Organelle redox autonomy during environmental stress

Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H₂O₂. Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi‐organelle oxidative change. Importantly, organelle‐specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions.     

Gibberellic Acid (GA3) Inhibits ROS Increase in Chloroplasts During Dark-Induced Senescence of Pelargonium Cuttings

The temporal and spatial changes in reactive oxygen species (ROS) during dark treatment of Pelargonium cuttings and the effect of gibberellic acid (GA₃) on ROS levels were studied. ROS-related fluorescence was detected in mitochondria and cytoplasm of epidermal cells and in chloroplasts. By monitoring dichlorofluorescein (DCF) fluorescence, an initial decrease in ROS was observed under darkness in the epidermal cell cytoplasm and the chloroplasts, which was followed by an increase on the third day. Following 3 days under darkness, the size and the structure of the chloroplasts also changed, and they became more sensitive to illumination as judged by a higher accumulation of ROS. Pretreatment of leaves with GA₃ did not prevent the structural changes in the chloroplasts, but it inhibited the increase in ROS levels in all cell compartments, including the chloroplasts. It is suggested that the inhibition of ROS increase by GA₃ prevented complete disintegration of chloroplasts during dark-induced senescence and thereby enabled the maintenance of chlorophyll levels in the tissue.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A + SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H₂O₂ treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge.     

Senescence-associated mRNAs that may participate in signal transduction and protein trafficking

The differential display technique was used to generate cDNA probes in order to identify mRNAs that are up-regulated during senescence of Arabidopsis leaves. Three mRNAs were examined that had not previously been associated with senescence. The steady-state levels of these mRNAs are detectable in small amounts in mature green leaves, but increase considerably as chlorophyll levels begin to decline. This relationship to senescence occurs under natural circumstances as well as when senescence is accelerated by leaf detachment in the dark or by addition of 1-aminocyclopropane-1-carboxylic acid (ACC). Retardation of senescence by benzyladenine slows the increase of the mRNAs. One of these mRNAs appears to code for a protein (Sec 13) that may be involved in vesicle formation at the endoplasmic reticulum. Another mRNA codes for a protein with WD‐repeat motif whose function is as yet unidentified, and the third codes for a putative calcium-dependent protein kinase. A fourth cDNA has also been cloned by subtractive hybridization from senescing Arabidopsis leaves that encodes vacuolar-processing enzyme ( γ VPE). Incubation of detached leaves in darkness also caused an abrupt elevation in the steady-state levels of the γVPE , similar to that of the senescing attached leaves. The possible functions of the gene products and their involvement in cellular and biochemical processes during senescence are discussed.     

Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer

The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.     

Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators

Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H(2)O(2)-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.     

Senescence Is Induced in Individually Darkened Arabidopsis Leaves, but Inhibited in Whole Darkened Plants

It has long been known that leaf senescence can be induced in many plant species by detaching leaves and placing them in the darkness. It recently has been shown that entire Arabidopsis plants placed in the darkness are not induced to senesce, as judged by visible yellowing and certain molecular markers. Here, we show that when individual Arabidopsis leaves are darkened, but not when entire plants are darkened, senescence is induced in the covered leaves. This induction of senescence is highly localized. The phenomenon is leaf age dependent in that it occurs more rapidly and strongly in older leaves than in younger ones, as is the case with many forms of induced senescence. Whole adult plants placed in darkness, in contrast, show delayed senescence, although seedlings lacking primary leaves do not. These observations imply that the light status of the entire plant affects the senescence of individual leaves. A model summarizing the results is presented.     

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.     

