The Arabidopsis SAFEGUARD1 suppresses singlet oxygen-induced stress responses by protecting grana margins.

Singlet oxygen (1O2), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent (flu) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu-like phenotype. In flu ex1 safe1, all 1O2-induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1O2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1O2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu-produced 1O2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs.

Dose-dependent effects of 1O2 in chloroplasts are determined by its timing and localization of production.

In plants, highly reactive singlet oxygen (1O2) is known to inhibit photosynthesis and to damage the cell as a cytotoxin. However, more recent studies have also proposed 1O2 as a signal. In plants under stress, not only 1O2 but also other reactive oxygen species (ROS) are generated simultaneously, thus making it difficult to link a particular response to the release of 1O2 and establish a signaling role for this ROS. This obstacle has been overcome by the identification of conditional mutants of Arabidopsis thaliana that selectively generate 1O2 and trigger various 1O2-mediated responses. In chloroplasts of these mutants, chlorophyll or its biosynthetic intermediates may act as a photosensitizer and generate 1O2. These 1O2-mediated responses are not only dependent on the dosage of 1O2 but also are determined by the timing and suborganellar localization of its production. This spatial- and temporal-dependent variability of 1O2-mediated responses emphasizes the importance of 1O2 as a highly versatile and short-lived signal that acts throughout the life cycle of a plant.

Singlet oxygen- and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2.

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.

Blocking the QB-binding site of photosystem II by tenuazonic acid, a non-host-specific toxin of Alternaria alternata, activates singlet oxygen-mediated and EXECUTER-dependent signalling in Arabidopsis.

Necrotrophic fungal pathogens produce toxic compounds that induce cell death in infected plants. Often, the primary targets of these toxins and the way a plant responds to them are not known. In the present work, the effect of tenuazonic acid (TeA), a non-host-specific toxin of Alternaria alternata, on Arabidopsis thaliana has been analysed. TeA blocks the QB -binding site at the acceptor side of photosystem II (PSII). As a result, charge recombination at the reaction centre (RC) of PSII is expected to enhance the formation of the excited triplet state of the RC chlorophyll that promotes generation of singlet oxygen ((1)O2). (1)O2 activates a signalling pathway that depends on the two EXECUTER (EX) proteins EX1 and EX2 and triggers a programmed cell death response. In seedlings treated with TeA at half-inhibition concentration (1)O2-mediated and EX-dependent signalling is activated as indicated by the rapid and transient up-regulation of (1)O2-responsive genes in wild type, and its suppression in ex1/ex2 mutants. Lesion formation occurs when seedlings are exposed to higher concentrations of TeA for a longer period of time. Under these conditions, the programmed cell death response triggered by (1)O2-mediated and EX-dependent signalling is superimposed by other events that also contribute to lesion formation.

Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway.

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.

Integration of stress-related and reactive oxygen species-mediated signals by Topoisomerase VI in Arabidopsis thaliana.

Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen ((1)O(2)) in chloroplasts of the flu mutant of Arabidopsis, reprogramming of nuclear gene expression reveals a rapid transfer of signals from the plastid to the nucleus. We have identified extraplastidic signaling constituents involved in (1)O(2)-initiated plastid-to-nucleus signaling and nuclear gene activation after mutagenizing a flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second-site mutations that lead to a constitutive up-regulation of the reporter gene or abrogate its (1)O(2)-dependent up-regulation. One of these mutants, caa39, turned out to be a weak mutant allele of the Topoisomerase VI (Topo VI) A-subunit gene with a single amino acid substitution. Transcript profile analysis of flu and flu caa39 mutants revealed that Topo VI is necessary for the full activation of AAA-ATPase and a set of (1)O(2)-responsive transcripts in response to (1)O(2). Topo VI binds to the promoter of the AAA-ATPase and other (1)O(2)-responsive genes, and hence could directly regulate their expression. Under photoinhibitory stress conditions, which enhance the production of (1)O(2) and H(2)O(2), Topo VI regulates (1)O(2)-responsive and H(2)O(2)-responsive genes in a distinct manner. These results suggest that Topo VI acts as an integrator of multiple signals generated by reactive oxygen species formed in plants under adverse environmental conditions.

A novel proteinase, SNOWY COTYLEDON4, is required for photosynthetic acclimation to higher light intensities in Arabidopsis.

Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis thaliana) mutant snowy cotyledon4 (sco4) revealed a mutation in a chloroplast-targeted protein that shares limited homology with CaaX-type endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photooxidative stress. The sco4 mutation impairs the acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in photosystem I function, decreased linear electron transfer, yet increased nonphotochemical quenching. SCO4 is localized to the chloroplasts, which suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveal that SCO4 is a member of an unknown group of higher plant-specific proteinases quite distinct from the well-described CaaX-type endopeptidases RAS Converting Enzyme1 (RCE1) and zinc metallopeptidase STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light.

