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Plant Physiol. Jan 2007; 143(1): 473–486.
PMCID: PMC1761957

GIGANTEA Acts in Blue Light Signaling and Has Biochemically Separable Roles in Circadian Clock and Flowering Time Regulation1,[C][W][OA]

Abstract

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

Circadian clocks, found widely in nature, act to coordinate biological processes with rhythmic changes in the environment. Light input plays an important role in adjusting the clock's phase as well as maintaining its pace, allowing for perception of both daily and seasonal information (Aschoff, 1979). The central biological oscillator interprets this environmental information and transmits the signal to a diverse set of outputs. A substantial portion of the Arabidopsis (Arabidopsis thaliana) transcriptome is clock regulated, with estimates ranging from 2% to 36% of expressed genes (Harmer et al., 2000; Schaffer et al., 2001; Michael and McClung, 2003a; Edwards et al., 2006; M.F. Covington and S.L. Harmer, personal communication).

In the current model of the Arabidopsis oscillator, TIMING OF CHLOROPHYLL A/B BINDING PROTEIN/PSEUDO RESPONSE REGULATOR1 (TOC1/PRR1) is believed to act as part of a central negative feedback loop with CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY; Alabadí et al., 2001). TOC1 positively regulates expression of CCA1 and LHY by an unknown mechanism, and CCA1 and LHY in turn negatively regulate TOC1 expression. CCA1 and LHY are homologous myb-like transcription factors thought to directly repress TOC1 expression by interacting with the evening element motif in the TOC1 promoter (Alabadí et al., 2001; Harmer and Kay, 2005). This gives rise to an antiphasic pattern of expression, with CCA1 and LHY messages peaking near dawn (Schaffer et al., 1998; Wang and Tobin, 1998) and TOC1 near dusk (Strayer et al., 2000). Mutation or altered expression of any of these three genes leads to severe circadian phenotypes, supporting their central roles in the clock (Millar et al., 1995; Schaffer et al., 1998; Wang and Tobin, 1998; Green and Tobin, 1999; Alabadí et al., 2002; Mizoguchi et al., 2002; Más et al., 2003a).

This simple model, however, does not accommodate all published data (Salomé and McClung, 2004); further, computer modeling suggests this loop is insufficient to drive the rhythms observed in Arabidopsis (Locke et al., 2005). It seems likely that the plant clock consists of multiple interlocking feedback loops, as has been suggested for animal circadian oscillators (Emery and Reppert, 2004). Proposed components of other Arabidopsis circadian loops include the TOC1 homologs PRR3, 5, 7, and 9 (Mizuno, 2004; Farré et al., 2005); EARLY FLOWERING4 (Kikis et al., 2005); LUX ARRHYTHMO/PHYTOCLOCK1 (Hazen et al., 2005; Onai and Ishiura, 2005); and GIGANTEA (GI; Locke et al., 2005; Mizoguchi et al., 2005; Gould et al., 2006). Computer modeling suggests that GI in particular may play an important role in the central clock, acting in series with or parallel to TOC1 (Locke et al., 2005; Mizoguchi et al., 2005; Gould et al., 2006).

GI was originally discovered as a late-flowering mutant (Rédei, 1962; Koornneef et al., 1991; Araki and Komeda, 1993) and positively regulates expression of the flowering time genes CONSTANS (CO) and FLOWERING LOCUS T (FT; Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000; Suarez-Lopez et al., 2001), suggesting a function for GI in clock output. One allele, gi-100, was isolated through a screen for photomorphogenic mutants, suggesting GI may also function in light input pathways involving phytochrome (PHY; Huq et al., 2000). Recent experiments suggest that GI has separable roles in the circadian clock and flowering time regulation (Mizoguchi et al., 2005; Gould et al., 2006).

Although circadian rhythms persist in the absence of environmental cues, in a natural environment, the clock must be reset by signals such as changes in light or temperature to allow for seasonal time measurement as well as daily coordination. Light input to the clock is mediated by PHY and cryptochrome (CRY) photoreceptors (Somers et al., 1998a; Devlin and Kay, 2000). Light input may also be carried out by ZEITLUPE (ZTL), a member of a small family of proteins with a distinctive assortment of domains (Nelson et al., 2000; Somers et al., 2000; Schultz et al., 2001). These proteins each have an F-box domain, an N-terminal LOV domain, and a series of six C-terminal kelch repeats. This domain structure suggests that ZTL might be a light-regulated protein involved in the proteasome-dependent degradation of protein substrates (Somers et al., 2000). Indeed, ZTL has been shown to cause the dark-dependent degradation of TOC1 through the proteasome (Más et al., 2003b; Han et al., 2004).

Most of the above components were isolated through forward-genetic screens, as is true of most clock components discovered in other model systems (Young and Kay, 2001). To isolate new clock components and gain insight into known regulatory components, we screened plants mutagenized with ethyl methane sulfonate (EMS) for mutants exhibiting clock dysfunction in constant darkness. We found new alleles of GI, LHY, and ZTL. Characterization of the new short period GI allele, gi-200, revealed that GI acts in both red and blue light input to the clock and that its roles in these pathways are distinct. Expression analysis in gi-200 and a gi T-DNA allele, gi-201, revealed that these mutations had minor effects on TOC1 expression, suggesting that GI is not solely responsible for promotion of TOC1 expression. No significant changes in GI expression levels were observed in toc1-2 mutants; moreover, epistasis analysis between gi and toc1 and ztl mutants suggests that GI and TOC1 proteins may act in parallel pathways. Finally, unlike most gi mutants, we found that gi-200 maintains its ability to relay timing signals from the clock to the day length-dependent flowering pathway through CO and FT. Combined with its short-period phenotype, this causes normal flowering in long days (LD) but early flowering in noninductive short days (SD). Thus, GI plays multiple and, in at least some cases, biochemically separable, roles in clock input, the central oscillator, and clock output pathways.

