RESUMEN
NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.
Asunto(s)
Chlamydomonas reinhardtii , Fotosíntesis , Reductasa de Tiorredoxina-Disulfuro , Aclimatación , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Oxidación-Reducción , Fotosíntesis/fisiología , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.
Asunto(s)
Acetatos/metabolismo , Chlamydomonas reinhardtii/genética , Secuenciación del Exoma , Mutación , Fotosíntesis/genética , Chlamydomonas reinhardtii/metabolismo , Eliminación de Gen , Duplicación de GenRESUMEN
The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6 f complex (Cytb6 f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6 f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6 f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6 f.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Complejo de Citocromo b6f/metabolismo , Tilacoides/metabolismo , Carotenoides/metabolismo , Transporte de Electrón , FotosíntesisRESUMEN
Chlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large-scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over-represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date.
Asunto(s)
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Fotosíntesis/genética , Proteínas de Plantas/metabolismo , Acetatos/metabolismo , Mutagénesis Insercional , Mutación , Proteínas de Plantas/genéticaRESUMEN
The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1 Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.
Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Liasas/genética , Transducción de Señal , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Clorofila/metabolismo , Regulación hacia Abajo , Liasas/metabolismo , Filogenia , Análisis de Secuencia de ADN , Tetrapirroles/metabolismoRESUMEN
The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.
Asunto(s)
Pigmentos Biliares/metabolismo , Chlamydomonas reinhardtii/fisiología , Procesos Fototróficos , Pigmentación , Transducción de Señal , Biliverdina/farmacología , Biocatálisis/efectos de los fármacos , Biocatálisis/efectos de la radiación , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/enzimología , Cloroplastos/efectos de la radiación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/efectos de la radiación , Genes de Plantas/genética , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hierro/farmacología , Luz , Mutación/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fenotipo , Procesos Fototróficos/efectos de los fármacos , Procesos Fototróficos/genética , Ficobilinas/biosíntesis , Ficocianina/biosíntesis , Pigmentación/efectos de los fármacos , Pigmentación/genética , Pigmentación/efectos de la radiación , Plantas Modificadas Genéticamente , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/efectos de la radiaciónRESUMEN
The GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light- or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to (1) O2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.
Asunto(s)
Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Tetrapirroles/biosíntesis , Protoporfirinas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/farmacología , Chlamydomonas reinhardtii/metabolismo , Mutación/genética , Proteínas de Plantas/metabolismo , Protones , Western Blotting , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Chlamydomonas reinhardtii/crecimiento & desarrollo , Clorofila/metabolismo , Transporte de Electrón/efectos de los fármacos , Electrones , Ferredoxinas/metabolismo , Fluorescencia , Cinética , Oxidación-Reducción/efectos de los fármacos , Oxígeno/metabolismo , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema I/metabolismoRESUMEN
Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these "GreenCut" proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700(+), and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Complejo de Citocromo b6f/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas de las Membranas de los Tilacoides/metabolismo , Chlamydomonas reinhardtii/genética , Clorofila/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/genética , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Complejo de Citocromo b6f/genética , Citocromos b6/genética , Citocromos b6/metabolismo , Citocromos f/genética , Citocromos f/metabolismo , Transporte de Electrón , Expresión Génica , Immunoblotting , Luz , Mutación , Oxidación-Reducción , Fotosíntesis/genética , Fotosíntesis/efectos de la radiación , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Plastoquinona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de las Membranas de los Tilacoides/genética , Tilacoides/metabolismoRESUMEN
In oxygenic photosynthesis, two photosystems work in tandem to harvest light energy and generate NADPH and ATP. Photosystem II (PSII), the protein-pigment complex that uses light energy to catalyze the splitting of water, is assembled from its component parts in a tightly regulated process that requires a number of assembly factors. The 2pac mutant of the unicellular green alga Chlamydomonas reinhardtii was isolated and found to have no detectable PSII activity, whereas other components of the photosynthetic electron transport chain, including photosystem I, were still functional. PSII activity was fully restored by complementation with the RBD1 gene, which encodes a small iron-sulfur protein known as a rubredoxin. Phylogenetic evidence supports the hypothesis that this rubredoxin and its orthologs are unique to oxygenic phototrophs and distinct from rubredoxins in Archaea and bacteria (excluding cyanobacteria). Knockouts of the rubredoxin orthologs in the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana were also found to be specifically affected in PSII accumulation. Taken together, our data suggest that this rubredoxin is necessary for normal PSII activity in a diverse set of organisms that perform oxygenic photosynthesis.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Complejo de Proteína del Fotosistema II/metabolismo , Rubredoxinas/química , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Chlamydomonas reinhardtii/metabolismo , Clorofila/química , Secuencia Conservada , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Fotosíntesis , Filogenia , Rubredoxinas/genética , Semillas/metabolismo , Especificidad de la Especie , Espectrofotometría , Synechocystis/metabolismoRESUMEN
Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.
