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Proc Natl Acad Sci U S A. Jul 12, 2011; 108(28): 11698–11703.
Published online Jun 27, 2011. doi:  10.1073/pnas.1106771108
PMCID: PMC3136272
Plant Biology

GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana


Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)–FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time.

Successful reproduction in higher plants depends on appropriate timing of flowering. Understanding the mechanisms underlying flowering time pathways can provide insight into the networks mediating the effects of environmental cues on developmental programs, and has important implications for crop production.

Plants use multiple environmental cues to determine the timing of flowering, such as temperature, quality and quantity of light, and day-length changes. Among these signals, day-length change is the most reliable because it occurs in regular and predictable cycles year after year. Photoperiodism refers to the rhythms of biological processes that are based on day-length changes and is found in many species including insects, birds, and mammals. Although the molecular mechanism for photoperiodism is not well-understood, biologists have identified several key elements governing this phenomenon in plants.

In the model plant Arabidopsis thaliana, flowering is accelerated when the length of daylight is prolonged compared with darkness (16 h of light and 8 h of darkness, designated as long day; LD). Time measurement in the photoperiodic flowering pathway is regulated by daytime expression of CONSTANS (CO) (1, 2). Under LD, CO expression coincides with light. CO protein is stabilized by light (a component of a process referred to as external coincidence) and it activates a downstream factor, FLOWERING LOCUS T (FT) (3). In short days (SD; 8 h of light and 16 h of darkness), the peak time of CO expression occurs after dusk, so that CO protein is not stabilized and is unable to activate FT (3, 4). Taken together, the timing of CO expression is crucial in the photoperiodic flowering pathway.

Previously we showed that GIGANTEA (GI) and FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1 (FKF1) are important in the regulation of the timing of CO gene expression (5). The expression of GI is under control of the circadian clock and peaks at the end of the day. We determined that GI and FKF1 form a complex in a light-dependent manner to mediate the degradation of CYCLING DOF FACTOR 1 (CDF1), a key CO repressor. Specifically, under LD conditions, the expression of GI and FKF1 peak at the same time, leading to the optimal formation of the GI–FKF1 complex (internal coincidence). Under SD conditions, the expression of GI peaks a few hours before the peak of FKF1 expression, resulting in low levels of the GI–FKF1 complex and the maintenance of CDF1. Thus, sufficient levels of the GI–FKF1 complex are required for the proper induction of CO, which in turn triggers flowering under LD conditions (6).

In addition to the COFT module, photoperiod-responsive flowering is modulated by multiple regulatory processes. Genetic analysis has indicated that GI alone may also play a role in inducing this function, separately or in parallel to its involvement in the COFT module (7). Jung et al. reported that GI regulates FT expression by regulating miR172 levels. They showed that TARGET OF EAT (TOE)1, one of the targets of miR172, regulates FT expression independently of CO (8). miR172 represses flowering through targeting multiple transcription factors such as SCHLAFMUTZE (SMZ) and its paralog SCHNARCHZAPFEN (SNZ) (9). SMZ has been shown to repress FT directly. SMZ also regulates expression of APETALA1 (AP1) and SUPRESSOR OF CONSTANS OVEREXPRESSION (SOC1), which regulate flowering time and floral development in shoot apical meristem (9). Kim et al. showed that EARLY FLOWERING 3 (ELF3) influences flowering by regulating the expression of multiple flowering genes such as GI and FT. Interestingly, the elf3-1 mutant displays an early flowering phenotype independently of CO expression (10). These reports suggest that photoperiodic flowering is regulated by complex mechanisms, including uncharacterized CO-independent pathways.

In addition to genetic analysis, expression patterns of flowering genes indicate the spatial complexity of the regulatory pathways in flowering. Plants perceive photoperiodic changes in leaves (11); however, the expression patterns of CO, FT, GI, and FKF1 are not identical throughout all leaf cell types. CO and FT are expressed exclusively in vascular bundles (12, 13), whereas GI and FKF1 can be detected in several additional tissues, including mesophyll. CDFs, the only known CO repressors, are expressed solely in vascular bundles (14). TOE1, which functions as an FT repressor downstream of GI, is expressed only in vascular tissue (8). On the other hand, several FT repressors, namely SHORT VEGETATIVE PHASE (SVP), FLOWERING LOCUS C (FLC), TEMPRANILLO (TEM)1, and TEM2, were detected in both mesophyll and vascular bundles (1517).

