Possible role of early flowering 3 (ELF3) in clock-dependent floral regulation by short vegetative phase (SVP) in Arabidopsis thaliana.

Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.

A loss-of-function mutation in the DWARF4/PETANKO5 gene enhances the late-flowering and semi-dwarf phenotypes of the Arabidopsis clock mutant lhy-12;cca1-101 under continuous light without affecting FLC expression.

The circadian clock plays important roles in the control of photoperiodic flowering in Arabidopsis. Mutations in the LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes (lhy;cca1) accelerate flowering under short days, whereas lhy;cca1 delays flowering under continuous light (LL). The lhy;cca1 mutant also exhibits short hypocotyls and petioles under LL. However, the molecular mechanisms underlying the regulation of both flowering time and organ lengths in the LHY/CCA1-dependent pathway are not fully understood. To address these questions, we performed EMS mutagenesis of the lhy-12;cca1-101 line and screened for mutations that enhance the lhy;cca1 phenotypes under LL. In this screen, we identified a novel allele of dwarf4 (dwf4) and named it petanko 5 (pta5). A similar level of enhancement of the delay in flowering was observed in these two dwf4 mutants when combined with the lhy;cca1 mutations. The lhy;cca1 and dwf4 mutations did not significantly affect the expression level of the floral repressor gene FLC under LL. Our results suggest that a defect in brassinosteroid (BR) signaling delayed flowering independent of the FLC expression level, at least in plants with the lhy;cca1 mutation grown under LL. The dwf4/pta5 mutation did not enhance the late-flowering phenotype of plants overexpressing SVP under LL, suggesting that SVP and BR function in a common pathway that controls flowering time. Our results suggest that the lhy;cca1 mutant exhibits delayed flowering due to both the BR signaling-dependent and -independent pathways under LL.

Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis.

The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.

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.

Arabidopsis clock-associated pseudo-response regulators PRR9, PRR7 and PRR5 coordinately and positively regulate flowering time through the canonical CONSTANS-dependent photoperiodic pathway.

Photoperiodism allows organisms to measure daylength, or external photoperiod, and to anticipate coming seasons. Daylength measurement requires the integration of light signal and temporal information by the circadian clock. In the long-day plant Arabidopsis thaliana, CONSTANS (CO) plays a crucial role in integrating the circadian rhythm and environmental light signals into the photoperiodic flowering pathway. Nevertheless, the molecular mechanism by which the circadian clock modulates the cyclic expression profile of CO is poorly understood. Here, we first showed that the clock-associated genes PSEUDO-RESPONSE REGULATOR (PRR) PRR9, PRR7 and PRR5 are involved in activation of CO expression during the daytime. Then, extensive genetic studies using CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) double mutants (cca1/lhy) and prr7/prr5 were conducted. The results suggested that PRR genes act coordinately in a manner parallel with and antagonistic to CCA/LHY, upstream of the canonical CO-FLOWERING LOCUS T (FT) photoperiodic flowering pathway. Finally, we provided evidence to propose a model, in which CCA1/LHY repress CO through GIGANTEA (GI), while PRR9, PRR7 and PRR5 activate CO predominantly by repressing CYCLING DOF FACTOR1 (CDF1) encoding a DNA-binding transcriptional repressor.

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.

Circadian rhythm of circumnutation in inflorescence stems of Arabidopsis.

We investigate the modulation of circumnutation in inflorescence stems of Arabidopsis to determine the circadian regulation of circumnutation. Under constant light conditions (LL), circumnutation speed in wild-type plants fluctuates, with the phase of the highest speed at subjective dawn; the period length is close to 24 h. toc1 appears to shorten the period and elf3 causes an arrhythmic phenotype in circumnutation speed in LL, suggesting that a common circadian clock may control both circumnutation speed and other circadian outputs. These results highlight for the first time a role for a circadian clock in the regulation of circumnutation based on genetic analysis of Arabidopsis.