RESUMEN
Light and temperature are two key environmental signals that profoundly affect plant growth and development, but underlying molecular mechanisms of how light and temperature signals affect the circadian clock are largely unknown. Here, we report that COR27 and COR28 are regulated not only by low temperatures but also by light signals. COR27 and COR28 are negative regulators of freezing tolerance but positive regulators of flowering, possibly representing a trade-off between freezing tolerance and flowering. Furthermore, loss-of-function mutations in COR27 and COR28 result in period lengthening of various circadian output rhythms and affect central clock gene expression. Also, the cor27 cor28 double mutation affects the pace of the circadian clock. Additionally, COR27 and COR28 are direct targets of CCA1, which represses their transcription via chromatin binding. Finally, we report that COR27 and COR28 bind to the chromatin of TOC1 and PRR5 to repress their transcription, suggesting that their effects on rhythms are in part due to their regulation of TOC1 and PRR5 These data demonstrate that blue light and low temperature-regulated COR27 and COR28 regulate the circadian clock as well as freezing tolerance and flowering time.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Relojes Circadianos/fisiología , Relojes Circadianos/efectos de la radiación , Luz , Proteínas Represoras/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Ritmo Circadiano/fisiología , Ritmo Circadiano/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteínas Represoras/genética , Temperatura , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited cryptochrome 2 interacts with the transcription factor CRYPTOCHROME-INTERACTING basic helix-loop-helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light-oxygen-voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Criptocromos/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Criptocromos/metabolismo , Flores/genética , Flores/metabolismo , Immunoblotting , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Flores , Hipocótilo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Inmunoprecipitación de Cromatina , Genes de Plantas , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Relojes Biológicos/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Mutación , Factores de Transcripción/metabolismoRESUMEN
The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four ß subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Quinasa de la Caseína II/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Luz , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Western Blotting , Quinasa de la Caseína II/genética , Regulación de la Expresión Génica de las Plantas , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Fosforilación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
Histone methylation plays an essential role in regulating chromatin structure and gene expression. Jumonji C (JmjC) domain-containing proteins are generally known as histone demethylases. Circadian clocks regulate a large number of biological processes, and recent studies suggest that chromatin remodeling has evolved as an important mechanism for regulating both plant and mammalian circadian systems. Here, we analyzed a subgroup of JmjC domain-containing proteins and identified Arabidopsis (Arabidopsis thaliana) JMJ30 as a novel clock component involved in controlling the circadian period. Analysis of loss- and gain-of-function mutants of JMJ30 indicates that this evening-expressed gene is a genetic regulator of period length in the Arabidopsis circadian clock. Furthermore, two key components of the central oscillator of plants, transcription factors CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, bind directly to the JMJ30 promoter to repress its expression, suggesting that JMJ30 regulates the pace of the circadian clock in close association with the central oscillator. JMJ30 represents, to our knowledge, the first JmjC domain-containing protein involved in circadian function, and we envision that this provides a possible molecular connection between chromatin remodeling and the circadian clock.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Relojes Circadianos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Metilación , Mutagénesis Insercional , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , ARN de Planta/genética , Nicotiana/genética , Nicotiana/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory ß-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Quinasa de la Caseína II/metabolismo , Relojes Circadianos , ADN Bacteriano/metabolismo , Subunidades de Proteína/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Quinasa de la Caseína II/genética , Relojes Circadianos/genética , Relojes Circadianos/efectos de la radiación , ADN Bacteriano/genética , Flores/genética , Flores/fisiología , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Luz , Mutagénesis Insercional/genética , Mutación/genética , Fosforilación/efectos de la radiación , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Our understanding of plant circadian rhythms has been advanced by two papers investigating the roles of the transcription factors CCA1 and LHY in the circadian oscillator.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Ritmo Circadiano/fisiología , Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Ritmo Circadiano/genética , Proteínas de Unión al ADN/genética , Genes de Plantas , Modelos Biológicos , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Factores de Transcripción/genéticaRESUMEN
