RESUMO
The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here we present a protein-DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes/genética , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Fatores de Transcrição E2F/metabolismo , Retroalimentação , Regulação da Expressão Gênica no Desenvolvimento/genética , Deficiências de Ferro , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Salinidade , Fatores de Tempo , Xilema/genética , Xilema/crescimento & desenvolvimento , Xilema/metabolismoRESUMO
A successful genetic dissection of the circadian regulation of behaviour has been achieved through phenotype-driven mutagenesis screens in flies and mice. Cloning and biochemical analysis of these evolutionarily conserved proteins has led to detailed molecular insight into the clock mechanism. Few behaviours enjoy the degree of understanding that exists for circadian rhythms at the genetic, cellular and anatomical levels. The circadian clock has so eagerly spilled her secrets that we may soon know the unbroken chain of events from gene to behaviour. It will likely be fruitful to wield this uncommon degree of knowledge to attack one of the most challenging problems in genetics: the basis of complex human behavioural disorders. We review here the genetic screens that provided the entreé into the heart of the circadian clock, the model of the clock mechanism that has resulted, and the prospects for using the homologues as candidate genes in studies of human circadian dysrhythmias.
Assuntos
Ritmo Circadiano/genética , Animais , Ritmo Circadiano/fisiologia , Drosophila , Feminino , Humanos , Masculino , Camundongos , Modelos Biológicos , RatosRESUMO
Cryptochromes are blue/UV-A-absorbing photoreceptor proteins discovered originally in plants and so named because their nature proved elusive in over a century of research. Now we know that the photoreceptor essential for proper seedling establishment in blue light has homologues in the animal kingdom - in insects, in mice and in humans. In recent months, evidence has emerged pointing to a common role for cryptochromes in all of these organisms in entraining the circadian clock, a biochemical timing mechanism running within cells, synchronizing metabolism to the daily light-dark cycle and having consequences on a much larger scale in the regulation of behaviour such as the sleep-wake cycle.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/fisiologia , Células Fotorreceptoras de Invertebrados , Células Fotorreceptoras/fisiologia , Animais , Arabidopsis , Proteínas de Arabidopsis , Criptocromos , Drosophila , Humanos , Camundongos , Receptores Acoplados a Proteínas GRESUMO
Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.
Assuntos
Arabidopsis/genética , Núcleo Celular/metabolismo , Indicadores e Reagentes/farmacocinética , Proteínas Luminescentes/farmacocinética , Sinais de Localização Nuclear , Células Fotorreceptoras , Fitocromo/farmacocinética , Fatores de Transcrição , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Cor , Escuridão , Proteínas de Fluorescência Verde , Hipocótilo/fisiologia , Immunoblotting , Iluminação , Proteínas Luminescentes/análise , Microscopia Confocal , Proteínas Nucleares/metabolismo , Estimulação Luminosa , Fitocromo/análise , Fitocromo B , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.
Assuntos
Arabidopsis/fisiologia , Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/fisiologia , Células Fotorreceptoras de Invertebrados , Células Fotorreceptoras , Fitocromo/fisiologia , Fatores de Transcrição , Arabidopsis/genética , Proteínas de Arabidopsis , Criptocromos , Flavoproteínas/genética , Luz , Mutação , Fitocromo/genética , Fitocromo A , Fitocromo B , Plantas Geneticamente Modificadas , Receptores Acoplados a Proteínas G , Transdução de SinaisRESUMO
Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period were used to monitor the circadian clock in explanted head, thorax, and abdominal tissues. The tissues (including sensory bristles in the leg and wing) showed rhythmic bioluminescence, and the rhythms could be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Drosophila/fisiologia , Proteínas Nucleares/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Células Quimiorreceptoras/fisiologia , Escuridão , Drosophila/genética , Proteínas de Drosophila , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Fluorescência Verde , Luz , Transdução de Sinal Luminoso , Luciferases/genética , Luminescência , Proteínas Luminescentes/genética , Proteínas Nucleares/genética , Proteínas Circadianas Period , Regiões Promotoras Genéticas , Receptores de Superfície Celular , Proteínas Recombinantes de FusãoRESUMO
Transgenic Arabidopsis plants expressing a luciferase gene fused to a circadian-regulated promoter exhibited robust rhythms in bioluminescence. The cyclic luminescence has a 24.7-hour period in white light but 30- to 36-hour periods under constant darkness. Either red or blue light shortened the period of the wild type to 25 hours. A phytochrome-deficient mutation lengthened the period in continuous red light but had little effect in continuous blue light, whereas seedlings carrying mutations that activate light-dependent pathways in darkness maintained shorter periods in constant darkness. These results suggest that both phytochrome- and blue light-responsive photoreceptor pathways control the period of the circadian clock.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/fisiologia , Relógios Biológicos , Ritmo Circadiano , Genes de Plantas , Luz , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema II , Proteínas de Plantas , Arabidopsis/genética , Relógios Biológicos/genética , Proteínas de Transporte/genética , Ritmo Circadiano/genética , Escuridão , Regulação da Expressão Gênica de Plantas , Complexos de Proteínas Captadores de Luz , Luciferases/genética , Luminescência , Plantas Geneticamente Modificadas , Proteínas Recombinantes de FusãoRESUMO
