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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., (© 2021 The Author(s).)

Low and high temperatures are life-threatening stress factors, diminishing plant productivity. One of the earliest responses of plants to stress is a rapid burst of reactive oxygen species (ROS) in chloroplasts. Widespread efforts over the past decade shed new light on the chloroplast as an environmental sensor, translating the environmental fluctuation into varying physiological responses by utilizing distinct retrograde (chloroplast-to-nucleus) signals. Recent studies have unveiled that chloroplasts mediate a similar unfolded/misfolded/damaged protein response (cpUPR) as observed in the endoplasmic reticulum and mitochondria. Although observing cpUPR is not surprising since the chloroplast is a prime organelle producing harmful ROS, the intertwined relationship among ROS, protein damage, and chloroplast protein quality controls (cpPQCs) with retrograde signaling has recently been reported. This finding also gives rise to critical attention on chloroplast proteins involved in cpPQCs, ROS detoxifiers, transcription/translation, import of precursor proteins, and assembly/maturation, the deficiency of which compromises chloroplast protein homeostasis (proteostasis). Any perturbation in the protein may require readjustment of proteostasis by transmitting retrograde signal(s) to the nucleus, whose genome encodes most of the chloroplast proteins involved in proteostasis. This review focuses on recent findings on cpUPR and chloroplast-targeted FILAMENTOUS TEMPERATURE-SENSITIVE H proteases involved in cpPQC and retrograde signaling and their impacts on plant responses to temperature stress.

We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles. For complete details on the use and execution of this protocol, please refer to Medina-Puche et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Chloroplasts mediate genetically controlled cell death via chloroplast-to-nucleus retrograde signaling. To decipher the mechanism, we examined chloroplast-linked lesion-mimic mutants of Arabidopsis ( Arabidopsis thaliana ) deficient in plastid division, thereby developing gigantic chloroplasts (GCs). These GC mutants, including crumpled leaf ( crl ), constitutively express immune-related genes and show light-dependent localized cell death (LCD), mirroring typical autoimmune responses. Our reverse genetic approach excludes any potential role of immune/stress hormones in triggering LCD. Instead, transcriptome and in silico analyses suggest that reactive electrophile species (RES) generated via oxidation of polyunsaturated fatty acids (PUFAs) or lipid peroxidation-driven signaling may induce LCD. Consistent with these results, the one of the suppressors of crl , dubbed spcrl4 , contains a causative mutation in the nuclear gene encoding chloroplast-localized FATTY ACID DESATURASE5 (FAD5) that catalyzes the conversion of palmitic acid (16:0) to palmitoleic acid (16:1). The loss of FAD5 in the crl mutant might attenuate the levels of RES and/or lipid peroxidation due to the reduced levels of palmitic acid-driven PUFAs, which are prime targets of reactive oxygen species. The fact that fad5 also compromises the expression of immune-related genes and the development of LCD in other GC mutants substantiates the presence of an intrinsic retrograde signaling pathway, priming the autoimmune responses in a FAD5-dependent manner., (© 2020 American Society of Plant Biologists. All rights reserved.)

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Singlet oxygen ( 1 O 2 ) is a potent oxidizing agent, principally generated by photosystem II (PSII) as a byproduct of photosynthesis. Hence, 1 O 2 damages PSII, especially the PSII reaction center (RC) proteins, promoting a process called PSII repair cycle. The hetero-hexameric FtsH protease, located in the thylakoid membrane, is essential in degrading these damaged PSII RC proteins, which defines the first step of the PSII repair. The loss of the central subunit of the FtsH protease, FtsH2 (VAR2), weakens the PSII repair, thereby impairing PSII proteostasis. A recent study demonstrated that the impaired proteostasis (or accumulation of damaged proteins) in the chloroplasts of the var2 mutant induces an unfolded/misfolded protein response (UPR)-like response, more appropriately referred to as a damaged protein response (DPR), as evident in the accumulation of proteins related to the protein quality control (PQC). Comparison of data from chloroplast proteomics data with RNA sequencing in the context of the UPR-like response suggests a plausible activation of retrograde signaling in the var2 mutant. Either through the enhanced level of 1 O 2 or by impairing the substrate-unfolding activity of FtsH2, the reinforced defect appears to induce stress-related genes via the stress hormone salicylic acid (SA). This finding suggests that impaired chloroplast proteostasis (specifically for PSII proteins) may activate the chloroplast-established isochorismate pathway to produce SA. If this assumption is correct, then SA serves as a retrograde signaling molecule. In this review, we will discuss the impact of chloroplast proteostasis on chloroplasts-to-nucleus communication.

