RFI2, a RING-domain zinc finger protein, negatively regulates CONSTANS expression and photoperiodic flowering

The red and far-red light-absorbing phytochromes interact with the circadian clock, a central oscillator that sustains a 24-h period, to measure accurately seasonal changes in day-length and regulate the expression of several key flowering genes. The interactions and subsequent signalling steps upstream of the flowering genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT) remain largely unknown. We report here that a photomorphogenic mutant, red and far-red insensitive 2-1 ( rfi2-1), flowered early particularly under long days. The rfi2-1 mutation also enhanced the expression of CO and FT under day/night cycles or constant light. Both co-2 and gigantea-2 (gi-2) were epistatic to rfi2-1 in their flowering responses. The gi-2 mutation was also epistatic to the rfi2-1 mutation in the expression of CO and hypocotyl elongation. However, the rfi2-1 mutation did not affect the expression of GI, a gene that mediates between the circadian clock and the expression of CO. Like many other flowering genes, the expression of RFI2 oscillated under day/night cycles and was rhythmic under constant light. The amplitude of the rhythmic expression of RFI2 was significantly reduced in phyB-9 or lhy-20 plants, and was also affected by the gi-2 mutation. As previously reported, the gi-2 mutation affects the period length and amplitude of CCA1 and LHY expression, and GI may act through a feedback loop to maintain a proper circadian function. We propose a regulatory step in which RFI2 represses the expression of CO, whereas GI may maintain the proper expression of RFI2 through its positive action on the circadian clock. The regulatory step serves to tune the circadian outputs that control the expression of CO and photoperiodic flowering.     

Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis

Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light-dark cycles applied to the imbibed seed and its period was shortened in the toc1-1 mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily groth arrest is abolished by the early flowering 3 (elf3) mutation, suggesting that this defect may cause its long-hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild-type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts wide-spread control over plant physiology.     

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.     

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.     

CCA1 and ELF3 Interact in the control of hypocotyl length and flowering time in Arabidopsis

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.     

The F-box protein ZEITLUPE confers dosage-dependent control on the circadian clock, photomorphogenesis, and flowering time

As an F-box protein, ZEITLUPE (ZTL) is involved in targeting one or more substrates for ubiquitination and degradation via the proteasome. The initial characterization of ZTL suggested a function limited largely to the regulation of the circadian clock. Here, we show a considerably broader role for ZTL in the control of circadian period and photomorphogenesis. Using a ZTL-specific antibody, we quantitated and characterized a ZTL dosage series that ranges from a null mutation to a strong ZTL overexpressor. In the dark, ztl null mutations lengthen circadian period, and overexpression causes arrhythmicity, suggesting a more comprehensive role for this protein in the clock than previously suspected. In the light, circadian period becomes increasingly shorter at higher levels of ZTL, to the point of arrhythmicity. By contrast, hypocotyl length increases and flowering time is delayed in direct proportion to the level of ZTL. We propose a novel testable mechanism by which circadian period and amplitude may act together to gate phytochrome B-mediated suppression of hypocotyl. We also demonstrate that ZTL-dependent delay of flowering is mediated through decreases in CONSTANS and FLOWERING LOCUS T message levels, thus directly linking proteasome-dependent proteolysis to flowering.     

Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.     

GIGANTEA and EARLY FLOWERING 4 in Arabidopsis exhibit differential phase-specific genetic influences over a diurnal cycle

The endogenous circadian clock regulates many physiological processes related to plant survival and adaptability. GIGANTEA (GI), a clock-associated protein, contributes to the maintenance of circadian period length and amplitude, and also regulates flowering time and hypocotyl growth in response to day length. Similarly, EARLY FLOWERING 4 (ELF4), another clock regulator, also contributes to these processes. However, little is known about either the genetic or molecular interactions between GI and ELF4 in Arabidopsis. In this study, we investigated the genetic interactions between GI and ELF4 in the regulation of circadian clock-controlled outputs. Our mutant analysis shows that GI is epistatic to ELF4 in flowering time determination, while ELF4 is epistatic to GI in hypocotyl growth regulation. Moreover, GI and ELF4 have a synergistic or additive effect on endogenous clock regulation. Gene expression profiling of gi, elf4, and gi elf4 mutants further established that GI and ELF4 have differentially dominant influences on circadian physiological outputs at dusk and dawn, respectively. This phasing of GI and ELF4 influences provides a potential means to achieve diversity in the regulation of circadian physiological outputs, including flowering time and hypocotyl growth.     

