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1.
Genes Cells ; 26(2): 83-93, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33341998

RESUMEN

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg2+ , and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24 hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.


Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Relojes Circadianos , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Luciferasas de Luciérnaga/metabolismo , Luminiscencia , Thermosynechococcus/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Bioensayo , Modelos Biológicos , Mutación/genética , Fosforilación
2.
Plant Cell Physiol ; 62(5): 883-893, 2021 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-33822207

RESUMEN

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.


Asunto(s)
Arabidopsis/fisiología , Relojes Circadianos/fisiología , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relojes Circadianos/genética , Proteínas de Unión al ADN/genética , Flores/genética , Luz , Mediciones Luminiscentes , Mutación , Plantas Modificadas Genéticamente , Temperatura , Factores de Transcripción/genética
3.
Mol Plant Microbe Interact ; 33(12): 1366-1380, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32876529

RESUMEN

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencing-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Pruebas Genéticas , Mutación , Inmunidad de la Planta , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Pruebas Genéticas/métodos , Luciferasas/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos , Inmunidad de la Planta/genética
4.
PLoS Genet ; 13(3): e1006645, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28333924

RESUMEN

The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.


Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas reinhardtii/efectos de la radiación , Relojes Circadianos/genética , Luz , Proteínas/genética , Proteínas Algáceas/metabolismo , Western Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Ritmo Circadiano/genética , Ritmo Circadiano/efectos de la radiación , Citoplasma/genética , Citoplasma/metabolismo , Expresión Génica/efectos de la radiación , Inmunohistoquímica , Proteínas Repetidas Ricas en Leucina , Mutación , Fosforilación/efectos de la radiación , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación
5.
Genes Cells ; 20(3): 173-90, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25492525

RESUMEN

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108 . KaiB10-108 was a monomer at 4 °C but a dimer at 35 °C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas CLOCK/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Ritmo Circadiano , Cianobacterias/metabolismo , Multimerización de Proteína , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Fosforilación , Temperatura
6.
Proc Natl Acad Sci U S A ; 110(33): 13666-71, 2013 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-23898163

RESUMEN

Although the circadian clock is a self-sustaining oscillator having a periodicity of nearly 1 d, its period length is not necessarily 24 h. Therefore, daily adjustment of the clock (i.e., resetting) is an essential mechanism for the circadian clock to adapt to daily environmental changes. One of the major cues for this resetting mechanism is light. In the unicellular green alga Chlamydomonas reinhardtii, the circadian clock is reset by blue/green and red light. However, the underlying molecular mechanisms remain largely unknown. In this study, using clock protein-luciferase fusion reporters, we found that the level of RHYTHM OF CHLOROPLAST 15 (ROC15), a clock component in C. reinhardtii, decreased rapidly after light exposure in a circadian-phase-independent manner. Blue, green, and red light were able to induce this process, with red light being the most effective among them. Expression analyses and inhibitor experiments suggested that this process was regulated mainly by a proteasome-dependent protein degradation pathway. In addition, we found that the other clock gene, ROC114, encoding an F-box protein, was involved in this process. Furthermore, we demonstrated that a roc15 mutant showed defects in the phase-resetting of the circadian clock by light. Taken together, these data strongly suggest that the light-induced degradation of ROC15 protein is one of the triggers for resetting the circadian clock in C. reinhardtii. Our data provide not only a basis for understanding the molecular mechanisms of light-induced phase-resetting in C. reinhardtii, but also insights into the phase-resetting mechanisms of circadian clocks in plants.


Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/fisiología , Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Luz , Secuencia de Bases , Relojes Circadianos/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Datos de Secuencia Molecular , Factores de Tiempo
7.
J Bacteriol ; 197(4): 676-87, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25313394

RESUMEN

Photoautotrophic bacteria have developed mechanisms to maintain K(+) homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K(+) uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K(+) (Kdp). The contributions of each of these K(+) transport systems to cellular K(+) homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K(+) uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K(+) uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K(+) depletion. Correspondingly, Kdp-mediated K(+) uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K(+) required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K(+) concentration under conditions of limited K(+) in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K(+) availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K(+) levels under fluctuating environmental conditions.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Synechocystis/metabolismo , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Operón , Potasio/metabolismo , Synechocystis/genética
8.
J Biol Chem ; 287(35): 29506-15, 2012 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-22722936

RESUMEN

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).