RESULTS

Screening and Initial Characterization of Mutants

A successful screen for clock mutants with altered expression of a circadian-regulated promoter luciferase reporter (CAB2::LUC) had previously been performed in constant light conditions (LL; Millar et al., 1995). To complement this earlier screen, we assayed mutagenized seedlings in constant dark conditions (DD). We monitored luciferase activity rhythms in Columbia-0 (Col) plants that expressed luciferase under the control of the COLD-CIRCADIAN RHYTHM-RNA BINDING 2/GLYCINE-RICH RNA-BINDING PROTEIN 7 (CCR2/GRP7) promoter (CCR2::LUC; Strayer et al., 2000). In contrast to CAB2::LUC, rhythmic CCR2::LUC activity persisted in plants even after several weeks in DD (Strayer et al., 2000; Supplemental Fig. S1). Therefore, we mutagenized Col CCR2::LUC seedlings with EMS, screened 10,000 M2 plants for alterations in period length (Supplemental Fig. S2), and identified mutant lines for further study. Prior to further phenotypic analysis, confirmed mutants were backcrossed four to five times to the parental Col CCR2::LUC strain to remove extraneous EMS-induced mutations. Mutant M3 plants were also outcrossed to Landsberg erecta (Ler) to establish populations for simple sequence length polymorphic mapping (Lukowitz et al., 2000; Jander et al., 2002).

Isolation of Genes Responsible for Short- and Long-Period Phenotypes

The mapping population of a short-period line demonstrated strong linkage to the top of chromosome I. Because of the proximity to the known clock locus LHY and the similar short-period phenotype observed for lhy-20 (Michael et al., 2003b), we sequenced the LHY locus in the mutant and identified a nonsense mutation, C2136T, predicted to cause a stop codon 359 amino acids after the translation start site (Supplemental Fig. S3). If translated, this would result in a truncated protein containing the myb-like DNA-binding domain but missing the C-terminal half of the wild-type gene product. The possibility that this mutation was responsible for the short-period phenotype was confirmed by lack of complementation by lhy-20 (Supplemental Table S1), and we therefore designated the nonsense allele lhy-100. Like lhy-20, period length is shortened in lhy-100 in constant red or blue light as well as in the dark (Supplemental Table S2), and lhy-100 exhibits a normal period response to increased fluence (data not shown).

Five long-period mutants demonstrated strong linkage to the bottom of chromosome V near the ZTL locus. ZTL mutants have been previously reported to have a long-period phenotype both in LL conditions and DD (Somers et al., 2000, 2004), consistent with the phenotype of these alleles (Supplemental Table S2). Surprisingly, three of the five mutants (ztl-100, ztl-101, and ztl-104), all isolated from different pools of M2 seeds, had alterations at the same nucleotide within the ZTL locus, C1977T. This mutation is predicted to cause a Pro to Ser mutation at amino acid 383, a residue conserved within the ZTL kelch repeats. In ztl-102, we found that the corresponding conserved Pro within a different kelch repeat was predicted to be altered, causing a P331L mutation. The fifth allele, ztl-103, had a G to A transition at nucleotide 1,680, causing a premature stop codon 280 amino acids past the translation initiation site at the start of the kelch repeats. In addition, we have characterized ztl-105 (SALK-069091), which contains a T-DNA insertion located near the beginning of the kelch repeats (Supplemental Fig. S3). ztl-105 failed to complement each of the EMS alleles (Supplemental Table S1). ZTL protein can be detected at near normal levels in ztl-101 and ztl-102 but is undetectable in ztl-103 and ztl-105 (D. Somers, personal communication). As previously described for almost all ztl mutants (Somers et al., 2000, 2004; Kevei et al., 2006), the alleles we characterized exhibited a steeper fluence response curve in red light than wild type (data not shown), indicating that all our alleles have defects in red light signaling to the clock.

An additional short-period mutant (Table I; Fig. 1) mapped to a 63-kb region on chromosome I encompassing the GI locus. Because GI has previously been implicated in clock function (Fowler et al., 1999; Park et al., 1999; Mizoguchi et al., 2005; Gould et al., 2006), the GI locus was sequenced as the most likely candidate gene within the region. A mutation (G3704A) was found that is predicted to induce a Ser to Ala (S932A) amino acid change (Supplemental Fig. S3). This Ser residue is conserved across monocots and dicots (Edwards et al., 2005), suggesting it may play an important role in GI function. Because the short-period phenotype of this mutant was not complemented by the T-DNA allele gi-201 (SALK-092757; Supplemental Table S1), we designated this mutant gi-200 (Supplemental Fig. S3). The T-DNA insertion in gi-201 is predicted to be within the second GI exon; if translated, the gi-201 protein product would contain only 66 of the normal 1,174 amino acids in the GI protein (Supplemental Fig. S3). No GI mRNA downstream of the insertion site could be detected in gi-201 plants by quantitative reverse transcription-PCR (qRT-PCR; Fig. 5D; data not shown), suggesting this is likely to be a null allele. Gene expression phenotypes similar to those seen for CCR2::LUC activity were also observed when we examined CCR2 mRNA levels by qRT-PCR in these gi mutants (Fig. 1D).

