Constitutive expression of CIR1 - Wiley Online Library

The Plant Journal (2007) 51, 512–525

doi: 10.1111/j.1365-313X.2007.03156.x

Constitutive expression of CIR1 (RVE2) affects several circadian-regulated processes and seed germination in Arabidopsis Xiangbo Zhang1,†, Yanhui Chen1,†, Zhi-Yong Wang2,3, Zhangliang Chen1,4, Hongya Gu1,4,* and Li-Jia Qu1,4,* National Laboratory for Protein Engineering and Plant Genetic Engineering, Peking–Yale Joint Research Center for Plant Molecular Genetics and AgroBiotechnology, College of Life Sciences, Peking University, Beijing 100871, China, 2 Department of Plant Biology, Carnegie Institution, Stanford, CA 94305, USA, 3 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China, and 4 The National Plant Gene Research Center (Beijing), Beijing 100101, China

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Received 9 February 2007; revised 4 April 2007; accepted 11 April 2007. * For correspondence (fax +86 10 6275 1841; email [email protected] or [email protected]). †These authors equally contribute to this work.

Summary Circadian clocks are endogenous auto-regulatory mechanisms that allow organisms, from bacteria to humans, to advantageously time a wide range of activities within 24 h environmental cycles. Here we report the identification and characterization of an MYB-related gene, designated Circadian 1 (CIR1), that is involved in circadian regulation in Arabidopsis. Expression of CIR1 is transiently induced by light and oscillates with a circadian rhythm. The rhythmic expression of CIR1 is controlled by the central oscillator. Constitutive expression of CIR1 resulted in a shorter period length for the rhythms of four central oscillator components, and much lower amplitude for the rhythms of central oscillator components CCA1 and LHY. Furthermore, CIR1 over-expression severely affected the circadian rhythms of its own RNA and those of the slave oscillator EPR1 and effector genes Lhcb and CAT3. Plants that constitutively expressed CIR1 displayed delayed flowering, longer hypocotyls and reduced seed germination in the dark. These results suggest that CIR1 is possibly part of a regulatory feedback loop that controls a subset of the circadian outputs and modulates the central oscillator. Keywords: MYB-related transcription factor, circadian rhythm, oscillator, hypocotyl elongation, seed germination.

Introduction Like other eukaryotes and many prokaryotes, plants have evolved an endogenous circadian clock to adapt to environmental changes associated with day/night cycles. The circadian clock generates self-sustained rhythms with a period of approximately 24 h. This allows an organism to anticipate the rhythmic changes in the natural environment and to synchronize their metabolic and physiological activities accordingly (Harmer et al., 2001). Circadian rhythms are entrained by environmental signals, such as light and temperature, but are sustained under constant conditions without external clues (McClung et al., 2002; Salome´ and McClung, 2004). The circadian clock is essential to the fitness of plants, because many developmental processes in plants 512

are regulated by circadian rhythms. Such processes include photoperiodic induction of flowering, rhythmic hypocotyl elongation, cotyledon and leaf movement, chloroplast movement and stomata opening (Barak et al., 2000; Dodd et al., 2005; Green et al., 2002; Harmer et al., 2000). It is believed that the circadian rhythm of gene expression is the basis for many of these metabolic and developmental processes. In addition to controlling multiple molecular and physiological responses, the circadian clock is itself a target of regulation by environmental response pathways. For instance, the circadian clock and light perception by photoreceptors are intimately related (Devlin and Kay, 2001). Light ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd

Circadian-related transcription factor CIR1 513 affects the period and amplitude of circadian rhythms (Johnson, 1994), whereas the circadian clock controls the activity of light signaling pathways (Millar and Kay, 1997; Roden and Carre, 2001). Conceptually, the circadian system consists of three main components: the input pathways that entrain the clock, the central oscillator (clock) that maintains the oscillation, and the output pathways that generate rhythms (McClung et al., 2002; Salome´ and McClung, 2004). However, to be biologically meaningful, the phase of the clock needs to be set by environmental cues, such as light and temperature, so that it is synchronized with the outside world and is set to local time. Therefore, the major function of the input pathways is to transduce environmental time-keeping signals to the oscillator. The output pathways provide links between the oscillator and the various biochemical and developmental pathways whose rhythms it controls (Barak et al., 2000; Hayama and Coupland, 2003; Strayer and Kay, 1999). Using the control of flowering by day length as an example, fluctuations in day length are recognized by plants to coordinate the initiation of flowering with changing seasons. The alteration of flowering time in response to day length (i.e. photoperiod) is mediated by complex interactions between environmental signals and the timekeeping mechanism associated with the circadian clock (Hayama and Coupland, 2003). Molecular analyses of circadian clocks in animals and cyanobacteria suggest that the oscillators include a group of positive and negative elements that form an auto-regulatory transcriptional/translational negative feedback loop (Dunlap, 1999; Harmer et al., 2001). In Arabidopsis, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) are proposed to encode molecular components of the circadian oscillator. The CCA1 gene and its closely related homolog LHY encode two transcription factors with single MYB (myeloblastosis gene family) repeats, and act redundantly in mediating the circadian rhythmicity of target genes (Alabadi et al., 2002; Mizoguchi et al., 2002; Schaffer et al., 1998; Wang and Tobin, 1998; Wang et al., 1997). The mRNA transcript levels of these genes oscillate in a similar pattern, peaking in the morning soon after dawn (Schaffer et al., 1998; Wang and Tobin, 1998). In addition, constitutive overexpression of either LHY or CCA1 disrupts many circadian rhythms, and loss of function of these two genes shortens the original circadian period by 2–3 h (Green and Tobin, 1999; Mizoguchi et al., 2002; Schaffer et al., 1998; Wang and Tobin, 1998). The TOC1 gene encodes a pseudo-response regulator (Makino et al., 2000; Strayer et al., 2000). The level of TOC1 mRNA peaks in the evening, and mutations in this gene result in a short-period phenotype (Mas et al., 2003; Millar et al., 1995; Somers et al., 1998; Strayer et al., 2000). Furthermore, the reciprocal regulation between CCA1, LHY and TOC1 provides a feedback loop that has been proposed to be essential for circadian rhythmicity in Arabidopsis, in which

