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. 2008 Feb;20(2):307–319. doi: 10.1105/tpc.107.055715

FIONA1 Is Essential for Regulating Period Length in the Arabidopsis Circadian Clock[W]

Jeongsik Kim a, Yumi Kim a, Miji Yeom a, Jin-Hee Kim a, Hong Gil Nam a,b,1
PMCID: PMC2276451  PMID: 18281507

Abstract

In plants, the circadian clock controls daily physiological cycles as well as daylength-dependent developmental processes such as photoperiodic flowering and seedling growth. Here, we report that FIONA1 (FIO1) is a genetic regulator of period length in the Arabidopsis thaliana circadian clock. FIO1 was identified by screening for a mutation in daylength-dependent flowering. The mutation designated fio1-1 also affects daylength-dependent seedling growth. fio1-1 causes lengthening of the free-running circadian period of leaf movement and the transcription of various genes, including the central oscillators CIRCADIAN CLOCK-ASSOCIATED1, LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, and LUX ARRHYTHMO. However, period lengthening is not dependent upon environmental light or temperature conditions, which suggests that FIO1 is not a simple input component of the circadian system. Interestingly, fio1-1 exerts a clear effect on the period length of circadian rhythm but has little effect on its amplitude and robustness. FIO1 encodes a novel nuclear protein that is highly conserved throughout the kingdoms. We propose that FIO1 regulates period length in the Arabidopsis circadian clock in a close association with the central oscillator and that the circadian period can be controlled separately from amplitude and robustness.

INTRODUCTION

Most eukaryotes and some prokaryotes have evolved rhythmic behaviors with a period of ∼24 h as an adaptive response to the daily cycling of environmental conditions such as light and temperature. This circadian rhythm is an endogenous process that occurs once an organism is entrained by environmental factors, and the cycling rhythm continues even under constant conditions. At the daily level, the circadian rhythm is thought to serve as a mechanism for an organism to anticipate and prepare for the predicted regular cycle of daily environmental factors (Harmer et al., 2000). The necessity for this clock system is suggested by its conservation in most organisms (Young and Kay, 2001), and its importance is underscored by the discovery that coincidence between endogenous and environmental period increases the biological fitness in plants (Green et al., 2002; Dodd et al., 2005). Furthermore, the circadian clock is used to measure changes in daylength, in order to determine and synchronize flowering time (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002). Seedling growth is another developmental process under the control of the circadian clock, and mutations affecting clock function induce aberrant hypocotyl elongation under both diurnal and constant conditions (Dowson-Day and Millar, 1999; Nozue et al., 2007).

The circadian clock is composed of the following three components: input that entrains or synchronizes the clock to environmental cycles; a central oscillator that forms a self-regulating loop and generates self-sustained rhythms; and a final output of rhythmic biological processes. Genetic mutations have been utilized extensively for the identification and functional characterization of circadian components. In Arabidopsis thaliana, a reciprocal feedback loop between CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/ LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB EXPRESSION1 (TOC1) is believed to function as a central oscillator (Alabadi et al., 2001).

However, proper function of the circadian clock system in Arabidopsis requires a number of other elements, such as GIGANTEA (GI), EARLY FLOWERING3 (ELF3), ZEITLUPE, EARLY FLOWERING4 (ELF4), TEJ, SENSITIVITY TO RED LIGHT RED1 (SRR1), LUX ARRHYTHMO (LUX)/PHYTOCLOCK1, TIME FOR COFFEE, LIGHT-INSENSITIVE PERIOD1, and PSEUDORESPONSE REGULATOR9, -7, and -5 (PRR9, PRR7, and PRR5) (reviewed in McClung, 2006; Ding et al., 2007; Kevei et al., 2007). Mutations in these clock elements can affect the period length and/or amplitude of the circadian rhythms (Park et al., 1999; Somers et al., 2000; Hicks et al., 2001; Doyle et al., 2002; Panda et al., 2002; Staiger et al., 2003; Farre et al., 2005; Hazen et al., 2005; Nakamichi et al., 2005; Onai and Ishiura, 2005; Salome and McClung, 2005b; Ding et al., 2007; Kevei et al., 2007). Some of these elements appear to participate in feedback loops that are interconnected with the CCA1/LHY-TOC1 central oscillator loop (Gardner et al., 2006; Locke et al., 2006; Zeilinger et al., 2006). GI, ELF4, and LUX make parallel evening feedback loops with CCA1/LHY, like TOC1 (Hazen et al., 2005; Kikis et al., 2005; Locke et al., 2005; Onai and Ishiura, 2005). The PRR9, PRR7, and PRR5 circuitry was suggested to form an additional regulatory loop with the CCA1/LHY-TOC1 loop (Farre et al., 2005; Nakamichi et al., 2005; Salome and McClung, 2005b).

Despite such progress in elucidating the regulatory mechanisms underlying circadian systems in plants, our understanding remains very limited and requires the further identification and functional study of regulatory elements. Recent network modeling and computational simulations have indicated that the Arabidopsis circadian clock comprises multiple oscillators interconnected with several feedback loop networks, predicting the requirement of additional circadian elements (Locke et al., 2006; Zeilinger et al., 2006).

We have been searching for the genetic elements of the circadian system, taking advantage of photoperiodic flowering mutants (Park et al., 1999). Here, we have identified the novel clock component, FIONA1 (FIO1), which is closely associated with the central oscillator and is critical for maintaining the correct period length but is not necessary for maintaining the amplitude of circadian rhythm.