Table I.
Phenotypes in different light conditions at 22°C
Figure 1.
CCR2 expression is altered in gi mutants. Plants were entrained for 8 d in 12-h-light/12-h-dark cycles at 22°C before release to LL at 22°C. A–C, Rhythmic CCR2::LUC expression was monitored in constant red light of 55 μ ...
Figure 5.
Expression of central clock components in constant white light. Plants were grown for 8 d in light/dark (12/12) cycles before release to constant white light of 55 μmol m−2 s−1. Tissue was collected for RNA extraction every 3 h. ...

GI Acts in Blue Light Signaling to the Clock

gi-200 plants exhibited a short-period CCR2::LUC expression phenotype under all light conditions tested (Table I; Fig. 1), similar to the gene expression phenotypes previously reported for gi-1 and gi-3 (Park et al., 1999; Mizoguchi et al., 2005): two alleles with premature stop codons near the 3′ end of GI (Fowler et al., 1999; Supplemental Fig. S3). Luciferase activity was greatly reduced in the T-DNA mutant gi-201 (Fig. 1A); however, nearly normal levels of CCR2 mRNA could be detected (Fig. 1D), indicating that CCR2 expression is not damped low in these plants and that luciferase activity is reduced through an unknown mechanism. For more accurate measurements, we monitored the luciferase activity of clusters of gi-201 plants and found that CCR2::LUC rhythms were lower amplitude, damped more rapidly, and had a slightly altered period relative to wild type (Fig. 1; Table I). These phenotypes are similar to gene expression phenotypes in the likely null alleles gi-2 and gi-11 (Park et al., 1999; Gould et al., 2006), further supporting gi-201 as a null allele.

Because it had previously been reported that gi-1 mutants displayed a low amplitude phenotype in constant white light (Fowler et al., 1999; Park et al., 1999; Sothern et al., 2002; Tseng et al., 2004; Mizoguchi et al., 2005) but not in DD (Park et al., 1999), whereas gi-3 rhythms are low amplitude in both conditions (Mizoguchi et al., 2005), we examined the light dependence of the gi-200 phenotype. Similar to previous reports for gi-1 (Park et al., 1999), the severity of the period and amplitude phenotypes of gi-200 plants was reduced when they were maintained in DD (Table I; data not shown); however, expression of CCR2::LUC still cycled with a short period.

To investigate the previous reports of variability in period phenotypes in gi-2 mutants (Park et al., 1999), we examined rhythmic CCR2::LUC expression in a variety of light conditions. In constant red light, the rhythmic amplitude of CCR2::LUC activity in gi-200 plants was significantly lower than that of wild type (P = 0.0013; Fig. 2A); however, in constant blue light, there was no difference in amplitude (P = 0.72; Fig. 2B). Interestingly, when held in red plus blue light, allowing for light perception through multiple pathways, gi-200 rhythms had an increased amplitude when compared to Col (P = 0.0013; Fig. 2C). Thus, the gi-200 clock phenotype is wavelength dependent, suggesting GI acts in both red and blue light input pathways to the clock and that these pathways may be differentially compromised by the gi-200 mutation. In contrast, the gi-201 T-DNA mutants displayed very low-amplitude rhythms that damped rapidly in constant red, constant blue, or the combination of constant red and blue light (Fig. 1). Interestingly, gi-201 displayed a slightly long-period phenotype in constant red light, but had a short period in constant blue light or the combination of red and blue light (Table I), further supporting functionally distinct roles for GI in blue and red light signaling.

Figure 2.
Amplitude of CCR2::LUC expression varies in gi-200 in different light conditions. Plants were entrained as described in Figure 1 and then released to constant red, blue, or red plus blue light. Luciferase activity was monitored in constant red ...

Although the circadian clock does not require light input to function, higher fluences of light cause the Arabidopsis clock to run with a shorter period, as is true for many other organisms (Aschoff, 1979). Because gi-1 was reported to demonstrate a lack of response to increasing red light (Park et al., 1999), we determined whether light input to the circadian clock was altered in the gi-200 mutant by examining the free-running period under various intensities of light. As previously reported (Somers et al., 1998a; Devlin and Kay, 2000), higher fluences of monochromatic red or blue light caused the clock to run with a shorter period in wild-type plants (Fig. 3). In contrast, there was no statistical difference (by one-way ANOVA or Student's t test) in the period length of gi-200 plants when grown in constant red, blue, or red plus blue light across a wide fluence range (Fig. 3). Additionally, a significant interaction between genotype and fluence could be detected (P < 0.05 in red and P < 0.01 in both blue and red plus blue; ANOVA for general linear fixed-effects model), indicating the response to both red, blue, and the combination of red plus blue is altered in gi-200 relative to wild type. Our study extends the role of GI beyond red light signaling to indicate a function for GI in blue light input to the clock.

Figure 3.
Fluence response of rhythmic CCR2::LUC expression is altered in gi-200. Plants were entrained as described in Figure 1 and then luciferase activity was monitored in plants transferred to continuous red (n = 11–12; A), blue (n = ...

