Role of Reactive Oxygen Species in the Initiation of Plant Retrograde Signaling

The interaction between the nucleus and the different organelles is important in the physiology of the plant.Reac-tive oxygen species(ROS)are a by-product of the oxidation of organic molecules to obtain energy by the need to carry out the electron transfer between the different enzymatic complexes.However,they also have a role in the generation of what is known as retrograde signaling.This signal comes from the different organelles in which the oxidation of molecules or the electron transference is taking place such as mitochondria and chloroplasts.Furthermore,ROS can also induce the release of signals from the apoplast.It seems that these signals plays a role communicating to the nucleus the current status of the different parts of the plant cell to induce a changes in gene expression.In this review,the molecular mechanism of ROS retrograde signaling is described.

A retrograde apoptotic signal originating in NGF-deprived distal axons of rat sympathetic neurons in compartmented cultures

以前的调查后退幸存由神经生长因素(NGF ) 和另外的 neurotrophins 发信号支持了多样的机制，但是所有建议机制有在普通 retrogradely 传给 neuronal 细胞身体的幸存信号的产生。我们报导在轴突发现被本地 NGF 发信号压制一个 retrograde apoptotic 信号。从远侧的轴突的 NGF 退却独自是足够的激活 pro-apoptotic 抄写因素， c-jun，在房间身体。直接提供 NGF 给房间身体，从而恢复导致 NGF 的幸存的来源发信号，不能阻止 NGF 退却从远侧的轴突引起的 c-jun 激活。这是 c-jun 没在房间身体由于幸存信号的损失被激活的证据。而且，与用番红花作原料而制成的植物盐基堵住 axonal 运输禁止了建议 retrogradely 搬运的幸存的一个 retrogradely 搬运的 pro-apoptotic 信号，而非损失发信号的 NGF 剥夺引起的 c-jun 激活，引起的 c-jun 激活。另外的实验证明 c-jun， pro-caspase-3 劈开，和 apoptosis 的激活被蛋白质 kinase C 禁止者， rottlerin 和 chelerythrine 堵住，仅仅当适用于建议他们堵住轴突特定的 pro-apoptotic 信号的远侧的轴突时。rottlerin 敏感的机制被发现调整肝糖 synthase kinase 3 (GSK3 ) 活动。siRNA 的效果击倒，并且 GSK3 的药理学抑制建议 GSK3 为 NGF 剥夺引起的 apoptosis 被要求并且可以作为轴突 apoptotic 信号的一个 retrograde 搬运人工作。在被 neurotrophins 压制的轴突表明系统的 retrograde 死亡的存在为 neurodevelopment 并且为为 neurodegenerative 疾病和 neurotrauma 发现治疗有宽广含意。

Role of axon resealing in retrograde neuronal death and regeneration after spinal cord injury

Spinal cord injury leads to persistent behavioral deficits because mammalian central nervous system axons fail to regenerate. A neuron's response to axon injury results from a complex interplay of neuron-intrinsic and environmental factors. The contribution of axotomy to the death of neurons in spinal cord injury is controversial because very remote axotomy is unlikely to result in neuronal death, whereas death of neurons near an injury may reflect environmental factors such as ischemia and inflammation. In lampreys, axotomy due to spinal cord injury results in delayed apoptosis of spinal-projecting neurons in the brain, beyond the extent of these environmental factors. This retrograde apoptosis correlates with delayed resealing of the axon, and can be reversed by inducing rapid membrane resealing with polyethylene glycol. Studies in mammals also suggest that polyethylene glycol may be neuroprotective, although the mechanism(s) remain unclear. This review examines the early, mechanical, responses to axon injury in both mammals and lampreys, and the potential of polyethylene glycol to reduce injury-induced pathology. Identifying the mechanisms underlying a neuron's response to axotomy will potentially reveal new therapeutic targets to enhance regeneration and functional recovery in humans with spinal cord injury.

1O2-Mediated and EXECUTER-Dependent Retrograde Plastid-to-Nucleus Signaling in Norflurazon-Treated Seedlings of Arabidopsis thaliana

Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded pro- teins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon （NF）, an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species （ROS） were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among oth- ers, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.