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.     

GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation

Circadian clocks are widespread in nature. In higher plants, they confer a selective advantage, providing information regarding not only time of day but also time of year. Forward genetic screens in Arabidopsis (Arabidopsis thaliana) have led to the identification of many clock components, but the functions of most of these genes remain obscure. To identify both new constituents of the circadian clock and new alleles of known clock-associated genes, we performed a mutant screen. Using a clock-regulated luciferase reporter, we isolated new alleles of ZEITLUPE, LATE ELONGATED HYPOCOTYL, and GIGANTEA (GI). GI has previously been reported to function in red light signaling, central clock function, and flowering time regulation. Characterization of this and other GI alleles has helped us to further define GI function in the circadian system. We found that GI acts in photomorphogenic and circadian blue light signaling pathways and is differentially required for clock function in constant red versus blue light. Gene expression and epistasis analyses show that TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) expression is not solely dependent upon GI and that GI expression is only indirectly affected by TOC1, suggesting that GI acts both in series with and in parallel to TOC1 within the central circadian oscillator. Finally, we found that the GI-dependent promotion of CONSTANS expression and flowering is intact in a gi mutant with altered circadian regulation. Thus GI function in the regulation of a clock output can be biochemically separated from its role within the circadian clock.     

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.     

Flowering in time: genes controlling photoperiodic flowering in Arabidopsis.

Successful sexual reproduction in plants relies upon the strict coordination of flowering time with favourable seasons of the year. One of the most important seasonal cues for the model plant Arabidopsis thaliana (Arabidopsis) is day length. Genes influencing flowering time in Arabidopsis have been isolated, some of which are involved in the perception and signalling of day length. This review discusses recent progress that has been made in understanding how Arabidopsis integrates environmental and internal signals to ensure a sharp transition to flowering and new insights on the role of the circadian clock in controlling the expression of genes that promote flowering in response to day length.     

Circadian timekeeping during early Arabidopsis development

The circadian coordination of organismal biology with the local temporal environment has consequences for fitness that may become manifest early in development. We directly explored the development of the Arabidopsis (Arabidopsis thaliana) clock in germinating seedlings by monitoring expression of clock genes. Clock function is detected within 2 d of imbibition (hydration of the dried seed). Imbibition is sufficient to synchronize individuals in a population in the absence of entraining cycles of light-dark or temperature, although light-dark and temperature cycles accelerate the appearance of rhythmicity and improve synchrony among individuals. Oscillations seen during the first 2 d following imbibition are dependent on the clock genes LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, ZEITLUPE, GIGANTEA, PSEUDO-RESPONSE REGULATOR7 (PRR7), and PRR9, although later circadian oscillations develop in mutants defective in each of these genes. In contrast to circadian rhythmicity, which developed under all conditions, amplitude was the only circadian parameter that demonstrated a clear response to the light environment; clock amplitude is low in the dark and high in the light. A circadian clock entrainable by temperature cycles in germinating etiolated seedlings may synchronize the buried seedling with the local daily cycles before emergence from the soil and exposure to light.     

EFO1 and EFO2, encoding putative WD-domain proteins, have overlapping and distinct roles in the regulation of vegetative development and flowering of Arabidopsis

From screening a population of Arabidopsis overexpression lines, two Arabidopsis genes were identified, EFO1 (early flowering by overexpression 1) and EFO2, that confer early flowering when overexpressed. The two genes encode putative WD-domain proteins which share high sequence similarity and constitute a small subfamily. Interestingly, the efo2-1 loss-of-function mutant also flowered earlier in short days and slightly earlier in long days than the wild type, while no flowering-time or morphological differences were observed in efo1-1 relative to the wild type. In addition, the efo2-1 mutation perturbed hypocotyl elongation, leaf expansion and formation, and stem elongation. EFO1 and EFO2 are both regulated by the circadian clock. Expression and genetic analyses revealed that EFO2 suppresses flowering largely through the action of CONSTANS (CO) and flowering locus T (FT), suggesting that EFO2 is a negative regulator of photoperiodic flowering. The growth defects in efo2-1 were augmented in efo1 efo2, but the induction of FT in the double mutant was comparable to that in efo2-1. Thus, while EFO2 acts as a floral repressor, EFO1 may not be directly involved in flowering, but the two genes do have overlapping roles in regulating other developmental processes. EFO1 and EFO2 may function collectively to serve as one of the converging points where the signals of growth and flowering intersect.