It has previously been reported that gi-1, gi-2, and gi-100 exhibit elongated hypocotyls in red light at all fluences tested and that gi-3 plants are tall when grown in blue light at elevated temperature (Huq et al., 2000; Paltiel et al., 2006). To further investigate the role of GI in photomorphogenesis, we examined hypocotyl elongation in many GI alleles under LL of a variety of wavelengths and fluences. All alleles tested, in both the Col and Ler backgrounds, had significantly elongated hypocotyls (P < 0.02 by Student's t test) when grown in constant white light or constant monochromatic red or blue light (Fig. 4) but were not significantly different from wild type when grown in DD (data not shown). The degree of significance varied across a wide range of fluences, but the same trend of long hypocotyls was observed in all wavelengths of LL examined (Supplemental Fig. S4). This tall hypocotyl phenotype of these six GI alleles in blue light further supports the role for GI in blue light signaling.

Figure 4.
The hypocotyl elongation response to many light conditions is altered in gi mutants. Plants were grown in constant white light of 4.3 μmol m−2 s−1, constant red light of 45 μmol m−2 s−1, or constant blue ...

Complex Interaction between GI and TOC1

Both GI and TOC1 have been suggested to function positively in regulation of CCA1 and LHY transcription (Fowler et al., 1999; Park et al., 1999; Alabadí et al., 2001; Mizoguchi et al., 2005). We therefore examined expression of CCA1 and LHY in gi mutants. Both expression levels and rhythmic amplitude of cycling were reduced for CCA1 and LHY in gi-201 and gi-200 plants, although the gi-200 phenotypes were less severe (Fig. 5, A and B). The short-period phenotype seen for CCR2::LUC and CCR2 mRNA rhythms (Fig. 1) is also apparent for CCA1 and LHY expression (Fig. 5, A and B) in gi-200.

Two models have recently placed GI within the central clock oscillator, one suggesting GI promotes TOC1 expression and thus acts in series with TOC1 (Locke et al., 2005) and the other suggesting that GI and TOC1 act in parallel pathways (Mizoguchi et al., 2005). To help clarify this issue, we examined TOC1 expression in gi mutants and GI expression in toc1 mutants. TOC1 expression levels were similar in gi-200 and wild-type plants but had a shorter period in gi-200 (Fig. 5C), consistent with the observed pattern of CCA1 and LHY expression (Fig. 5, A and B). Interestingly, TOC1 message was also easily detected in the putative null allele gi-201, with mRNA levels damping toward the median level of wild-type expression rather than to trough levels as would be expected if GI were the only factor promoting TOC1 expression (Fig. 5C). Therefore, factors other than GI must positively regulate TOC1 gene regulation. However, these observations are consistent with the possibility that GI normally provides a portion of TOC1-activating activity (Locke et al., 2005; Supplemental Fig. S5, A and B).

We also examined GI expression in the strong loss-of-function allele toc1-2. Because it has previously been reported that overexpression of TOC1 causes GI message levels to damp to low levels (Makino et al., 2002), it was of interest to observe that GI message levels were very similar to wild type in toc1-2 mutants, although they revealed the expected short-period phenotype (Fig. 5D). A recent model has suggested that TOC1 negatively regulates GI expression, leading to the prediction that GI levels would damp high in a toc1 loss-of-function mutant (Locke et al., 2005; Supplemental Fig. S5D). In contrast, our data suggest that TOC1 does not directly regulate GI expression levels.

To further examine the relationship between TOC1 and GI, we generated double gi-200 toc1-2 mutants and compared their phenotypes to those of the single mutants. We observed that in both monochromatic red and blue light, the short-period phenotypes of gi-200 and toc1-2 were nearly additive in the double mutant (Fig. 6, A and B; Table I). Similarly, the tall hypocotyl phenotype seen in both single mutants in constant red light was more exaggerated in the double mutant (data not shown). Furthermore, gi-201 toc1-2 mutants are largely sterile, a phenotype not present in either single mutant (data not shown). Thus, a number of phenotypes are more extreme in the double mutant than in either single mutant, suggesting the two proteins have parallel functions rather than acting solely in series with each other.

Figure 6.
CCR2::LUC expression in gi-200 toc1-2 (A and B) and gi-200 ztl-105 (C and D) double mutants. Plants were entrained as described in Figure 1 and then monitored in constant red light of 55 μmol m−2 s−1 (n = 33–48; ...

TOC1 is targeted for degradation by the F-box protein ZTL; in ztl mutants, there is a sustained accumulation of TOC1 protein and a consequent increase in free-running period length (Más et al., 2003b). Consistent with the regulated degradation of TOC1 being an important function of ZTL, the short-period phenotype of a toc1 loss-of-function allele is epistatic to the long-period ztl-1 phenotype in plants mutant for both genes (Más et al., 2003b). We generated gi-200 ztl-105 double mutants to further explore the relationship between GI, TOC1, and ZTL. ztl-105 is a T-DNA insertion allele (SALK-069091) that produces no detectable ZTL message by northern blot or RT-PCR (data not shown) nor any detectable ZTL protein (D. Somers, personal communication). The free-running period estimates for gi-200 ztl-105 double mutants held in constant red or blue light were intermediate between the estimates for either single mutant but were more similar to ztl-105 (Fig. 6, C and D; Table I). Additionally, we found that the hypocotyl lengths of the double mutants in constant red light were slightly shorter than Col but taller than ztl-105 (data not shown), again demonstrating an additive rather than epistatic effect of the two mutations. Thus, unlike TOC1, a GI allele is not epistatic to ZTL, further suggesting that TOC1 and GI may act in parallel.