Retrograde signaling in plants: A critical review focusing on the GUN pathway and beyond

Plastids communicate their developmental and physiological status to the nucleus via retrograde signaling,allowing nuclear gene expression to be adjusted appropriately.Signaling during plastid biogenesis and responses of mature chloroplasts to environmental changes are designated“biogenic”and“operational”controls,respectively.A prominent example of the investigation of biogenic signaling is the screen for gun(genomes uncoupled)mutants.Although the first five gun mutants were identified 30 years ago,the functions of GUN proteins in retrograde signaling remain controversial,and that of GUN1 is hotly disputed.Here,we provide background information and critically discuss recently proposed concepts that address GUN-related signaling and some novel gun mutants.Moreover,considering heme as a candidate in retrograde signaling,we revisit the spatial organization of heme biosynthesis and export from plastids.Although this review focuses on GUN pathways,we also highlight recent progress in the identification and elucidation of chloroplast-derived signals that regulate the acclimation response in green algae and plants.Here,stress-induced accumulation of unfolded/misassembled chloroplast proteins evokes a chloroplast-specific unfolded protein response,which leads to changes in the expression levels of nucleus-encoded chaperones and proteases to restore plastid protein homeostasis.We also address the importance of chloroplast-derived signals for activation of flavonoid biosynthesis leading to production of anthocyanins during stress acclimation through sucrose non-fermenting 1-related protein kinase 1.Finally,a framework for identification and quantification of intercompartmental signaling cascades at the proteomic and metabolomic levels is provided,and we discuss future directions of dissection of organelle-nucleus communication.

Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants.Previous studies have established that tetrapyrrole biosynthesis(TPB)and plastid gene expression(PGE)play essential roles in plastid retrograde signaling during early chloroplast biogenesis;however,their functional relationship remains unknown.In this study,we generated a series of rice TPB-related gun(genome uncoupled)mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon(NF;a noncompetitive inhibitor of carotenoid biosynthesis)and/or lincomycin(Lin;a specific inhibitor of plastid translation).We show that under NF treatment,expression of plastid-encoded polymerase(PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants.We further demonstrate that the derepressed expression of PEPtranscribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome.In addition,we show that expression of photosynthesis-associated nuclear genes(PhANGs)and PEP-transcribed genes is correlated in the rice TPB-related gun mutants,with or without NF or Lin treatment.A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants.Moreover,we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants.Further,we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling.Taken together,our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

Induced Deactivation of Genes Encoding Chlorophyll Biosynthesis Enzymes Disentangles Tetrapyrrole- Mediated Retrograde Signaling

In photosynthetic organisms, tetrapyrrole-mediated retrograde signals are proposed to contribute to a bal- anced nuclear gene expression （NGE） in response to metabolic activity in chloroplasts. We followed an experimental short- term approach that allowed the assessment of modified NGE during the first hours of specifically modified enzymatic steps of the Mg branch of tetrapyrrole biosynthesis, when pleiotropic effects of other signals can be avoided. In response to 24-h-induced silencing of CHLH, CHLM, and CHL27 encoding the CHLH subunit of Mg chelatase, the Mg protoporphyrin methyltransferase and Mg protoporphyrin monomethylester cyclase, respectively, deactivated gene expression rapidly led to reduced activity of the corresponding enzymes and altered Mg porphyrin levels. But NGE was not substantially altered. When these three genes were continuously inactivated for up to 4 d, changes of transcript levels of nuclear genes were determined. CHL27 silencing for more than 24h results in necrotic leaf lesions and modulated transcript levels of oxidative stress-responsive and photosynthesis-associated nuclear genes （PhANGs）. The prolonged deactivation of CHLH and CHLM results in slightly elevated transcript levels of PhANGs and tetrapyrrole-associated genes. These time-resolved studies indicate a complex scenario for the contribution of tetrapyrrole biosynthesis on NGE mediated by IO2-induced signaling and feedback-regulated ALA synthesis.

Meta-Analysis of Retrograde Signaling in Arabidopsis thaliana Reveals a Core Module of Genes Embedded in Complex Cellular Signaling Networks

Plastid-to-nucleus signaling is essential for the coordination and adjustment of cellular metabolism in response to environmental and developmental cues of plant cells. A variety of operational retrograde signaling path- ways have been described that are thought to be triggered by reactive oxygen species, photosynthesis redox imbalance, tetrapyrrole intermediates, and other metabolic traits. Here we report a meta-analysis based on transcriptome and pro- tein interaction data. Comparing the output of these pathways reveals the commonalities and peculiarities stimulated by six different sources impinging on operational retrograde signaling. Our study provides novel insights into the interplay of these pathways, supporting the existence of an as-yet unknown core response module of genes being regulated under all conditions tested. Our analysis further highlights affiliated regulatory cis-elements and classifies abscisic acid and auxin-based signaling as secondary components involved in the response cascades following a plastidial signal. Our study provides a global analysis of structure and interfaces of different pathways involved in plastid-to-nucleus signaling and a new view on this complex cellular communication network.