To better understand the molecular mechanisms of the photoperiodic flowering pathway, we investigated the effect of tissue-specific expression of GI. We show that GI expression in mesophyll can rescue the flowering deficiency in gi-2 loss-of-function mutants. In addition, under SD conditions, GI increased FT expression without inducing the expression of CO. We also determined that GI binds to several known FT repressors that are expressed not only in vascular bundles but also in mesophyll. Finally, we found that GI binds to FT promoter regions. Taken together, our findings show that GI is capable of directly activating FT expression, acting alone and independently of CO.


Effects of Tissue-Specific Expression of GI on Flowering Time.

GI is expressed in many plant tissues, including vascular bundles, mesophyll, apical shoot meristem, and root (18). However, the function of GI in those tissues is not well-understood. To shed more light on how GI influences photoperiodic flowering, we expressed a GI transgene in the gi-2 loss-of-function mutant under tissue-specific promoters. For this, we took advantage of previously defined tissue-specific promoters: LIGHT-HARVESTING COMPLEX B2.1 (pLhCB2.1) and CHLOROPHYLL A/B-BINDING PROTEIN 3 (pCAB3) for mesophyll specificity (19, 20), SUCROSE TRANSPORTER 2 (pSUC2) and SULFATE TRANSPORTER 1:3 (pSultr1:3) for expression in vascular bundles (21, 22), ECERIFERUM 6 (pCER6) for epidermis expression (23), SHOOT-MERISTEMLESS (pSTM) for expression in the apical shoot meristem (24), and At3g25820/At3g25830 (pAt3g25820/At3g25830) for root-specific expression (25).

After selecting transformants on agar plates, we transferred T1 generation plants (first generation transformants derived from transformed plants) to soil and assayed them under long-day conditions. We recorded the total number of rosette and cauline leaves when plants started to bolt (beginning of flowering). Typically, wild-type plants form only about 15 leaves at bolting, whereas gi-2 mutants show a significant late-flowering phenotype, forming about 60 leaves. Overexpression of GI under the cauliflower mosaic virus 35S promoter promotes flowering even earlier than wild type, at about 12 leaves. In this set of experiments, we have shown that GI expression in mesophyll or vascular bundles rescues the late-flowering phenotype of gi-2 (Fig. 1A and Fig. S1). GI expressed in apical shoot meristem promoted flowering time with statistical significance (Student's t test, P < 0.01). However, the effect was much weaker than that of GI expression in mesophyll or vascular bundles. GI expression in epidermis or root did not have a significant effect (Fig. 1A). In this study, we focused on GI function in leaf tissue because the photoperiodic signal is perceived in leaves (11).

Fig. 1.
Mesophyll or vascular bundle-specific expression of GI can rescue the late-flowering phenotype of gi-2 (loss-of-function mutant). (A) Total leaf number was counted under LD conditions (16 h of light/8 h of darkness) for all T1 transgenic lines. Data are ...

GI Expression in Mesophyll or Vascular Bundles Rescues the Late-Flowering Phenotype of gi-2 Under Both LD and SD Conditions.

