EXECUTER1- and EXECUTER2-dependent transfer of stress signals from the plastid to the nucleus of Arabidopsis thaliana
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE10509 | GEO | 2008/02/14
SECONDARY ACCESSION(S): PRJNA107969
REPOSITORIES: GEO
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Project description:Shortly after the release of singlet oxygen (1O2), 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. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment. Experiment Overall Design: Two individual biologica replicates, each containing material of five mature plants of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, respectively, were used for the microarray analysis. Plants were germinated on soil and kept under continuous light until the beginning of bolting and then transferred to the dark for 8 h. Dark-incubated mature plants were reilluminated for 30 min and subsequently harvested for RNA extraction. Experiment Overall Design: The EX1 (At4g33630) T-DNA insertion line SALK 002088 and EX2 (At1g27510) T-DNA insertion line SALK 012127 were obtained from the European Arabidopsis Stock Centre (NASC). Homozygous mutant lines were identified by PCR analysis by using T-DNA-, EX1- and EX2-specific primers. Both T-DNA-lines were crossed with a flu Col-0 line that had been obtained by 5 backcrosses of flu1-1 in Landsberg erecta with wild-type Columbia. The ex1/flu and ex2/flu mutant lines were crossed, and within the segregating F2 population triple mutants were identified by PCR-based genotyping. For the cultivation of mature plants, seeds of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, all in Col-0 ecotype, were sown on soil and plants were grown under continuous light (100 mol m 2 s 1).
2008-04-08 | E-GEOD-10509 | biostudies-arrayexpress
Project description:Singlet oxygen (1O2), the main ROS produced in chloroplasts, has long been recognized as a cytotoxin that inhibits photosynthesis and damages the cell. However, more recent findings have proposed 1O2 as a highly versatile signal that induces various stress responses. A signaling role for 1O2 was first documented in the conditional fluorescent (flu) mutant of Arabidopsis. Release of 1O2 in flu mutant induces bleach/die of young seedlings and growth inhibition of mature plants. However, all these drastic phenotypic changes can be suppressed by mutation of EXCUTER1 (EX1) gene. The Arabidopsis YDL1 was identified during a screen of revertant mutant from EMS mutagenized flu/ex1. Loss of function of SAFE1 leads to highly expression of 1O2-indluced genes in flu/ex1 mutant, mimicking the flu mutant. We found that YDL1 localized in the stroma of chloroplast, and suppressed 1O2-mediated stress responses via inhibiting 1O2-induced damage on the grana margins.
2020-04-17 | GSE131610 | GEO
Project description:Retrograde signaling from the chloroplast to the nucleus is necessary to regulate the chloroplast proteome during development and fluctuating environmental conditions. Although the specific chloroplast process(es) that must occur and the nature of the signal(s) that exits the chloroplast are not well understood, previous studies using drug inhibitors of chloroplast biogenesis have revealed that normal chloroplast development is required to express Photosynthesis Associated Nuclear Genes (PhANGs). In an attempt to determine which specific steps in chloroplast development are involved in retrograde signaling, we analyzed Arabidopsis mutants defective in the six genes encoding sigma factor (Sig) proteins that are utilized by the plastid-encoded RNA polymerase to transcribe specific sets of plastid genes. Here, we demonstrate that both Sig2 and Sig6 have partially redundant roles in not only plastid transcription, but also tetrapyrrole synthesis and retrograde signaling to control PhANG expression. Normal PhANG expression can be partly restored in the sig2 mutant by increasing heme synthesis. Furthermore, there is a genetic interaction between Sig and GUN (genomes uncoupled) genes to generate chloroplast-retrograde signals. These results demonstrate that defective plastid transcription is the source of at least two retrograde signals to the nucleus; one involving tetrapyrrole synthesis and the other involving the accumulation of an unknown plastid transcript. We also propose that the study of sig mutants (with defects in the expression of specific plastid genes) provides a new genetic system, which avoids the use of harsh inhibitors and their potential side effects, to monitor developmental retrograde signaling and to elucidate its mechanisms.
