Drought effects on carbon and nitrogen metabolism of pea nodules can be mimicked by paraquat: evidence for the occurrence of two regulation pathways under oxidative stresses

Biological nitrogen fixation (BNF) is dramatically affected by environmental constraints such as water stress or heavy metals. It has been reported that these stresses induce the over-production of reactive oxygen species (ROS) and, in turn, oxidative stress that may be responsible for the above-mentioned BNF decline at the molecular level. Oxidative stress, occurring under different environmental stresses, has been widely related to physiological damage. However, a direct relationship between oxidative stress and the decline of BNF, independently from any other cellular damage resulting from adverse environmental situations, has yet to be demonstrated. In order to study the likely in vivo relationship between ROS and BNF inhibition in the legume-Rhizobium symbiosis, two paraquat (PQ) doses, 1 (LPQ) and 10 (HPQ) mmol m(-3), were applied to pea roots for 96 h in order to exacerbate ROS production. Whole-plant physiology and nodule metabolism parameters were determined every 24 h to monitor the evolution of plant responses to ROS. LPQ provoked BNF decline, which was preceded by a prior decrease in sucrose synthase (SS) activity. However, HPQ gave rise to a faster and more pronounced BNF inhibition, which coincided with a decline in SS and also with a reduction in leghaemoglobin (Lb) content. These results indicate a likely involvement of ROS in the effects of environmental stresses on BNF. Furthermore, these results support the occurrence of two regulation pathways for BNF under oxidative stress, one of these involving carbon shortage and the other involving Lb/oxygen flux.     

Binding of a 50-kD Protein to a U-Rich Sequence in an mRNA Encoding a Proline-Rich Protein That Is Destabilized by Fungal Elicitor

The mRNA encoding the bean proline-rich protein PvPRP1 has been shown previously to be destabilized in elicitor-treated cells. In this study, we identified a 50-kD protein in cellular extracts that binds specifically to the PvPRP1 mRNA by UV cross-linking assays. Using 32P-labeled RNAs transcribed in vitro from a series of 5[prime] deleted PvPRP1 cDNA clones, we demonstrated that the PvPRP1 mRNA binding protein (PRP-BP) binds to a 27-nucleotide U-rich (~60%) domain in the 3[prime] untranslated region. Poly(U) and, to a lesser extent, poly(A-U) competed for the PRP-BP binding activity. PRP-BP activity is redox regulated in vitro, as shown by the effects of sulfhydryl-modifying reagents on the RNA binding activity. Treatment of cellular extracts with the reducing agents DTT and [beta]-mercaptoethanol increased binding activity, whereas treatment with the oxidizing agent diamide and the alkylating agent N-ethylmaleimide inhibited binding. In extracts from elicitor-treated cells, PRP-BP activity increased approximately fivefold prior to rapid PvPRP1 mRNA degradation. The increase in PRP-BP activity was apparently due to post-translational regulation because control and elicitor-treated cell extracts supplemented with DTT showed high comparable levels of RNA binding activity. The kinetics of PRP-BP activation after elicitor treatment and its capacity for redox regulation in vitro suggested that PRP-BP may function in the elicitor-induced destabilization of PvPRP1 mRNA.     

An approach to evaluate two-electron reduction of 9,10-phenanthraquinone and redox activity of the hydroquinone associated with oxidative stress

Quinones are widely used as medicines or redox agents. The chemical properties are based on the reactions against an electron donor. 9,10-Phenanthraquinone (PQ), which is a quinone contaminated in airborne particulate matters, forms redox cycling, not Michael addition, with electron donors. Redox cycling of PQ contributes to its toxicity, following generation of reactive oxygen species (ROS). Detoxification of quinones is generally thought to be two-electron reduction forming hydroquinones. However, a hydroquinone of PQ, 9,10-dihydroxyphenanthrene (PQH(2)), has been never detected itself, because it is quite unstable. In this paper, we succeeded in detecting PQH(2) as its stable derivative, 9,10-diacetoxyphenanthrene (DAP). However, higher concentrations of PQ (>4 microM) form disproportionately with PQH(2), producing the 9,10-phenanthraquinone radical (PQ(-)) which is a one-electron reducing product of PQ. In cellular experiments using DAP as a precursor of PQH(2), it was shown that PQH(2) plays a critical role in the oxidative protein damage and cellular toxicity of PQ, showing that two-electron reduction of PQ can also initiate redox cycling to cause oxidative stress-dependent cytotoxicity.     

Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain

Paraquat (PQ(2+)) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ(2+)-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ(2+)-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ(2+)-induced hydrogen peroxide (H(2)O(2)) production was measured by fluorometric and polarographic methods. The production of H(2)O(2) was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ(2+)-induced H(2)O(2) production, (b) PQ(2+)-induced H(2)O(2) production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H(2)O(2) production by PQ(2+), and (d) the mechanism by which PQ(2+) generates H(2)O(2) depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ(2+)-induced H(2)O(2) production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease.     