Alteration of 'normal' levels of ultraviolet-B light (UV-B, 280-320 nm) can affect plant chemical composition as well as growth; however, little is known about how plants perceive UV-B light. We have carried out fluence response curves, and demonstrated that the growth inhibition of etiolated Arabidopsis thaliana seedlings by low fluence UV light is specific to UV-B and not UV-A (320-390 nm). The response shows reciprocity between duration and intensity, at least over a limited range, and thus depends only on photon fluence and not on photon flux. The action spectrum for this response indicates a peak of maximum effectiveness at 290 nm, and response spectra at different fluences indicate that the most effective wavelength at 30,000 micromol m(-2) is 290 nm, whereas 300 nm light was the most effective at 100,000 micromol m(-2). This response occurs in mutant seedlings deficient in cryptochrome, phytochrome or phototropin, suggesting that none of the known photoreceptors is the major UV-B photoreceptor. Some null mutants in DNA repair enzymes show hypersensitivity to UV-B, suggesting that even at low fluence rates, direct damage to DNA may be one component of the response to UV-B.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Plantones/efectos de la radiación , Rayos Ultravioleta , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Reparación del ADN , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Mutación , Plantones/crecimiento & desarrollo , Transducción de SeñalRESUMEN
Circadian rhythms are generated by endogenous central oscillators that respond to input from the environment and regulate rhythmic outputs. In Arabidopsis, more than a dozen components that affect rhythms have been identified and used to propose models of the central oscillator. However, none has been shown to fulfill one of the expected characteristics of an oscillator component: that a pulse of its expression shifts the phase of circadian rhythms. Here we show that a pulse of the proposed oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) causes dramatic phase shifts in rhythms of expression of the circadian reporter CAB2::LUC, as well as of the clock-associated genes TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI). These results demonstrate that pulses of either CCA1 or LHY are capable of resetting the circadian clock. In contrast, a pulse of TOC1 expression did not elicit phase shifts. Control of TOC1 protein level is in part posttranscriptional; thus a pulse of TOC1 protein could be induced only at times when it is already high. Our work also shows that the ethanol-inducible system can be useful for achieving relatively short (<8 h) pulses of gene expression in seedlings.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/genética , Etanol/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/fisiología , ADN de Plantas/biosíntesis , ADN de Plantas/genética , Luminiscencia , ARN de Planta/biosíntesis , ARN de Planta/genética , Factores de Transcripción/genéticaRESUMEN
I summarize my scientific journey from my first interest in science to my career investigating how plants use the phytochrome photoreceptor to regulate what genes they express. I then describe how this work led to an understanding of how circadian rhythms function in plants and to the discovery of CCA1, a component of the plant central oscillator.
RESUMEN
The Alc-inducible system is a simple, yet effective, "gene switch" that can be used to transiently induce gene expression in Arabidopsis. Here we provide a protocol for using the Alc-inducible system to give a pulse in expression of a circadian clock gene in transgenic seedlings. The line we use as an example harbors an Alc-inducible copy of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene (Alcâ·CCA1). Alcâ·CCA1 seedlings are grown on solid MS medium and subsequently treated with ethanol vapor. Because the ethanol is quickly absorbed into the medium upon exposure, the seedlings are moved to fresh plates following treatment to avoid continuous induction. After the induction, the seedlings are harvested over a time-course for future total RNA and/or protein extraction that can be used for subsequent gene expression analyses.