nitiation of ER Body Formation and Indole Glucosinolate Metabolism by the Plastidia Retrograde Signaling Metabolite, MEcPP

Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub（s） involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate （MEcPP）, a precursor of plastidial isoprenoids and a stress- specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum （ER） body formation and induction of indole glucosinolate （IGs） metabolism selectively, via tran- scriptional regulation of key regulators NAIl for ER body formation and MYB51/122 for IGs biosynthesis）. The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COil-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the sup- pressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard- wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.

Retrograde Signaling and Photoprotection in a gun4 Mutant of Chlamydomonas reinhardtii

GUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. Here, we report the first characterization of a gun4 insertion mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll as compared to wild-type and accumulates ProtolX. In contrast to the increase in LHCtranscription, the accumulation of most LHC proteins is drastically diminished, implying posttranscriptional down-regulation in the absence of transcriptional coordination. We found that 803 genes change their expression level in gun4 as compared to wild-type, by RNA-Seq, and this wide-ranging effect on transcription is apparent under physiological conditions. Besides LHCs, we identified transcripts encoding enzymes of the tetrapyrrole pathway and factors involved in signal transduction, transcription, and chromatin remodeling. Moreover, we observe perturbations in electron transport with a strongly decreased PSI-to-PSII ratio. This is accompanied by an enhanced activ- ity of the plastid terminal oxidase （PTOX） that could have a physiological role in decreasing photosystem II excitation pressure.

Coordinated regulation of the mitochondrial retrograde response by circadian clock regulators and ANAC017

Mitochondrial retrograde signaling(MRS)supports photosynthetic function under a variety of conditions.Induction of mitochondrial dysfunction with myxothiazol(a specific inhibitor of the mitochondrial bc1 complex)or antimycin A(an inhibitor of the mitochondrial bc1 complex and cyclic electron transport in the chloroplast under light conditions)in the light and dark revealed diurnal control of MRS.This was evidenced by(1)significantly enhanced binding of ANAC017 to promoters in the light compared with the dark in Arabidopsis plants treated with myxothiazol(but not antimycin A),(2)overlap in the experimentally determined binding sites for ANAC017 and circadian clock regulators in the promoters of ANAC013 and AOX1a,(3)a diurnal expression pattern for ANAC017 and transcription factors it regulates,(4)altered expression of ANAC017-regulated genes in circadian clock mutants with and without myxothiazol treatment,and(5)a decrease in the magnitude of LHY and CCA1 expression in an ANAC017-overexpressing line and protein–protein interaction between ANAC017 and PIF4.This study also shows a large difference in transcriptome responses to antimycin A and myxothiazol in the dark:these responses are ANAC017 independent,observed in shoots and roots,similar to biotic challenge and salicylic acid responses,and involve ERF and ZAT transcription factors.This suggests that antimycin A treatment stimulates a second MRS pathway that is mediated or converges with salicylic acid signaling and provides a merging point with chloroplast retrograde signaling.

加味大柴胡汤预防胆总管结石ERCP术后胰腺炎的临床研究

目的探讨加味大柴胡汤预防胆总管结石内镜逆行胰胆管造影术(ERCP)后胰腺炎的效果。方法选取2020年10月—2022年1月80例行ERCP手术的胆总管结石患者,根据ERCP术后胰腺炎防治方法不同分为观察组和对照组各40例。对照组术前术后予吲哚美辛纳肛,观察组在对照组基础上联合加味大柴胡汤灌肠。统计2组胰腺炎发生率、症状改善时间、住院时间及不良反应,比较治疗前后中医证候积分、JAK2/STAT3信号通路蛋白、肠道屏障功能指标水平。结果术后24 h,观察组胰腺炎发生率为2.50%低于对照组的17.50%(P<0.05)。术后24 h 2组腹胀、腹痛积分及JAK2、STAT3、D-乳酸、内毒素、二胺氧化酶水平均低于术前,且观察组低于对照组(P<0.05,P<0.01)。观察组症状改善时间、住院时间较对照组短(P<0.01)。2组不良反应发生率比较差异无统计学意义(P>0.05)。结论加味大柴胡汤可有效防治ERCP术后胰腺炎,改善肠道屏障功能,缩短住院时间,且安全性高,作用机制可能与下调JAK2/STAT3信号通路蛋白有关。