Singlet oxygen ( 1 O 2 ) is a prime cause of photo-damage of the photosynthetic apparatus. The chlorophyll molecules in the photosystem II reaction center and in the light-harvesting antenna complex are major sources of 1 O 2 generation. It has been thought that the generation of 1 O 2 mainly takes place in the appressed regions of the thylakoid membranes, namely, the grana core, where most of the active photosystem II complexes are localized. Apart from being a toxic molecule, new evidence suggests that 1 O 2 significantly contributes to chloroplast-to-nucleus retrograde signalling that primes acclimation and cell death responses. Interestingly, recent studies reveal that chloroplasts operate two distinct 1 O 2 -triggered retrograde signalling pathways in which β-carotene and a nuclear-encoded chloroplast protein EXECUTER1 play essential roles as signalling mediators. The coexistence of these mediators raises several questions: their crosstalk, source(s) of 1 O 2 , downstream signalling components, and the perception and reaction mechanism of these mediators towards 1 O 2 . In this review, we mainly discuss the molecular genetic basis of the mode of action of these two putative 1 O 2 sensors and their corresponding retrograde signalling pathways. In addition, we also propose the possible existence of an alternative source of 1 O 2 , which is spatially and functionally separated from the grana core., (© 2018 John Wiley & Sons Ltd.)

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are an important microbe-associated molecular pattern (MAMP) that triggers immune responses in plants and animals. A previous genetic screen in Arabidopsis ( Arabidopsis thaliana ) identified LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE), a B-type lectin S -domain receptor kinase, as a sensor of LPS. However, the LPS-activated LORE signaling pathway and associated immune responses remain largely unknown. In this study, we found that LPS trigger biphasic production of reactive oxygen species (ROS) in Arabidopsis. The first transient ROS burst was similar to that induced by another MAMP, flagellin, whereas the second long-lasting burst was induced only by LPS. The LPS-triggered second ROS burst was found to be conserved in a variety of plant species. Microscopic observation of the generation of ROS revealed that the LPS-triggered second ROS burst was largely associated with chloroplasts, and functional chloroplasts were indispensable for this response. The lipid A moiety, the most conserved portion of LPS, appears to be responsible for the second ROS burst. Surprisingly, the LPS- and lipid A-triggered second ROS burst was only partially dependent on LORE. Together, our findings provide insight on the LPS-triggered ROS production and the associated signaling pathway., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Formation of singlet oxygen ((1)O2) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, (1)O2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of (1)O2 was first documented in the fluorescent (flu) mutant of Arabidopsis It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by (1)O2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of (1)O2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of (1)O2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and (1)O2-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.

Enhanced levels of singlet oxygen ((1)O(2)) in chloroplasts trigger programmed cell death. The impact of (1)O(2) production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates (1)O(2) upon a dark/light shift. The onset of (1)O(2) production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than (1)O(2) directly. In flu seedlings, (1)O(2)-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. (1)O(2)-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of (1)O(2) in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.

Retrograde plastid-to-nucleus signaling tightly controls and coordinates the nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activity. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens target plastid-specific but not extraplastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the constitutive activator of AAA-ATPase (caa33) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division, and thereby perturbing plastid homeostasis. This results in preconditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the capacity of the plant to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the susceptibility of the plant to stress, and emphasizes the need to consider the possible impact of preconditioning on retrograde plastid-to-nucleus signaling., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The NADPH-dependent protochlorophyllide (Pchlide) oxidoreductase (POR) is unique because it is a photoenzyme that requires light for its catalytic activity and uses Pchlide itself as a photoreceptor. In Arabidopsis, there are three structurally related PORs, denoted PORA, PORB, and PORC. The import of one of them, PORA, into plastids of cotyledons is substrate dependent. This substrate dependence is demonstrated in intact seedlings of wild-type Arabidopsis and two mutants, xantha2, which is devoid of Pchlide, and flu, which upon redarkening rapidly accumulates Pchlide. In true leaves, PORA uptake does not require the presence of Pchlide. The organ specificity of the substrate-dependent import of PORA reveals a means of controlling plastid protein translocation that is closely associated with a key step in plant development, the light-dependent transformation of cotyledons from a storage organ to a photosynthetically active leaf.

