RESUMO
The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Carbono/metabolismo , Fosfatos/metabolismo , Dióxido de Carbono/metabolismo , Vitamina B 6 , Piridoxina/metabolismo , Fosfato de Piridoxal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nitrogênio/metabolismoRESUMO
Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA, was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation.
Assuntos
Ferredoxinas/fisiologia , Ferro/metabolismo , Fotossíntese/fisiologia , Synechocystis/fisiologia , Adaptação Fisiológica , Clorofila/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , Homeostase/genética , Synechocystis/genética , Synechocystis/metabolismoRESUMO
Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbors up to 5% of genes that were most likely acquired through horizontal gene transfer. These genes probably contributed to G.sulphuraria's adaptation to its extreme habitats, resulting in today's polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42�C/46�C to 28�C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon return to optimal growth temperature. Numerous genes acquired by horizontal gene transfer displayed temperature-dependent expression changes, indicating that these genes contributed to adaptive evolution in G.sulphuraria.
Assuntos
Rodófitas/metabolismo , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Temperatura Baixa , Resposta ao Choque Frio/genética , Resposta ao Choque Frio/fisiologia , Transferência Genética Horizontal/genética , Transferência Genética Horizontal/fisiologia , Filogenia , Rodófitas/genética , Rodófitas/fisiologia , Biologia de Sistemas/métodosRESUMO
Photorespiration is frequently considered a wasteful and inefficient process. However, mutant analysis demonstrated that photorespiration is essential for recycling of 2-phosphoglycolate in C3 and C4 land plants, in algae, and even in cyanobacteria operating carboxysome-based carbon (C) concentrating mechanisms. Photorespiration links photosynthetic C assimilation with other metabolic processes, such as nitrogen and sulfur assimilation, as well as C1 metabolism, and it may contribute to balancing the redox poise between chloroplasts, peroxisomes, mitochondria and cytoplasm. The high degree of metabolic interdependencies and the pleiotropic phenotypes of photorespiratory mutants impedes the distinction between core and accessory functions. Newly developed synthetic bypasses of photorespiration, beyond holding potential for significant yield increases in C3 crops, will enable us to differentiate between essential and accessory functions of photorespiration.
Assuntos
Luz , Processos Fotoquímicos , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Respiração Celular/efeitos da radiação , Glicolatos/metabolismo , Nitrogênio/metabolismoRESUMO
Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5'-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitrogênio/metabolismo , Fosfato de Piridoxal/análogos & derivados , Piridoxamina/análogos & derivados , Piridoxaminafosfato Oxidase/metabolismo , Vitamina B 6/metabolismo , Compostos de Amônio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Vias Biossintéticas , Homeostase , Metaboloma , Modelos Biológicos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Piridoxamina/química , Piridoxamina/metabolismo , Piridoxaminafosfato Oxidase/genética , Reprodução , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Análise de Sequência de RNA , Vitamina B 6/químicaRESUMO
In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Manganês/metabolismo , Proteínas das Membranas dos Tilacoides/metabolismo , Tilacoides/metabolismo , Arabidopsis/genética , Cálcio/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismoRESUMO
The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Ácidos Glicéricos/metabolismo , Glicólise/genética , Glicólise/fisiologia , Plastídeos/genéticaRESUMO
The essential micronutrient manganese (Mn) functions as redox-active cofactor in active sites of enzymes and, thus, is involved in various physiological reactions. Moreover, in oxygenic photosynthetic organisms, Mn is of special importance, since it is central to the oxygen-evolving complex in photosystem II. Although Mn is an essential micronutrient, increased amounts are detrimental to the organism; thus, only a small window exists for beneficial concentrations. Accordingly, Mn homeostasis must be carefully maintained. In contrast to the well-studied uptake mechanisms in cyanobacteria, it is largely unknown how Mn is distributed to the different compartments inside the cell. We identified a protein with so far unknown function as a hypothetical Mn transporter in the cyanobacterial model strain Synechocystis sp. PCC 6803 and named this protein Mnx for Mn exporter. The knockout mutant Δmnx showed increased sensitivity toward externally supplied Mn and Mn toxicity symptoms, which could be linked to intracellular Mn accumulation. 54Mn chase experiments demonstrated that the mutant was not able to release Mn from the internal pool. Microscopic analysis of a Mnx::yellow fluorescent protein fusion showed that the protein resides in the thylakoid membrane. Heterologous expression of mnx suppressed the Mn-sensitive phenotype of the Saccharomyces cerevisiae mutant Δpmr1 Our results indicate that Mnx functions as a thylakoid Mn transporter and is a key player in maintaining Mn homeostasis in Synechocystis sp. PCC 6803. We propose that Mn export from the cytoplasm into the thylakoid lumen is crucial to prevent toxic cytoplasmic Mn accumulation and to ensure Mn provision to photosystem II.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Homeostase , Manganês/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Citoplasma/metabolismo , Regulação Bacteriana da Expressão Gênica , Técnicas de Silenciamento de Genes , Transporte de Íons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Espectrometria de Massas/métodos , Microscopia de Fluorescência , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Synechocystis/genética , Tilacoides/metabolismoRESUMO
Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.