GI Functions in the Central Clock and in Regulation of a Clock Output Are Biochemically Separable

Previously characterized lesions in GI cause a near complete loss of photoperiodism, resulting in late flowering in inductive photoperiods (Rédei, 1962; Koornneef et al., 1991; Araki and Komeda, 1993; Park et al., 1999; Huq et al., 2000). Consistent with these results, the T-DNA allele gi-201 flowered late in LD but with the normal number of leaves in SD (Fig. 7A). But to our surprise, we found that under inductive photoperiods, gi-200 flowered normally and actually flowered earlier than wild type under SD (Fig. 7A). Therefore, although gi-200 mutants have a similar clock phenotype to the previously characterized gi-1 and gi-3 alleles, gi-200 plants completely lack the late-flowering phenotype that gave GI its name.

Figure 7.
gi-200 retains the ability to stimulate flowering through CO and FT. A, Leaf number at bolting in LD and SD; *, the leaf number is significantly different from the appropriate wild-type control (P < 0.05 [n = 15–18]). B, ...

The early flowering phenotype in gi-200 plants is reminiscent of that seen in the short-period mutant toc1-1, which is caused by the inappropriately early phase of CO expression and the consequent up-regulation of FT expression in SD (Yanovsky and Kay, 2002). Hypothesizing that the short-period phenotypes of gi-200 and toc1-2 might have a similar effect on the phase of CO expression, we used qRT-PCR to examine CO message in these plants. gi-200 and toc1-2 displayed near wild-type levels of CO transcript (Fig. 7B), whereas CO levels were decreased in gi-201, similar to previous reports for the late-flowering mutant gi-3 (Suarez-Lopez et al., 2001). Notably, CO expression was detectable late in the day in gi-200 and toc1-2 mutants but not in Col or gi-201 plants (Fig. 7B, see time points 7 and 31). FT mRNA was very low in Col and gi-201 but was similarly high in gi-200 and toc1-2 plants, consistent with their early flowering phenotypes in SD (Fig. 7, C and D). Thus, a coincidence between light and CO expression in both toc1-2 and gi-200 in SD is correlated with high FT expression and early flowering. The flowering phenotype in gi-200 mutants is therefore due to a change in the phase in CO expression and thus results from the short-period clock phenotype of these plants rather than being caused directly by a change in CO expression levels. This indicates gi-200 retains its normal biochemical function in CO regulation but lacks the ability to properly regulate circadian timing, demonstrating that the roles of GI in clock function and CO regulation are separable.

DISCUSSION

GI Functions in Red and Blue Light Signaling

GI has previously been reported to be involved in red light signaling, both in photomorphogenesis and input to the circadian clock (Park et al., 1999; Huq et al., 2000). We now report phenotypes showing defects in blue light signaling in gi mutants. All the gi mutants we examined showed a decrease in photomorphogenesis in constant blue light (Fig. 4; Supplemental Fig. S4), consistent with a previous report that gi-3 shows defects in blue light-induced photomorphogenesis under unusual temperature conditions (Paltiel et al., 2006) and suggesting that multiple domains of GI are required to inhibit hypocotyl elongation in the light. In addition, the change in free-running period of the circadian clock in response to increased blue light intensity is compromised in gi-200 (Fig. 3), indicating that GI functions in blue light input to the circadian clock. Because CRYs are the blue light receptors involved in photomorphogenesis and input to the clock (Ahmad and Cashmore, 1993; Lin et al., 1998; Devlin and Kay, 2000), it is likely that GI is involved in CRY signaling. However, the flattening of the fluence response curve to blue light seen in gi-200 (Fig. 3) was not observed in cry1 cry2 double mutants (Devlin and Kay, 2000), indicating that the gi light input defects are not solely due to a decrease in blue light signaling through CRY.

GI has been proposed to act within the central clock, either as a positive regulator of TOC1 expression or in parallel with TOC1 (Locke et al., 2005; Mizoguchi et al., 2005; Gould et al., 2006). Like gi mutants, toc1 loss-of-function alleles also have long hypocotyls in red light (Más et al., 2003a). However, the semidominant short-period toc1-1 mutant has no hypocotyl phenotype in red light despite its circadian phenotype (Somers et al., 1998b). These data suggest TOC1 plays biochemically distinct roles in PHY signaling and central clock function. Because gi mutants have clock (but not hypocotyl) phenotypes even in DD (Park et al., 1999; Mizoguchi et al., 2005; Table I; data not shown), GI's functions in red and blue light signaling are likely distinct from its role in the central circadian oscillator. Notably, all gi mutants examined had similar hypomorphic hypocotyl phenotypes (Fig. 4; Supplemental Fig. S4) but disparate and often antimorphic circadian phenotypes (Table I; Park et al., 1999; Mizoguchi et al., 2005). In particular, we found that the long-period clock phenotype of the likely null gi-201 was recessive, while the short-period phenotype of the EMS allele gi-200 was semidominant (Supplemental Table S1), consistent with the short-period phenotype of plants overexpressing GI (Mizoguchi et al., 2005). Taken together, these data support the hypothesis that the biochemical roles of GI in light signaling and the circadian clock are separable (Mizoguchi et al., 2005). The observation that gi but not toc1 mutants have hypocotyl phenotypes in blue light (Más et al., 2003a; Fig. 4; Supplemental Fig. S4) also suggests that GI and TOC1 have independent functions, at least in blue light signaling.