Singlet oxygen (1O2) is an excited state of molecular oxygen with an electron spin shift in the molecular orbitals, which is extremely unstable and highly reactive. In plants, 1O2 is primarily generated as a byproduct of photosynthesis in the photosystem II reaction center (PSII RC) and the light-harvesting antenna complex (LHC) in the grana core (GC). This occurs upon the absorption of light energy when the excited chlorophyll molecules in the PSII transfer the excess energy to molecular oxygen, thereby generating 1O2. As a potent oxidant, 1O2 promotes oxidative damage. However, at sub-lethal levels, it initiates chloroplast-to-nucleus retrograde signaling to contribute to plant stress responses, including acclimation and cell death. The thylakoid membranes comprise two spatially separated 1O2 sensors: β-carotene localized in the PSII RC in the GC and the nuclear-encoded chloroplast protein EXECUTER1 (EX1) residing in the non-appressed grana margin (GM). Finding EX1 in the GM suggests the existence of an additional source of 1O2 in the GM and the presence of two distinct 1O2-signaling pathways. In this review, we mainly discuss the genesis and impact of 1O2 in plant physiology. [ABSTRACT FROM AUTHOR]

Chloroplast–nucleus communication takes place via processes called anterograde and retrograde signaling pathways. Discovery of the retrograde signaling pathways from the chloroplasts to the nucleus also raised an intriguing proposition that chloroplasts may serve as environmental sensors since multitudes of environmental factors disturb chloroplastic homeostasis. Certain chloroplastic perturbations, mostly impairing transcription/translation, are coupled to the repression of photosynthesis-associated nuclear genes (PhANGs), thus finely coordinating photosynthetic and chloroplastic homeostasis. The unbiased forward genetic screen in Arabidopsis leads to the identification of six independent loci called GENOMES UNCOUPLED (GUN), whose inactivation was found to de-repress the expression of PhANGs under certain conditions promoting retrograde signaling. Of the six GUNs, five encode proteins associated with tetrapyrrole biosynthesis and one, namely GUN1, encodes a member of the pentatricopeptide repeat protein family. Despite the fact that GUN1 plays a role as a central signaling mediator for retrograde communication, the molecular details of GUN1 protein still remain to be elucidated. Here, we recapitulate our current understanding of the GUN1-mediated retrograde signaling pathway and propose a possible mode of action of GUN1 in the chloroplasts together with different aspects of GUN1 protein activity that deserve further investigation. [ABSTRACT FROM AUTHOR]

Cellular protein homeostasis (proteostasis) is maintained through the balance between de novo synthesis and proteolysis. The unfolded/misfolded protein response (UPR) that is triggered by stressed endoplasmic reticulum (ER) also plays an important role in proteostasis in both plants and animals. Although ER-triggered UPR has been extensively studied in plants, the molecular mechanisms underlying mitochondrial and chloroplastic UPRs are largely uncharacterized despite the fact that these organelles are sites of production of harmful reactive oxygen species (ROS), which damage proteins. In this study, we demonstrate that chloroplasts of the Arabidopsis yellow leaf variegation 2 (var2) mutant, which lacks the metalloprotease FtsH2, accumulate damaged chloroplast proteins and trigger a UPR-like response, namely the accumulation of a suite of chloroplast proteins involved in protein quality control (PQC). These PQC proteins include heat-shock proteins, chaperones, proteases, and ROS detoxifiers. Given that FtsH2 functions primarily in photosystem II proteostasis, the accumulation of PQC-related proteins may balance the FtsH2 deficiency. Moreover, the apparent up-regulation of the cognate transcripts indicates that the accumulation of PQC-related proteins in var2 is probably mediated by retrograde signaling, indicating the occurrence of a UPR-like response in var2. [ABSTRACT FROM AUTHOR]

Seedlings of Arabidopsis have been exposed to norflurazon. Depending on the developmental stage at which seedlings were first exposed to the inhibitor, enhanced production of reactive oxygen species occurred and, among others, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced.Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded proteins. 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 others, 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. [ABSTRACT FROM AUTHOR]

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen (1O2) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator ( soldat), were identified that abrogate 1O2-mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/ flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of 1O2-responsive genes after a dark-to-light shift they do not show a 1O2-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/ flu seedlings seems to antagonize a subsequent 1O2-mediated cell death response without suppressing 1O2-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as ‘plastid signaling’. [ABSTRACT FROM AUTHOR]