The chloroplast division mutant caa33 of Arabidopsis thaliana reveals the crucial impact of chloroplast homeostasis on stress acclimation and retrograde plastid-to-nucleus signaling.

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.

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LHCB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings.

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression.

Singlet oxygen-mediated signaling in plants: moving from flu to wild type reveals an increasing complexity.

Singlet oxygen ((1)O2)-mediated signaling has been established in the conditional fluorescent (flu) mutant of Arabidopsis. In the dark, the flu mutant accumulates free protochlorophyllide (Pchlide), a photosensitizer that in the light generates (1)O2. The release of (1)O2 leads to growth inhibition of mature plants and bleaching of seedlings. These (1)O2-mediated responses depend on two plastid proteins, EXECUTER (EX) 1 and 2. An ex1/ex2/flu mutant accumulates in the dark Pchlide and upon illumination generates similar amounts of (1)O2 as flu, but (1)O2-mediated responses are abrogated in the triple mutant. The (1)O2- and EX-dependent signaling pathway operates also in wild type placed under light stress. However, it does not act alone as in flu, but interacts with other signaling pathways that modulate (1)O2-mediated responses. Depending on how severe the light stress is, (1)O2- and EX-dependent signaling may be superimposed by (1)O2-mediated signaling that does not depend on EX and is associated with photo-oxidative damage. Because of its high reactivity and short half-life, (1)O2 is unlikely to be a signal that is translocated across the chloroplast envelope, but is likely to interact with other plastid components close to its site of production and to generate more stable signaling molecules during this interaction. Depending on the site of (1)O2 production and the severity of stress, different signaling molecules may be expected that give rise to different (1)O2-mediated responses.

The cytoskeleton and the peroxisomal-targeted snowy cotyledon3 protein are required for chloroplast development in Arabidopsis.

Here, we describe the snowy cotyledon3 (sco3-1) mutation, which impairs chloroplast and etioplast development in Arabidopsis thaliana seedlings. SCO3 is a member of a largely uncharacterized protein family unique to the plant kingdom. The sco3-1 mutation alters chloroplast morphology and development, reduces chlorophyll accumulation, impairs thylakoid formation and photosynthesis in seedlings, and results in photoinhibition under extreme CO(2) concentrations in mature leaves. There are no readily apparent changes to chloroplast biology, such as transcription or assembly that explain the disruption to chloroplast biogenesis. Indeed, SCO3 is actually targeted to another organelle, specifically to the periphery of peroxisomes. However, impaired chloroplast development cannot be attributed to perturbed peroxisomal metabolic processes involving germination, fatty acid ß-oxidation or photorespiration, though there are so far undescribed changes in low and high CO(2) sensitivity in seedlings and young true leaves. Many of the chloroplasts are bilobed, and some have persistent membranous extensions that encircle other cellular components. Significantly, there are changes to the cytoskeleton in sco3-1, and microtubule inhibitors have similar effects on chloroplast biogenesis as sco3-1 does. The localization of SCO3 to the periphery of the peroxisomes was shown to be dependent on a functional microtubule cytoskeleton. Therefore, the microtubule and peroxisome-associated SCO3 protein is required for chloroplast development, and sco3-1, along with microtubule inhibitors, demonstrates an unexpected role for the cytoskeleton and peroxisomes in chloroplast biogenesis.

The Endocrine Treatment Landscape for Patients with HR+ HER2- Early-stage Breast Cancer in Germany Before the Introduction of CDK4/6 Inhibitor Therapy - A Real-World Analysis.

Introduction: While premenopausal patients with HR+ HER2- early breast cancer are treated with tamoxifen +/- ovarian suppression with a GnRH analog or an aromatase inhibitor (AI) + GnRH, the majority of postmenopausal women receive an AI due to its higher efficacy compared to tamoxifen. As the introduction of CDK4/6 inhibitors into the treatment of early-stage breast cancer with a higher risk of recurrence will probably result in a shift in the endocrine treatment landscape, the question is what treatment did potential candidates for CDK4/6 inhibitors in Germany receive before CDK4/6 inhibitors were available. Patients and Methods: As part of a retrospective multicenter analysis, anonymized data were collected of patients with HR+ HER2- early-stage breast cancer who received endocrine therapy in the period between 10/2021 and 03/2022. Potential candidates for CDK4/6 inhibitor treatment were classified into different risk cohorts using the inclusion criteria of the NATALEE and monarchE trials. Results: The data of 238 patients from 29 different centers were analyzed. While 20.6% of patients met the monarchE criteria, the subgroup which met the NATALEE inclusion criteria consisted of 46.2% of patients. 53.8% of patients did not meet the inclusion criteria for either the NATALEE or the monarchE trial. More than half of the patients did not receive chemotherapy. 28.6% of patients in the whole cohort were premenopausal. 67.6% of premenopausal women received neo-/adjuvant chemotherapy. 61.8% of premenopausal patients received tamoxifen as adjuvant endocrine therapy, 19.1% received an AI + GnRH and 10.3% were treated with tamoxifen + GnRH. Conclusion: Despite the high percentage of premenopausal patients who received aggressive treatment in the form of chemotherapy, only one third of premenopausal patients received GnRH in addition to their standard endocrine therapy. Studies carried out at a later point in time and registry studies will be necessary to see how the endocrine therapy landscape in Germany has changed following the introduction of CDK4/6 inhibitors.