The interactive regulation between clock genes is central for oscillator function. Here, we show interactions between the Arabidopsis clock genes LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), and TIMING OF CAB EXPRESSION 1 (TOC1). The MYB transcription factors LHY and CCA1 negatively regulate TOC1 expression. We show that both proteins bind to a region in the TOC1 promoter that is critical for its clock regulation. Conversely, TOC1 appears to participate in the positive regulation of LHY and CCA1 expression. Our results indicate that these interactions form a loop critical for clock function in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/fisiologia , Relógios Biológicos/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Plantas , Modelos Genéticos , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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
Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion. TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light. CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion. PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal. Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/metabolismo , Proteínas de Insetos/metabolismo , Luz , Células Fotorreceptoras de Invertebrados , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Criptocromos , Citoplasma/metabolismo , Escuridão , Dimerização , Drosophila , Flavoproteínas/genética , Proteínas de Fluorescência Verde , Proteínas de Insetos/genética , Proteínas Luminescentes , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Leveduras/genética , Leveduras/metabolismoRESUMO
Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.
Assuntos
Arabidopsis/fisiologia , Relógios Biológicos/genética , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Transcrição Gênica , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Metabolismo dos Carboidratos , Perfilação da Expressão Gênica , Genes de Plantas , Luz , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Enxofre/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
Photoperiodic responses, such as the daylength-dependent control of reproductive development, are associated with a circadian biological clock. The photoperiod-insensitive early-flowering 3 (elf3) mutant of Arabidopsis thaliana lacks rhythmicity in two distinct circadian-regulated processes. This defect was apparent only when plants were assayed under constant light conditions. elf3 mutants retain rhythmicity in constant dark and anticipate light/dark transitions under most light/dark regimes. The conditional arrhythmic phenotype suggests that the circadian pacemaker is intact in darkness in elf3 mutant plants, but the transduction of light signals to the circadian clock is impaired.
Assuntos
Arabidopsis/fisiologia , Ritmo Circadiano , Luz , Fotoperíodo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Escuridão , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Movimento , Mutação , Fenótipo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Folhas de Planta/fisiologia , Plantas Geneticamente ModificadasRESUMO
Photoperiodic responses in plants include flowering that is day-length-dependent. Mutations in the Arabidopsis thaliana GIGANTEA (GI) gene cause photoperiod-insensitive flowering and alteration of circadian rhythms. The GI gene encodes a protein containing six putative transmembrane domains. Circadian expression patterns of the GI gene and the clock-associated genes, LHY and CCA1, are altered in gi mutants, showing that GI is required for maintaining circadian amplitude and appropriate period length of these genes. The gi-1 mutation also affects light signaling to the clock, which suggests that GI participates in a feedback loop of the plant circadian system.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Arabidopsis/fisiologia , Ritmo Circadiano , Genes de Plantas , Proteínas de Plantas/genética , Clonagem Molecular , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Escuridão , Retroalimentação , Regulação da Expressão Gênica de Plantas , Luz , Dados de Sequência Molecular , Mutação , Fotoperíodo , Folhas de Planta/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Estruturas Vegetais/fisiologia , Deleção de Sequência , Fatores de Transcrição/genéticaRESUMO
The circadian oscillator generates a rhythmic output with a period of about 24 hours. Despite extensive studies in several model systems, the biochemical mode of action has not yet been demonstrated for any of its components. Here, the Drosophila CLOCK protein was shown to induce transcription of the circadian rhythm genes period and timeless. dCLOCK functioned as a heterodimer with a Drosophila homolog of BMAL1. These proteins acted through an E-box sequence in the period promoter. The timeless promoter contains an 18-base pair element encompassing an E-box, which was sufficient to confer dCLOCK responsiveness to a reporter gene. PERIOD and TIMELESS proteins blocked dCLOCK's ability to transactivate their promoters via the E-box. Thus, dCLOCK drives expression of period and timeless, which in turn inhibit dCLOCK's activity and close the circadian loop.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas Nucleares/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Relógios Biológicos , Proteínas CLOCK , Linhagem Celular , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Dimerização , Drosophila , Retroalimentação , Expressão Gênica , Sequências Hélice-Alça-Hélice , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.