RESULTS

The fio1-1 Mutation Alters Daylength-Dependent Flowering and Hypocotyl Growth

Many Arabidopsis mutations with aberrant clock function contain a defect in the control of photoperiodic flowering, leading to early- or late-flowering phenotypes (Hicks et al., 1996; Suarez-Lopez et al., 2001; Doyle et al., 2002; Yanovsky and Kay, 2002). In order to identify new clock components, we screened an ethyl methanesulfonate–mutagenized seed pool of Arabidopsis for mutants with altered flowering time. The mutant that exhibited the earliest flowering timing was designated fiona1-1 (fiona means “flowering” in Korean) and was chosen for further characterization (Figure 1A). Flowering time is controlled by multiple pathways, including the autonomous, photoperiodic, and vernalization- and gibberellic acid–dependent pathways (Mouradov et al., 2002). Since the circadian clock is associated with photoperiodic flowering control, we examined fio1-1 seedlings for altered photoperiodic flowering responses by comparing the flowering time under long-day (LD; 16 h of light/8 h of dark; 16L/8D) and short-day (SD; 10 h light/14 h dark; 10L/14D) conditions. Under both LD and SD conditions, the fio1-1 mutants flowered earlier than wild-type plants and produced less than half of the leaves at the bolting stage (Figure 1B). This analysis showed that fio1-1 is one of the earliest flowering mutants to have been reported for Arabidopsis under LD conditions. Comparison between fio1-1 and wild-type plants revealed a difference in flowering time responses under SD and LD conditions. The SD:LD ratios of leaf numbers at the bolting stage were 5.8 and 4.7 for the wild type and fio1-1, respectively; the SD:LD ratios of time to bolting were 3.3 and 2.7, respectively (Figure 1B).

Figure 1.

Figure 1.

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The fio1-1 Mutation Causes Daylength-Dependent Phenotypes under LD and SD Conditions.

(A) Whole plant morphology of the wild type (Col) and fio1-1 grown for 28 d under LD (16L/8D) conditions. Bar = 1 cm.

(B) Flowering time of Col-0 and fio1-1 seedlings under LD (16L/8D) and SD (10L/14D) conditions. Flowering time was expressed as rosette leaf number and days to bolting (opening of the first flower). Data are means ± 95% confidence interval (95% CI) of 16 plants for each condition. The white and gray bars represent flowering time under LD and SD conditions, respectively.

(C) Seedlings after 7 d of growth under LD (16L/8D) and SD (8L/16D) conditions. Bar = 0.5 cm.

(D) Hypocotyl growth of Col, fio1-1, and elf3-1 seedlings under LD (16L/8D), SD (8L/16D), and LL (24L/0D) conditions. Data are means ± 95% confidence interval of 15 plants for each condition. The black, white, and gray bars represent hypocotyl length of Col, fio1-1, and elf3-1, respectively.

In Arabidopsis, hypocotyl growth is also dependent on photoperiod (Nozue et al., 2007). Thus, we compared the hypocotyl growth of wild-type and fio1-1 plants under constant light (LL; 24L/0D), LD (16L/8D), and SD (8L/16D) conditions to support our hypothesis that FIO1 affects the photoperiod response in Arabidopsis. The ratios of hypocotyl length between the wild type and fio1-1 were 1.1, 1.4, and 1.7 under LL, LD, and SD conditions, respectively (Figures 1C and 1D). Furthermore, 1.2 was the largest ratio in hypocotyl growth observed under LL at various fluence rates and represents a much smaller difference than found under LD or SD conditions (see Supplemental Figure 1 online). The fact that the ratio was significantly different in LD and SD, and that it was reduced in LL, suggests that fio1-1 affects photoperiodic hypocotyl growth rather than light signaling–dependent hypocotyl growth. The same trend was observed in the elf3-1 mutant reported to affect photoperiodic hypocotyl growth (Zagotta et al., 1996). Differences observed in photoperiod-dependent flowering time and hypocotyl growth of the fio1-1 mutant indicate that FIO1 is involved in the photoperiodic control of those phenotypes.

fio1-1 Increases the Expression of CONSTANS, a Key Regulator of the Photoperiodic Flowering Pathway

The photoperiodic flowering pathway is regulated specifically by CONSTANS (CO), and changes in CO expression or phase lead to alterations in photoperiodic flowering via regulation of the expression of its immediate target gene, FLOWERING LOCUS T (FT) (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002). To confirm that fio1-1 alters the photoperiodic flowering pathway, we examined the expression of CO and FT in fio1-1 and wild-type plants under LD (16L/8D) (Figures 2A and 2B) and SD (10L/14D) (Figures 2C and 2D) conditions. Relative to the wild type, the CO expression of fio1-1 seedlings was elevated under both LD and SD conditions, particularly during the dark period of the diurnal cycle (Figures 2A and 2C). In addition, the fio1-1 mutants exhibited elevated FT expression at all time points of the diurnal cycle under both LD and SD conditions (Figures 2B and 2D). These results indicate that the fio1-1 mutation affects the photoperiodic flowering pathway, which in turn suggests the possibility that FIO1 performs a circadian clock–associated function.

Figure 2.

Figure 2.

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The fio1-1 Mutation Enhances CO and FT Expression.

(A) and (B) Expression of CO (A) and FT (B) in the wild type (Col) and fio1-1 under LD (16L/8D) conditions.

(C) and (D) Expression of CO (C) and FT (D) in Col and fio1-1 under SD (10L/14D) conditions.

Ten-day-old seedlings were harvested at Zeitgeber time 1 (ZT1) and at 3-h intervals thereafter. Expression levels of CO and FT were determined by real-time PCR, and values were normalized against actin (ACT2) expression. Closed circles, Col-0; open circles, fio1-1. The white and black boxes at the bottom of each graph represent light and dark periods, respectively. Data are shown as means ± se from four ([A] and [B]) and six ([C] and [D]) independent experiments.

The fio1-1 Mutation Affects Period Lengths of Various Circadian Output Rhythms under Constant Light Conditions

Since changes in the photoperiodic response of fio1-1 suggest that it might exhibit a defect in circadian clock function, we examined circadian leaf movement, a well-established circadian rhythmic response in Arabidopsis (Hicks et al., 1996). Seedlings were entrained for 10 d under 12L/12D and then transferred to free-running conditions in LL. Consistent with previous reports (Park et al., 1999), wild-type plants exhibited a robust rhythmic movement, with a free-running period of 25.3 ± 0.1 h (Figure 3A). Under the same conditions, fio1-1 also exhibited a robust rhythmic movement, but with a free-running period of 27.8 ± 0.4 h, which is 2.5 h longer than in the wild type. We also assessed the robustness (integrity of rhythmicity) of the circadian rhythm in individual seedlings by measuring relative amplitude error (RAE) using fast Fourier transform nonlinear least square (FFT-NLLS) analysis (Figure 3B). In this assessment, the rhythmic pattern of each seedling was compared with its best-fit cosine curve, and the amplitude errors relative to the best fit were calculated; a smaller RAE indicates a more robust rhythm. For the leaf movement rhythm, fio1-1 seedlings exhibited RAE values of ∼0.2, which is similar to those of the wild type. Therefore, while period lengthening was clearly apparent in the traces from leaf movements, the movement itself was as persistent in the fio1-1 mutant as in the wild type (Figures 3A and 3B).