'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings.

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.

Arabidopsis light-dependent NADPH: protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development: an addendum.

Recently the porA-1 null mutant of Arabidopsis thaliana has been identified, which contains an insertion of the Dissociation (Ds) element in the PORA gene (Paddock et al. in Plant Mol Biol 78:447-460, 2012). Light-grown porA-1 seedlings suffer from a drastically reduced chlorophyll content and a developmental arrest beyond the cotyledon stage, suggesting that PORA is not only transiently involved in initiating chlorophyll synthesis during illumination of etiolated seedlings but is also essential for normal growth and plant development. Here we report the presence of a second Ds element in this porA-1 mutant line that inactivates the Speechless gene required for stomata formation. Similar to porA-1, speechless seedlings are severely impaired in their development. Our results suggest that the lack of stomata in porA-1 may contribute to the dwarfed phenotype of the mutant and thus emphasizes the need to re-address the proposed role of PORA during plant development by studying a porA mutant that retains its stomata formation.

Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development.

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.

(1)O2-mediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid.

Plastid development in seedlings of Arabidopsis thaliana is affected by the transfer of (1)O(2)-mediated retrograde signals from the plastid to the nucleus and changes in nuclear gene expression during late embryogenesis. The potential impact of these mechanisms on plastid differentiation is maintained throughout seed dormancy and becomes effective only after seed germination. Inactivation of the 2 nuclear-encoded plastid proteins EXECUTER1 and EXECUTER2 blocks (1)O(2)-mediated retrograde signaling before the onset of dormancy and impairs normal plastid formation in germinating seeds. This long-term effect of (1)O(2) retrograde signaling depends on the recruitment of abscisic acid (ABA) during seedling development. Unexpectedly, ABA acts as a positive regulator of plastid formation in etiolated and light-grown seedlings.

Modulation of O-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling.

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in (1)O(2)-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that (1)O(2)-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other (1)O(2)-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting (1)O(2)-mediated retrograde signaling with other signaling pathways.

A mutation in the Arabidopsis mTERF-related plastid protein SOLDAT10 activates retrograde signaling and suppresses (1)O(2)-induced cell death.

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ((1)O(2)) 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 (1)O(2)-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 (1)O(2)-responsive genes after a dark-to-light shift they do not show a (1)O(2)-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent (1)O(2)-mediated cell death response without suppressing (1)O(2)-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as 'plastid signaling'.

Enzymatic, but not non-enzymatic, 1O2-mediated peroxidation of polyunsaturated fatty acids forms part of the EXECUTER1-dependent stress response program in the flu mutant of Arabidopsis thaliana.

The conditional flu mutant of Arabidopsis accumulates excess amounts of protochlorophyllide within plastid membranes in the dark and generates singlet oxygen upon light exposure. By varying the length of the dark period, the level of the photosensitizer protochlorophyllide may be modulated, and conditions have been established that either endorse the cytotoxicity of (1)O(2) or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of (1)O(2): the impact of the EXECUTER1 mutation and the prevalence of either non-enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings, toxic effects of (1)O(2) prevail and non-enzymatic lipid peroxidation proceeds rapidly. In contrast, in light-grown flu plants that were subjected to an 8 h dark/light shift, lipid peroxidation occurred almost exclusively enzymatically. The resulting oxidation product, 13-hydroperoxy octadecatrienoic acid (13-HPOT), serves as a substrate for synthesis of 12-oxo phytodienoic acid (OPDA) and jasmonic acid (JA), both of which are known to control various metabolic and developmental processes in plants. Inactivation of the EXECUTER1 protein abrogates not only (1)O(2)-mediated cell death and growth inhibition of flu plants, but also enzymatic lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect (1)O(2)-mediated growth inhibition and cell death. Hence, JA and OPDA do not act as second messengers during (1)O(2) signaling, but form an integral part of a stress-related signaling cascade activated by (1)O(2) that encompasses several signaling pathways known to be activated by abiotic and biotic stressors.

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development.

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a (1)O(2)-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by (1)O(2) but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether (1)O(2) was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the (1)O(2)-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that (1)O(2)-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development.