TOC1 acts as a positive regulator of CCA1 and LHY, and CCA1 and LHY as negative regulators of TOC1 (Alabadi et al., 2001). This reciprocal control mechanism forms the basis of the central oscillator. Recently, a novel MYB protein EARLYPHYTOCHROME-RESPONSIVE 1 (EPR1), which is highly similar to CCA1 and LHY, has been characterized (Kuno et al., 2003). Constitutive expression of EPR1 delays flowering, and inhibits the circadian rhythms of Lhcb gene expression. It also feedback-inhibits the rhythmic expression of its own RNA, but has no effect on hypocotyl elongation or the central oscillator. The circadian expression of EPR1 requires the central oscillator, suggesting that EPR1 is probably a slave oscillator that contributes to the fine-tuning of output pathways (Kuno et al., 2003). More recently, another mutant, lux, has been identified, in which CCA1 and LHY are repressed and TOC1 is activated (Hazen et al., 2005). Further analysis has shown that LUX encodes an MYB domain protein that is necessary for the activation of CCA1 and LHY expression, suggesting that LUX is essential for circadian rhythms (Hazen et al., 2005). It has been proposed that expression of approximately 6% of Arabidopsis genes would maintain circadian oscillation under constant conditions (Harmer et al., 2000; Schaffer et al., 2001). The vast number of circadian-regulated genes in Arabidopsis reflects the vital role of circadian rhythms in plant growth and development. This is consistent with the fact that the expression of many genes oscillates rhythmically, including those involved in photosynthesis, light signaling, nitrogen assimilation, sulfur metabolism, and responses to cold and pathogens. In addition, many physiological processes are controlled by circadian rhythms, such as flowering, movement of cotyledons and leaves, and opening of stomata (Harmer et al., 2000). Expression of the genes involved in various physiological processes must be coordinated with the various environmental cues and developmental requirements. Therefore, the circadian rhythms created by the central oscillator composed of CCA1, LHY and TOC1 must be differentially modified. Cloning and characterization of EPR1 and LUX not only substantiate the circadian system in plants, but also raise the possibility that regulation of the central oscillator is much more complex than expected (Hazen et al., 2005; Kuno et al., 2003). A regulatory network consisting of many positive and negative feedback regulatory loops derived from the central oscillator has been proposed in fruit flies and mammals (Allada, 2003; Emery and Reppert, 2004; Harmer et al., 2001; Roenneberg and Merrow, 2003). Whether a similar regulatory network exists in plants, and how the circadian output pathways are fine-tuned, are questions that remain to be answered. Here, we report the cloning and characterization of an MYB-related gene, designated Circadian 1 (CIR1), that encodes a single MYB repeat protein. The expression of CIR1 is transiently induced by light and oscillates with a

ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 512–525

514 Xiangbo Zhang et al.

We have been studying the function of the MYB-related transcription factors in Arabidopsis (Chen et al., 2006), one of which, designated CIR1 (previously RVE2, Chaudhury et al., 1999), has been isolated and identified. The cDNA of CIR1 encodes a 287 amino acid protein with a single MYB domain that shows > 70% identity to the MYB domains of CCA1, LHY and EPR1 (Figure 1). However, unlike CCA1 and LHY, which share extensive similarity outside the MYB domain, CIR1 exhibited no significant sequence similarity with either the central oscillators CCA1 and LHY or the slave oscillator EPR1 in regions other than the MYB domain (Figure 1). Therefore, it would be interesting to investigate the specific role of CIR1 in the circadian system.