2012-09-18 | E-MTAB-4338 | biostudies-arrayexpress
Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Keywords: Single time point comparison
2008-03-13 | GSE10812 | GEO
Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Experiment Overall Design: Arabidopsis thaliana rosette leaves were harvested after 2 h of reillumination following a 8h dark period for RNA extraction and hybridization on Affymetrix ATH1 microarrays. The entire experiment was performed six times, providing independent biological replicates. For each of the six experiments, all four lines, wild-type, thylakoidal ascorbate peroxidase overexpressor (over tAPX, line 14/2), flu mutant and flu plants overexpressing thylakoidal ascorbate peroxidase were grown for 3 weeks under continuous light at 90 mmol. m-2 . s-1, transferred to the dark for 8 h, and reilluminated for 120 min before the rosette leaves of at least 10 plants per line were harvested. Total RNAs from three separate biological experiments were pooled (= 1 biological rep.) for the preparation of cDNA and the subsequent synthesis of biotin-labeled complementary RNA as recommended by Affymetrix.
2008-04-05 | E-GEOD-10812 | biostudies-arrayexpress
Project description:The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins, and plastid protein homeostasis is maintained through the balance between de novo protein synthesis and proteolysis. Intracellular communication pathways, including the pastid-to-nucleus retrograde communication, and the protein homeostasis machinery shape the chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is functional to the maintenance of a heathy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this study, we have addressed this complex regulatory chloroplast quality-control pathway by modulating the expression of two nuclear genes encoding the plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escaping strategy from stress conditions. On the contrary, the overaccumulation of PRPL4 protein is kept under control by the increased accumulation of plastid chaperones and other components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of the molecular mechanisms underlying chloroplast retrograde communication and provides new insight into cellular responses to impaired plastid protein homeostasis.
2023-05-12 | PXD034479 | Pride
Project description:Regulation of expression of genes encoding chloroplast components is critical to the autotrophic plant and never more so than in the cotyledons of the de-etiolating seedling. Many chloroplast proteins are nuclear-encoded and a retrograde signal from the chloroplasts (the Plastid Signal) modulates nuclear transcription. However, not all chloroplast-targeted genes are subject to this control and not all plastid-dependent nuclear genes are chloroplast-targeted. We therefore aim to provide the most comprehensive screen yet of which genes are affected by plastid-signalling. To specifically knock-out positive plastid signalling in light-grown cotyledons, the herbicide Norflurazon (NF) is supplied in the growth medium, causing a carotenoid deficiency that leaves the chloroplasts vulnerable to photobleaching. This blocks the expression of a subset of nuclear genes, such as Lhcb and HEMA1. Two pairs of RNAs will directly compare the transcription in seedlings grown under continuous white light with and without NF. A third RNA will also be compared from a mutant that shows a degree of constitutive positive plastid signalling. These two mutants act synergistically to counteract the effect of NF on nuclear transcription. The gun1,gun5 double mutant maintains a significantly higher level of Lhcb and HEMA1 expression in the presence of photobeached chloroplasts than the NF-treated wild-type. This transcriptome set will therefore complement RNA1 (wild-type+NF) and indicate which of the genes identified from the RNA1/RNA2 comparison are subject to the particular gun1/gun5 plastid signalling pathway(s). The growth of conditions of the seedlings (namely on MS medium supplemented with 1.5% sucrose, for 3 days under continuous WL following 2 days germination in darkness) has been chosen from the results of our own recent studies using Northern blotting techniques that show these conditions to maximise the respective NF and gun mutant effects on Lhcb and HEMA1 gene expression. This experiment is part one of a two-part study to compare the transcriptional output of this NF-affected pathway with that of a newly discovered FR-mediated pathway. A subsequent array experiment will assess the nuclear response as affected by this FR/ phyA-input pathway and the two sets of array data will be compared and contrasted. Note: Col-0 wild-type (NASC code N1092). gun1,gun5 double mutant(obtained from Enriquez Lopez-Juez, Royal Holloway, University of London.(Mochizuki et al. 2001 PNAS 98: 2053-2058). This line cannot be donated by us as it is the IP of Joanne Chory (SALK Institute, USA). Treatment = a herbicide (Norflurazon) application which leads to chloroplast photobleaching and hence down regulation of nuclear genes dependent on plastid signalling from intact chloroplasts.