To confirm the rescue of gi-2 by GI expression in mesophyll or vascular bundles, we examined the flowering phenotype in homozygous T4 generations (Fig. 1 B and C). We selected two independent plant lines for both mesophyll- and vasculature-specific GI expression (pLhCB2.1::GI/gi-2 #1 and #3; pSUC2::GI/gi-2 #1 and #2). In all four lines, GI expression levels were up-regulated (Fig. S2). GI expression in pLhCB2.1::GI/gi-2 #1 is about 10 times higher than in wild type at zeitgeber time (ZT) 12 (the time of lowest expression) and about 100 times higher than in wild type at ZT0 (the highest expression time point). pLhCB2.1::GI/gi-2 #3 has 2 times higher expression of GI than wild type at ZT12 and about 20 times higher at ZT0. pSUC2::GI/gi-2 #1 shows almost the same expression of GI as wild type at ZT20, and about 6 times higher than wild type at ZT0. pSUC2::GI/gi-2 #3 expressed GI at levels 2 times higher than wild type at ZT20 and about 8 times higher than wild type at ZT0. We studied the flowering phenotype under LD and SD at both 16 °C and 23 °C, because recent findings have demonstrated the importance of ambient temperature in flowering pathways (15).

Under LD conditions, as shown in Fig. 1B, transgenic lines expressing GI in mesophyll or vascular bundles show phenotypes similar to the wild type at both 16 °C and 23 °C. Flowering time for all transgenic plants under LD is delayed at 16 °C compared with 23 °C. This indicates that transgenic plants that express GI in mesophyll or vascular tissues are still sensitive to lower temperature (typical for wild-type plants). Under SD conditions, GI expression in mesophyll or vascular tissues also rescued the late-flowering phenotype of gi-2 at both temperature conditions, with temperature sensitivity preserved (Fig. 1C).

FT mRNA Levels Are Elevated in a CO-Independent Manner by the Ectopic Expression of GI.

GI was shown to influence flowering time through activation of the COFT module (26). Thus, we checked how tissue-specific expression of GI influences both CO and FT expression levels. To this end, we collected wild type, gi-2 loss-of-function mutants, and our transgenic plants every 4 h over a 24-h period. These plants were grown under four different conditions (LD at 16 °C, SD at 16 °C, LD at 23 °C, and SD at 23 °C) and expression levels of CO and FT were measured by quantitative (q)PCR (Fig. 2 A and B and Fig. S3 A and B).

Fig. 2.
Effects of tissue-specific expression of GI on CO and FT gene expression at 16 °C under LD and SD. CO and FT expression levels were analyzed by qPCR in 10-d-old seedlings on samples collected every 4 h. ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL ...

Under LD conditions, wild-type plants show cyclic expression of CO, with peaks around ZT20 at 16 °C and ZT16 at 23 °C. FT expression peaks at ZT16 at both 16 °C and 23 °C. In gi-2 mutants under both temperature conditions, CO expression is constantly dampened and FT expression is not induced. Plants that overexpress GI showed an increase of both CO and FT expression. GI expression in mesophyll or vascular tissue showed a similar pattern of CO expression as wild-type plants at both 16 °C and 23 °C. On the other hand, GI expression in mesophyll or vascular tissue increased FT expression between ZT4 and ZT8 whereas maintaining an expression peak at ZT16 (Fig. 2A and Fig. S3A).

Under SD conditions, wild-type plants show cyclic expression of CO at both 16 °C and 23 °C, as is observed under LD conditions. However, FT is not expressed, because CO expression does not coincide with light under SD conditions (3). In a gi-2 mutant, neither CO nor FT expression is induced. Plants expressing GI in mesophyll or vascular tissue did not show any induction of CO compared with wild-type plants, or they showed a similar pattern as the gi-2 mutant. However, these transgenic lines showed significantly high FT expression compared with that in wild-type plant or gi-2 mutant. FT induction without increase of CO expression was detected at both 16 °C and 23 °C (Fig. 2B and Fig. S3B). These data indicate that the transgenic plants rescued the late-flowering phenotype under SD conditions, mostly by increasing FT expression in the absence of CO induction.

GI Expression in Mesophyll or Vascular Bundles Can Rescue the Late-Flowering Time of co-101 Mutants.