The integration of glutathione homeostasis and redox signaling

Formation of reactive oxygen species (ROS) is a common feature of abiotic and biotic stress reactions. ROS need to be detoxified to avoid deleterious reactions, but at the same time, the increased formation of ROS can also be exploited for redox signaling. Glutathione, as the most abundant low-molecular weight thiol in the cellular redox system, is used for both detoxification of ROS and transmission of redox signals. Detoxification of H(2)O(2) through the glutathione-ascorbate cycle leads to a transient change in the degree of oxidation of the cellular glutathione pool, and thus a change in the glutathione redox potential. The shift in the glutathione redox potential can be sensed by glutaredoxins (GRXs), small ubiquitous oxidoreductases, which reversibly transfer electrons between the glutathione redox buffer and thiol groups of target proteins. While very little is known about native GRX target proteins and their behavior in vivo, it is shown here that reduction-oxidation-sensitive GFP (roGFP), when expressed in plants, is an artificial target protein of GRXs. The specific interaction of roGFP with GRX results in continuous formation and release of the roGFP disulfide bridge depending on the actual redox potential of the cellular glutathione buffer. Ratiometric analysis of redox-dependent fluorescence allows dynamic imaging of the glutathione redox potential. It was hypothesized that a similar equilibration occurs between the glutathione buffer and native target proteins of GRXs. As a consequence, even minor deviations in the glutathione redox potential due to either depletion of reduced glutathione (GSH) or increasing oxidation can be exploited for fine tuning the activity of target proteins. The integration of the glutathione buffer with redox-active target proteins is a local reaction in specific subcellular compartments. This observation emphasizes the importance of subcellular compartmentalization in understanding the biology of the cellular redox system in plants.     

Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity

To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss- and gain-of-function GSH content in vitro. We monitored the redox events in the cytosol and mitochondria using reduction-oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H(2)O(2) or the Trx reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, E(hc) (GSSG/2GSH), respectively. In contrast, N-acetyl-L-cysteine (NAC) treatment in H9c2 cells, or overexpression of either the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) or GCL modifier subunit (GCLM) in human embryonic kidney 293 T (HEK293T) cells, led to 3- to 4-fold increase of GSH and caused 7 or 12 mV more reducing E(hc), respectively. This condition paradoxically increased the level of mitochondrial oxidation, as demonstrated by redox shifts in mitochondrial roGFP and Trx2. Lastly, either NAC treatment (EC(50) 4 mM) or either GCLC or GCLM overexpression exhibited increased cytotoxicity and the susceptibility to the more reducing milieu was achieved at decreased levels of ROS. Taken together, our findings reveal a novel mechanism by which GSH-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.     

RoGFP1 is a quantitative biosensor in maize cells for cellular redox changes caused by environmental and endogenous stimuli

Reduction–oxidation-sensitive green fluorescent proteins (roGFPs) have been demonstrated to be valuable tools in sensing cellular redox changes in mammalian cells and model plants, yet have not been applied in crops such as maize. Here we report the characteristics of roGFP1 in transiently transformed maize mesophyll protoplasts in response to environmental stimuli and knocked-down expression of ROS-scavenging genes. We demonstrated that roGFP1 in maize cells ratiometrically responds to cellular redox changes caused by H₂O₂ and DTT, as it does in mammalian cells and model plants. Moreover, we found that roGFP1 is sensitive enough to cellular redox changes caused by genetic perturbation of single ROS genes, as exemplified by knocked-down expression of individual ZmAPXs, in maize protoplasts under controlled culture conditions and under stress conditions imposed by H₂O₂ addition. These data provide evidence that roGFP1 functions in maize cells as a biosensor for cellular redox changes triggered by genetic lesion of single ROS genes even under stress conditions, and suggest a potential application of roGFP1 in large-scale screening for maize mutants of ROS signaling involved in development and stress resistance.     

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (E_GSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) and roGFP2-Orp1 (for H₂O₂) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Methyl viologen-induced photo-oxidative stress increases hydrogen peroxide and oxidation of glutathione in chloroplasts, cytosol, and mitochondria, as well as autonomous oxidation in mitochondria.     

植物过氧化物酶体在活性氧信号网络中的作用

过氧化物酶体是高度动态、代谢活跃的细胞器,主要参与脂肪酸等脂质的代谢及产生和清除不同的活性氧(reactive oxygen species, ROS)。ROS是细胞有氧代谢的副产物。当胁迫长期作用于植物,过量的ROS会引起氧胁迫,损害细胞结构和功能的完整性,导致细胞代谢减缓,活性降低,甚至死亡;但低浓度的ROS则作为分子信号,感应细胞ROS/氧化还原变化,从而触发由环境因素导致的过氧化物酶体动力学以及依赖ROS信号网络改变而产生快速、特异性的应答。ROS也可以通过直接或间接调节细胞生长来控制植物的发育,是植物发育的重要调节剂。此外,过氧化物酶体的动态平衡由ROS、过氧化物酶体蛋白酶及自噬过程调节,对于维持细胞的氧化还原平衡至关重要。本文就过氧化物酶体中ROS的产生和抗氧化剂的调控机制进行综述,以期为过氧化物酶体如何感知环境变化,以及在细胞应答中,ROS作为重要信号分子的研究提供参考。