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.

Alternaria TeA toxin activates a chloroplast retrograde signaling pathway to facilitate JAdependent pathogenicity

The chloroplast is a critical battleground in the arms race between plants and pathogens.Among microbe-secreted mycotoxins,tenuazonic acid(TeA),produced by the genus Alternaria and other phytopathogenic fungi,inhibits photosynthesis,leading to a burst of photosynthetic singlet oxygen(1O2)that is implicated in damage and chloroplast-to-nucleus retrograde signaling.Despite the signicant crop damage caused by Alternaria pathogens,our understanding of the molecular mechanism by which TeA promotes pathoge-nicity and cognate plant defense responses remains fragmentary.We now reveal that A.alternata induces necrotrophic foliar lesions by harnessing EXECUTER1(EX1)/EX2-mediated chloroplast-to-nucleus retro-grade signaling activated by TeA toxin–derived photosynthetic 1O2 in Arabidopsis thaliana.Mutation of the 1O2-sensitive EX1-W643 residue or complete deletion of the EX1 singlet oxygen sensor domain compro-mises expression of 1O2-responsive nuclear genes and foliar lesions.We also found that TeA toxin rapidly induces nuclear genes implicated in jasmonic acid(JA)synthesis and signaling,and EX1-mediated retro-grade signaling appears to be critical for establishing a signaling cascade from 1O2 to JA.The present study sheds new light on the foliar pathogenicity of A.alternata,during which EX1-dependent 1O2 signaling in-duces JA-dependent foliar cell death.

Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2

Stress-induced retrograde signal transmission from the plastids to the nucleus has long puzzled plant biologists.To address this,we performed a suppressor screen of the ceh1 mutant,which contains elevated 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate(MEcPP)levels,and identified the gain-of-function mutant impα-9,which shows reversed dwarfism and suppressed expression of stress-response genes in the ceh1 background despite heightened MEcPP.Subsequent genetic and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in Arabidopsis SKP1-like 1(ASK1)abundance,a pivotal component in the proteasome degradation pathway.This increase in ASK1 prompts the degradation of IMPα-9.Moreover,we uncovered a protein-protein interaction between IMPα-9 and TPR2,a transcriptional co-suppressor and found that a reduction in IMPα-9 levels coincides with a decrease in TPR2 abundance.Significantly,the interaction between IMPα-9 and TPR2 was disrupted in impα-9 mutants,highlighting the critical role of a single amino acid alteration in maintaining their association.Disruption of their interaction results in the reversal of MEcPP-associated phenotypes.Chromatin immunoprecipitation coupled with sequencing analyses revealed that TPR2 binds globally to stress-response genes and suggested that IMPα-9 associates with the chromatin.They function together to suppress the expression of stress-response genes under normal conditions,but this suppression is alleviated in response to stress through the degradation of the suppressing machinery.The biological relevance of our discoveries was validated under high light stress,marked by MEcPP accumulation,elevated ASK1 levels,IMPα-9 degredation,reduced TPR2 abundance,and subsequent activation of a network of stress-response genes.In summary,our study collectively unveils fresh insights into plant adaptive mechanisms,highlighting intricate interactions among retrograde signaling,the proteasome,and nuclear transport machinery.

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.

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.

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.

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.

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.

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

Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glyco- gen synthase kinase 3 （GSK3） activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotro- phins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.

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.

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.

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.

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.

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.

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.

An update on the function and regulation of methylerythritol phosphate and mevalonate pathways and their evolutionary dynamics

Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development,and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate(MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues.Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments.

The Translational Apparatus of Plastids and Its Role in Plant Development

Chloroplasts （plastids） possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco （plastid-encoded components） and Arabidopsis （nucleus-encoded components）. This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant develop- ment at various levels, presumably via retrograde signaling pathway（s）. In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.

Inactivation of Mitochondrial Complex Ⅰ Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function

At12Cys-1 （At5g64400） and At12Cys-2 （At5g09570） are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosoh Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 orAtl2cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1：At12cys.2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I ＋ lU. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth But Not of Photosynthesis in Arabidopsis

Mitogen-activated protein kinase （MAPK） pathways regulate signal transduction from different cellular com- partments and from the extracellular environment to the nucleus in all eukaryotes. One of the best-characterized MAPKs in Arabidopsis thaliana is MPK4, which was shown to be a negative regulator of systemic-acquired resistance. The mpk4 mutant accumulates salicylic acid （SA）, possesses constitutive expression of pathogenesis-related （PR） genes, and has an extremely dwarf phenotype. We show that suppression of SA and phylloquinone synthesis in chloroplasts by knocking down the IC51 gene （by crossing it with the icsl mutant） in the mpk4 mutant background did not revert mpk4-impaired growth. However, it did cause changes in the photosynthetic apparatus and severely impaired the quantum yield of pho- tosystem Ih Transmission microscopy analysis revealed that the chloroplasts＇ structure was strongly altered in the mpk4 and mpk4/icsl double mutant. Analysis of reactive oxygen species （ROS）-scavenging enzymes expression showed that suppression of SA and phylloquinone synthesis in the chloroplasts of the mpk4 mutant caused imbalances in ROS homeo- stasis which were more pronounced in mpk4/icsl than in mpk4. Taken together, the presented results strongly suggest that MPK4 is an ROS/hormonal rheostat hub that negatively, in an SA-dependent manner, regulates immune defenses, but at the same time positively regulates photosynthesis, ROS metabolism, and growth. Therefore, we concluded that MPK4 is a complex regulator of chloroplastic retrograde signaling for photosynthesis, growth, and immune defenses in Arabidopsis.

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.