逆行信使NO在突触长时程增强过程中的作用

NO作为一种重要的生物信使分子,广泛存在于外周和中枢神经系统(CNS)。近年来,有关NO在CNS中的作用研究已取得一定进展,尤其在神经突触可塑性方面,它参与了长时程增强(LTP)过程的诱导和维持。作为一种逆行信使,通过一系列信号通路或分子,如:NO-cGMP-PKG、激发内源性ADP核糖苷化及影响C-FOS的表达,最终参与LTP的产生和维持。

THF_1 mutations lead to increased basal and wound-induced levels of oxylipins that stimulate anthocyanin biosynthesis via COI_1 signaling in Arabidopsis

Mutants defective in chloroplast development or photosynthesis are liable to accumulate higher levels of anthocyanin in photo-oxidative stress.However,regulatory mechanisms of anthocyanin biosynthesis in the mutants remain unclear.Here,we investigated the mechanism by which the deletion of thylakoid formation1（THF1） leads to an increased level of anthocyanin in Arabidopsis thaliana L.Physiological and genetic evidence showed that the increased level of anthocyanin in thf1 is dependent on coronatine-insensitive1（COM）signaling.Our data showed that thf1 had higher levels of basalα-linolenic acid（α-LeA）,and methyl jasmonate（JA）-induced α-LeA and 12-oxophytodienoic acid（OPDA） than the wild type（WT）.Consistently,expression levels of phospholipase genes including pPLAIIα and PLA-Iγ1 were elevated in thf1.Furthermore,inhibition of lipase activity by bromoenol lactone,a specific inhibitor of plant pPLA,led to producing identical levels of anthocyanins in WT and thf1 plants.Interestingly,OPDA biosynthesis was triggered by light illumination in isolated chloroplasts,indicating that new protein import into chloroplasts is not required for OPDA biosynthesis.Thus,we conclude that the elevated anthocyanin accumulation in thf1 is attributed to an increase in JA levels.This JA-mediated signaling to coordinate plant metabolism and growth in stress may be conserved in other photosensitive mutants.

Anterograde and Retrograde Regulation of Nuclear Genes Encoding Mitochondrial Proteins during Growth, Development, and Stress

Mitochondrial biogenesis and function in plants require the expression of over 1000 nuclear genes encoding mitochondrial proteins （NGEMPs）. The expression of these genes is regulated by tissue-specific, developmental, internal, and external stimuli that result in a dynamic organelle involved in both metabolic and a variety of signaling processes. Although the metabolic and biosynthetic machinery of mitochondria is relatively well understood, the factors that regu- late these processes and the various signaling pathways involved are only beginning to be identified at a molecular level. The molecular components of anterograde （nuclear to mitochondrial） and retrograde （mitochondrial to nuclear） signaling pathways that regulate the expression of NGEMPs interact with chloroplast-, growth-, and stress-signaling pathways in the cell at a variety of levels, with common components involved in transmission and execution of these signals. This positions mitochondria as important hubs for signaling in the cell, not only in direct signaling of mitochondrial function per se, but also in sensing and/or integrating a variety of other internal and external signals. This integrates and optimizes growth with energy metabolism and stress responses, which is required in both photosynthetic and non-photosynthetic cells.

Identification of Target Genes and Transcription Factors Implicated in Translation-Dependent Retrograde Sig- naling in Arabidopsis

Changes in organellar gene expression （OGE） trigger retrograde signaling. The molecular dissection of OGE-dependent retrograde signaling based on analyses of mutants with altered OGE is complicated by compensatory responses that mask the primary signaling defect and by secondary effects that influence other retrograde signaling pathways. Therefore, to identify the earliest effects of altered OGE on nuclear transcript accumulation, we have induced OGE defects in adult plants by ethanol-dependent repression of PRORS1, which encodes a prolyl-tRNA synthetase located in chloroplasts and mitochondria. After 32 h of PRORS1 repression, the translational capacity of chloroplasts was reduced, and this effect subsequently intensified, while basic photosynthetic parameters were still unchanged at 51 h. Analysis of changes in whole-genome transcriptomes during exposure to ethanol revealed that induced PRORS1 silencing affects the expression of 1020 genes in all. Some of these encode photosynthesis-related proteins, including several down-regulated light-harvesting chlorophyll a/b binding （LHC） proteins. Interestingly, genes for presumptive endoplasmic reticulum pro- teins are transiently up-regulated. Furthermore, several NAC-domain-containing proteins are among the transcription factors regulated. Candidate cis-acting elements which may coordinate the transcriptional co-regulation of genes sets include both G-box variants and sequence motifs with no similarity to known plant c/s-elements.

