Project description:The unicellular, free-living, nonphotosynthetic chlorophycean alga Polytomella parva, closely related to Chlamydomonas reinhardtii and Volvox carteri, contains colorless, starch-storing plastids. The P. parva plastids lack all light-dependent processes but maintain crucial metabolic pathways. The colorless alga also lacks a plastid genome, meaning no transcription or translation should occur inside the organelle. Here, using an algal fraction enriched in plastids as well as publicly available transcriptome data, we provide a proteomic characterization of the P. parva plastid, ultimately identifying several plastid proteins, both by mass spectrometry and bioinformatic analyses. Altogether these results led us to propose a plastid proteome for P. parva, i.e., a set of proteins that participate in carbohydrate metabolism; in the synthesis and degradation of starch, amino acids and lipids; in the biosynthesis of terpenoids and tetrapyrroles; in solute transport and protein translocation; and in redox homeostasis. This is the first detailed plastid proteome from a unicellular, free-living colorless alga.
Project description:Plastids are endosymbiotic organelles containing their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid-encoded polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplasts DNA using ptChIP-seq. We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex pattern of binding to DNA with preferential association at genes encoding rRNA, tRNA and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with the levels of RNA accumulation, which allowed estimating the quantitative contribution of transcription to RNA accumulation.
Project description:Plastids emit signals that broadly affect cellular processes. Based on previous genetic analyses, we propose that plastid signaling regulates the downstream components of a light signaling network and that these interactions coordinate chloroplast biogenesis with both the light environment and development by regulating gene expression. We tested these ideas by analyzing light-regulated and plastid-regulated transcriptomes. We found that the plastid is a major regulator of light signaling, attenuating the expression of more than half of all light-regulated genes in our dataset and changing the nature of light regulation for a smaller fraction of these light-regulated genes. Our analyses provide evidence that light and plastid signaling are interactive processes and are consistent with these interactions serving as major drivers of chloroplast biogenesis and function. Four biological replicates were grown separately under the same conditions. Arabidopsis seedlings were grown in the presence (+Lin) or absence (-Lin) of lincomycin in 0.5 µmol m-2 s-1 blue plus red (BR) light for 6 days. After 6 days of growth in 0.5 µmol m-2 s-1 of BR light, seedlings were transferred to 60 µmol m-2 s-1 BR light. 50-100 seedlings were collected before (0 h) and 0.5 h, 1 h, 4 h, and 24 h after the 0.5 to 60 µmol m-2 s-1 BR-fluence-rate shift for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Plastids emit signals that broadly affect cellular processes. Based on previous genetic analyses, we propose that plastid signaling regulates the downstream components of a light signaling network and that these interactions coordinate chloroplast biogenesis with both the light environment and development by regulating gene expression. We tested these ideas by analyzing light-regulated and plastid-regulated transcriptomes. We found that the plastid is a major regulator of light signaling, attenuating the expression of more than half of all light-regulated genes in our dataset and changing the nature of light regulation for a smaller fraction of these light-regulated genes. Our analyses provide evidence that light and plastid signaling are interactive processes and are consistent with these interactions serving as major drivers of chloroplast biogenesis and function.
Project description:We report that phosphatidylglycerol (PG) biosynthesis in plastid is required for plastid gene expression mediated by plastid-encoded RNA polymerase and light-induced expression of nuclear-encoded photosynthesis-associated genes. A transcription factor GOLDEN-LIKE1 was also found to be involved in the downregulation of nuclear photosynthesis genes in responce to PG deficiency.
Project description:<p>The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to be evolved from the putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.</p>
Project description:Plastids communicate with the nucleus through retrograde signaling pathways that coordinate nuclear and plastid gene expression to support plant development and environmental adaptation. However, how plastid regulatory factors are dynamically controlled to modulate these signals remains largely unknown. Here we identify a proteostasis mechanism that regulates plastid retrograde signaling through the ubiquitin–26S proteasome system. We show that the nuclear-encoded plastid RNA-editing factor MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) is targeted for degradation by a Skp1–Cullin1–F-box (SCF) ubiquitin ligase assembled with the MORF2-INTERACTING F-BOX (MIF) protein. MIF physically associates with MORF2 and promotes its ubiquitylation and proteasome-dependent turnover. Genetic and physiological analyses reveal that this pathway balances early plant development. MIF overexpression suppresses seedling growth and disrupts chloroplast structure and function, whereas mif null mutants or MORF2 overaccumulation retard seed germination and reduce photosynthetic efficiency. Transcriptomic profiling and RNA-editing analyses further demonstrate that MIF-dependent MORF2 turnover modulates plastid RNA editing and retrograde signaling pathways. Together, our results uncover a previously unrecognized cytoplasmic regulatory layer that links ubiquitin-mediated proteostasis to plastid RNA editing and retrograde signaling. This mechanism enables plants to coordinate seed germination and chloroplast development during early seedling establishment.
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.