Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Insulin‐like growth factor‐1 abrogates microglial oxidative stress and TNF‐α responses to spreading depression

Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability, which includes increased SD susceptibility. SD also elevates tumor necrosis factor‐α (TNF‐α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF‐α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin‐like growth factor‐1 (IGF‐1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF‐1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF‐1 abrogated the SD‐induced TNF‐α increase. Furthermore, TNF‐α application increased microglial but not astrocytic OS, an effect abrogated by IGF‐1. Next, we showed that SD increased SD susceptibility, and does so via TNF‐α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF‐α signaling. Thus, IGF‐1 mitigation of microglial ROS and TNF‐α responses may be targets for novel therapeutics development to prevent SD, and perhaps migraine.

     

Induction of oxidative stress causes functional alterations in mouse urothelium via a TRPM8‐mediated mechanism: implications for aging

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five‐month‐old(young) and 24‐month‐old (aged) mice were used. H₂O₂, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H₂O₂‐induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N‐acetyl‐cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8‐mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.     

p38 MAPK pathway is involved in high glucose-induced thioredoxin interacting protein induction in mouse mesangial cells

Excessive reactive oxygen species (ROS) play a key role in the pathogenesis of diabetic nephropathy. The thioredoxin (TRX) system, a major thiol antioxidant system, regulates the reduction of intracellular ROS. Here we show that high glucose (HG) inhibits TRX ROS-scavenging function through p38 mitogen-activated protein kinase (MAPK)-mediated induction of thioredoxin interacting protein (TXNIP) in mouse mesangial cells (MMCs). Knockdown of TXNIP in MMCs reversed HG-induced reduction of TRX activity and inhibited HG-induced activation of p38 MAPK and increased synthesis of TGF-β1 and fibronectin. These data suggest that HG-induced overexpression of TXNIP in MMCs, which may be via the p38 MAPK pathway.     

Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of quiescent cells to oxidative stress

Oxidative stress has been postulated to be involved in aging and age-related degenerative diseases. Cell death as a result of oxidative stress plays an important role in the age related diseases. Using human diploid fibroblasts (HDF) as model to study the mechanism of cell death induced by oxidative stress, a condition was standardized to induce apoptosis in the early passage sub-confluent HDFs by a brief exposure of cells to 250 M hydrogen peroxide. It was observed that p38 MAP kinase (MAPK) was activated soon after the treatment followed by over-expression of Bax protein in cells undergoing apoptosis. An interesting finding of the present study is that the confluent, quiescent HDFs were resistant to cell death under identical condition of oxidative stress. The contact-inhibited quiescent HDFs exhibited increased glutathione level following H₂O₂-treatment, did not activate p38 MAP kinase, or over-express Bax, and were resistant to cell death. These findings indicated that there was a correlation between the cell cycle and sensitivity to oxidative stress. This is the first report to our knowledge that describes a relationship between the quiescence state and anti-oxidative defense. Furthermore, our results also suggest that the p38MAPK activation-Bax expression pathway might be involved in apoptosis induced by oxidative stress.     

The intracellular domain of p75NTR as a determinant of cellular reducing potential and response to oxidant stress

The low-affinity neurotrophin receptor, p75NTR, has been found to be pro- or anti-apoptotic depending upon the cell in which it is expressed. Reactive oxygen species play a major role in apoptosis induction and enactment. Using two polyclonal PC12 populations that, respectively, do or do not express p75NTR, this paper demonstrates that p75NTR expression confers resistance to oxidant stress upon PC12 cells maintained in serum-containing medium. The effect of p75NTR on cell survival is mimicked in p75-negative cells by expression of constructs that produce the p75NTR intracellular domain (ICD) or p75NTR with the extracellular domain deleted (DeltaECD), suggesting that binding of an extracellular ligand to p75NTR is not required. Our studies further document that the differential sensitivity to oxidant stress is serum-dependent and associated with differential oxidation of glutathione between p75-positive and p75-negative cells. These results suggest that the role of p75NTR in determining the consequences and treatment of age-related disorders and conditions in which reactive oxygen species are involved may require neither the extracellular receptor domain nor, by inference, the cognate extracellular ligands of this neurotrophin receptor.     

Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16^ink4a, and p21^waf1, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(S,R)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins .     

