A Mutant Impaired in the Production of Plastome-Encoded Proteins Uncovers a Mechanism for the Homeostasis of Isoprenoid Biosynthetic Enzymes in Arabidopsis Plastids

Departament de Genètica Molecular de Plantes, Centre for Research on Agricultural Genomics, 08034 Barcelona, Spain.
The Plant Cell (Impact Factor: 9.34). 06/2008; 20(5):1303-15. DOI: 10.1105/tpc.108.058768
Source: PubMed


The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states.

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Available from: Manuel Rodríguez-Concepción, Jan 13, 2015
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    • "The possibility of other pathways delivering proteins to the Clp protease, however, remains open. As shown above (Fig 1A), DXS levels increase in mutants defective in Clp protease activity such as clpr1[20]. If DXS is targeted to the Clp protease for degradation, we would also expect a post-translational upregulation of DXS enzyme levels in mutants impaired in the adaptors and chaperones that deliver the protein to the Clp catalytic core. "
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    ABSTRACT: The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid.
    Full-text · Article · Jan 2016 · PLoS Genetics
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    • "As this apparent protection of DXR from degradation could be confirmed in isolated chloroplasts (Fig. 8B), we hypothesize that the substrate-like tight binding of fosmidomycin to the active site of DXR [47] either shields protease-sensitive sites or causes a conformational change of DXR rendering it resistant to chloroplast protease(s), e.g. Clp protease [26], [32]. Thus, fosmidomycin inhibits but also stabilizes the DXR enzyme. "
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    ABSTRACT: In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.
    Full-text · Article · May 2013 · PLoS ONE
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    • "Thus, the assembly of photosynthetic complexes and the build-up of thylakoid membranes and plastoglobules in chloroplasts increase the capacity to sequester the newly synthesized carotenoid molecules. Defects in chloroplast development hence result in a decreased accumulation of carotenoids, even under conditions in which an enhanced supply of their isoprenoid precursors is available (Sauret-Güeto et al., 2006; Flores-Perez et al., 2008b). Differentiation of chloroplasts into chromoplasts involves the development of larger plastoglobules and/or carotenoid-sequestering structures of different shapes, allowing the deposition of massive amounts of carotenoids in a matrix of lipoproteins (Deruere et al., 1994; Vishnevetsky et al., 1999; Simkin et al., 2007; Walter and Strack, 2011). "
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    ABSTRACT: Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology.
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