seedlings (Figure 2b), suggesting that CIR1 may be regulated by phytochromes. We also monitored CIR1 expression under different light/dark photoperiod conditions in wild-type Arabidopsis seedlings. The levels of CIR1 RNA exhibited a circadian oscillation under continuous light (LL) conditions in wildtype seedlings (Figure 2c). Peaks in RNA levels occurred at 44 and 60 h in LL, which is slightly earlier than those of CCA1 and LHY at around subjective dawn (Schaffer et al., 1998; Wang and Tobin, 1998). Similar circadian rhythmic expression patterns of CIR1 were also observed in continuous darkness (DD), with peaks at 8 and 36 h (Figure 2d). These data suggest that the expression of CIR1 is under circadian control. Furthermore, we found that CIR1 expression continued to oscillate strongly during light–dark cycles, with a peak at 20 h under both long-day (LD, 16 h light/8 h dark) and short-day (SD, 8 h light/16 h dark) conditions (Figure 2e). However, the main differences between LD and SD conditions occurred at dawn, i.e. the onset of light, when the CIR1 mRNA was more abundant under LD conditions. The level of CIR1 mRNA decreased more rapidly around 24 h under SD than LD conditions (Figure 2e). The oscillation of CIR1 changed with the photoperiod, i.e. higher levels of CIR1 maintained the correct coincidence with the light period under LD but not SD conditions (Figure 2e). This suggests an important role of CIR1 in the flowering process based on the external coincidence model of photoperiodic control of flowering (Hayama and Coupland, 2003; Yanovsky and Kay, 2002).

Expression of CIR1 is regulated by light and oscillates with a circadian rhythm

Rhythmic expression of CIR1 is regulated by the central oscillator, but not the slave oscillator EPR1

To examine whether the expression of CIR1 was induced by light, as is true for CCA1 (Wang and Tobin, 1998; Wang et al., 1997), we analyzed the level of CIR1 mRNA in etiolated seedlings after their transfer to continuous white light. Expression of CIR1 was quickly induced, with a peak after 1 h of white light treatment, and then declined rapidly over the following 8 h (Figure 2a). Moreover, CIR1 expression was also quickly induced by red light treatments in etiolated

To elucidate the role of CIR1 in the circadian system, we first examined the expression of CIR1 in CCA1 overexpression (CCA1-ox) plants (Wang and Tobin, 1998). The rhythmic expression of CIR1 was severely disrupted in CCA1-ox plants (Figure 3a), suggesting that the circadian rhythm of CIR1 expression is under the regulation of the central oscillator CCA1. We then examined CIR1 expression in the lhy-12 cca1-1 double mutant. We found that,

circadian rhythm controlled by the central oscillator CCA1. Constitutive expression of CIR1 inhibited rhythmic expression of the endogenous CIR1 gene, altered the period length of the central oscillator, reduced the amplitude of CCA1 and LHY, and severely affected the rhythm of the EPR1, Lhcb and CAT3 genes. Furthermore, over-expression of CIR1 delayed photoperiodic flowering, reduced the expression of CONSTANS (CO) and FLOWERING LOCUS T (FT), increased hypocotyl elongation, and inhibited seed germination in the dark. These results suggest that CIR1 is possibly part of a regulatory feedback loop that controls a subset of the circadian outputs and modulates the central oscillator. Results CIR1 encodes an MYB-related protein

Figure 1. Sequence analysis of the CIR1 cDNA. Sequence alignment of the predicted amino acid sequences of CIR1, CCA1, LHY and EPR1. The amino acid sequence of the MYB domain is underlined. Identical amino acid residues are in black boxes, asterisks represent the positions of conserved Trp and Ala residues in the MYB domain, and dashes indicate gaps.


Circadian-related transcription factor CIR1 515 Figure 2. Expression pattern analysis of CIR1 in light and the associated circadian rhythms. (a) CIR1 RNA is induced by white light in etiolated seedlings. Arabidopsis seedlings were grown in the dark for 6 days, transferred to continuous white light, and harvested at the specified time point after transfer. RNA samples (approximately 15 lg) were analyzed on gel blots by hybridization with the probes of CIR1 genes. (b) Time course of CIR1 RNA induction by red light. Six-day-old etiolated seedlings were given 1.5 min of red-light treatment, and then shifted to the dark. Samples were collected at the specified times after the start of the dark incubation. CIR1 RNA was analyzed by quantitative real-time PCR. Values shown are the means SD of three experiments. UBQ10 was used as an internal control. (c,d,e) Northern blot analysis on RNA extracted from wild-type seedlings entrained to 12 h light: 12 h dark photoperiod LD and then shifted at time zero into continuous light (c) or continuous dark (d), and from wild-type seedlings grown under LD and SD conditions (e). Time zero corresponds to normal dawn (c) or dusk (d). Quantification of CIR1 mRNA levels after normalization to the rRNA levels is also shown. These data are the mean of two replicate experiments, with error bars representing the range. The open and shaded bars represent light and dark photoperiods, respectively.

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under LL conditions, the transcripts of CIR1 cycled with only one earlier and broader peak at about 12 h, and no obvious regular oscillations of CIR1 were observed after 24 h (Figure 3b). These data indicate that the rhythmic expression of CIR1 is under the control of the central oscillator mediated by CCA1 and LHY. However, we found that rhythmic expression of CIR1 was not altered in EPR1over-expressing plants under LL conditions (Figure 3c). This suggests that, unlike CCA1, the slave oscillator EPR1 is not involved in the regulation of CIR1 rhythmic expression. Constitutive expression of CIR1 affects fine-tuning regulation of both the central oscillators and circadian output pathways To further investigate the role of CIR1 in the circadian system, we obtained one of the T-DNA insertion mutants of this gene (cir1, SALK_051843) from the Arabidopsis Biological

