Dataset Information

CLPC1 maintains chloroplast proteome and RNA homeostasis

ABSTRACT: In chloroplasts, homeostasis of the proteome maintained by protein synthesis, importation, and degradation is critical to the development of chloroplasts. The caseinolytic protease complex component CLPC1, a member of the HSP100 family of chaperones, not only facilitates the translocation of pre-proteins into chloroplasts, but also mediates protein degradation via its associated caseinolytic protease complex (CLPPs). Although CLPC1 has been shown to affect the abundance of some chloroplast proteins, its overall functions and coordination with other protease systems in maintaining proteome homeostasis have not been fully elucidated. Its possible roles in gene expression are also unclear. Here we used the iTRAQ technique to profile the chloroplast proteomes of the clpc1 mutant and its various complementation lines. Whereas proteins involved in photosynthesis were found to be dramatically decreased in the clcp1 mutant, many involved in chloroplast transcription and translation were increased. Expression of the full-length CLPC1 protein, but not of the N-terminal truncated form (ΔN), in clpc1 restored the level of most of these proteins to that of the wild type. Interestingly, the ΔN complementation line could also restore certain proteins to the wild type level. We found that nearly half of previously suggested candidate substrates of the CLPS1, another CLPP component, were mis-regulated in clpc1, indicating that CLPC1 greatly affects CLPS1 targets. We provide the first experimental evidence to show that CLPC1 profoundly affects gene transcription in chloroplasts. Sense transcripts of many chloroplast genes are up-regulated whereas antisense transcripts down-regulated in the clpc1 mutant. We propose that this altered transcript landscape is a result of the transcription and degradation dynamics conditioned by the changed proteomic profile in the mutant. Our study reveals that CLPC1 and likely the CLPP proteasome complex have a greater than previously recognized role in coordinating and maintaining the proteome and transcriptome homeostasis in chloroplasts.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: shoudong zhang

LAB HEAD: Liming Xiong

PROVIDER: PXD000869 | Pride | 2018-10-19

REPOSITORIES: Pride

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The caseinolytic protease complex component CLPC1 in Arabidopsis maintains proteome and RNA homeostasis in chloroplasts.

Zhang Shoudong S Zhang Huoming H Xia Yiji Y Xiong Liming L

BMC plant biology 20180912 1

<h4>Background</h4>Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation.<h4>Results</h4>We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression o ...[more]

PMID: 30208840

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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

Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310.

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	20130503iTRAQ_a01.raw	Raw
	20130503iTRAQ_a02.raw	Raw
	20130503iTRAQ_a03.raw	Raw
	20130503iTRAQ_a04.raw	Raw
	20130503iTRAQ_a05.raw	Raw