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Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Proteins and Nuclear Responses for High-Light Acclimation

ABSTRACT: Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5`-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast CML49 KO and SAP3 KO 0' and 60' of low light (8µE) to high light (800µE) transfer in comparison to Col-0

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE211805 | GEO | 2022/08/25

REPOSITORIES: GEO

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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.

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

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Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Proteins and Nuclear Responses for High-Light Acclimation

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