Resources Center for further analysis. The T-DNA is inserted in the 5th exon of the CIR1 gene (Supplementary Figure S1). No transcripts of CIR1 were detected in the cir1 mutant using RNA gel blots, suggesting that cir1 is a loss-of-function null mutant (Figure 4a). We also generated transgenic lines in which CIR1 was constitutively over-expressed under the control of the CaMV 35S promoter. Four homozygous lines with single-copy T-DNA insertions, i.e. CIR1-ox-7, CIR1-ox-10, CIR1-ox-55 and CIR1-ox-58, were obtained. Northern blots showed that CIR1-ox-10 had the highest expression level of CIR1, whereas CIR1-ox-7 had the lowest level (Figure 4b). We next tested whether loss-of-function or constitutive expression of CIR1 affected the circadian expression of central/slave oscillator components (i.e. CCA1, LHY, TOC1, LUX and EPR1) and downstream effectors (Lhcb and CAT3) (Millar and Kay, 1991; Zhong and McClung, 1996). We found that oscillations of the expression of these seven genes in cir1 were robust, with similar period and phase to those of


516 Xiangbo Zhang et al. (b)

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Figure 3. Expression of CIR1 in relation to other components in the circadian system. (a) CIR1 RNA levels in wild-type and CCA1-ox (line ox-38) plants after shifting from 12 h light:12 h dark photoperiods into constant light. (b) CIR1 RNA levels in Ler and lhy-12 cca1-1 plants after shifting from 12 h light:12 h dark photoperiods into constant light. (c) RT-PCR analysis in the left panel reveals the increased EPR1 level in the EPR1-ox line. UBQ10 was used as a positive internal control. The right panel shows CIR1 RNA levels in wild-type and EPR1-ox plants after shifting from 12 h light:12 h dark photoperiods into constant light. The open and shaded bars represent light and dark photoperiods, respectively. These data are the mean of two replicate experiments, with error bars representing the range.

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Figure 4. Identification of CIR1 loss-of-function mutant cir1 and over-expression lines. (a) Northern blot of cir1. RNA from wild-type and cir1 seedlings entrained in 16 h/8 h LD and then transferred to constant light was analyzed on gel blots by hybridization with a 32P-labelled CIR1 probe. (b) RNA gel-blot analysis of wild-type (WT) and four transgenic lines overexpressing CIR1. Each lane contains 15 lg of total RNA.)

wild-type plants (Supplementary Figure S2). In spite of this, the expression of CCA1, LHY, TOC1 and LUX was found to oscillate with a shorter period length in the CIR1-ox-10 plants than in wild-type plants, and showed no subsequent, overt oscillations after 72 h under LL conditions (Figure 5a). In particular, the peak levels of CCA1 and LHY mRNA were reduced by more than threefold in the CIR1-ox-10 plants, leading to much lower amplitudes of the oscillation for these two transcripts (Figure 5a). Moreover, no regular circadian rhythm of EPR1, Lhcb or CAT3 was observed in CIR1-ox-10, suggesting that the rhythmic expression of these genes was profoundly affected (Figure 5b,c). These data suggest that, under LL conditions, constitutive expression of CIR1 affects the normal function of both the feedback loop and the output pathways. We then tested whether there was an auto-regulated negative-feedback inhibition loop for CIR1 expression, as in other circadian genes (Dunlap, 1999). In contrast to the robust oscillation of CIR1 in wild-type plants, the endogenous CIR1 transcripts in CIR1-ox-10 remained at a constant low level at all time points tested (Figure 5d). This indicates that the rhythmicity of endogenous CIR1 expression is abolished by constitutive expression of CIR1. We therefore


Circadian-related transcription factor CIR1 517

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Figure 5. Effect of constitutive expression of CIR1 on other components in the circadian system. (a) Expression of the central oscillator components CCA1, LHY, TOC1 and LUX in wild-type seedlings (blue lines) and CIR1-ox-10 seedlings (red lines) entrained in 12:12 LD for 8 days and then transferred to continuous light. Quantification of the gel blots was normalized to the signal probed by UBQ10. The bar represents the light conditions. (b) Expression of downstream effector genes Lhcb and CAT3 in wild-type and CIR1-ox-10 lines. (c) Expression of slave oscillator EPR1 in wild-type and CIR1-ox-10 lines. (d) Gel-blot analysis of the endogenous CIR1 expression in wild-type and CIR1-ox-10 lines. The blot was hybridized with RNA probes containing the sequence of the CIR1 3¢ untranslated region, which is not included in the CIR1-ox transgene. Bars represent photoperiods. These data are the mean of three replicate experiments, with error bars representing the range.