Assuntos
Arabidopsis/metabolismo , Fitol/metabolismo , Tocoferóis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Vias Biossintéticas , Clorofila/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Mutação , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Filogenia , Fitol/química , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Tocoferóis/químicaRESUMO
Diatoms, albeit being only distantly related with higher plants, harbor a plant-like cryptochrome (CryP) that was proposed to act as a photoreceptor required for the regulation of some photosynthetic proteins. Plant cryptochromes are involved in the regulation of developmental processes relevant only to multicellular organisms. Their role in the unicellular diatoms to date is mostly enigmatic. To elucidate the function of this plant-like cryptochrome in a unicellular species, we examined the role of CryP in the regulation of transcription in the diatom Phaeodactylum tricornutum by comparative RNA-seq of wild type and CryP knock-down mutants, under prolonged darkness and one hour after onset of blue light. In total, mRNAs of 12,298 genes were identified and more than 70% of the genes could be sorted into functional bins. CryP influenced groups of transcripts in three different ways: some transcripts displayed altered expression under blue light only, others independent of the light condition, and, surprisingly, some were influenced by CryP only in darkness. Genes regulated in any condition were distributed over almost all functional categories. CryP exerted an influence on two other photoreceptors: the genes encoding phytochrome and CPF1, another cryptochrome, which were down-regulated by CryP independent of the light condition. However, the regulatory responses of the affected photoreceptors on transcriptional output were independent. The influence of CryP on the expression of other photoreceptors hints to the existence of a regulatory signaling network in diatoms that includes several cryptochromes and phytochrome, whereby CryP acts as a regulator of transcript abundance under light as well as in darkness.
Assuntos
Criptocromos/genética , Diatomáceas/fisiologia , Transcriptoma , Criptocromos/metabolismo , Escuridão , Diatomáceas/genética , Perfilação da Expressão Gênica , Luz , Mutação , Análise de Sequência de RNARESUMO
Photorespiration is essential for all organisms performing oxygenic photosynthesis. The evolution of photorespiratory metabolism began among cyanobacteria and led to a highly compartmented pathway in plants. A molecular understanding of photorespiration in eukaryotic algae, such as glaucophytes, rhodophytes, and chlorophytes, is essential to unravel the evolution of this pathway. However, mechanistic detail of the photorespiratory pathway in red algae is scarce. The unicellular red alga Cyanidioschyzon merolae represents a model for the red lineage. Its genome is fully sequenced, and tools for targeted gene engineering are available. To study the function and importance of photorespiration in red algae, we chose glycolate oxidase (GOX) as the target. GOX catalyses the conversion of glycolate into glyoxylate, while hydrogen peroxide is generated as a side-product. The function of the candidate GOX from C. merolae was verified by the fact that recombinant GOX preferred glycolate over L-lactate as a substrate. Yellow fluorescent protein-GOX fusion proteins showed that GOX is targeted to peroxisomes in C. merolae The GOX knockout mutant lines showed a high-carbon-requiring phenotype with decreased growth and reduced photosynthetic activity compared to the wild type under ambient air conditions. Metabolite analyses revealed glycolate and glycine accumulation in the mutant cells after a shift from high CO2 conditions to ambient air. In summary, or results demonstrate that photorespiratory metabolism is essential for red algae. The use of a peroxisomal GOX points to a high photorespiratory flux as an ancestral feature of all photosynthetic eukaryotes.