Figure 3.

Figure 3.

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The fio1-1 Mutation Lengthens the Free-Running Period of Circadian Output Rhythms under LL Conditions.

(A) and (B) Assay of circadian leaf movement under LL conditions. Seedlings were entrained for 10 d under a 12L/12D cycle and then transferred to LL (20 μmol·m−2·s−1 ). Leaf movement data (A) represent means ± se of at least 10 seedlings. Each value was normalized to the minimum length of each leaf during 0 to 144 h of free running. The white and gray regions indicate subjective light and dark periods, respectively. Period versus RAE (B) was calculated by the BRASS program. Period lengths are shown as partially variance-weighted periods, which were estimated from 24 to 144 h of free running using the BRASS program.

(C) to (F) Luminescence assays of CAB2 ([C] and [D]) and CCR2 ([E] and [F]) promoter activity in Col-0 and fio1-1 seedlings under LL conditions. Seedlings harboring CAB2:LUC ([C] and [D]) or CCR2:LUC ([E] and [F]) were entrained for 7 d under 12L/12D and then transferred to continuous white light (15 μmol·m−2·s−1 ). Data represent means ± se, which were normalized to the mean expression level during 0 to 96 h of free running for the CAB2:LUC (C) (n = 24) and CCR2:LUC (E) (n = 32) bioluminescence rhythms. RAEs were calculated with the BRASS program using the CAB2:LUC (D) and CCR2:LUC (F) bioluminescence rhythms. Period lengths are shown as partially variance-weighted periods, which were estimated from 24 to 96 h of free running using the BRASS program.

Luciferase (LUC) activity is a noninvasive method for monitoring clock function and integrity (Millar et al., 1995). We assayed the circadian expression patterns of CHLOROPHYLL a/b BINDING PROTEIN2 promoter:LUC (CAB2:LUC) and COLD- AND CIRCADIAN-REGULATED2 promoter:LUC (CCR2:LUC) in the wild type and fio1-1 (Figures 3C to 3F). CAB2:LUC expression peaked in the early morning, with period lengths of 23.9 ± 0.1 and 26.7 ± 0.2 h for the wild type and fio1-1, respectively (Figures 3C and 3D). CCR2:LUC expression peaked at dusk, with period lengths of 24.2 ± 0.1 and 27.2 ± 0.1 h for the wild type and fio1-1, respectively (Figures 3E and 3F). Thus, in fio1-1, the period for gene expression controlled by both promoters was lengthened by ∼3 h under LL conditions, which is consistent with the period lengthening observed in leaf movement rhythm. Similar robustness as that seen with leaf movement rhythms was also observed in both CAB2:LUC and CCR2:LUC luminescence assays (Figures 3D and 3F).

While the luminescence assay measures the promoter activities of the two genes, we also tested whether fio1-1 affects the period length of clock-controlled accumulation of the endogenous mRNAs. For this purpose, we performed RNA gel blot analysis for CAB2 and CCR2 (see Supplemental Figure 2 online). In the wild type, the two genes showed a clock-controlled accumulation pattern of endogenous mRNAs, as reported previously (Kreps and Simon, 1997). In fio1-1, it was apparent that the period length of the cyclic accumulation of mRNAs was lengthened for both genes. The result was consistent with the effect of the fio1-1 mutation on the promoter activities of the two genes (Figures 3C and 3E). Thus, fio1-1 affects the period of various output rhythms, including leaf movement, promoter activity, and mRNA accumulation, which suggests that FIO1 is an important component of the circadian clock and not merely a subordinate output.

FIO1 Controls Free-Running Circadian Rhythms in Continuous Darkness

Once entrained by a daily cycle of light and darkness, the Arabidopsis circadian clock exhibits a robust circadian rhythmic activity under continuous light (LL) or darkness (DD) (Millar and Kay, 1991). Some clock components play distinct roles under the two free-running conditions (Hicks et al., 1996; Covington et al., 2001; Mas et al., 2003; Hazen et al., 2005). The above data show that FIO1 is involved in the control of circadian rhythm in LL. We investigated whether or not FIO1 functions in DD by assaying circadian rhythmic expression of CCR2:LUC or CATALASE3 promoter:LUC (CAT3:LUC) in fio1-1 seedlings. Under DD, a longer circadian period was observed for both reporters in fio1-1. CCR2:LUC expression exhibited mean periods of 25.1 ± 0.1 and 27.1 ± 0.2 h in the wild type and fio1-1, respectively (Figures 4A and 4B). CAT3:LUC also demonstrated a longer period in fio1-1 (26.9 ± 0.4 h) than in the wild type (25.1 ± 0.4 h; see Supplemental Figure 3 online). Although the period difference between the mutant and the wild type was ∼0.5 to 1 h shorter in DD than in LL, the period length change was clearly observed in fio1-1 mutants in DD for both reporters. Also, the persistence of the circadian rhythm for fio1-1 mutants in DD was similar to that in LL, and they exhibited similar RAEs to wild-type plants (Figure 4B; see Supplemental Figure 3B online). These data show that FIO1 is involved in controlling circadian rhythm under both free-running DD and LL conditions. This further supports the suggestion that FIO1 plays an important role in circadian clock activity.

Figure 4.

Figure 4.

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fio1-1 Lengthens the Period of Free-Running Rhythm Irrespective of Ambient Light or Temperature Conditions.

(A) Luminescence of the CCR2:LUC reporter under continuous darkness (DD). Seedlings harboring CCR2:LUC were entrained for 7 d under a 12L/12D cycle and then transferred to DD. Data represent mean values ± se (n = 23), which were normalized to the mean expression level during 0 to 120 h of free running. The gray and dark gray regions indicate subjective light and dark periods, respectively.