OsTRXh1 regulates the redox state of the apoplast and influences stress responses in rice

The plant cell apoplast is the compartment beyond the cell plasmalemma, including the cell wall and intercellular space. Many environmental elements can trigger reactive oxygen species (ROS) burst at the plasma membrane which then alters the redox state of the apoplast. Recently, h-type thioredoxin (Trx), OsTRXh1, was identified to be involved in apoplastic redox state regulation in rice. OsTRXh1 is conserved redox-active Trx and can be secreted into the extracellular regions. Through transgenic rice plant, we found that OsTRXh1 regulated ROS accumulation in apoplast and influenced plant development and stress responses. This provides new insights into apoplastic redox state regulation pathway and expands our understanding of h-type Trxs function.     

Oxidative stress in biological systems and its relation with pathophysiological functions: the effect of physical activity on cellular redox homeostasis

The body of evidence from the past three decades demonstrates that oxidative stress can be involved in several diseases. This study aims to summarise the current state of knowledge on the association between oxidative stress and the pathogenesis of some characteristic to the biological systems diseases and aging process. This review also presents the effect of physical activity on redox homeostasis. There is strong evidence from studies for participation of reactive oxygen and nitrogen species in pathogenesis of acute and chronic diseases based on animal models and human studies. Elevated levels of pro-oxidants and various markers of the oxidative stress and cells and tissues damage linked with pathogenesis of cancer, atherosclerosis, neurodegenerative diseases hypertension, diabetes mellitus, cardiovascular disease, atherosclerosis, reproductive system diseases, and aging were reported. Evidence confirmed that inflammation contributes widely to multiple chronic diseases and is closely linked with oxidative stress. Regular moderate physical activity regulates oxidative stress enhancing cellular antioxidant defence mechanisms, whereas acute exercise not preceded by training can alter cellular redox homeostasis towards higher level of oxidative stress. Future studies are needed to clarify the multifaceted effects of reactive oxygen/nitrogen species on cells and tissues and to continue study on the biochemical roles of antioxidants and physical activity in prevention of oxidative stress-related tissue injury.     

A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

Glycolytic pyruvate regulates P‐Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state

ABC transporters like P‐glycoprotein (P‐gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non‐malignant cells and tumor tissue. Aim: To investigate the effect of glycolysis and the tissue redox state on P‐gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU‐145), glioma cells (Gli36), and the human cervix carcinoma cell line KB‐3‐1 transfected with a P‐gp–EGFP fusion gene that allows monitoring of P‐gp expression in living cells. During cell culture of DU‐145, Gli36, and KB‐3‐1 tumor spheroids P‐gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2‐deoxy‐D ‐glucose (2‐DDG) downregulated P‐gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi‐quantitative immunohistochemisty in DU‐145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB‐3‐1 tumor spheroids. Consequently endogenous ROS generation in DU‐145 tumor spheroids was increased in the presence of either IA or 2‐DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P‐gp expression as well as efflux of the P‐gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2‐DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P‐gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state. J. Cell. Biochem. 109: 434–446, 2010. © 2009 Wiley‐Liss, Inc.     

17‐β estradiol attenuates the pro‐oxidant activity of corticotropin‐releasing hormone in macroendothelial cells

Corticotropin‐releasing hormone, which is the predominant regulator of neuroendocrine responses to stress, attenuates inflammation through stimulation of glucocorticoid release. Enhanced corticotropin‐releasing hormone expression has been detected in inflammatory cells of the vascular endothelium, where it acts as a local regulator of endothelial redox homeostasis. Estrogens have beneficial effects on endothelial integrity and function, though the mechanism underlying their antioxidative effect remains as yet largely unknown. We therefore investigated the effect of 17β‐estradiol on pro‐oxidant action of corticotropin‐releasing hormone in vitro in macroendothelial cells, and, more specifically, the role of 17β‐estradiol on corticotropin‐releasing hormone‐induced activities/release of the antioxidant enzymes namely, endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that 17β‐estradiol abolished the stimulatory effect of corticotropin‐releasing hormone on intracellular reactive oxygen species levels and counteracted its inhibitory effect on endothelial nitric oxide synthase activity and nitric oxide release. In addition, 17β‐estradiol significantly induced superoxide dismutase and catalase activity, an effect that was not significantly influenced by corticotropin‐releasing hormone. Finally, 17β‐estradiol significantly increased glutathione levels and the glutathione/glutathione + glutathione disulfide ratio, an action that was partially blocked by corticotropin‐releasing hormone. Our results reveal that 17β‐estradiol counterbalances corticotropin‐releasing hormone‐mediated pro‐inflammatory action and thereby maintains the physiological threshold of the endothelial cell redox environment. These observations may be of importance, considering the protective role of estrogen in the development of atherosclerosis.     