518 Xiangbo Zhang et al. suggest that CIR1 negatively self-regulates its own expression, similar to the regulation patterns of other typical circadian oscillators (Kuno et al., 2003; Schaffer et al., 1998; Wang and Tobin, 1998). Alteration of CIR1 expression affects flowering time and/or hypocotyl elongation One of the developmental responses regulated by circadian rhythms is photoperiodic flowering (Simpson and Dean, 2002). We observed a slightly earlier-flowering phenotype in cir1 when measured by either days of bolting or leaf number at the time of bolting under LD conditions (Figure 6a), although the hypocotyl length of cir1 was similar to that of wild-type (data not shown). We then examined the expression of CO and FT in cir1 plants. We found that, although the rhythmic expression of CO was almost unaffected in cir1 compared with that of the wildtype, expression of FT increased in the cir1 background at almost every time point under LD conditions. This is

consistent with the slightly early-flowering phenotype of cir1 (Figure 6b). In contrast to cir1, the four CIR1-over-expressing lines showed obvious delayed-flowering and longer-hypocotyl phenotypes under LD conditions. These phenotypes were tightly correlated with the expression level of CIR1 (Figures 7a and 8a). We next analyzed expression of the genes known to control flowering time in the CIR1-ox-10 plants. The results showed that, in the CIR1-ox-10 plants, expression of CO and FT, two of the key genes in the photoperiod pathway, decreased at almost every time point under LD conditions (Figure 7b). This suggests that the delayed-flowering phenotype resulted from downregulation of CO and FT in the CIR1-ox lines. Further analysis with scanning electron microscopy (SEM) showed that the hypocotyl cells were approximately 2.5 times longer in CIR1-ox-10 than in wild-type plants grown under LD conditions. Therefore, altered cell elongation (rather than cell division) contributes to the changes in hypocotyl length (Figure 8b).

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Figure 6. Loss of function of CIR1 results in a slightly earlier-flowering phenotype. (a) Early-flowering phenotypes of cir1 (left panel) and flowering time assay (right panel). Asterisks represent statistically significant differences. (b) Expression levels of CO and FT transcripts in the wild-type and cir1 determined by quantitative real-time PCR. Expression levels were normalized against UBQ10 control. Data represent three independent trials. The open and closed boxes represent light and dark periods, respectively.


Circadian-related transcription factor CIR1 519 Figure 7. Constitutive expression of CIR1 resulted in late-flowering phenotype. (a) Late-flowering phenotypes of transgenic Arabidopsis over-expressing CIR1 (left panel) and flowering time assay (right panel). Asterisks represent statistically significant differences. (b) Expression levels of CO and FT transcripts in the wild-type and CIR1-ox-10 determined by quantitative real-time PCR. Expression levels were normalized against UBQ10 control. Data represent three independent trials. The open and closed boxes represent light and dark periods, respectively.

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Figure 8. Hypocotyl elongation assay on CIR1-ox seedlings. (a) Hypocotyl length comparison between wild-type and CIR1-ox seedlings under LD conditions. Average hypocotyl length (SD) was determined for seedlings grown for 6 days under LD 16:8 light cycles. (b) Scanning electron microscopy image (left panel) and cell length measurements (right panel) of the hypocotyl cells of 6-day-old CIR1-ox-10 seedlings under LD conditions. Bars = 100 lm. The error bar represents SE. Asterisks represent statistically significant differences.

Seed germination in the dark was severely affected by constitutive expression of CIR1 Light is one of the important factors that regulate seed germination (Neff et al., 2000; Sullivan and Deng, 2003). When performing hypocotyl assays, we noticed that CIR1ox seeds did not germinate well in the dark. The germination rate of CIR1-ox seeds was also negatively correlated with the expression level of CIR1. When kept in total darkness for 6 days, < 50% of the seeds from CIR1-ox lines germinated compared to 90% of wild-type seeds (Figure 9a). In CIR1-ox-10, the proportion of seeds that germinated was only about 10% (Figure 9a). We also examined the effect of continuous irradiation with red, farred, blue or white light on germination in these plants. Continuous irradiation with white and red light for 3 days

could primarily rescue the low-germination phenotype in the CIR1-ox lines, whereas continuous irradiation with blue light for 3 days could only partially rescue these phenotypes (Figure 9a). However, continuous irradiation with far-red light for 3 days had no effect on the rescue of germination (Figure 9a). These data suggest that constitutive expression of CIR1 may affect PhyB- and/or Cry-regulated seed germination. It has been proposed that light regulates seed germination by regulating gibberellin (GA) biosynthesis (GarciaMartinez and Gil, 2002; Kamiya and Garcia-Martinez, 1999). To determine whether the reduced germination of the CIR1-ox seeds in the dark was caused by reduced GA or increased abscisic acid (ABA) levels, we examined whether treatment with GA4 or ABA biosynthesis inhibitors could rescue germination of CIR1-ox plants in the dark. CIR1-ox


520 Xiangbo Zhang et al. Figure 9. Germination assay of CIR1-ox seeds. (a) Germination rates of various CIR1-ox seeds in complete darkness and under various light conditions. The light intensities were as follows: red light (R, 20 lmol m)2 sec)1), far-red light (FR, 15.5 lmol m)2 sec)1), blue light (BL, 16.2 lmol m)2 sec)1) and white light (WL, 30– 60 lmol m)2 sec)1). (b) Germination rates of various CIR1-ox seeds treated with various concentrations of GA4 and norflurazon (NF) in total darkness. For GA4 treatment, the control is 0.02% ethanol. For NF treatment, the control is 0.1% DMSO. (c) Germination rates of various CIR1-ox seeds treated with NF + GA4 in the dark. The concentrations of NF and GA4 were 100 and 10 lM, respectively. The control is 0.02% ethanol + 0.1% DMSO.