Asunto(s)
Chlamydomonas reinhardtii/genética , Genoma de Planta/genética , Genómica/métodos , Fotosíntesis/genética , Filogenia , Proteínas de Plantas/genética , Aclimatación/genética , Secuencia de Bases , Genoma de Planta/fisiología , Mutación/genética , FenotipoRESUMEN
Plants, algae and cyanobacteria need to regulate photosynthetic light harvesting in response to the constantly changing light environment. Rapid adjustments are required to maintain fitness because of a trade-off between efficient solar energy conversion and photoprotection. The xanthophyll cycle, in which the carotenoid pigment violaxanthin is reversibly converted into zeaxanthin, is ubiquitous among green algae and plants and is necessary for the regulation of light harvesting, protection from oxidative stress and adaptation to different light conditions(1,2). Violaxanthin de-epoxidase (VDE) is the key enzyme responsible for zeaxanthin synthesis from violaxanthin under excess light. Here we show that the Chlorophycean VDE (CVDE) gene from the model green alga Chlamydomonas reinhardtii encodes an atypical VDE. This protein is not homologous to the VDE found in plants and is instead related to a lycopene cyclase from photosynthetic bacteria(3). Unlike the plant-type VDE that is located in the thylakoid lumen, the Chlamydomonas CVDE protein is located on the stromal side of the thylakoid membrane. Phylogenetic analysis suggests that CVDE evolved from an ancient de-epoxidase that was present in the common ancestor of green algae and plants, providing evidence of unexpected diversity in photoprotection in the green lineage.
Asunto(s)
Proteínas Bacterianas/genética , Chlamydomonas reinhardtii/fisiología , Evolución Molecular , Oxidorreductasas/genética , Fotosíntesis , Proteínas Bacterianas/metabolismo , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Oxidorreductasas/metabolismo , Filogenia , Tilacoides/metabolismoRESUMEN
Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.DOI: http://dx.doi.org/10.7554/eLife.02286.001.
Asunto(s)
Adaptación Fisiológica , Chlamydomonas reinhardtii/metabolismo , Fosfoproteínas/metabolismo , Oxígeno Singlete/metabolismo , Chlamydomonas reinhardtii/genética , Regulación de la Expresión Génica , Transducción de SeñalRESUMEN
Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis--the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Elementos de Facilitación Genéticos/genética , Mutación , Oxidorreductasas/genética , Supresión Genética/genética , Alelos , Secuencia de Aminoácidos , Carotenoides/metabolismo , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/metabolismo , ADN de Plantas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Conformación ProteicaRESUMEN
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
Asunto(s)
Proteínas Algáceas/genética , Proteínas Algáceas/fisiología , Evolución Biológica , Chlamydomonas reinhardtii/genética , Genoma , Animales , Chlamydomonas reinhardtii/fisiología , Cloroplastos/metabolismo , Biología Computacional , ADN de Algas/genética , Flagelos/metabolismo , Genes , Genómica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Fotosíntesis/genética , Filogenia , Plantas/genética , Proteoma , Análisis de Secuencia de ADNRESUMEN
Mutants with defects in the cytochrome (cyt) b6/f complex were analyzed for their effect on the expression of a subgroup of nuclear genes encoding plastid-localized enzymes participating in chlorophyll biosynthesis. Their defects ranged from complete loss of the cytb6/f complex to point mutations affecting specifically the quinone-binding QO site. In these seven mutants, light induction of the tetrapyrrole biosynthetic genes was either abolished or strongly reduced. In contrast, a normal induction of chlorophyll biosynthesis genes was observed in mutants with defects in photosystem II, photosystem I, or plastocyanin, or in wild-type cells treated with 3-(3'4'-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl benzoquinone. We conclude that the redox state of the plastoquinone pool does not control light induction of these chlorophyll biosynthetic genes. The signal that affects expression of the nuclear genes appears to solely depend on the integrity of the cytb6/f complex QO site. Since light induction of these genes in Chlamydomonas has recently been shown to involve the blue light receptor phototropin, the results suggest that cytb6/f activity regulates a plastid-derived factor required for their expression. This signaling pathway differs from that which regulates state transitions, since mutant stt7, lacking a protein kinase involved in phosphorylation of the light-harvesting complex II, was not altered in the expression of the chlorophyll biosynthetic genes.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Clorofila/biosíntesis , Complejo de Citocromo b6f/fisiología , Animales , Chlamydomonas reinhardtii/genética , Transporte de Electrón/fisiología , Regulación de la Expresión Génica , Luz , Mutación , Fotosíntesis/fisiología , Transducción de Señal/fisiologíaRESUMEN
The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.