GI Acts within the Central Clock

Rhythmic luciferase activity quickly damped in gi-201 plants transferred to LL, a more extreme phenotype than observed in the gi-200 mutant (Fig. 1) and consistent with the suggestion that GI may act in the central clock along with TOC1, CCA1, and LHY (Locke et al., 2005; Mizoguchi et al., 2005). TOC1 and GI are both evening-phased genes whose expression is negatively regulated by CCA1 and LHY (Fowler et al., 1999; Alabadí et al., 2001). Further analogies with TOC1 are suggested by the wavelength-dependent phenotype of gi-200 plants: like toc1 loss-of-function mutants (Más et al., 2003a), gi-200 demonstrates a more severe clock phenotype in constant red than in constant blue light (Figs. 1, ,2,2, and 6, A and B). Finally, both TOC1 and GI are required for normal peak levels of CCA1 and LHY expression (Fig. 5, A and B; Fowler et al., 1999; Park et al., 1999; Alabadí et al., 2001; Mizoguchi et al., 2002).

This and other data was incorporated into a recent model of the plant circadian oscillator. In this model, a component termed Y positively regulates TOC1 expression, and the expression of Y is in turn negatively regulated by TOC1, LHY, and CCA1. It has been proposed that GI represents all or part of component Y (Locke et al., 2005). Our data indicate that GI is not the only protein that positively regulates TOC1 expression and thus cannot constitute all of the Y activity, because TOC1 mRNA levels do not show a marked reduction in the strong gi-201 mutant (Fig. 5C). However, the apparent damping of TOC1 levels toward the median in this mutant is consistent with GI providing a portion of Y activity (Supplemental Fig. S5, A and B). On the other hand, a different prediction of the two-loop model is not supported by our data. The model predicts that a strong loss-of-function toc1 allele would cause expression of Y to be high and arrhythmic (Locke et al., 2005; Supplemental Fig. S5D), but we found GI transcript levels were very similar in toc1-2 and wild-type plants (Fig. 5D; Supplemental Fig. S5C). This suggests that either the decreased GI expression seen in TOC1 overexpressing plants (Makino et al., 2002) is a nonspecific effect or that another factor acts redundantly with TOC1 to inhibit GI expression.

The circadian phenotype of gi-200 toc1-2 double mutants is more severe than that observed in either single mutant and is in fact nearly additive (Fig. 6, A and B; Table I). Because neither allele is a null, this result must be interpreted with caution. However, combined with the observation that overexpression of TOC1 causes a long-period while overexpression of GI causes a short-period phenotype (Más et al., 2003a; Mizoguchi et al., 2005), the extreme short-period phenotype in the double mutant suggests that GI has functions in the circadian clock independent of regulation of TOC1 expression. This possibility is reinforced by the observation that CCA1 and LHY message levels are reduced in both gi-200 and gi-201 (Fig. 5, A and B) despite the different period phenotypes of these mutants (Table I; Fig. 1). Furthermore, 35S::GI plants demonstrate near wild-type levels of CCA1 and LHY despite their short-period phenotype (Mizoguchi et al., 2005). Because loss of LHY or CCA1 function causes a shortened period (Green and Tobin, 1999; Michael et al., 2003b), these data suggest that GI plays a role within the central clock that is independent of the TOC1/CCA1/LHY feedback loop. We therefore propose that GI acts both in series and in parallel to TOC1 within the central oscillator.

Further distinctions between TOC1 and GI were revealed by epistasis analysis with ZTL. The double gi-200 ztl-105 mutant has a long-period phenotype (Fig. 6; Table I) unlike the toc1-like short-period phenotype seen in toc1 ztl double mutants (Más et al., 2003b). It therefore seems unlikely that accumulation of GI protein contributes significantly to the ztl long-period phenotype. This conclusion is underscored by the observation that plants that constitutively overexpress GI have a short period, in contrast to the long period seen in ztl mutants (Somers et al., 2000; Mizoguchi et al., 2005). These data suggest that GI is not a substrate for ZTL-mediated degradation and that the dark-induced proteolysis observed for GI (David et al., 2006) is likely directed by another mechanism.

GI Has Separable Functions within the Circadian Clock and in the Regulation of a Clock Output

GI has been implicated in flowering time regulation, red light signaling, and central clock function (Rédei, 1962; Koornneef et al., 1991; Araki and Komeda, 1993; Huq et al., 2000; Locke et al., 2005; Mizoguchi et al., 2005; Gould et al., 2006). Because both plant responses to red light and photoperiodic control of flowering are regulated by the clock, it has been difficult to determine whether all gi phenotypes are secondary to disruption of clock function. This issue has been clarified by our characterization of gi-200 mutants. These plants have a low-amplitude, short-period phenotype similar to gi-1 and gi-3 (Park et al., 1999; Tseng et al., 2004; Mizoguchi et al., 2005); however, gi-1 and gi-3 flower late in LD, while gi-200 flowers normally in inductive photoperiods (Fig. 7A). The late flowering of gi-3 plants is due to reduced levels of CO expression (Suarez-Lopez et al., 2001), whereas CO levels are similar to wild type in gi-200 plants (Fig. 7B). gi-200, however, demonstrates a phase shift in CO expression due to the increased pace of the clock (Fig. 7B), indirectly causing early flowering in SD. Thus, gi-200 has a defect in clock function but is nonetheless able to promote CO expression in LD. This shows that the genetically separable functions of GI in the circadian clock and flowering time regulation (Mizoguchi et al., 2005) in fact reflect distinct biochemical requirements for GI activity in these two pathways.

