RESUMO
The high-value carotenoid astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type-like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.
Assuntos
Nicotiana/genética , Nicotiana/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Riboswitch/genética , Xantofilas/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Plantas Geneticamente ModificadasRESUMO
In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fotossíntese , Tilacoides/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos , Microscopia Eletrônica de Transmissão , Mutação , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Plântula , Homologia de Sequência de Aminoácidos , Tilacoides/ultraestruturaRESUMO
During plant photosynthesis, photosystems I (PSI) and II (PSII), located in the thylakoid membranes of the chloroplast, use light energy to mobilize electron transport. Different modes of electron flow exist. Linear electron flow is driven by both photosystems and generates ATP and NADPH, whereas cyclic electron flow (CEF) is driven by PSI alone and generates ATP only. Two variants of CEF exist in flowering plants, of which one is sensitive to antimycin A (AA) and involves the two thylakoid proteins, PGR5 and PGRL1. However, neither the mechanism nor the site of reinjection of electrons from ferredoxin into the thylakoid electron transport chain during AA-sensitive CEF is known. Here, we show that PGRL1 accepts electrons from ferredoxin in a PGR5-dependent manner and reduces quinones in an AA-sensitive fashion. PGRL1 activity itself requires several redox-active cysteine residues and a Fe-containing cofactor. We therefore propose that PGRL1 is the elusive ferredoxin-plastoquinone reductase (FQR).
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Ferredoxinas/metabolismo , Proteínas de Membrana/metabolismo , Fotossíntese , Quinona Redutases/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Sequência Conservada , Cisteína/metabolismo , Transporte de Elétrons , Ferro/metabolismo , Proteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/metabolismo , Tiorredoxinas/metabolismo , Tilacoides/metabolismoRESUMO
In vascular plants, the chloroplast NAD(P)H dehydrogenase complex (NDH-C) is assembled from five distinct subcomplexes, the membrane-spanning (subM) and the luminal (subL) subcomplexes, as well as subA, subB, and subE. The assembly process itself is poorly understood. Vascular plant genomes code for two related intrinsic thylakoid proteins, photosynthesis-affected mutant68 (PAM68), a photosystem II assembly factor, and photosynthesis-affected mutant68-like (PAM68L). As we show here, inactivation of Arabidopsis thaliana PAM68L in the pam68l-1 mutant identifies PAM68L as an NDH-C assembly factor. The mutant lacks functional NDH holocomplexes and accumulates three distinct NDH-C assembly intermediates (subB, subM, and subA+L), which are also found in mutants defective in subB assembly (ndf5) or subM expression (chlororespiratory reduction4-3 mutant). NDH-C assembly in the cyanobacterium Synechocystis sp PCC 6803 and the moss Physcomitrella patens does not require PAM68 proteins, as demonstrated by the analysis of knockout lines for the single-copy PAM68 genes in these species. We conclude that PAM68L mediates the attachment of subB- and subM-containing intermediates to a complex that contains subA and subL. The evolutionary appearance of subL and PAM68L during the transition from mosses like P. patens to flowering plants suggests that the associated increase in the complexity of the NDH-C might have been facilitated by the recruitment of evolutionarily novel assembly factors like PAM68L.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , NADPH Desidrogenase/metabolismo , Fotossíntese , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Bryopsida/metabolismo , Proteínas de Cloroplastos/genética , NADPH Desidrogenase/genética , Filogenia , Synechocystis/metabolismoRESUMO
Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/ultraestrutura , Immunoblotting , Membranas Intracelulares/ultraestrutura , Lipídeos/análise , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Fosforilação , Fotossíntese , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tilacoides/ultraestruturaRESUMO
The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.
Assuntos
Dosagem de Genes , Genomas de Plastídeos , Sequências Repetidas Invertidas , Nicotiana , Nicotiana/genética , Sequências Repetidas Invertidas/genética , Plastídeos/genética , Tamanho do Genoma , Variações do Número de Cópias de DNA , Genoma de PlantaRESUMO
Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tilacoides/metabolismo , Prótons , Antiporters/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Cloroplastos/metabolismo , LuzRESUMO
The conversion of light energy to chemical energy by photosynthesis requires the concerted action of large protein complexes in the thylakoid membrane. Recent work has provided fundamental insights into the three-dimensional structure of these complexes, but how they are assembled from hundreds of parts remains poorly understood. Particularly little is known about the biogenesis of the cytochrome b6f complex (Cytb6f), the redox-coupling complex that interconnects the two photosystems. Here we report the identification of a factor that guides the assembly of Cytb6f in thylakoids of chloroplasts. The protein, DE-ETIOLATION-INDUCED PROTEIN 1 (DEIP1), resides in the thylakoid membrane and is essential for photoautotrophic growth. Knock-out mutants show a specific loss of Cytb6f, and are defective in complex assembly. We demonstrate that DEIP1 interacts with the two cytochrome subunits of the complex, PetA and PetB, and mediates the assembly of intermediates in Cytb6f biogenesis. The identification of DEIP1 provides an entry point into the study of the assembly pathway of a crucial complex in photosynthetic electron transfer.
Assuntos
Arabidopsis , Complexo Citocromos b6f , Arabidopsis/genética , Arabidopsis/metabolismo , Complexo Citocromos b6f/genética , Complexo Citocromos b6f/metabolismo , Citocromos b/metabolismo , Estiolamento , Fotossíntese , Tilacoides/metabolismoRESUMO
Recent work has revealed that both plants and animals transfer genomes between cells. In plants, horizontal transfer of entire plastid, mitochondrial, or nuclear genomes between species generates new combinations of nuclear and organellar genomes, or produces novel species that are allopolyploid. The mechanisms of genome transfer between cells are unknown. Here, we used grafting to identify the mechanisms involved in plastid genome transfer from plant to plant. We show that during proliferation of wound-induced callus, plastids dedifferentiate into small, highly motile, amoeboid organelles. Simultaneously, new intercellular connections emerge by localized cell wall disintegration, forming connective pores through which amoeboid plastids move into neighboring cells. Our work uncovers a pathway of organelle movement from cell to cell and provides a mechanistic framework for horizontal genome transfer.
Assuntos
Genoma Mitocondrial , Genomas de Plastídeos , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Plantas/genética , Plastídeos/genéticaRESUMO
The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.
Assuntos
Clorofila/análogos & derivados , Cloroplastos/enzimologia , Complexo Citocromos b6f/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Clorofila/química , Clorofila/metabolismo , Complexo Citocromos b6f/química , Dimerização , Proteínas de Plantas/química , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I.