(B) Period versus RAE plot of the luminescence rhythms of the CCR2:LUC reporter under DD. The plot was determined from the data in (A) using the BRASS software. Period lengths are shown as partially variance-weighted periods.

(C) and (D) FRC for free-running rhythms under a wide range of fluence rates in red (C) and blue (D) light. Seedlings containing CAB2:LUC were entrained for 7 d under a 12L/12D cycle and then transferred to continuous red or blue light at different fluence rates. The free-running period lengths represent partially variance-weighted periods ± se (n = 24 to 36), which were determined from expression levels during 24 to 96 h of free running under each light condition. At the lowest intensity of red and blue light, period calculations were performed on data collected from groups of four seedlings.

(E) Luminescence of CCR2:LUC in LL following temperature entrainment. Seedlings harboring CCR2:LUC were entrained for 7 d under 12-h high (22°C)/12-h low (12°C) temperature cycles and then transferred to constant temperature (22°C) under LL (15 μmol·m−2·s−1 ). Data represent mean values ± se (n = 22 to 24), which were normalized to the mean expression level during 0 to 96 h of free running. The hatched regions indicate subjective cold periods under LL conditions.

(F) Period versus RAE plot of the luminescence rhythms of the CCR2:LUC reporter in LL following temperature entrainment. The plot was determined from the data in (E) using the BRASS software. Period lengths are shown as partially variance-weighted periods, which were estimated from 24 to 96 h of free running.

(G) Temperature compensation under various ambient temperatures. Seedlings harboring CCR2:LUC were entrained as described in (E) and then transferred to each ambient temperature indicated. Free-running period lengths are shown as partially variance-weighted periods ± se (n = 24 to 36), which were estimated from 24 to 96 h of free running under each condition.

FIO1 Is Not an Input Component of the Circadian Clock

Since our results suggest that FIO1 is not a subordinate output of the circadian clock, we investigated whether it is as an input or a central oscillator–related component. The fluence rate response curve (FRC) is the change in circadian period length in response to fluence rates of monochromatic light (Somers et al., 1998a, 1998b). FRC is a well-established method for discriminating between input and central oscillator components. Mutations in light input components but not in central oscillator components alter the slope of the period response to fluence rates in FRC under a specific light (Somers et al., 2000; Kevei et al., 2007). We used the CAB2:LUC reporter to examine the effect of fio1-1 on the period length of free-running rhythms under different fluence rates of red and blue light.

Under red light conditions, the period of wild-type plants decreased gradually with increasing fluence rates. Although fio1-1 mutants exhibited longer periods than wild-type plants throughout the fluence rates tested, they showed a similar gradual decrease in period length (Figure 4C). Even under higher fluence rates of red light, the wild type and fio1-1 exhibited similar slopes in a graph of period response versus fluence rate (P > 0.15; see Supplemental Figure 4 online). Increasing the fluence of blue light also induced a shorter period in the wild type. However, over a wide range of blue light fluences, fio1-1 consistently exhibited a period that was 2 to 3 h longer than that of the wild type (Figure 4D). These findings suggest that it is unlikely that FIO1 is involved in the red or blue light–dependent input pathways of the circadian clock system.

The fio1-1 Mutation Affects the Period of a Temperature-Entrained Circadian Clock

Daily changes in ambient temperature provide important environmental cues for entrainment of the circadian clock (Figure 4E) (Salome and McClung, 2005a). We used CCR2:LUC to examine the effect of the fio1-1 mutation on a temperature-entrained circadian clock. Temperature entrainment of the circadian clock was unaffected by the fio1-1 mutation, which exhibited a robust circadian rhythm that was similar to that in the wild type (Figures 4E and 4F). However, the mutation caused period lengthening, with the wild type and fio1-1 exhibiting free-running periods of 23.9 ± 0.1 and 25.8 ± 0.1 h, respectively. This finding is similar to that observed in the light-entrained circadian rhythm, where period lengthening of 2 to 3 h was observed in the mutant (Figure 3). These data together show that FIO1 is involved in controlling the circadian period: whether it is entrained by light or temperature, whether it is in free-running conditions of continuous light (red or blue) or darkness, and whether the phase of the output rhythm is in the morning (CAB2) or in the evening (CCR2).

The fio1-1 Mutation Does Not Affect Temperature Compensation by the Circadian Clock

Although temperature is an important environmental signal for entraining the circadian clock, it exerts a limited influence on period length under free-running conditions, and this is termed temperature compensation (Pittendrigh, 1954; Rensing and Ruoff, 2002). Recently, some of the molecular mechanisms underlying this process were identified; GI and FLOWERING LOCUS C appear to contribute to the temperature compensation mechanism in Arabidopsis (Edwards et al., 2006; Gould et al., 2006). We examined whether or not the fio1-1 mutation exhibits any defects in its temperature compensation mechanism (Figure 4G). Wild-type Arabidopsis showed a period change of <2 h in the range of growing temperatures between 13 and 27°C. At the free-running ambient temperature of 13°C, fio1-1 exhibited a 2.1-h longer period than the wild type. This difference was consistently maintained up to the free-running ambient temperature of 27°C. These data demonstrate that the period lengthening effect observed in fio1-1 is not temperature-specific and that it is unlikely that FIO1 is involved in temperature compensation.

fio1-1 Affects the Period Length of Expression of Central Oscillator Genes

The above data support the suggestion that FIO1 is closely associated with the central oscillator. In Arabidopsis, the central circadian oscillator is thought to comprise the CCA1/LHY-TOC1 and -LUX negative feedback loops (Alabadi et al., 2001; Hazen et al., 2005; Onai and Ishiura, 2005). We tested the effect of fio1-1 on the expression patterns of these central oscillators. As shown in Figure 5, fio1-1 caused period lengthening of all four of these genes, CCA1, LHY, TOC1, and LUX. In addition, the fio1-1 mutation lengthened the expression period of GI, ELF4, PRR9, and PRR7, which function in other feedback loops that are interconnected with the central oscillator loop (see Supplemental Figure 5 online). Thus, FIO1 controls the period of the central oscillator, supporting the suggestion that FIO1 functions in the Arabidopsis circadian clock in close association with the central oscillator. Control of the period of the central oscillator by FIO1 may explain why the fio1-1 mutation affects period length in various levels of outputs.