Similar to gi-200, a previous study reported that gi-611 mutants had a short free-running period and flowered early in SD, although the basis for the flowering phenotype was not explored (Gould et al., 2006). gi-611 is altered at Leu-281 with a change to Phe. Another allele, gi-596, exhibited a long period with normal flowering in LD (Gould et al., 2006). Like gi-200, gi-596 is a missense allele with Ser-191 changed to Phe. gi-200 has a similar alteration, with Ser-932 changed to Ala, but has a short-period phenotype. Therefore, the normal function of GI within the circadian clock requires sequences at both the N and C termini of the protein. Ser-191 is conserved in all known GI homologs, both in monocots and dicots. Ser-932 is conserved in all dicot sequences and those of most monocots, but in two Lemna homologs, the corresponding residue is a Gly. These Ser residues might be important for the structure or activity of GI or might be the sites of posttranslational modification (Tseng et al., 2004).

Comparison of the phenotypes of gi mutants may help shed light on its mode of action in the clock. Period is lengthened when GI is not present, as in gi-2 or gi-201 (Park et al., 1999; Table I). However, two mutants with premature stop codons near the C terminus (gi-1 and gi-3) both have short-period phenotypes (Park et al., 1999; Mizoguchi et al., 2005) similar to plants overexpressing GI (Mizoguchi et al., 2005). This suggests that the N terminus of GI acts to stimulate the pace of the clock and that the C terminus may function to block N-terminal activity. Alteration of a C-terminal residue in gi-200 leads to a semidominant short-period phenotype, suggesting that the Ser932A mutation impairs the putative negative regulatory role of the C terminus.

CONCLUSION

The isolation and characterization of new alleles of known clock-associated genes has provided insights into the functioning of GI in the circadian system. GI has a wavelength-dependent role in circadian clock function, acting in both red and blue light signaling to the clock. Characterization of a missense allele revealed that its action in the clock is biochemically distinct from its regulation of the flowering time pathway. In fact, it may be that most plant clock proteins also act in non-clock-dependent processes (Más et al., 2003b; Kevei et al., 2006). Further biochemical characterization of GI function will shed light on the mechanisms underlying its diverse roles in plant signaling.

MATERIALS AND METHODS

Mutagenesis and Screening

Arabidopsis (Arabidopsis thaliana; Col ecotype) plants were transformed with the CCR2::LUC reporter construct (Strayer et al., 2000), which confers gentamycin resistance. Seeds homozygous for CCR2::LUC were mutagenized with EMS by soaking 2 g of seeds in 0.25% EMS at 21°C for 19 h. Approximately 10,000 M1 plants were grown in pots of about 100 plants, each pot producing one pool of M2 seeds. M2 seeds were sterilized and plated on Murashige and Skoog containing 3% Suc and stratified for 3 to 4 d at 4°C before release to 12:12 light:dark cycles. Five days postgermination, seedlings were transferred to 96-well Packard plates and entrained for three more days. Seedlings were then assayed in DD in a Packard multiwell scintillation counter for 6 d (see Harmer and Kreps [2001] for more details).

Data collected from the assay were analyzed for rhythmicity following the luciferase activity analysis methods found in Plautz et al. (1997). Due to the high frequency of arrhythmic plants in DD, only plants with period estimates greater than 2 sds from the variance weighted mean of the population were isolated and allowed to self fertilize for the M3 generation. Screening of approximately 10,000 m2 plants yielded nearly 200 M3 families, of which 14 exhibited heritable alterations in period length in DD. Only 10 mutant lines demonstrated sufficiently robust phenotypes to allow for further characterization. Mutant lines were outcrossed to Ler for mapping and backcrossed to the parental Col CCR2::LUC line at least four times to remove extraneous EMS mutations before further characterization. Mutations were confirmed with cleavable amplified polymorphic sequence markers according to Supplemental Protocols S1.

SALK T-DNA Alleles

T-DNA insertion mutants ztl-105 (SALK-069091) and gi-201 (SALK-092757) were obtained from the Arabidopsis Biological Resources Center (Alonso et al., 2003). See Supplemental Protocols S1 on the Web for the primer sequences used for genotyping.

Period Phenotype Assay

Seeds were plated on Murashige and Skoog (MP Biomedicals) 0.7% agar (Sigma A1296) plates containing 3% Suc (EMD chemicals) and 75 mg/L gentamycin (EMD chemicals) and stratified at 4°C for 4 d before 12:12 light/dark entrainment at 22°C. After 6 d in light/dark cycles, plants were sprayed with 3 mm d-luciferin (Biosynth AG) and monitored with a cooled CCD camera (either an ORCA II ER [Hamamatsu] or a DU434-BV [Andor Technology]). Plants were grown at a constant 22°C. Light was provided by red and/or blue LED SnapLites (Quantum Devices) or cool-white fluorescent bulbs. Neutral density filters (RoscoLux no. 98 and no. 398) were used to obtain the different fluence levels for the period length fluence response curves. Images were analyzed with MetaMorph (Molecular Devices) software, and the pattern of luciferase activity was fit to a cosine wave through Fourier Fast Transform-Non-Linear Least Squares (Plautz et al., 1997) allowing for estimates of period length, amplitude, and phase.

Hypocotyl Analysis

Hypocotyl length was assayed by sowing seeds on Murashige and Skoog agar plates containing 3% Suc before stratification at 4°C for 4 d. After a 4-h white light pulse (100 μmol m−2 s−1), seeds were released to constant white light (cool-white fluorescent bulbs, Sylvania and Phillips) or constant red or blue light (LED SnapLites, Quantum Devices) at 22°C under neutral density filters (RoscoLux no. 98 and no. 398) for 6 d before collection. On day 6, seedlings were transferred to transparencies and scanned. Individual measurements were obtained from the scans using the Seed Vigor Index System (Hoffmaster et al., 2003) with program modifications implemented by K. Fujimura and L. Xu (unpublished data).