Chloroplast ROS and stress signaling

Chloroplasts overproduce reactive oxygen species(ROS)under unfavorable environmental conditions,and these ROS are implicated in both signaling and oxidative damage.There is mounting evidence for their roles in translating environmental fluctuations into distinct physiological responses,but their targets,signaling cascades,and mutualism and antagonism with other stress signaling cascades and within ROS signaling remain poorly understood.Great efforts made in recent years have shed new light on chloroplast ROS-directed plant stress responses,from ROS perception to plant responses,in conditional mutants of Arabidopsis thaliana or under various stress conditions.Some articles have also reported the mechanisms underlying the complexity of ROS signaling pathways,with an emphasis on spatiotemporal regulation.ROS and oxidative modification of affected target proteins appear to induce retrograde signaling pathways to maintain chloroplast protein quality control and signaling at a whole-cell level using stress hormones.This review focuses on these seemingly interconnected chloroplast-to-nucleus retrograde signaling pathways initiated by ROS and ROS-modified target molecules.We also discuss future directions in chloroplast stress research to pave the way for discovering new signaling molecules and identifying intersectional signaling components that interact in multiple chloroplast signaling pathways.

活性氧诱发的植物亚细胞间通讯

在正常发育和应激条件下,活性氧(reactive oxygen species,ROS)均会在植物细胞的不同细胞器中产生,如线粒体呼吸作用和叶绿体光合作用都是ROS的重要来源。ROS虽然对细胞存在潜在的毒害,但是适量的ROS可以作为信号分子激活信号传导通路引起细胞器内多种生理反应,从而协调亚细胞器的代谢功能及相互作用。尽管这些过程已被广泛研究,但ROS在植物亚细胞器间的通讯过程所发挥的作用则相对零散。综述了ROS的种类及其产生途径,重点介绍了植物细胞如何感知并响应ROS以及细胞器间的ROS信号传递过程。这些结果将有助于理解ROS作为信号分子在线粒体、叶绿体和细胞核信号交流中的作用。

Mitochondrial Perturbation Negatively Affects Auxin Signaling

Mitochondria are crucial players in the signaling and metabolic homeostasis of the plant cell. The molecular components that orchestrate the underlying processes, however, are largely unknown. Using a chemical biology approach, we exploited the responsiveness of Arabidopsis UDP-glucosyltransferase-encoding UGT74E2 towards mitochondrial per- turbation in order to look for novel mechanisms regulating mitochondria-to-nucleus communication. The most potent inducers of UGT74E2 shared a （2-furyl）acrylate （FAA） substructure that negatively affected mitochondrial function and was identified before as an auxin transcriptional inhibitor. Based on these premises, we demonstrated that perturbed mitochondria negatively affect the auxin signaling machinery. Moreover, chemical perturbation of polar auxin transport and auxin biosynthesis was sufficient to induce mitochondrial retrograde markers and their transcript abundance was constitutively elevated in the absence of the auxin transcriptional activators ARF7 and ARF19.

EXECUTER2 modulates the EXECUTER1 signalosome through its singlet oxygen-dependent oxidation

Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling.The Arabidopsis thaliana EXECUTER1(EX1)protein,a chloroplast-localized singlet oxygen(^(1)O_(2))sensor,undergoes tryptophan(Trp)643 oxidation by^(1)O_(2),a chloroplast-derived and light-dependent reactive oxygen species.The indole side chain of Trp is vulnerable to^(1)O_(2),leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease.The perception of^(1)O_(2)via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling.In this study,we discovered that the EX1-like protein EX2 also undergoes^(1)O_(2)-dependent Trp530 oxidation and FtsH-dependent turnover,which attenuates^(1)O_(2)signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation.Consistent with this finding,the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis,whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss-of-function mutants of EX2.Intriguingly,phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward^(1)O_(2).Collectively,these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own^(1)O_(2)-dependent oxidation,providing a new mechanistic insight into the regulation of EX1-mediated^(1)O_(2)signaling.