Assuntos
Drosophila/genética , Luciferases/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Ritmo Circadiano , DNA Complementar , Proteínas de Drosophila , Genes Reporter , Luciferases/metabolismo , Medições Luminescentes , Proteínas Circadianas Period , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Recombinantes de FusãoRESUMO
BACKGROUND: The AP-1 family of transcription factors has been implicated in the control of the expression of many genes in response to environmental signals. Previous studies have provided temporal profiles for c-fos expression by taking measurements from many animals at several points in time, but these studies provide limited information about dynamic changes in expression. Here, we have devised a method of continuously measuring c-fos expression. RESULTS: A transgenic mouse line expressing the human c-fos promoter linked to the firefly luciferase reporter gene (fos/luc) was generated to continuously monitor c-fos gene expression. A second transgenic mouse line expressing luciferase under the control of the cytomegalovirus promoter (CMV/luc) served as a control. Luminescence originating from identifiable brain regions was imaged from fos/luc brain slice cultures. Expression of the fos/luc transgene accurately reflected transcriptional responses of the endogenous c-fos gene. Dynamic changes in fos/luc expression in suprachiasmatic nuclei (SCN) explant cultures were monitored continuously, and luminescence showed almost 24 hour rhythms lasting up to five circadian cycles. In contrast, bioluminescence monitored from CMV/luc SCN explant cultures was not rhythmic. CONCLUSION: The fos/luc transgenic mouse will be useful for long-term, non-invasive monitoring of c-fos transcriptional responses to the changing cellular environment. Circadian rhythms in c-fos expression can be monitored non-invasively in real time from the SCN, clearly demonstrating that c-fos transcription is regulated by the circadian clock.
Assuntos
Ritmo Circadiano , Proteínas Proto-Oncogênicas c-fos/biossíntese , Núcleo Supraquiasmático/metabolismo , Animais , Citomegalovirus/genética , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
An intriguing property of many organisms is their ability to exhibit rhythmic cellular events that continue independently of environmental stimuli. These rhythmic processes are generated by an endogenous mechanism known as the biological clock. We wished to determine whether Arabidopsis thaliana will serve as a model plant system for a molecular genetic dissection of the circadian clock. To this end, we investigated the expression of Arabidopsis chlorophyll a/b-binding protein (cab) genes throughout the circadian cycle. Steady-state mRNA levels of the cab2 and cab3 genes showed a dramatic circadian cycling in plants shifted from light/dark cycles to constant darkness, whereas the cab1 mRNA level exhibited little or no cycling under the same conditions. Analysis of cab promoter fusions in transgenic tobacco revealed that both the cab1 and cab2 5[prime] upstream regions confer circadian-regulated expression on a chloramphenicol acetyltransferase (cat) reporter gene. In vitro nuclear run-on transcription assays also indicated that the transcription of the cab1 and cab2 genes is circadian regulated in Arabidopsis. Taken together, these data suggest that a post-transcriptional mechanism influences cab1 mRNA levels in Arabidopsis. The identification of circadian-regulated cis-acting elements in the cab1 and cab2 upstream regions will provide powerful tools for both molecular and genetic analysis of the higher plant circadian clock.
RESUMO
Higher plant CAB genes encode chlorophyll a/b binding proteins that are part of light-harvesting complexes in chloroplasts. Transcription of the Arabidopsis CAB2 (lhcb1*1) gene is under the control of a circadian oscillator and exhibits high amplitude diurnal oscillations that persist within a period close to 24 hr in the absence of environmental time cues. Initial deletion studies in transgenic tobacco have demonstrated that the region between -111 and -38 of the CAB2 promoter sequence confers circadian regulation to a luciferase (luc) reporter gene. We dissected this element further and characterized five DNA binding complexes from Arabidopsis whole-cell extracts that bind within this region of the promoter and may be components of the signal transduction pathway for the control of transcription by the circadian clock. The in vivo analysis of cab2::luc fusion constructs in transgenic Arabidopsis demonstrated that a circadian-regulated element lies within a 36-bp sequence that overlaps a conserved CCAAT box and contains binding sites for three putative transcription factors.
RESUMO
To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.