Potent inhibition of gastric cancer cells by a natural compound via inhibiting TrxR1 activity and activating ROS-mediated p38 MAPK pathway

Thioredoxin reductase 1 (TrxR1) has emerged as a potential target for cancer therapy, because it is overexpressed in several types of cancers and associated with increased tumour growth and poor patient prognosis. Alantolactone (ALT), a natural sesquiterpene lactone originated from traditional folk medicine Inula helenium L., has been reported to exert antitumor activity in various tumours. However, the effect of ALT on human gastric cancer cells and its underlying mechanism remains unknown. In this study, we showed that ALT inhibited cell proliferation and induced cell apoptosis in gastric cancer cells. Mechanistically, our data found that ALT induced reactive oxygen species (ROS) production by inhibiting TrxR1 activity, resulting in the activation of p38 mitogen-activated protein kinase (MAPK) pathway and eventually cell apoptosis in gastric cancer cells. And the effects of ALT were reversed by pre-treatment with NAC (a scavenger of ROS). Further investigation revealed that ALT displayed synergistic lethality with erastin against gastric cancer cells, which demonstrating combined inhibition of TrxR1 and glutathione (GSH) leads to a synergistic effect in gastric cancer cells. More importantly, ALT treatment markedly reduced the activity of TrxR1 in vivo and inhibited the growth of gastric cancer xenografts without exhibiting significant toxicity. Taken together, these findings suggest that ALT may be used as a novel therapeutic agent against human gastric cancer.     

Analysis of catalase mutants underscores the essential role of CATALASE2 for plant growth and day length‐dependent oxidative signalling

Three genes encode catalase in Arabidopsis. Although the role of CAT2 in photorespiration is well established, the importance of the different catalases in other processes is less clear. Analysis of cat1, cat2, cat3, cat1 cat2, and cat2 cat3 T‐DNA mutants revealed that cat2 had the largest effect on activity in both roots and leaves. Root growth was inhibited in all cat2‐containing lines, but this inhibition was prevented by growing plants at high CO₂, suggesting that it is mainly an indirect effect of stress at the leaf level. Analysis of double mutants suggested some overlap between CAT2 and CAT3 functions in leaves and CAT1 and CAT2 in seeds. When plants had been grown to a similar developmental stage in short days or long days, equal‐time exposure to oxidative stress caused by genetic or pharmacological inhibition of catalase produced a much stronger induction of H₂O₂ marker genes in short day plants. Together, our data (a) underline the importance of CAT2 in basal H₂O₂ processing in Arabidopsis; (b) suggest that CAT1 and CAT3 are mainly “backup” or stress‐specific enzymes; and (c) establish that day length‐dependent responses to catalase deficiency are independent of the duration of oxidative stress.     

Stable overexpression of DJ‐1 protects H9c2 cells against oxidative stress under a hypoxia condition

It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ‐1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ‐1 overexpression attenuates simulated ischemia/reperfusion (sI/R)‐induced oxidative stress. A rat cDNA encoding DJ‐1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6‐fold increase in DJ‐1 protein expressing. Subsequently, the DJ‐1 gene‐transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ‐1 efficiently attenuated sI/R‐induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ‐1 inhibited sI/R‐induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ‐1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R‐induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ‐1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Novel molecular insights into the anti‐oxidative stress response and structure–function of a salt‐inducible cyanobacterial Mn‐catalase

KatB, a salt‐inducible Mn‐catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological–biochemical function of KatB at the molecular level. Anabaena overexpressing the wild‐type KatB protein (KatBWT) detoxified H₂O₂ efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F‐2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X‐ray crystallography‐based analysis showed that F‐2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F‐2, through its interaction with F‐66 and W‐43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn‐catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.     

Knockdown of endogenous RNF4 exacerbates ischaemia‐induced cardiomyocyte apoptosis in mice

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H₂O₂/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H₂O₂/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.