Asunto(s)
Arabidopsis/genética , Relojes Circadianos/genética , Regulación de la Expresión Génica de las Plantas , Plantones/genética , Alcoholes/farmacología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regiones Promotoras GenéticasRESUMEN
Circadian rhythms are a universal way for organisms, ranging from prokaryotes to humans, to maintain coordination with the daily changes of light and temperature. It is known that a functional circadian clock confers enhanced fitness. In both animals and plants, diverse physiological processes are affected by the clock and more than 10% of transcripts show a circadian rhythm. Recent advances in the field have revealed a link between circadian regulated gene expression and dynamic changes in chromatin. Jumonji C (JmjC) domain-containing proteins have been shown to be involved in chromatin remodeling, acting as histone demethylases. The recent discovery that a JmjC-domain containing protein functions as a novel clock component suggests that histone modification has evolved as an important mechanism at the core of the circadian machinery.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Relojes Circadianos/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Modelos BiológicosRESUMEN
The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment. Heterodimerization of central clock components is an important way of controlling clock function in several different circadian systems. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are Myb-related proteins that function in or close to the central oscillator in Arabidopsis (Arabidopsis thaliana). Single mutants of cca1 and lhy have a phenotype of short-period rhythms. cca1 lhy double mutants show an even shorter period phenotype than the cca1 single mutant, suggesting that CCA1 and LHY are only partially functionally redundant. To determine whether CCA1 and LHY act in parallel or synergistically in the circadian clock, we examined their expression in both light-grown and etiolated seedlings. We have shown that LHY and CCA1 bind to the same region of the promoter of a Light-harvesting chlorophyll a/b protein (Lhcb, also known as CAB). CCA1 and LHY can form homodimers, and they also colocalize in the nucleus and heterodimerize in vitro and in vivo. In Arabidopsis, CCA1 and LHY physically interact in a manner independent of photoperiod. Moreover, results from gel filtration chromatography indicate that CCA1 and LHY are present in the same large complex in plants. Taken together, these results imply that CCA1 and LHY function synergistically in regulating circadian rhythms of Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Proteínas de Unión a Clorofila , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Complejos de Proteína Captadores de Luz/genética , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Complejo de Proteína del Fotosistema I/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Factores de Transcripción/genéticaRESUMEN
The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes--Lhcb1*1 and Lhcb1*3--and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/fisiología , Arabidopsis/efectos de los fármacos , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efectos de la radiación , Northern Blotting , Sequías , Etilenos/farmacología , Flores/efectos de los fármacos , Flores/fisiología , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/efectos de los fármacos , Hipocótilo/fisiología , Hipocótilo/efectos de la radiación , Luz , Mutación , Fenotipo , Fosfoproteínas/fisiología , Reguladores del Crecimiento de las Plantas/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/efectos de la radiación , Protoplastos/efectos de los fármacos , Protoplastos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
The circadian clock controls numerous physiological and molecular processes in organisms ranging from fungi to human. In plants, these processes include leaf movement, stomata opening, and expression of a large number of genes. At the core of the circadian clock, the central oscillator consists of a negative autoregulatory feedback loop that is coordinated with the daily environmental changes, and that generates the circadian rhythms of the overt processes. Phosphorylation of some of the central oscillator proteins is necessary for the generation of normal circadian rhythms of Drosophila, humans, and Neurospora, where CK1 and CK2 are emerging as the main protein kinases involved in the phosphorylation of PER and FRQ. We have previously shown that in Arabidopsis, the protein kinase CK2 can phosphorylate the clock-associated protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) in vitro. The overexpression of one of its regulatory subunits, CKB3, affects the regulation of circadian rhythms. Whether the effects of CK2 on the clock were due to its phosphorylation of a clock component had yet to be proven. By examining the effects of constitutively expressing a mutant form of the Arabidopsis clock protein CCA1 that cannot be phosphorylated by CK2, we demonstrate here that CCA1 phosphorylation by CK2 is important for the normal functioning of the central oscillator.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Ritmo Circadiano/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Quinasa de la Caseína II , Cartilla de ADN , Cinética , Fosfoproteínas/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Circadian rhythms have been demonstrated in organisms across the taxonomic spectrum. In view of their widespread occurrence, the adaptive significance of these rhythms is of interest. We have previously shown that constitutive expression of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene in Arabidopsis plants (CCA1-ox) results in loss of circadian rhythmicity. Here, we demonstrate that these CCA1-ox plants retain the ability to respond to diurnal changes in light. Thus, transcript levels of several circadian-regulated genes, as well as CCA1 itself and the closely related LHY, oscillate robustly if CCA1-ox plants are grown under diurnal conditions. However, in contrast with wild-type plants in which transcript levels change in anticipation of the dark/light transitions, the CCA1-ox plants have lost the ability to anticipate this daily change in their environment. We have used CCA1-ox lines to examine the effects of loss of circadian regulation on the fitness of an organism. CCA1-ox plants flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts. In addition, we demonstrate that two other circadian rhythm mutants, LHY-ox and elf3, have low-viability phenotypes. Our findings demonstrate the adaptive advantage of circadian rhythms in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Ritmo Circadiano/genética , Factores de Transcripción/genética , Aclimatación/genética , Aclimatación/fisiología , Arabidopsis/fisiología , Supervivencia Celular , Ritmo Circadiano/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Oscuridad , Luz , Fotoperiodo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Tallos de la Planta/fisiología , ARN de Planta/genética , ARN de Planta/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the phase or period of circadian rhythms. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement. We conclude that circadian rhythms of ethylene production are not critical for rhythmic growth.