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seeds still had a much lower germination rate than wild-type seeds on GA4 medium, although GA4 increased the germination rates of both wild-type and CIR1-ox seeds in darkness (Figure 9b,c). This suggests that the poor germination of CIR1-ox seeds is only partially caused by reduced GA levels. Similarly, 100 lM of norflurazon (NF), an inhibitor of ABA biosynthesis, slightly increased the germination rate of both CIR1-ox and wild-type seeds in the dark (Figure 9b,c). Treatment with both GA4 and NF was up to 2.5 times more effective in improving the germination rate than treatment with each chemical alone, but failed to rescue the germination of CIR1-ox to the same level as wild-type seeds (Figure 9c). These data suggest that the low germination of CIR1-ox seeds is mainly due to alteration of a pathway for light regulation of seed germination. Discussion Like most organisms, plants have evolved an endogenous biological clock to coordinate their physiological and

developmental activities with changing environmental conditions. In this study, we demonstrate that CIR1 is possibly involved in regulating a subset of circadian clock output pathways and modulating the central oscillators, based on the following evidence. Firstly, the transcript level of CIR1 showed a circadian oscillation under continuous conditions. Secondly, constitutive expression of CIR1 abolished the circadian rhythm of its own RNA, indicating that CIR1 is part of a feedback loop that contributes to its own oscillation. Thirdly, the rhythmicity of CIR1 expression was abolished in CCA1-ox lines and severely affected in lhy-12 cca1-1 double mutants. In addition, we found a potential evening element (ATAATATCT) at positions -328 to -320 in the promoter region of CIR1 (data not shown). Taken together, these results suggest that CCA1 (and/or LHY) is possibly involved in the regulation of CIR1 oscillation, resulting in an eveningphased expression of CIR1. Fourthly, constitutive expression of CIR1 considerably reduced the amplitudes of the CCA1 and LHY oscillations, similar to the CCA1 and LHY


Circadian-related transcription factor CIR1 521 oscillations in toc1-2 (Alabadi et al., 2001). This suggests that oscillation of the central oscillators was also affected by the constitutive expression of CIR1. In terms of the effect on TOC1, constitutive expression of CIR1 shortened the period of TOC1 expression. This differed from the effect of constitutive expression of CCA1 or LHY, which altered the TOC1 transcript level to low and constant (Alabadi et al., 2001). The effect of CIR1 on CCA1/LHY and TOC1 is also different to that of the slave oscillator EPR1, which is controlled by but has no effect on the central oscillator components (Kuno et al., 2003). Constitutive expression of CIR1 severely altered the rhythmic expression of EPR1, whereas constitutive expression of EPR1 had no obvious effect on CIR1 expression. These results suggest that, like CCA1/LHY and TOC1, CIR1 is possibly involved in regulating EPR1 rhythmicity. Finally, the rhythmic expression of several downstream effector genes such as Lhcb, CAT3, CO and FT was also greatly affected in CIR1-ox lines. These data suggest that the normal function of the circadian system is severely affected in CIR1-ox plants. The fact that the expression of TOC1 was not upregulated in the CIR1-ox lines, a phenomenon that was also observed in the lhy-12 cca1-1 double mutant, also suggests that the central oscillator regulation loop is more complicated than we expected. Recently, a multiple-loop model of the circadian oscillator has been proposed. In this model, the plant clock consists of coupled morning and evening loops (Locke et al., 2005, 2006; Zeilinger et al., 2006). The fact that CIR1 over-expression has a greater effect on rhythmic expression of CCA1 and LHY than on that of TOC1 suggests that CIR1 might mainly influence the morning (CCA1/LHY) feedback loop of this model rather than the evening (TOC1) loop. Light signals perceived by photoreceptors entrain the circadian oscillator by regulating the expression of its components (Crosthwaite et al., 1995; Devlin and Kay, 2001; Fankhauser and Staiger, 2002; Zeng et al., 1997). The observation that CIR1 expression is quickly induced by light suggests that CIR1 is an early light-responsive gene. This is supported by the identification of a light-responsive element G-box motif (CACGTG) at positions -279 to -274 in the CIR1 promoter (relative to the putative translational start site) (data not shown). Hypocotyl elongation is regulated by the circadian clock (Dowson-Day and Millar, 1999; Thain et al., 2004). In wildtype Arabidopsis, the rate of hypocotyl elongation shows a circadian rhythm, with maximum elongation at subjective dusk and growth arrest at subjective dawn (Dowson-Day and Millar, 1999). In CCA1-ox plants, hypocotyls elongate constantly at a rate similar to the maximum rate of wild-type, with no obvious arrest at subjective dawn. The lack of rhythmic growth arrest is believed to be the cause of the long-hypocotyl phenotype of CCA1-ox (Thain et al., 2004). Similar to CCA1-ox plants (Wang and Tobin, 1998), seed-