Figure 5.

Figure 5.

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fio1-1 Affects the Period of Cyclic mRNA Accumulation for Central Oscillator Genes.

Wild-type (Col) and fio1-1 seedlings were entrained for 10 d in a 12L/12D cycle and then transferred to LL at ZT0. Plants were harvested for 3 d at 3-h intervals from ZT25. Transcript levels of CCA1 (A), LHY (B), TOC1 (C), and LUX (D) were estimated by real-time PCR and normalized against actin (ACT2) expression. The white and gray regions indicate subjective light and dark periods, respectively. Similar results were obtained in two independent experiments, one set of which is shown here.

FIO1 Encodes a Novel Nuclear Protein That is Highly Conserved across a Wide Variety of Organisms

fio1-1 is a single-locus, recessive nuclear mutation, and it was subjected to map-based cloning (see Supplemental Figure 6A online). Cleaved-amplified polymorphic sequence mapping located the FIO1 locus on the BAC clone F26H11. Sequencing of candidate genes on this clone identified a G-to-A substitution in the open reading frame of At2g21070 in the fio1-1 mutant (Figure 6A). To determine whether or not this single-nucleotide change was responsible for the mutant phenotype, we transformed fio1-1 plants with a full-length genomic DNA expression cassette containing the wild-type At2g21070 sequence. The cassette comprised a 2-kb upstream sequence, the putative protein coding region, and 0.5 kb of downstream sequence. The early-flowering phenotype of the fio1-1 mutant was rescued in multiple independent T1 transgenic lines (see Supplemental Figure 7A online), two of which (lines 1 and 5) were subjected to further examination. Flowering time under LD conditions and leaf movement rhythm were both completely restored in homozygous T3 progeny from these transgenic lines (see Supplemental Figures 7B to 7D online). This result confirms that a single mutation in the open reading frame of At2g21070 is responsible for both the circadian and flowering phenotypes of the fio1-1 mutant. Thus, At2g21070 was designated FIO1, and it contains eight exons that encode a protein of 483 amino acids (Figures 6A and 6B). Interestingly, the mutation in fio1-1 occurs at the splice acceptor site of the fourth exon. Analysis of FIO1 cDNA clones from the fio1-1 mutant revealed that an alternative splice site occurs 15 bp downstream of the original splice acceptor site (see Supplemental Figure 6B online). Use of this alternative site results in the deletion of five amino acids from the putative protein sequence of wild-type FIO1. Since the fio1-1 mutation is recessive and the presence of a wild-type copy completely restored the mutation phenotypes, fio1-1 is likely a loss-of-function or a reduced-function mutation.

Figure 6.

Figure 6.

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FIO1 Is a Nuclear Protein with a Highly Conserved DUF890 Domain.

(A) Genomic architecture for FIO1 and position of the mutation in fio1-1. The exons (black boxes) and 5′ or 3′ untranslated regions (white boxes) of FIO1 are shown. The fio1-1 mutation is in the splicing junction at the start of the fourth exon of At2g21070 and is indicated by an asterisk.

(B) Deduced amino acid sequence encoded by FIO1. The DUF890 domain is found among the methyltransferase superfamily and is underlined. The five amino acids deleted in fio1-1 are indicated by a box.

(C) Localization of FIO1-eGFP in Arabidopsis protoplasts. Images show bright-field, GFP, and red fluorescent protein (RFP) fluorescence, as well as a merged picture of Arabidopsis protoplasts transfected with CsVMVpro:FIO1-eGFP and CsVMVpro:H2B-RFP. H2B indicates histone 2B.

FIO1 appears to be a single-copy gene that encodes a novel protein. A database search of Arabidopsis sequences did not identify any related proteins. However, FIO1 homologs are found in many other organisms, from prokaryotes to eukaryotes (see Supplemental Figure 8 online), and they exhibit relatively high levels of conservation. Although their function remains unknown, a Pfam (for Protein families database of alignments and hidden Markov models) database search indicated that these FIO1-related proteins share a DUF890 domain (Figure 6B; see Supplemental Figure 8 online; http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF05971), which is found in a group of proteins belonging to the methyltransferase superfamily. FIO1 appears to be a nucleus-localized protein, because transfection of cassava vein mosaic virus (CsVMV) promoter: FIO1-eGFP (for green fluorescent protein) fusion constructs into Arabidopsis protoplast resulted in a green fluorescence signal in the nucleus (Figure 6C). The same fusion protein also localized in the nucleus of transgenic plants harboring the construct. The construct was biologically active, since it was able to complement the fio1-1 mutant phenotypes (see Supplemental Figure 9A online).

The FIO1 Transcript Level Exhibits neither Circadian nor Feedback Regulation

Clock components are often themselves regulated by the circadian clock at the transcriptional level and may also be controlled by feedback regulation (Harmer et al., 2000; Edwards et al., 2006). Since FIO1 controls the period length of a variety of circadian rhythms as a component closely related to the central oscillator function, we were interested in testing the possible circadian control or feedback regulation of FIO1 transcription. We first examined FIO1 transcript levels in wild-type plants under diurnal conditions. The result (see Supplemental Figure 10A online) showed that the FIO1 transcript level is neither diurnally regulated nor induced by light. Next, we examined transcript levels of FIO1 during free-running LL conditions, but again we found no detectable cycling pattern (see Supplemental Figure 10B online). We further examined FIO1 promoter activity in detail by monitoring LUC activity in transgenic plants harboring a fusion between the FIO1 promoter and the LUC reporter gene. In contrast with the CCR2 promoter, which exhibited a robust rhythm under both diurnal and free-running continuous light conditions, the FIO1 promoter showed only a minor, if any, circadian rhythmic pattern, based on its high RAE values (see Supplemental Figures 10C and 10D online). These results indicate that FIO1 expression is not noticeably regulated by the circadian clock in Arabidopsis.