2022-11-21 | E-NASC-40 | biostudies-arrayexpress
Project description:Regulation of expression of genes encoding chloroplast components is critical to the autotrophic plant and never more so than in the cotyledons of the de-etiolating seedling. Many chloroplast proteins are nuclear-encoded and a retrograde signal from the chloroplasts (the Plastid Signal) modulates nuclear transcription. However, not all chloroplast-targeted genes are subject to this control and not all plastid-dependent nuclear genes are chloroplast-targeted. We therefore aim to provide the most comprehensive screen yet of which genes are affected by plastid-signalling. To specifically knock-out positive plastid signalling in light-grown cotyledons, the herbicide Norflurazon (NF) is supplied in the growth medium, causing a carotenoid deficiency that leaves the chloroplasts vulnerable to photobleaching. This blocks the expression of a subset of nuclear genes, such as Lhcb and HEMA1. Two pairs of RNAs will directly compare the transcription in seedlings grown under continuous white light with and without NF. A third RNA will also be compared from a mutant that shows a degree of constitutive positive plastid signalling. These two mutants act synergistically to counteract the effect of NF on nuclear transcription. The gun1,gun5 double mutant maintains a significantly higher level of Lhcb and HEMA1 expression in the presence of photobeached chloroplasts than the NF-treated wild-type. This transcriptome set will therefore complement RNA1 (wild-type+NF) and indicate which of the genes identified from the RNA1/RNA2 comparison are subject to the particular gun1/gun5 plastid signalling pathway(s). The growth of conditions of the seedlings (namely on MS medium supplemented with 1.5% sucrose, for 3 days under continuous WL following 2 days germination in darkness) has been chosen from the results of our own recent studies using Northern blotting techniques that show these conditions to maximise the respective NF and gun mutant effects on Lhcb and HEMA1 gene expression. This experiment is part one of a two-part study to compare the transcriptional output of this NF-affected pathway with that of a newly discovered FR-mediated pathway. A subsequent array experiment will assess the nuclear response as affected by this FR/ phyA-input pathway and the two sets of array data will be compared and contrasted. Note:; Col-0 wild-type (NASC code N1092). gun1,gun5 double mutant(obtained from Enriquez Lopez-Juez, Royal Holloway, University of London.(Mochizuki et al. 2001 PNAS 98: 2053-2058). This line cannot be donated by us as it is the IP of Joanne Chory (SALK Institute, USA). Treatment = a herbicide (Norflurazon) application which leads to chloroplast photobleaching and hence down regulation of nuclear genes dependent on plastid signalling from intact chloroplasts. Experimenter name = Alex McCormac; Experimenter phone = 023 80594297; Experimenter fax = 023 80594319; Experimenter address = School of Biological Sciences; Experimenter address = Division of Cell Sciences; Experimenter address = University of Southampton; Experimenter address = Bassett Crescent East; Experimenter address = Southampton; Experimenter zip/postal_code = SO16 7PX; Experimenter country = UK Experiment Overall Design: 6 samples were used in this experiment
2008-06-14 | E-GEOD-5726 | biostudies-arrayexpress
Project description:Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system, and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism and expression of the plastid and nuclear genomes is poorly understood. Here a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf de-etiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids and soluble metabolites, and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during de-etiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.
2019-03-09 | GSE128049 | GEO
Project description:Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates ACS1 as a true retrograde signal impacting expression of hundreds of nuclear genes, including suppression of most genes encoding plastid ribosomal proteins.
2021-06-02 | GSE152252 | GEO