Asunto(s)
Ciclos de Actividad/fisiología , Proteínas Bacterianas/metabolismo , Relojes Circadianos/fisiología , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Cianobacterias/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Multimerización de Proteína , Proteínas Bacterianas/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Cianobacterias/genética , Mutación , Estructura Cuaternaria de Proteína
9.
Genes Cells ; 17(5): 398-419, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22512339

RESUMEN

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation-enhancing activity of KaiC, ultimately resulting in genome-wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the domain structure of an orthodox histidine kinase and that its C-terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature- and phosphorylation state-dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N-terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation-enhancing activity of KaiC requires the C-terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.


Asunto(s)
Proteínas Bacterianas/metabolismo , Relojes Circadianos/fisiología , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cianobacterias/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación
10.
J Bacteriol ; 194(24): 6828-36, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23043001

RESUMEN

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.


Asunto(s)
Acuaporinas/metabolismo , Presión Osmótica , Synechocystis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Tamaño de la Célula , Relojes Circadianos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Cloruro de Mercurio/farmacología , Concentración Osmolar , Oxígeno/metabolismo , Cloruro de Sodio/farmacología , Sorbitol/farmacología , Synechocystis/genética , Agua/metabolismo
11.
J Biol Chem ; 286(28): 25224-35, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21558269

RESUMEN

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism.


Asunto(s)
Acuaporinas/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Glucosa/metabolismo , Synechocystis/metabolismo , Acuaporinas/genética , Proteínas Bacterianas/genética , Membrana Celular/genética , Citosol/metabolismo , Eliminación de Gen , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Concentración Osmolar , Vía de Pentosa Fosfato/fisiología , Synechocystis/genética
12.
Genes Cells ; 14(1): 1-16, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19032344

RESUMEN

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.


Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/química , Synechococcus/metabolismo , Transactivadores/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Bioensayo , Proteínas CLOCK , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Dimerización , Regulación Bacteriana de la Expresión Génica , Mediciones Luminiscentes , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Desnaturalización Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Temperatura , Transactivadores/genética , Transactivadores/metabolismo
13.
Mol Cell Biol ; 26(3): 863-70, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16428442

RESUMEN

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.


Asunto(s)
Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Ritmo Circadiano/genética , Genes Protozoarios/genética , Animales , Secuencia de Bases , Cloroplastos/enzimología , ADN de Cloroplastos/genética , Expresión Génica , Genes Reporteros , Luciferasas/análisis , Luciferasas/genética , Mediciones Luminiscentes , Datos de Secuencia Molecular , Temperatura
14.
PLoS One ; 8(11): e80200, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244649

RESUMEN

The cyanobacterial circadian clock oscillator is composed of three clock proteins--KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC(1mer)) and formed a complex with KaiB. The addition of ATP plus Mg(2+) (Mg-ATP), but not that of ATP only, to the KaiB-KaiC(1mer) complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC(1mer) complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg(2+) (Mg-ATP), KaiC became a homohexameric ATPase (KaiC(6mer)) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC(6mer) lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC(6mer) is required for formation of the KaiB-KaiC(6mer) complex. KaiC(6mer) that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC(6mer) with bound ADP-Pi.


Asunto(s)
Proteínas Bacterianas/genética , Relojes Circadianos/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Cianobacterias/genética , Regulación Bacteriana de la Expresión Génica , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cationes Bivalentes , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Cianobacterias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Magnesio/química , Magnesio/metabolismo , Fosforilación , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Front Plant Sci ; 4: 31, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23460029

RESUMEN

Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes.

16.
Channels (Austin) ; 7(4): 238-42, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23764907

RESUMEN

Synechocystis sp strain PCC 6803 contains one gene encoding a putative large conductance mechanosensitive channel homolog [named SyMscL (slr0875)]. However, it is unclear whether SyMscL contributes to the adaptation to hypoosmotic stress in Synechocystis. Here we report the in vivo characteristics of SyMscL. SyMscL was mainly expressed in the plasma membrane of Synechocystis. Cell volume monitoring using stopped-flow spectrophotometry showed that ΔsymscL cells swelled more rapidly than wild-type cells under hypoosmotic stress conditions. Expression of symscL was under circadian control, and its peak corresponded to the beginning of subjective night. These results indicate that SyMscL functioned as one component of the osmotic homeostatic regulatory system of the cell coordinating the response of Synechocystis to daily metabolic osmotic fluctuations and environmental changes.


Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Canales Iónicos/metabolismo , Presión Osmótica , Synechocystis/fisiología , Proteínas Bacterianas/genética , Ritmo Circadiano , Ambiente , Espacio Intracelular/metabolismo , Canales Iónicos/genética , Transporte de Proteínas , Synechocystis/citología , Synechocystis/genética , Synechocystis/metabolismo
17.
J Biol Chem ; 284(24): 16513-16521, 2009 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-19372598

RESUMEN

Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.


Asunto(s)
Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Synechocystis/enzimología , Tilacoides/enzimología , Ritmo Circadiano/fisiología , Escherichia coli/genética , Homeostasis/fisiología , Concentración de Iones de Hidrógeno , Consumo de Oxígeno/fisiología , Fotosíntesis/fisiología , Potasio/metabolismo , Protones , Sodio/metabolismo , Synechocystis/genética , Tilacoides/genética
18.
Genes Dev ; 22(7): 918-30, 2008 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-18334618

RESUMEN

The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components.


Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Ritmo Circadiano/genética , Mutación , Proteínas Algáceas/fisiología , Secuencia de Aminoácidos , Animales , Northern Blotting , Chlamydomonas reinhardtii/fisiología , Cloroplastos/metabolismo , Ritmo Circadiano/fisiología , Clonación Molecular , ADN de Cloroplastos/genética , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Insercional , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido
19.
Plant J ; 47(1): 49-62, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16740149

RESUMEN

The low-beta-amylase1 (lba1) mutant of Arabidopsis thaliana has reduced sugar-induced expression of Atbeta-Amy and shows pleiotropic phenotypes such as early flowering; short day-sensitive growth; and seed germination that is hypersensitive to glucose and abscisic acid and resistant to mannose. lba1 was a missense mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay (NMD), which eliminates mRNAs with premature termination codons (PTCs), and replaces highly conserved Gly851 of UPF1 with Glu. Expression of the wild-type UPF1 in lba1 rescued not only the reduced sugar-inducible gene expression, but also early flowering and altered seed-germination phenotypes. Sugar-inducible mRNAs were over-represented among transcripts decreased in sucrose-treated lba1 compared with Col plants, suggesting that UPF1 is involved in the expression of a subset of sugar-inducible genes. On the other hand, transcripts increased in lba1, which are likely to contain direct targets of NMD, included mRNAs for many transcription factors and metabolic enzymes that play diverse functions. Among these, the level of an alternatively spliced transcript of AtTFIIIA containing PTC was 17-fold higher in lba1 compared with Col plants, and it was reduced to the level in Col by expressing the wild-type UPF1. The lba1 mutant provides a good tool for studying NMD in plants.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Carbohidratos/fisiología , ARN Helicasas/fisiología , ARN Mensajero/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Codón sin Sentido , Análisis Mutacional de ADN , ADN Complementario , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , ARN Helicasas/genética , Transducción de Señal , Factor de Transcripción TFIIIA/metabolismo , beta-Amilasa/metabolismo
20.
Genes Cells ; 10(10): 963-72, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16164597

RESUMEN

Previously, we screened 50 000 seedlings of Arabidopsis thaliana carrying a P(GI)::LUC+ bioluminescence reporter gene mutagenized with ethylmethanesulfonate for mutants with phenotypes of extensively altered circadian rhythms, and identified three loci, PHYTOCLOCK 1 (PCL1), PCL2 and PCL3, whose mutations cause arrhythmia. Here we succeeded to clone the PCL1 gene and show that the PCL1 gene encodes a novel DNA binding protein belonging to the GARP protein family and is essential for a functional clock oscillator in A. thaliana. The PCL1 gene satisfies the requirements for the clock oscillator gene: (i) pcl1 null mutations caused arrhythmia in multiple circadian outputs, including expression of potential clock genes TOC1, CCA1 and LHY, and flowering lacked a photoperiodic response; (ii) PCL1 expression showed circadian rhythm in both constant light and constant dark; (iii) over-expression of the PCL1 gene gradually caused arrhythmicity in all the multiple circadian outputs examined; and (iv) the PCL1 gene controlled its own expression via negative feedback. Therefore, the PCL1 gene is the clock oscillator gene essential to the generation of clock oscillation in the higher plant.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ritmo Circadiano/genética , Proteínas de Unión al ADN/genética , Secuencia de Aminoácidos , Arabidopsis/fisiología , Relojes Biológicos/genética , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Mutación , Estimulación Luminosa , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Homología de Secuencia de Aminoácido , Factores de Transcripción
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