma membrane bound NADPH oxidase enzyme has been evidenced as the ROS producing system in various plant-microorganisms interactions. We very recently reported, that a protein of the 14-3-3 family was able to interact directly with the C-terminus part of this NADPH oxidase, and that modification of its expression in tobacco cells led to reduced amount of ROS production upon elicitation. In this addendum, we summarize this work, present additional results, and propose an hypothetic model of regulation of this oxidase in a plant defense context.Key Words: ROS, NADPH oxidase, regulation, 14-3-3, PP2C, two-hybrid     

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.

Abstract

Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

     

Phospholipase D-mediated Activation of IQGAP1 through Rac1 Regulates Hyperoxia-induced p47phox Translocation and Reactive Oxygen Species Generation in Lung Endothelial Cells

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47^phox to cell periphery, and ROS production. Exposure of [³²P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O₂ and 5% CO₂) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [³²P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of /reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [³²P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47^phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47^phox translocation and ROS formation in human lung endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes, and macrophages) generate superoxide ()² instrumental in the killing of invading pathogens solely by NADPH oxidase (1-3). Deficiency of results in the genetically inherited disorder chronic granulomatous disease, a condition in which the affected individuals are susceptible to infection (4). Phagocytic NADPH oxidase is activated when cytosolic p47^phox, p67^phox, and Rac2 translocate to the phagosomes and plasma membrane and form a complex with integral membrane cytochrome b₅₅₈, which, in turn, is a Nox2 (gp91^phox)/p22^phox heterodimer (5, 6). Assembly of phagocytic NADPH oxidase is initiated by two signals. The first is the phosphorylation of multiple serine and tyrosine residues in the p47^phox domain, which leads to unmasking of p47^phox SH3 domains that bind to a proline-rich target in the C terminus of p22^phox (7-10). The interaction between p47^phox and p22^phox seems to be an essential requirement for the translocation of other cytosolic components of the oxidase. The second signal is the binding of GTP to Rac2, which leads to the dissociation of Rac from Rho-GDI and binding to p67^phox, followed by translocation of p67^phox/GTP-Rac2 to the membrane (11). Nonphagocytic cells express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5, and most of the other cytosolic phagocytic oxidase components (12); however, the oxidative output of non-phagocytes is much smaller compared with the phagocytes. A recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to actin, thereby enhancing ROS production and contributing to cell motility in ECs (13). The one or more mechanisms responsible for differences in the oxidative burst between the phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial NADPH oxidase, which in part is mediated by ERK, p38 MAPK (14, 15), and Src (16), and hyperoxia-induced p47^phox tyrosine phosphorylation and translocation to cell periphery is dependent on Src (16). Further, tyrosine phosphorylation of cortactin mediated by Src is essential for hyperoxia-induced p47^phox translocation and /ROS generation in HPAECs (17). In addition to Src, phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of p47^phox and p22^phox in neutrophils both in vivo and in vitro (18-20). PA is generated in mammalian cells via de novo biosynthesis or hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD) (21-25). Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced the oxidative burst that correlated with PA accumulation, and inclusion of short-chain primary alcohols attenuated the NADPH oxidase mediated /ROS generation, suggesting a potential role for PLD in the regulation of NADPH oxidase (12, 26, 27). However, the downstream targets of PLD that signal NADPH oxidase activation have not been fully characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is downstream of PLD in hyperoxia-induced ROS generation. In addition, we show that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine phosphorylation of cortactin and p47^phox translocation to cell periphery. Thus, our results define a novel molecular mechanism for hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated p47^phox membrane translocation via Rac1/IQGAP1/Src/cortactin signaling cascade.     

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration

‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars.     

