Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.     

Characterization of Circadian-associated pseudo-response regulators: I. Comparative studies on a series of transgenic lines misexpressing five distinctive PRR Genes in Arabidopsis thaliana

Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.     

Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.     

ELF4 is required for oscillatory properties of the circadian clock

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 (ELF4) locus of Arabidopsis (Arabidopsis thaliana) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LONG ELONGATED HYPOCOTYL (LHY)-TIMING OF CAB EXPRESSION1 (TOC1) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4, therefore, can be considered a component of the central CCA1/LHY-TOC1 feedback loop in the plant circadian clock.     

Conserved expression profiles of circadian clock-related genes in two Lemna species showing long-day and short-day photoperiodic flowering responses

The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light-dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution.     

Genetic linkages between circadian clock-associated components and phytochrome-dependent red light signal transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Plants measure the time

All eukaryotes, including plants, and most prokaryotes have developed elaborate mechanisms to anticipate external environmental changes associated with the Earth’s rotation. These mechanisms are mediated by a circadian clock, which regulates several physiological and biochemical processes. Microarray experiments using Affymetrix chips that included about 8000 of the 27000 Arabidopsis genes have demonstrated that as much as 6% of that genome may be under the control of this clock. While our understanding of such mechanisms is lagging, molecular genetics studies of Arabidopsis have allowed us to make great progress toward identifying and characterizing components of the plant circadian clock since its first component was isolated in 1995. The generation of 24-h rhythms by this clock appears to rely on mechanisms similar to those found in other organisms. However, an entirely different set of molecular components are recruited to perform these functions in Arabidopsis. In this review, we introduce useful and powerful approaches for identifying clock-associated genes and determining how they can act together in the interlocking feedback loops that comprise this particular clock.     

Phylogenetic footprint of the plant clock system in angiosperms: evolutionary processes of <Emphasis Type="Italic">Pseudo-Response Regulator</Emphasis>s

Background  

Plant circadian clocks regulate many photoperiodic and diurnal responses that are conserved among plant species. The plant circadian clock system has been uncovered in the model plant, Arabidopsis thaliana, using genetics and systems biology approaches. However, it is still not clear how the clock system had been organized in the evolutionary history of plants. We recently revealed the molecular phylogeny of LHY/CCA1 genes, one of the essential components of the clock system. The aims of this study are to reconstruct the phylogenetic relationships of angiosperm clock-associated PRR genes, the partner of the LHY/CCA1 genes, and to clarify the evolutionary history of the plant clock system in angiosperm lineages.     

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock

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 β-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.     

Sensitive to freezing6 integrates cellular and environmental inputs to the plant circadian clock

The sensitive to freezing6 (sfr6) mutant of Arabidopsis (Arabidopsis thaliana) is late flowering in long days due to reduced expression of components in the photoperiodic flowering pathway in long-day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was overrepresented in promoters of genes down-regulated in sfr6 plants. Analysis of leaf movement rhythms found sfr6 plants showed a sucrose (Suc)-dependent long period phenotype; unlike wild-type Arabidopsis, the clock in sfr6 plants did not have a shorter rhythm in the presence of Suc. Other developmental responses to Suc were unaltered in sfr6 plants, suggesting insensitivity to Suc is restricted to the clock. We investigated the effect of sfr6 and Suc upon clock gene expression over 24 h. The sfr6 mutation resulted in reduced expression of the clock components CIRCADIAN CLOCK ASSOCIATED1, GIGANTEA, and TIMING OF CAB1. These changes occurred independently of Suc supplementation. Wild-type plants showed small increases in clock gene expression in the presence of Suc; this response to Suc was reduced in sfr6 plants. This study shows that large changes in level and timing of clock gene expression may have little effect upon clock outputs. Moreover, although Suc influences the period and accuracy of the Arabidopsis clock, it results in relatively minor changes in clock gene expression.