Flowering Time Analysis

Seeds were stratified in water for 4 d before sowing directly to soil. Flats were then placed in either LD of 16 h light and 8 h dark (approximately 100 μmol m−2 s−1) or SD of 8 h light and 16 h dark (approximately 200 μmol m−2 s−1) provided by cool-white fluorescent bulbs (Sylvania and Phillips). After germination, flats were weeded, allowing only one plant per pot and monitored daily for bolting. When a 1-cm bolt was present, the number of rosette leaves was noted.

qRT-PCR Assay for Expression of CCA1, LHY, TOC1, GI, CCR2, and PP2a

Approximately 40 plants of each genotype for each time point were germinated on Whatman filter paper atop Murashige and Skoog agar, 3% Suc plates and entrained in 12/12 light/dark cycles under cool-white fluorescent bulbs (55 μmol m−2 s−1) for 8 d before release to LL (55 μmol m−2 s−1) and sample collection at 3-h intervals. Total RNA was prepared with TRIzol reagent (Invitrogen), and 3 μg each were used for cDNA synthesis from oligo(dT)(18) with SuperScript II Reverse Transcriptase (Invitrogen) following manufacturer's protocol. Real-time qRT-PCR was performed using an iCycler (Bio-Rad) in 40 mm Tris-HCL, pH 8.4, 100 mm KCl, 6 mm MgCl2, 8% glycerol, 20 nm fluorescein, 0.4× SYBR Green I (Molecular Probes), 1× bovine serum albumin (New England Biolabs), 1.6 mm dNTPs, 2.5 μm each primer, and 10% diluted cDNA using Taq polymerase. Samples were run in duplicate and starting quantity was estimated from critical thresholds compared to the standard curve of amplification. Data presented are normalized to PP2a expression level. All primer sets contain one primer that bridges an intron to prevent genomic amplification, melt curve analysis was performed following amplification to confirm specificity of products over primer dimers, and a no-RT control was used to ensure products detected were from cDNA rather than genomic. See Supplemental Protocols S1 for primer sequences.

FT and CO Expression Analysis

cDNA samples were generated as for qRT-PCR analysis except that plants were entrained in SD (8:16) under cool-white fluorescent bulbs (200 μmol m−2 s−1) for 8 d before collection. cDNA samples were diluted 1:5 for FT expression and 1:20 for CO expression. FT expression was monitored by PCR using FT and UBQ10 specific primers (see Supplemental Protocols S1 for primer sequences). We found 30 cycles for FT and 20 cycles for UBQ-10 to be within the log-linear phase of amplification on a template dilution series. RT-PCR products were visualized on agarose gels with ethidium bromide staining and quantified using ImageQuant (GE Healthcare) software. CO expression was monitored by qRT-PCR as described above (see Supplemental Protocols S1 for primer sequences).

Supplemental Data

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

  • Supplemental Protocols S1. See this section for a description of the cleavable amplified polymorphic sequence markers used to genotype the EMS alleles described in this study, primer sequences used for genotyping lines obtained from Arabidopsis Biological Resource Center, the sequences of primers used in qRT-PCR experiments, the sequences of primers used for semiquantitative RT-PCR, and supplemental references.
  • Supplemental Figure S1. CCR2::LUC plants maintain rhythmic luciferase activity after more than 2 weeks in DD.
  • Supplemental Figure S2. Period distribution of EMS mutagenized Col CCR2::LUC population.
  • Supplemental Figure S3. Location of ZTL, LHY, and GI mutations utilized in this study.
  • Supplemental Figure S4. Fluence response of hypocotyl phenotypes in gi mutants grown in constant red, blue, or white light.
  • Supplemental Figure S5. Comparison of predicted and observed effects of toc1 and gi mutations on gene expression.
  • Supplemental Table S1. Complementation testing of gi, ztl, and lhy mutants.
  • Supplemental Table S2. CCR2::LUC expression phenotypes in ztl and lhy mutants.

Note Added in Proof

After acceptance of this article, two papers were published extending the two-loop model of the plant circadian clock to three loops (Locke JCW, Kozma-Bognár L, Gould PD, Fehér B, Kevei E, Nagy F, Turner MS, Hall A, Millar AJ [2006] Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana. Mol Syst Biol 2: 59; Zeilinger MN, Farré EM, Taylor SR, Kay SA, Doyle FJ [2006] A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9. Mol Syst Biol 2: 58). Locke et al. (2006) also conclude that GI likely constitutes only a portion of “Y” activity.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank S.A. Kay, in whose lab this work was initiated with support of National Institutes of Health grant GM056006, and D.E. Somers, for communicating data prior to publication. We also thank Kikuo Fujimura and Lijie Xu for providing hypocotyl analysis software, M.S. Waugh and M.F. Covington for technical assistance, J.N. Maloof, K. Nozue, and M.F. Covington for critical reading of the manuscript and helpful discussions, and J.N. Maloof for statistical advice.

Notes

1This work was supported by the National Science Foundation (graduate research fellowship to E.L.M.T.) and by the National Institutes of Health (grant no. R01GM069418 to S.L.H.).

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.plantphysiol.org) is: Stacey L. Harmer (ude.sivadcu@remrahls).

[C]Some figures in this article are displayed in color online but in black and white in the print edition.

[W]The online version of this article contains Web-only data.

[OA]Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.088757

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