The role of sucrose synthase in the response of soybean nodules to drought

Experiments were carried out to investigate the effects of droughtstress on enzymatic activities related to carbon and nitrogenmetabolism in soybean nodules. Gradual drought stress, imposedby withholding water nutrients, resulted in declines in thewater potential of leaves and nodules consistent with a significantdecline in N2 fixation. However, the amounts of nitrogenasecomponents 1 and 2 were virtually unaffected by drought stress.Similarly, no significant changes could be detected in aspartateaminotransferase, phosphoenolpyruvate carboxylase, glutaminesynthetase or alkaline invertase activities throughout the experiment.In contrast, sucrose synthase (SS), one of the enzymes involvedin sucrose metabolism in legume nodules, declined dramaticallyin activity and in content within a few days of withholdingwater. Coincident with this decline in SS activity were significantincreases in the nodule contents of sucrose, total free aminoacids and ureides. The amounts of proline, however, did notincrease until some days later. It is suggested that SS mayplay a key role in the regulation of nodule carbon metabolismand, therefore, of nitrogen fixation under drought stress conditions. Key words: Glycine max, soybean, nodule metabolism, drought stress, sucrose synthase     

Copper is a potent inhibitor of both the canonical and non-canonical NFκB pathways

Copper is an essential trace element that plays key roles in many metabolic processes. Homeostatic regulation of intracellular copper is normally tightly controlled, but deregulated copper levels are found in numerous metabolic and neurodegenerative diseases, as well as in a range of neoplasms. There are conflicting reports regarding the exact role of copper in the regulation of NFκB-responsive genes, specifically whether copper leads to increased activation of the NFκB pathways, or downregulation. Here we show that increased intracellular levels of copper, using the ionophore clioquinol, leads to a potent inhibition of NFκB pathways, induced by multiple distinct stimuli. Addition of copper to cells inhibits ubiquitin-mediated degradation of IκBα by preventing its phoshorylation by the upstream IKK complex. Intriguingly, copper-dependent inhibition of NFκB can be reversed by the addition of the reducing agent, N-acetylcysteine (NAC). These results suggest that the oxidative properties of excess copper prevent NFκB activation by blocking IκBα destruction, and that NFκB activity should be assessed in diseases associated with copper excess.     

Cancer chemoprevention: a radical perspective

Cancer chemopreventive agents block the transformation of normal cells and/or suppress the promotion of premalignant cells to malignant cells. Certain agents may achieve these objectives by modulating xenobiotic biotransformation, protecting cellular elements from oxidative damage, or promoting a more differentiated phenotype in target cells. Conversely, various cancer chemopreventive agents can encourage apoptosis in premalignant and malignant cells in vivo and/or in vitro, which is conceivably another anticancer mechanism. Furthermore, it is evident that many of these apoptogenic agents function as prooxidants in vitro. The constitutive intracellular redox environment dictates a cell's response to an agent that alters this environment. Thus, it is highly probable that normal cells, through adaption, could acquire resistance to transformation via exposure to a chemopreventive agent that promotes oxidative stress or disrupts the normal redox tone of these cells. In contrast, transformed cells, which typically endure an oxidizing intracellular environment, would ultimately succumb to apoptosis due to an uncontrollable production of reactive oxygen species caused by the same agent. Here, we provide evidence to support the hypothesis that reactive oxygen species and cellular redox tone are exploitable targets in cancer chemoprevention via the stimulation of cytoprotection in normal cells and/or the induction of apoptosis in transformed cells.     

Glutathione and modulation of cell apoptosis

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.     

Inhibition of fatty acid oxidation enhances oxidative protein folding and protects hepatocytes from endoplasmic reticulum stress

The unfolded protein response (UPR) signals protein misfolding in the endoplasmic reticulum (ER) to effect gene expression changes and restore ER homeostasis. Although many UPR-regulated genes encode ER protein processing factors, others, such as those encoding lipid catabolism enzymes, seem unrelated to ER function. It is not known whether UPR-mediated inhibition of fatty acid oxidation influences ER function or, if so, by what mechanism. Here we demonstrate that pharmacological or genetic inhibition of fatty acid oxidation renders liver cells partially resistant to ER stress-induced UPR activation both in vitro and in vivo. Reduced stress sensitivity appeared to be a consequence of increased cellular redox potential as judged by an elevated ratio of oxidized to reduced glutathione and enhanced oxidative folding in the ER. Accordingly, the ER folding benefit of inhibiting fatty acid (FA) oxidation could be phenocopied by manipulating glutathione recycling during ER stress. Conversely, preventing cellular hyperoxidation with N-acetyl cysteine partially negated the stress resistance provided by blocking FA oxidation. Our results suggest that ER stress can be ameliorated through alteration of the oxidizing environment within the ER lumen, and they provide a potential logic for the transient regulation of metabolic pathways by the UPR during stress.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Redox-dependent downregulation of Rho by Rac

Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.