To determine whether FIO1 expression is subject to feedback regulation, we examined transgenic plants that overexpress a FIO1-GFP fusion transcript. The FIO1-GFP fusion protein is functional in vivo, since it can complement the fio1-1 mutant phenotypes, although its ectopic overexpression induced no detectable changes in circadian clock activity (see Supplemental Figure 9 online). We observed no significant difference in the endogenous FIO1 transcript levels between the wild-type and transgenic lines, although the expression of FIO1-GFP was >100-fold greater than that of the endogenous gene (see Supplemental Figures 10E and 10F online). Furthermore, FIO1 expression was very similar in fio1-1 and wild-type plants (see Supplemental Figures 10A and 10B online). These data suggest that FIO1 expression is not subjected to feedback regulation.

DISCUSSION

FIO1 Is Closely Associated with the Central Oscillator of the Circadian Clock

Under constant light, the fio1-1 mutation caused lengthening of the free-running period of a variety of circadian output rhythms, including leaf movement, promoter activity of CAB2 and CCR2, and transcript levels of CAB2 and CCR2 (Figure 3). Although these outputs oscillate with different phases, their circadian periods were lengthened in a similar manner in fio1-1. The fio1-1 mutation also lengthened the circadian period for CCR2 and CAT3 promoter activity in constant darkness (Figures 4A and 4B; see Supplemental Figure 3 online). These results suggest that FIO1 is important for maintaining the correct free-running periods of several circadian outputs in Arabidopsis. In addition, the fio1-1 mutation caused lengthening of the expression period for genes in the central oscillator loop (CCA1, LHY, TOC1, and LUX) (Figure 5) as well as for genes in other feedback loops (GI, ELF4, PRR9, and PRR7) (see Supplemental Figure 5 online) that are interconnected with the central oscillator loop. These results imply that FIO1 is not just a simple output component but is either involved in the circadian input pathway or closely linked to the central oscillator.

FIO1 is more likely an element within or near the central oscillator, since the period lengthening effect of the fio1-1 mutation was not influenced greatly by the quality (red or blue) or quantity of the ambient light under parametric entrainment conditions (continuous light) or by the ambient temperature (Figure 4). Light input components in a circadian system can also be distinguished by a distinct phase shift response to a light pulse (nonparametric entrainment) (Covington et al., 2001). Since parametric and nonparametric entrainment share the same mechanisms in Arabidopsis (Devlin and Kay, 2001), the parametric assay we employed can sufficiently support our idea that FIO1 is not acting as a simple light input element for the circadian clock but rather functions as a central oscillator–associated component.

Nevertheless, FIO1 does not fulfill the classical criteria for a central oscillator, for the following reasons: it does not follow a circadian rhythm and is not subject to feedback regulation (see Supplemental Figure 10 online); ectopic overexpression of FIO1 causes no detectable effects on the circadian clock (see Supplemental Figure 9 online); and the fio1-1 mutation has only a minimal effect on the transcript levels of the central oscillator genes or other clock components (Figure 5; see Supplemental Figure 5 online).

FIO1 Controls the Period Length of Circadian Rhythm Rather Than Amplitude and Robustness

The characteristics of circadian rhythms include phase, period, amplitude, and robustness. Among these characteristics, period and amplitude are the two key factors that determine a cycle. The fio1-1 mutation affected one of these two factors, period length. However, it did not alter the amplitude of the circadian output rhythms, such as leaf movement rhythm, or CAB2:LUC and CCR2:LUC luminescence assays during free-running conditions (Figure 3). This phenotype is consistent with the finding that the fio1-1 mutation increases the period of central oscillator gene expression but has little effect on the amplitude of expression (Figure 5).

Robustness, another characteristic of circadian rhythm, was determined using RAE, a measure of variability within a rhythm that provides an indication of the regularity of a rhythmic pattern. Small (close to 0) and large (close to 1) RAEs indicate robust and weak rhythms, respectively. The robustness of wild-type Arabidopsis seedlings is high, with low RAE values. The fio1-1 mutants exhibited RAE values similar to those of the wild type (Figures 3B, 3D, 3F, 4B, and 4F). Thus, the fio1-1 mutation did not affect the robustness of the circadian rhythm. On the other hand, it is clear that the fio1-1 mutation exhibits a high genetic penetrance, since its RAE value was not greater than that of the wild type.

As a central oscillator–associated factor, it appears that FIO1 is involved primarily in controlling the circadian period and has very little influence on the amplitude and robustness of the circadian rhythm. This observation leads to the notion that at least in Arabidopsis, period and amplitude may be controlled by separate genetic elements. This idea is supported by a few previous findings. Mutations have been observed in the central oscillator genes CCA1 and LHY and in the central oscillator–associated gene TEJ, which exhibit clearly altered periods but which have little effect on the amplitude and robustness of output rhythms or on the expression of other central oscillator genes (Mizoguchi et al., 2002; Panda et al., 2002). An even more striking finding was a prr7 prr9 double mutant that displayed a very long period of up to 35 h but without any detectable alteration in circadian amplitude or the expression of central oscillator genes (Farre et al., 2005; Salome and McClung, 2005b).

The fio1-1 Mutation Affects Daylength-Dependent Phenotypes

Many Arabidopsis circadian rhythm mutants exhibit daylength-dependent phenotypes. Since the fio1-1 mutation affected both photoperiodic phenotypes and circadian rhythm, it is possible that FIO1 is another genetic element involved in integrating the two biological processes. fio1-1 exerted a more pronounced effect on daylength-dependent flowering under SD than LD. A similar daylength-dependent flowering response has been observed in period-altering circadian mutations such as srr1, tej, and toc1 (Strayer et al., 2000; Panda et al., 2002; Staiger et al., 2003). It is interesting that these mutants show no direct correlation between the lengths of their circadian periods and their daylength-dependent flowering phenotypes. srr1 and toc1 exhibit short periods, whereas tej and fio1-1 display long periods. However, all of these mutants show an early-flowering phenotype that is more pronounced in SD than LD.

