Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.     

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.     

A complex genetic interaction between Arabidopsis thaliana TOC1 and CCA1/LHY in driving the circadian clock and in output regulation

It has been proposed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) together with TIMING OF CAB EXPRESSION 1 (TOC1) make up the central oscillator of the Arabidopsis thaliana circadian clock. These genes thus drive rhythmic outputs, including seasonal control of flowering and photomorphogenesis. To test various clock models and to disclose the genetic relationship between TOC1 and CCA1/LHY in floral induction and photomorphogenesis, we constructed the cca1 lhy toc1 triple mutant and cca1 toc1 and lhy toc1 double mutants and tested various rhythmic responses and circadian output regulation. Here we report that rhythmic activity was dramatically attenuated in cca1 lhy toc1. Interestingly, we also found that TOC1 regulates the floral transition in a CCA1/LHY-dependent manner while CCA1/LHY functions upstream of TOC1 in regulating a photomorphogenic process. This suggests to us that TOC1 and CCA1/LHY participate in these two processes through different strategies. Collectively, we have used genetics to provide direct experimental support of previous modeling efforts where CCA1/LHY, along with TOC1, drives the circadian oscillator and have shown that this clock is essential for correct output regulation.     

Cytokinin affects circadian-clock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner

In higher plants, many developmental processes, such as photomorphogenesis and flowering, are coregulated by light and the phytohormone cytokinin. Interactions between light and cytokinin pathways are presumably mediated by common signaling intermediates. However, the molecular mechanism of these interactions remains unclear. Here, we report that cytokinin specifically induces the expression of the Arabidopsis circadian oscillator genes LATE ELONGATED HYPOCOTYL ( LHY ) and CIRCADIAN CLOCK-ASSOCIATED 1 ( CCA1 ) but represses the expression of TIMING OF CAB EXPRESSION 1 in a light-dependent manner. Consistent with these observations, cytokinin causes a shifted phase of the circadian clock. Mutant studies showed that the altered clock oscillation modulated by cytokinin is dependent on phytochrome B ( PHYB ) and Arabidopsis RESPONSE REGULATOR 4 ( ARR4 ). Whereas overexpression of LHY or CCA1 renders plants slightly more sensitive to cytokinin, phyB and a lhy/cca1 double mutant are less sensitive to the hormone. These results suggest that cytokinin affects the circadian clock oscillation in a PHYB - and ARR4 -dependent manner and that cytokinin signaling is also regulated by light-signaling components, including PHYB , LHY and CCA1 . Therefore, phyB, ARR4 and the circadian oscillator may function as signaling intermediates to integrate light and cytokinin pathways.     

Comparative overviews of clock-associated genes of Arabidopsis thaliana and Oryza sativa

In higher plants, circadian rhythms are highly relevant to a wide range of biological processes. To such circadian rhythms, the clock (oscillator) is central, and recent intensive studies on the model higher plant Arabidopsis thaliana have begun to shed light on the molecular mechanisms underlying the functions of the central clock. Such representative clock-associated genes of A. thaliana are the homologous CCA1 and LHY genes, and five PRR genes that belong to a small family of pseudo-response regulators including TOC1. Others are GI, ZTL, ELF3, ELF4, LUX/PCL1, etc. In this context, a simple question arose as to whether or not the molecular picture of the model Arabidopsis clock is conserved in other higher plants. Here we made an effort to answer the question with special reference to Oryza sativa, providing experimental evidence that this model monocot also has a set of highly conserved clock-associated genes, such as those designated as OsCCA1, OsPRR-series including OsTOC1/OsPRR1, OsZTLs, OsPCL1 as well as OsGI. These results will provide us with insight into the general roles of plant circadian clocks, such as those for the photoperiodic control of flowering time that has a strong impact on the reproduction and yield in many higher plants.     

Interaction between the Late Elongated hypocotyl (LHY) and Early flowering 3 (ELF3) genes in the Arabidopsis circadian clock

The circadian clock governs rhythms with 24 hours that allow organisms to anticipate daily changing environmental time cues. In Arabidopsis, the circadian clock is conceptually composed of three parts; input pathways for light and temperature signals, oscillators and output pathways for physiological processes including leaf movement, gene expression rhythms and flowering time. Oscillators consist of three interlocking loops, named morning, central and evening loops. Components of the central oscillator contain LHY, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION (TOC1). The oscillator can be reset by light signals through input pathways. Genetic studies have revealed the components involved in light input pathways. The elf3 (early flowering 3) mutant was isolated by insensitivity to photoperiod showing long hypocotyls, elongated petioles and pale leaves characteristic of plants defective in light perception. Therefore the ELF3 has been proposed to act on light input pathways. The aim of this study is to test whether LHY and ELF3 encode interacting components of a circadian light input pathway. To address this possibility, lhy-1 elf3-1 (LHY overexpressing-mutant X ELF3 loss of function-mutant) and lhy-11 elf3-1 (LHY loss of function-mutant X ELF3 loss of function-mutant) double mutants were constructed. Their visual phenotypes and CAB (Chlorophyll a/b binding protein) expression patterns demonstrate that LHY may function downstream of ELF3 and that this interaction is disrupted when LHY expression is placed under the control of the 35S promoter. In addition, ELF3 is required for vigorous rhythms of LHY gene expression and LHY protein levels.