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.     

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.     

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.     

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.     

PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock

Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endogenous biological clocks to local time. Although much is known about entrainment of the Arabidopsis thaliana clock to photocycles, the determinants of thermoperception and entrainment to thermocycles are not known. The Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, including the clock component TIMING OF CAB EXPRESSION 1/PRR1, are related to bacterial, fungal, and plant response regulators but lack the conserved Asp that is normally phosphorylated by an upstream sensory kinase. Here, we show that two PRR family members, PRR7 and PRR9, are partially redundant; single prr7-3 or prr9-1 mutants exhibit modest period lengthening, but the prr7-3 prr9-1 double mutant shows dramatic and more than additive period lengthening in the light and becomes arrhythmic in constant darkness. The prr7-3 prr9-1 mutant fails both to maintain an oscillation after entrainment to thermocycles and to reset its clock in response to cold pulses and thus represents an important mutant strongly affected in temperature entrainment in higher plants. We conclude that PRR7 and PRR9 are critical components of a temperature-sensitive circadian system. PRR7 and PRR9 could function in temperature and light input pathways or they could represent elements of an oscillator necessary for the clock to respond to temperature signals.     

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.