RESUMO
The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/fisiologia , Ritmo Circadiano/fisiologia , Proteínas Nucleares/fisiologia , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Arabidopsis/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Clonagem Molecular , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Plantas Geneticamente ModificadasRESUMO
The Arabidopsis genes CONSTANS-LIKE 1 (COL1) and CONSTANS-LIKE 2 (COL2) are predicted to encode zinc finger proteins with approximately 67% amino acid identity to the protein encoded by the flowering-time gene CONSTANS (CO). We show that the circadian clock regulates expression of COL1 and COL2 with a peak in transcript levels around dawn. We analyzed transgenic plants misexpressing COL1, COL2 and CO. Unlike CO, altered expression of COL1 and COL2 in transgenic plants had little effect on flowering time. However, analysis of circadian phenotypes in the transgenic plants showed that over-expression of COL1 can shorten the period of two distinct circadian rhythms. Experiments with the highest COL1 over-expressing line indicate that its circadian defects are fluence rate-dependent, suggesting an effect on a light input pathway(s).
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Ritmo Circadiano , Proteínas de Ligação a DNA/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Dedos de Zinco , Arabidopsis/fisiologia , Relógios Biológicos , Northern Blotting , Proteínas de Ligação a DNA/metabolismo , Medições Luminescentes , Fenótipo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/análise , Fatores de Transcrição/metabolismoRESUMO
The toc1 mutation causes shortened circadian rhythms in light-grown Arabidopsis plants. Here, we report the same toc1 effect in the absence of light input to the clock. We also show that TOC1 controls photoperiodic flowering response through clock function. The TOC1 gene was isolated and found to encode a nuclear protein containing an atypical response regulator receiver domain and two motifs that suggest a role in transcriptional regulation: a basic motif conserved within the CONSTANS family of transcription factors and an acidic domain. TOC1 is itself circadianly regulated and participates in a feedback loop to control its own expression.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Relógios Biológicos/genética , Ritmo Circadiano/genética , Proteínas de Plantas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/fisiologia , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Retroalimentação , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fenótipo , Fotoperíodo , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Sequências Repetitivas de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
The circadian rhythms we observe originate from the circadian regulation of gene expression. Common control points, like transcription and protein phosphorylation, are used to effect this regulation, both within the clock and in output pathways. Recent work has advanced our understanding of these processes in plants and other models. Also, specific photoreceptors that mediate light entrainment of the clock have been identified in Arabidopsis.
Assuntos
Arabidopsis/fisiologia , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Cloroplastos/genética , Cloroplastos/efeitos da radiação , Luz , Fosforilação , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genéticaAssuntos
Doença das Coronárias/diagnóstico , Teste de Esforço/normas , Idoso , Viés , Angiografia Coronária , Doença das Coronárias/diagnóstico por imagem , Doença das Coronárias/fisiopatologia , Estudos Transversais , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Probabilidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The cycling bioluminescence of Arabidopsis plants carrying a firefly luciferase fusion construct was used to identify mutant individuals with aberrant cycling patterns. Both long- and short-period mutants were recovered. A semidominant short-period mutation, timing of CAB expression (toc1), was mapped to chromosome 5. The toc1 mutation shortens the period of two distinct circadian rhythms, the expression of chlorophyll a/b-binding protein (CAB) genes and the movements of primary leaves, although toc1 mutants do not show extensive pleiotropy for other phenotypes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/fisiologia , Relógios Biológicos/genética , Proteínas de Transporte/genética , Ritmo Circadiano/genética , Genes de Plantas , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema II , Proteínas de Plantas , Arabidopsis/genética , Cruzamentos Genéticos , Escuridão , Regulação da Expressão Gênica de Plantas , Luz , Complexos de Proteínas Captadores de Luz , Luciferases/genética , Luminescência , Movimento , Mutação , Fenótipo , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes de FusãoRESUMO
We developed a versatile, efficient genetic transfer method for Synechococcus sp. strains PCC 7942 and PCC 6301 that exceeds natural transformation efficiencies by orders of magnitude. As a test case, we complemented a histidine auxotroph and identified a hisS homolog of PCC 7942 as the complementing gene.
Assuntos
Conjugação Genética , Cianobactérias/genética , Técnicas de Transferência de Genes , Transformação Genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Bacterianos , Escherichia coli/genética , Teste de Complementação Genética , Vetores Genéticos , Histidina/biossíntese , Dados de Sequência MolecularRESUMO
We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem II protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAI message following light/dark entrainment supported the reporter data. The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock.