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.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na⁺] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets.Reactive oxygen species (ROS) contribute to neuronal damage and have been linked to excitotoxicity.^{1, 2, 3, 4} An increase in ROS generation has also been identified in acute neurologic disease such as stroke,^5,6 and recent evidence indicates that this may contribute to neuronal damage in seizures and epilepsy.^{7, 8, 9, 10} However, ROS measurements during seizure-like activity were predominantly performed in homogenates, extracellular fluids or brain regions with no clear demonstration of whether the ROS were of neuronal origin.^9,11,12 Moreover, these studies lacked the necessary temporal resolution to determine accurately the evolution of ROS generation during and after prolonged seizure activity. Such obstacles can be overcome by live cell imaging of ROS, which has emerged as a powerful tool to study disease mechanisms.¹³If seizure activity induces ROS production in neurons, then a critical question is which sources of ROS production are triggered by such activity. Previous studies have suggested that mitochondria are the primary source of ROS generation in seizure models.^8,14 However, there are alternative sources of ROS, in particular the enzymes NADPH oxidase and xanthine oxidase (XO). How these contribute to excitotoxicity during seizure activity is uncertain. That these enzymes may have an important role in seizure-induced ROS generation is suggested by two observations: (1) NMDA receptors have a pivotal role in seizure-induced neuronal damage¹⁵ and (2) direct pharmacologic activation of NMDA receptors can activate NADPH oxidase, increasing free radical production and consequently neuronal death.^5,16,17 There is also burgeoning evidence of a role for NADPH oxidase activation in chronic brain pathology secondary to psychosocial stress, which leads to the development of neuropathologic alterations, and also in neurodegenerative disease.^18,19Acute activation of NADPH oxidase in neurons has mainly been shown after direct pharmacologic activation of NMDA receptors via exposure to high levels of NMDA and this activation is calcium-dependent.^16,17 More recently, activation of NADPH oxidase has been shown during seizure activity.^9,20 These pathways also involved NMDA receptor activation and upregulation of NMDA receptor subunits NR1 and NR2B. Nonetheless, these studies used chemoconvulsant epilepsy models, which, in themselves, may have an impact on ROS generation. The mechanisms and relevance of activation of NADPH oxidase during seizure activity independent of chemoconvulsants is unclear, especially given the presence of alternative sources of ROS production. Moreover, XO may also represent a major potential source of ROS during periods of increased metabolism, such as that occuring during seizures. We have therefore asked whether NMDA receptor activation has a role in seizure-induced ROS production and which sources and mechanisms of ROS production are involved in its time course during seizure-like activity.Here, we demonstrate increased ROS generation during seizure-like activity. This is activity-dependent, but it is maintained by a Ca²⁺-independent pathway involving the activation of NMDA receptors, NADPH oxidase and XO at a later phase. Blocking NADPH oxidase and XO prevented seizure-induced neuronal cell death in vitro. We thus provide compelling evidence that these ROS-generating pathways are appropriate targets for preventing neuronal death in seizures.     

Downregulation of Blood-Brain Barrier Phenotype by Proinflammatory Cytokines Involves NADPH Oxidase-Dependent ROS Generation: Consequences for Interendothelial Adherens and Tight Junctions

Background and Objectives

Blood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.

Methods

The present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.

Results

In response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766).

Conclusion

A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.     

Nitric oxide-mediated cytosolic glucose-6-phosphate dehydrogenase is involved in aluminum toxicity of soybean under high aluminum concentration

Aims

Glucose-6-phosphate dehydrogenase (G6PDH) has been reported to be involved in resistance to various environmental stresses. However, the role of G6PDH in aluminum (Al) toxicity remains unclear.

Methods

Physiological and biochemical methods together with histochemical analysis were used to investigate the participation of G6PDH in Al-induced inhibition of root growth.

Results

Exposure to high Al concentration caused a significant increase in the activities of total and cytosolic G6PDH in roots of soybean. Al-induced inhibition of root growth and oxidative stress were alleviated by a G6PDH inhibitor. Reactive oxygen species (ROS) accumulation in Al-treated root apexes could be abolished by a NADPH oxidase inhibitor. Furthermore, treatment with a G6PDH inhibitor reduced NADPH content and NADPH oxidase activity in Al-treated root apexes. Further investigation demonstrates that nitric oxide (NO) mediates Al-induced increase in cytosolic G6PDH activity by modulating the expression of genes encoding cytosolic G6PDH. In addition, nitrate reductase pathway is mainly responsible for Al-induced NO production in root apexes.

Conclusions

These results indicate that NADPH produced by NO-modulated cytosolic G6PDH in root apexes is responsible for ROS accumulation mediated by NADPH oxidase under Al stress, subsequently suffering from oxidative stress and thus causing the inhibition of root elongation.

     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

The role of anthocyanin in photoprotection and its relationship with the xanthophyll cycle and the antioxidant system in apple peel depends on the light conditions

The synthesis of anthocyanin, the xanthophyll cycle, the antioxidant system and the production of active oxygen species (AOS) were compared between red and non‐red apple cultivars, in response to either long‐term sunlight exposure (high light intensity) during fruit development, or to exposure of bagged fruits to lower light intensity late in fruit development. During fruit development of red and non‐red apples, the xanthophyll cycle pool size decreased much more in red apple peel late in development. With accumulation of AOS induced by long‐term sunlight exposure, enhancement of the antioxidant system was found. However, this change became significantly lower in red apple than non‐red apple as fruit developed, which might serve to accelerate the anthocyanin synthesis in red apple peel. When, late in fruit development, bagged fruits were exposed to sunlight, the accumulation of AOS was lower in red apple peel than in non‐red peel. This could be due to the higher anthocyanin concentration in the red peels. Meanwhile, compared with that in non‐red cultivar, the xanthophyll cycle and the antioxidant system in red apple peel were protected first but then down‐regulated by its higher anthocyanin concentration during sunlight exposure. In conclusions, red and non‐red apples peel possess different photoprotective mechanisms under high light conditions. The relationship between anthocyanin synthesis and the xanthophyll cycle, and the antioxidant system, depends on the light conditions that fruit undergoes.