The fio1-1 mutation exhibits altered CO expression, which indicates that FIO1 mediates the photoperiodic flowering pathway for the regulation of flowering time. FIO1 is also involved in the control of period length in the circadian clock. The daily circadian rhythm was suggested to be integrated with the daylength-dependent phenotypes through the external coincidence mechanism, by which the circadian clock controls photoperiodic flowering time through coincidence between the particular phase of CO expression and light period (Yanovsky and Kay, 2002). For example, the cca1, lhy, and toc1 mutants all display a short circadian period. Under SD conditions, they also exhibit a noticeable shift in the phase of CO expression into the light period, and these elevated CO levels in this period lead to early flowering (Mizoguchi et al., 2002, 2005; Yanovsky and Kay, 2002). Similar to the cca1, lhy, and toc1 mutants, fio1-1 exhibits early flowering, but it may control flowering time differently. The fio1-1 mutation causes a long period, but it does not appear to have an effect on the phase of CO transcription in LD and SD (Figures 2A and 2C).

Molecular Nature of FIO1

FIO1 encodes a nuclear protein that is highly conserved among a wide variety of organisms that exhibit circadian behavior, including plants, Caenorhabditis elegans, Drosophila, zebrafish, and mammals (see Supplemental Figure 8 online). This observation is consistent with the idea that parts of the circadian clock have been conserved throughout evolution (Young and Kay, 2001; Wijnen and Young, 2006). However, FIO1-related proteins have also been found in organisms that are not known to display circadian activity, including Escherichia coli and yeast (see Supplemental Figure 8 online) (Wijnen and Young, 2006). Furthermore, no FIO1-related proteins have been identified in Neurospora or cyanobacteria, which have well-described circadian behaviors. Thus, the FIO1-related proteins do not represent a universal element of circadian clocks. Most plant clock elements are unique to plants and are not found in other organisms. However, a few, such as FIO1, TEJ, and SRR1, are distributed widely across kingdoms (Panda et al., 2002; Staiger et al., 2003). Therefore, it will be interesting to determine whether or not the FIO1 homologs perform circadian clock functions in other organisms.

FIO1 and its homologs share a conserved DUF890 domain, which is found in the methyltransferase superfamily. In addition, a crystal structure (2H00) for the human homolog of FIO1 has been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The crystal structure indicates that the human homolog can bind S-adenosyl-l-homocysteine, a reaction product of methyl transfer from S-adenosyl-l-methionine (SAM). Thus, it is possible that FIO1 may function as an S-adenosyl-l-methionine–dependent methyltransferase.

The molecular structure of the human homolog of FIO1 suggests an intriguing possibility: that Arabidopsis FIO1 functions as a methyltransferase in the plant circadian system. Considering that protein methylation plays a critical role in many cellular processes (Loenen, 2006), it is highly plausible that this process is also involved in circadian regulation. The eventual identification of its target proteins may enable the elucidation of the exact function that FIO1 performs within the circadian clock.

METHODS

Plant Materials and Transgenics

fio1-1 is an early-flowering mutant that was isolated from 40,000 ethyl methanesulfonate–mutagenized M2 Arabidopsis thaliana ecotype Columbia (Col-0) plants and then backcrossed three times to the wild type. All experiments were performed using this line, and all of the transgenic plants and mutants were from the Col-0 background. The elf3-1 mutant was used in this study after the gl1-1 mutation in elf3-1 gl1-1 (CS3793; ABRC) was removed via a backcross to wild-type Col-0.

fio1-1 mutants carrying CAB2:LUC (Millar et al., 1995) and CCR2:LUC (Strayer et al., 2000) were selected from F2 segregating lines using derived cleaved-amplified polymorphic sequence mapping (Neff et al., 2002). Mapping was performed by PCR using the primers 5′-CTCAATTTGGTGACAATGTTGTTTTGTCTGCA-3′ and 5′-AACACCGAGTAGTACAGCTGGTTC-3′, followed by digestion with PstI; the fio1-1 mutants remained undigested.

Flowering Time and Hypocotyl Length Determination

For flowering time measurement, seed imbibition (4°C for 3 d) was followed by growth on soil (a 1:1:1 mix of vermiculite, perlite, and peat moss) in a controlled culture room under either LD conditions (16L/8D; 100 to 150 μmol·m−2·s−1; F48T12/CW/VHO; Philips) or SD conditions (10L/14D; 100 to 150 μmol·m−2·s−1; TLD32w/850RS; Philips). White light fluence rates were monitored using a basic quantum meter (BQM; Spectrum Technologies). Flowering time was determined as the number of days before the first flower opened and the number of rosette leaves at flowering. Results represent means ± 95% confidence interval from 16 plants.

For hypocotyl length measurement, seed imbibition (4°C for 3 d) was followed by growth for 7 d on half-strength Gamborg's B5 medium supplemented with 1% sucrose under LD, SD, or LL conditions (16L/8D, 8/16D, or 24L/0D, respectively; 80 to 120 μmol·m−2·s−1; TLD18w/865; Philips). Hypocotyl length was measured using the Scion Image Beta 4.0.2 program (Scion), and results represent means ± 95% confidence interval from 15 seedlings.

Luminescence and Leaf Movement Assays

To measure LUC activity, plants were entrained for 6 to 10 d with 12L/12D cycles under white light. Luminescence activity was measured as described previously (Michael and McClung, 2002; Somers et al., 2004) with minor modifications. Detailed methods are provided in the Supplemental Methods online. Luminescence images were recorded with low-light video imaging using a Peltier-cooled CCD slow-scan camera (Versarray; Roper Scientific). Image processing and quantification were performed using the MetaVue software program (Universal Imaging). Data were imported into the Biological Rhythms Analysis Software System (BRASS; available from http://www.amillar.org) and analyzed with the FFT-NLLS suite of programs, as described previously (Plautz et al., 1997; Somers et al., 2004). Period lengths are shown as partially variance-weighted periods ± se, which were estimated using bioluminescence data obtained from 24 to 96 h or from 24 to 120 h under free-running conditions. For the lowest intensities of red or blue light, period calculations were performed on data collected from groups of four seedlings, and results represent the partially variance-weighted periods ± se of between 24 and 48 plants.