Assuntos
Oxirredutases do Álcool/metabolismo , Fotossíntese/fisiologia , Rodófitas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/fisiologia , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Clorofila A , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Inativação de Genes , Genes de Plantas/genética , Glicolatos/metabolismo , Ácido Láctico/metabolismo , Peroxissomos/enzimologia , Fotossíntese/genética , Reação em Cadeia da Polimerase em Tempo Real , Rodófitas/enzimologia , Rodófitas/genética , Rodófitas/fisiologia , Frações Subcelulares/enzimologiaRESUMO
Synechocystis sp PCC 6803 has four genes encoding flavodiiron proteins (FDPs; Flv1 to Flv4). Here, we investigated the flv4-flv2 operon encoding the Flv4, Sll0218, and Flv2 proteins, which are strongly expressed under low inorganic carbon conditions (i.e., air level of CO(2)) but become repressed at elevated CO(2) conditions. Different from FDP homodimers in anaerobic microbes, Synechocystis Flv2 and Flv4 form a heterodimer. It is located in cytoplasm but also has a high affinity to membrane in the presence of cations. Sll0218, on the contrary, resides in the thylakoid membrane in association with a high molecular mass protein complex. Sll0218 operates partially independently of Flv2/Flv4. It stabilizes the photosystem II (PSII) dimers, and according to biophysical measurements opens up a novel electron transfer pathway to the Flv2/Flv4 heterodimer from PSII. Constructed homology models suggest efficient electron transfer in heterodimeric Flv2/Flv4. It is suggested that Flv2/Flv4 binds to thylakoids in light, mediates electron transfer from PSII, and concomitantly regulates the association of phycobilisomes with PSII. The function of the flv4-flv2 operon provides many ß-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving PSII.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Transporte de Elétrons/fisiologia , Óperon/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/genética , Transporte de Elétrons/genética , Óperon/genética , Complexo de Proteína do Fotossistema II/genética , SynechocystisRESUMO
Photorespiratory metabolism is essential in all oxygenic photosynthetic organisms. In plants, it is a highly compartmentalized pathway that involves chloroplasts, peroxisomes, mitochondria and the cytoplasm. The metabolic pathway itself is well characterized, and the enzymes required for its function have been identified. However, very little information is available on the transport proteins that catalyze the high metabolic flux between the involved compartments. Here we show that the A BOUT DE SOUFFLE (BOU) gene, which encodes a mitochondrial carrier, is involved in photorespiration in Arabidopsis. BOU was found to be co-expressed with photorespiratory genes in leaf tissues. The knockout mutant bou-2 showed the hallmarks of a photorespiratory growth phenotype, an elevated CO(2) compensation point, and excessive accumulation of glycine. Furthermore, degradation of the P-protein, a subunit of glycine decarboxylase, was demonstrated for bou-2, and is reflected in strongly reduced glycine decarboxylase activity. The photorespiration defect in bou-2 has dramatic consequences early in the seedling stage, which are highlighted by transcriptome studies. In bou-2 seedlings, as in shm1, another photorespiratory mutant, the shoot apical meristem organization is severely compromised. Cell divisions are arrested, leading to growth arrest at ambient CO(2) . Although the specific substrate for the BOU transporter protein remains elusive, we show that it is essential for the function of the photorespiratory metabolism. We hypothesize that BOU function is linked with glycine decarboxylase activity, and is required for normal apical meristems functioning in seedlings.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Dióxido de Carbono/metabolismo , Glicina Desidrogenase (Descarboxilante)/metabolismo , Proteínas de Membrana Transportadoras/genética , Meristema/genética , Aminoácidos/análise , Aminoácidos/metabolismo , Arabidopsis/citologia , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Respiração Celular , Perfilação da Expressão Gênica , Teste de Complementação Genética , Glicina/metabolismo , Luz , Proteínas de Membrana Transportadoras/metabolismo , Meristema/citologia , Meristema/fisiologia , Meristema/efeitos da radiação , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fotossíntese , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Plantas Geneticamente Modificadas , Plântula/citologia , Plântula/genética , Plântula/fisiologia , Plântula/efeitos da radiaçãoRESUMO
RNA-seq data in publicly available repositories enable the efficient reanalysis of transcript abundances in existing experiments. Graphical user interfaces usually only allow the visual inspection of a single gene and of predefined experiments. Here, we describe how experiments are selected from the Sequence Read Archive or the European Nucleotide Archive, how data is efficiently mapped onto a reference transcriptome, and how global transcript abundances and patterns are inspected. We exemplarily apply this analysis pipeline to study the expression of photorespiration-related genes in photosynthetic organisms, such as cyanobacteria, and to identify conditions under which photorespiratory transcript abundances are enhanced.