lings constitutively expressing CIR1 have longer hypocotyls than wild-type seedlings, a phenotype that is associated with mutations that impair light transduction pathways or circadian clock function (Dowson-Day and Millar, 1999). Whether the longer-hypocotyl phenotype of CIR1-ox plants is caused by the same mechanism as in CCA1-ox lines is yet to be investigated. For many plant species, light is required for seed germination (Taiz and Zeiger, 1998). In most Arabidopsis ecotypes, seed germination is improved by red light treatment and inhibited by far-red light (Wang, 2005). Seeds of CIR1-ox germinated normally under white light but not in complete darkness, suggesting a possible disturbance of light-regulated germination in CIR1-ox plants. This low-germination phenotype was fully rescued by red light and partially rescued by blue light, but was not rescued by far-red light, consistent with an important role of phyB in regulating light-induced germination (Casal and Sanchez, 1998; Neff et al., 2000; Sullivan and Deng, 2003). Blue light could partially rescue the low-germination phenotype, suggesting that it may regulate germination directly or via interconnection with phytochromes (Ahmad and Cashmore, 1997; Casal, 2000; Casal and Boccalandro, 1995; Casal and Mazzella, 1998; Mas et al., 2000; Neff and Chory, 1998). A few downstream genes involved in phytochrome promotion of seed germination, e.g. PIL5 and HFR1, have been reported (Oh et al., 2004; Yang et al., 2003). Relationships between CIR1 and these genes have yet to be established. It is not clear whether CIR1’s function in seed germination is related to the circadian clock, but, given the intimate relationship between light signaling and the circadian clock, it is not surprising that one protein plays roles in both systems. Both GA and ABA have important roles in seed germination (Bentsink and Koornneef, 2002; Bewley, 1997; Lovegrove and Hooley, 2000). Light can facilitate germination by promoting GA biosynthesis (Garcia-Martinez and Gil, 2002; Kamiya and Garcia-Martinez, 1999). Therefore, it is possible that the germination defect in CIR1-ox plants is caused by altered levels of GA or ABA. However, we found that GA4 and NF only partially rescued the low-germination phenotype in the dark. In contrast, both red and white light were able to fully rescue the germination phenotype. Thus, CIR1-ox plants appear to have a light requirement for germination that cannot be bypassed by treatment with GA or inhibition of ABA. However, our previous study showed that the transcription of CIR1 is upregulated upon treatment with GA and ABA (Chen et al., 2006). Therefore, whether GA and ABA signaling pathways are involved in the germination phenotypes of the CIR1-ox plants needs to be further clarified. Moreover, as TOC1 has been identified as an ABI3-interacting protein (Kurup et al., 2000), whether there is an involvement of the circadian clock in GA and ABA signaling also deserves further investigation.


522 Xiangbo Zhang et al. Constitutive expression of CIR1 results in a similar phenotype in terms of flowering and hypocotyl elongation as the CCA1/LHY-over-expressing plants. A similar lateflowering phenotype was also displayed by EPR1-overexpressing plants. Taking the sequence similarity between these proteins into consideration, it is not surprising that these proteins have redundant functions. This is also supported by the weak-flowering phenotype of cir1, and the lack of obvious phenotypes in other circadian-regulated processes, such as CCG (circadian-controlled gene) expression and hypocotyl elongation. The mild effect of CCA1 or LHY loss of function on CCG expression and flowering control further confirmed the functional redundancy between these genes (Green and Tobin, 1999; Mizoguchi et al., 2002). Constitutive expression of EPR1 does not result in a longer-hypocotyl phenotype. In addition, constitutive expression of CIR1 has distinct functions in seed germination. Taken together, these results suggest that the modes of action of these proteins are at least partially different. Based on phylogenetic analysis of CCA1-like MYB-related proteins, CIR1 is clustered with CCA1, LHY and EPR1 in clade I, forming a monophyletic group supported by a high bootstrap value (Chen et al., 2006). In clade I, there also is another monophyletic group of genes, the function of which has not yet been reported (Supplementary Figure S3). It is most likely that, although the functions of these CCA1-like genes in clade I differentiated after they duplicated from a common ancestor, partial function redundancy remains among certain genes. The properties of these homologous genes that determine the master/peripheral/slave relationship, and the function specification between different oscillators remain to be elucidated in the future. Taken together, our study shows that constitutive expression of CIR1 affects a subset of clock outputs, including the photoperiodic flowering pathway, hypocotyl elongation and expression of the Lhcb and CAT3 genes. It also affects fine-tuning of the central oscillator’s rhythmicity, and mediates light-dependent seed germination. These results indicate that plant circadian clock consists of not only a central oscillator but also peripheral/ancillary and slave oscillators that regulate specific circadian rhythms. The sequence homology between components of the central, peripheral/ancillary and slave oscillators suggests that gene duplication has contributed to the expansion and sophistication of the plant circadian clock systems during evolution.

An aliquot of extracted RNA was treated with DNase I (Promega, http://www.promega.com/), and then reverse-transcribed in a 20 ll reaction volume using Superscript II RNase H- reverse transcriptase according to the manufacturer’s instructions (Invitrogen; http:// www.invitrogen.com/). The cDNA product was diluted to appropriate concentrations for real-time PCR assays. All reactions were performed in triplicate using a DNA Engine Opticon 2 for Continuous Fluorescence Detector (MJ Research Incorporated, http://www.mjr.com) as described by Guo et al. (2006). UBQ10 was used as the internal reference, and expression levels are normalized to the levels of the control.