For analysis of leaf movement rhythms, seedlings were entrained for 10 d on a 12L/12D cycle under white light and then transferred to continuous white light (20 to 25 μmol·m−2·s−1; FCL32SD/30; Byul-pyo). Images under LL were recorded hourly for 6 d using a USB-based camera system (KDC205; Kocom). Leaf movement was assessed by measuring the distance from tip to tip of the first and second leaves on a horizontal axis using Leaf Movement Analysis (LMA) software. Results represent means ± se of between 10 and 18 plants. LMA software that was written in Visual Basic 6.0 (Microsoft) is available upon request (nam@postech.ac.kr).

Analysis of Gene Expression

To analyze gene expression under diurnal conditions, seedlings were entrained for 10 d under each indicated photocycle and were collected at ZT1 and at 3-h intervals thereafter. ZT0 is defined as the beginning of the first light period following entrainment. For analysis of gene expression under the control of the circadian clock, wild-type and fio1-1 seedlings were entrained for 10 d under 12L/12D and then placed in continuous light. Samples were harvested at ZT25 and then every 3 h for 3 d.

For real-time PCR, total RNA was isolated from tissues using WelPrep (Join Bio-Innovation) and treated with DNase I (Ambion). Total RNA (0.75 μg) was reverse-transcribed in a 10-μL reaction using an oligo(dT15) primer and ImProm II reverse transcriptase (Promega). Following a 12-fold dilution, 3 μL of diluted cDNA was amplified by real-time PCR using SYBR Premix Extaq (Takara) and with an ABI 7300 real-time PCR system (Applied Biosystems). The following PCR conditions were used: 94°C for 2 min, followed by 40 cycles of 94°C for 15 s and 60°C for 34 s. We used previously described gene-specific primers for amplification of the following genes: CO, FT, CCA1, LHY, and TOC1 (Mockler et al., 2004); ACT2 (Hall et al., 2003); and LUX (Edwards et al., 2006). Fold changes in gene expression were calculated using the comparative CT method (Livak and Schmittgen, 2001), with normalization against ACT2 expression. Relative values of expression were determined against the maximum value of wild-type samples. Experiments were repeated at least twice.

Cellular Localization of FIO1

The CsVMVpro:FIO1-eGFP construct was prepared using attL × attR (LR) recombination (Gateway; Invitrogen). A FIO1 cDNA clone was generated by PCR using the primers 5′-GTCGACATGCGGAGTGGGAAGAAG-3′ and 5′-CTCGAGTACCGGCAAAATTTGGACT-3′, which resulted in removal of the termination codon. The FIO1 entry clone was prepared by digesting the FIO1 cDNA clone with XhoI and SalI and then ligating the FIO1 fragment into similarly digested pENTR 1A (Invitrogen). Correct construction of the clones was confirmed by sequencing. CsVMVpro:FIO1-eGFP was established by LR recombination reaction using the FIO1 entry clone and the Gateway version of pCsVMV-eGFP-N-999 according to the manufacturer's instructions (Verdaguer et al., 1998).

For transient expression in Arabidopsis, 6 × 104 mesophyll cell protoplasts were cotransfected with 40 μg of CsVMVpro:FIO1-eGFP and the control construct CsVMVpro:H2B-RFP, as described previously (Hwang and Sheen, 2001). Transfected protoplasts were incubated under dim white light conditions at 23°C for 12 to 16 h. GFP and RFP signals were observed by epifluorescence microscopy (AxioVert 200; Carl Zeiss), recorded using CoolSnap HQ (Roper Scientific), and pseudocolored with Photoshop 7.0 (Adobe Systems).

Accession Numbers

Sequence data for the genes (or proteins) described in this article can be found in the Arabidopsis Genome Initiative and GenBank/DDBJ/EMBL data libraries under the following accession numbers: FIO1 (At2g21070), CAB2 (At1g29920), CCR2 (At2g21660), CCA1 (At2g46830), LHY (At1g01060), TOC1 (At5g61380), LUX (At3g46640), CO (At5g15840), FT (At1g65480), ACT2 (At5g09810), H2B (At3g45980), and Hs_FIO1 (for human homolog of FIO1, CAD89999).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Fluence Rate Response Curve for Hypocotyl Elongation under Constant White Light.

  • Supplemental Figure 2. Clock-Controlled Expression of CAB2 and CCR2 under LL Conditions.

  • Supplemental Figure 3. CAT3:LUC Bioluminescence Rhythms in fio1-1 under DD Conditions.

  • Supplemental Figure 4. Fluence Rate Response Curve for Free-Running Rhythms under High Fluence Rates in Red Light.

  • Supplemental Figure 5. fio1-1 Affects the Period of Cyclic Expression for Clock-Controlled Genes, Including GI, ELF4, PRR9, and PRR7.

  • Supplemental Figure 6. Map-Based Cloning of FIO1.

  • Supplemental Figure 7. The FIO1 Gene Complements the fio1-1 Mutant Phenotypes.

  • Supplemental Figure 8. Multiple Alignment of FIO1 and Homologous Proteins.

  • Supplemental Figure 9. Circadian Rhythms in FIO1 Overexpression Lines.

  • Supplemental Figure 10. No Detectable Regulation of FIO1 mRNA Levels by the Clock or Feedback Inhibition.

  • Supplemental Table 1. List of PCR-Based Molecular Markers.

  • Supplemental Methods.

Supplementary Material

[Supplemental Data]
plntcell_tpc.107.055715_index.html (699B, html)

Acknowledgments

We acknowledge the assistance of the ABRC, which provided elf3-1 gl1-1. We thank Steve A. Kay for the pZPXomegaLuc+ vector and the CAB2:LUC and CCR2:LUC reporter lines, and C. Robertson McClung for the CAT3:LUC reporter line. We appreciate the contributions of Andrew J. Millar with respect to the BRASS program. We also thank K.H. Suh, B.H. Kim, and Y.S. Park for excellent technical assistance. This research was supported by a Korea Science and Engineering Foundation grant funded by the Korean government (Grant R15-2004-033-05002-0).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hong Gil Nam (nam@postech.ac.kr).

[W]

Online version contains Web-only data.

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