Assuntos
RNA-Seq , Software , Transcriptoma , RNA-Seq/métodos , Transcriptoma/genética , Perfilação da Expressão Gênica/métodos , Biologia Computacional/métodos , Bases de Dados Genéticas , Cianobactérias/genética , Cianobactérias/metabolismo , Fotossíntese/genética , Análise de Sequência de RNA/métodosRESUMO
The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.
Assuntos
Carbono/química , Regulação Bacteriana da Expressão Gênica , Óperon , RNA Antissenso/genética , Synechocystis/genética , Sequência de Bases , Cianobactérias , DNA Complementar/metabolismo , Elétrons , Glucose/química , Ferro/química , Cinética , Luz , Dados de Sequência Molecular , Estresse Oxidativo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismoAssuntos
Ciclo do Carbono , Fotossíntese , Plantas/genética , Evolução Biológica , Mutação , Plantas/metabolismoRESUMO
The D1 protein of photosystem II in the thylakoid membrane of photosynthetic organisms is encoded by psbA genes, which in cyanobacteria occur in the form of a small gene family. Light-dependent up-regulation of psbA gene expression is crucial to ensure the proper replacement of the D1 protein. To gain a high level of gene expression, psbA transcription can be enhanced by several orders of magnitude. Recent transcriptome analyses demonstrated a high number of cis-encoded antisense RNAs (asRNAs) in bacteria, but very little is known about their possible functions. Here, we show the presence of two cis-encoded asRNAs (PsbA2R and PsbA3R) of psbA2 and psbA3 from Synechocystis sp. PCC 6803. These asRNAs are located in the 5' untranslated region of psbA2 and psbA3 genes. Their expression becomes up-regulated by light and down-regulated by darkness, similar to their target mRNAs. In the PsbA2R-suppressing strain [PsbA2R(-)], the amount of psbA2 mRNA was only about 50% compared with the control strain. Likewise, we identified a 15% lowered activity of photosystem II and a reduced amount of the D1 protein in PsbA2R(-) compared with the control strain. The function of PsbA2R in the stabilization of psbA2 mRNA was shown from in vitro RNase E assay when the AU box and the ribosome-binding site in the 5' untranslated region of psbA2 mRNA were both covered by PsbA2R. These results add another layer of complexity to the mechanisms that contribute to psbA gene expression and show PsbA2R as a positively acting factor to achieve a maximum level of D1 synthesis.
Assuntos
Regulação Bacteriana da Expressão Gênica , Complexo de Proteína do Fotossistema II/metabolismo , RNA Antissenso/genética , Synechocystis/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escuridão , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Luz , Dados de Sequência Molecular , Mutagênese , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Synechocystis/efeitos da radiação , Tilacoides/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Photorespiration is inevitable for oxygenic photosynthesis. It has fascinated researchers over decades because of its multicompartmental organization. Recently, Lin and Tsay identified a vacuole glycerate transporter contributing to photorespiratory metabolism under short-term nitrogen depletion. This key finding adds a fifth interacting subcellular compartment and extends the photorespiratory metabolic repair module.
Assuntos
Fotossíntese , Vacúolos , Vacúolos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oxigênio/metabolismo , Nitrogênio/metabolismoRESUMO
Pseudomonas sp. strain MM223, Pseudomonas sp. strain MM227, and Rheinheimera sp. strain MM224 were isolated from a muddy soil sample from the edge of a pond. Here, we present whole-genome sequences and phylogenetic classifications for all three bacterial isolates.
RESUMO
The bacterial strains Pseudomonas sp. strain MM221 and Pseudoarthrobacter sp. strain MM222 were isolated from a sandy soil sample. Here, we report on their complete genome sequences, including a circular plasmid for MM221, which were assembled after sequencing with an Oxford Nanopore Technologies flow cell.