Experimental procedures

Measurement of flowering time

Plant materials and growth conditions

Arabidopsis plants were grown under long-day (LD, 16 h light/8 h dark) or short-day (SD, 8 h light/16 h dark) conditions. The time of bolting was determined as the day when the plant had a bolt of 1 cm, and then the number of rosette and cauline leaves was

Arabidopsis thaliana (Columbia ecotype) was used for all experiments described unless otherwise indicated. Seed germination and

seedling growth were performed as described previously (Qin et al., 2005). Circadian-related experiments were carried out as described by Fan et al. (2005). Light-grown plants were grown at 22C in a growth chamber (Percival Scientific, CU36 L5, http://www.percivalscientific.com/) with a light intensity of 150–200 lmol m)2 sec)1. The colored-light growth chamber (Percival Scientific, E-30LED2/3) had intensities of 16.2 lmol m)2 sec)1 for blue light (470 nm), 15.5 lmol m)2 sec)1 for far-red light (730 nm) and 108.5 lmol m)2 sec)1 for red light (660 nm).

Isolation of CIR1 cDNA The full-length ORF of CIR1 was amplified by RT-PCR from wild-type Arabidopsis seedlings using primers 5¢-TCTTCTTCAGCTTCAGATTT-3¢ and 5¢-AACTTTGGAGTGATCTTACG-3¢. The PCR product was cloned into pGEM-T (Promega; http://www.promega.com/) and sequenced. This construct was designated pGEM-T-CIR1, and served as the template for subsequent cloning of CIR1 into other vectors.

Nuclear localization analysis A BglII site and a SpeI site were introduced into CIR1 in pGEM-T-CIR1 by PCR, using primers 5¢-AGATCTATGGCTATGCAGGAACGTTGTG-3¢ (BglII site underlined) and 5¢-ACTAGTTCACCA-CAAAGGATATGATAATTTTAC-3¢ (SpeI site underlined). The BglII and SpeI-digested PCR product was cloned into pRTL2-GFP for a nuclearlocalizationassayasdescribedpreviously(Yiet al.,2002).

RNA extraction and RNA gel-blot analysis For all experiments, we used whole seedlings grown on plates. RNA extraction and Northern blot analysis were carried out as described previously (Qu et al., 2003). Radioactivity images were visualized using a phosphorimager (Molecular Dynamics, http:// www.ump.com/mdynamic.html), and band intensities were quantified using IMAGEQUANT software (Molecular Dynamics, http://www.ump.com/mdynamic.html). Values were normalized to rRNA.

cDNA synthesis and quantitative real-time PCR


Circadian-related transcription factor CIR1 523 counted. Thirty to forty plants were measured, and the mean was calculated for each measurement.

Measurement of hypocotyl length Arabidopsis seedlings were grown on plates containing 1 · MS medium supplemented with 1% sucrose at 22C for 5 days with the appropriate light quality and fluence rate. Images of 5–10 seedlings were produced using an anatomic microscope (Leica MZ FL III, http://www.leica-microsystems.com/) before measurement of hypocotyl lengths using SPOT IMAGE software (Diagnostic Instruments Inc., http://www.diaginc.com).

Germination assay Seeds lots used in germination assays were harvested from plants grown under the same environmental conditions and stored dry for more than 2 months. For light-related seed germination assays, triplicates of 50 seeds for each line were surface-sterilized, plated on aqueous agar medium (0.6% agar) and kept at 4C for 24 h. The seeds were illuminated for 3 days with red, far-red, blue or white light at the intensities indicated above, and then incubated in darkness for 3 days. Radicle protrusion was used as the criterion for germination. For the GA and ABA-related seed germination assays, seeds were plated on aqueous agar medium with appropriate concentrations of GA4 or the ABA biosynthesis inhibitor NF, first at 4C for 24 h, and then at 22C for 6 days in the dark. The solvents used for the hormones (DMSO or ethanol) are used as mock treatment.

Preparation of hypocotyl samples for scanning electron microscopy Seedlings were fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde) at least for 24 h and then transferred to 95% ethanol overnight. The chosen specimens were dehydrated in the following ethanol and isoamyl acetate series: 95% ethanol, 100% ethanol, 75% ethanol + 25% isoamyl acetate, 50% ethanol + 50% isoamyl acetate, 25% ethanol + 75% isoamyl acetate, and 100% isoamyl acetate. The specimens were CO2-critical point-dried and coated with gold for scanning electron microscope observation.

Acknowledgements We thank Professor Elaine Tobin (University of California at Los Angeles, USA) for kindly providing CCA1-ox seeds, and Professor George Coupland (Max Planck Institute, Cologne, Germany) for lhy12 cca1-1 seeds. We thank Professor Xing Wang Deng (Yale University, New Haven, CT, USA), Professor Hongwei Guo (Peking University, Beijing, China) and Dr Hongwei Xue (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China) for helpful suggestions and valuable discussions on this manuscript. We also thank Professor M. Liu, L. Zhang and J. Wei (Peking University) for technical assistance. The work was supported by the National Natural Science Foundation of China (grant number 30625002), and the Excellent Young Teachers Program of Ministry of Education (to L.-J.Q.).

Supplementary Material The following supplementary material is available for this article online:

Figure S1. T-DNA insertion site in cir1. Figure S2. Effect of C1R1 loss-of-function on other components in the circadian system. Figure S3. Phylogenetic analysis for CCA1-like sequences. Table S1. Primer used in the real-time PCR and northern blot analysis.

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