To examine the possibility that GI can induce flowering in a CO-independent manner, we expressed GI in mesophyll (pLhCB2.1::GI) or vascular bundles (pSUC2::GI) in the background of a co null mutation [co-101; T-DNA insertion line (12)]. In the absence of CO (co-101), plants show a late-flowering phenotype by producing around 48 leaves when they bolt. This late-flowering time was reduced to about 38 leaves by overexpression of GI under the 35S promoter or by tissue-specific expression of GI in mesophyll (pLhCB2.1::GI/co-101) or vascular bundles (pSUC2::GI/co-101) (Fig. S4). This result shows that GI can accelerate flowering in the absence of CO, tested here under LD in a T1 generation. However, the rescue of the late-flowering phenotype by the ectopic expression of GI was less effective in co-101 than that in the gi-2 mutant background.

GI Expression in Mesophyll Induces FT Expression in This Tissue.

FT is activated by CO and expressed exclusively in vascular bundles (12). However, FT expression is regulated in both mesophyll and vasculature by FT repressors (e.g., SVP) (15). In our hands, GI expression in mesophyll rescued the late-flowering phenotype under SD conditions, possibly by up-regulating FT expression. Therefore, we examined whether FT expression is induced in mesophyll in transgenic lines that express GI in mesophyll. For this, we expressed β-Glucuronidase (GUS) under the control of the 7-kbp promoter of FT in the pLhCB2.1::GI/gi-2 #1 and #3 plant lines. It was shown that the 5.7-kbp sequence upstream of the FT translational start site contains all regulatory elements required to mediate spatial and temporal expression of FT in response to photoperiod (27). We performed GUS staining of 3-wk-old T1 plants grown under SD conditions (Fig. 3A). In wild-type plants, FT expression was observed in vascular bundles only. In transgenic lines that express GI in mesophyll, FT was coexpressed in this tissue, confirming that the expression of GI protein in mesophyll can induce FT expression in the same tissue.

Fig. 3.
GI up-regulates FT expression in mesophyll and binds to FT repressors. (A) GUS staining. FT is up-regulated in mesophyll in pLhCB2.1::GI/gi-2 #3. pFT::GUS expression in wild type (Left) and in a plant line that expresses GI in mesophyll (pLhCB2.1::GI/gi-2 ...

Interactions Between GI and FT Repressors.

To further examine the possibility that GI regulates FT expression in mesophyll, we examined direct interactions between GI and FT repressors. We focused on SVP, FLC, TEM1, and TEM2, because these four repressors were shown to be expressed not only in vascular bundles but also in mesophyll, and they all bind to FT promoter regions (1517). We first examined interactions by yeast two-hybrid assays (Fig. S5). We found that the GI N-terminal region interacts with all of the FT repressors. We next used a tobacco transient expression assay to examine the physical interactions of these proteins in vivo. Briefly, we coexpressed HA-tagged GI with GFP-tagged SVP, FLC, TEM1, or TEM2 under the 35S promoter. After harvesting tissues, we fractionated plant lysates to separate cytosolic and nuclear-enriched material. We used anti-GFP antibody to immunoprecipitate tagged repressors and examined whether HA-GI coprecipitated with anti-HA antibody. We found that three out of the four repressors, namely SVP, TEM1, and TEM2, did coprecipitate HA-GI in nuclear but not in cytosolic fractions (Fig. 3 B and C). FLC did not coprecipitate HA-GI (Fig. 3B, both fractions tested). SVP, TEM1, and TEM2 repress FT expression by binding FT promoter regions (1517), which suggests that GI binds directly to these FT repressors in the nucleus, where they function.

GI Is Recruited to FT Promoter Regions.

To further elucidate how GI regulates FT expression in a CO-independent manner, we examined the physical interaction of GI protein with FT promoter regions. We performed chromatin immunoprecipitation assays (ChIP) using endogenous GI promoter-driven GFP-tagged GI lines (pGI::GFP-GI/gi-2 #27). Plant tissues were collected at ZT13 under LD. To detect the enrichment of promoter regions, we used primer pairs to amplify the five genomic regions as reported previously (15) (Fig. 4C). FT repressors SVP, FLC, TEM1, and TEM2 were shown to bind to FT promoter regions, including the 5′ UTR regions (1517). We determined that amplicons 4 and 5, which are close to or include the 5′ UTR region, were significantly enriched (Fig. 4A), indicating that GI may bind there. This result suggests that GI activates FT gene expression independently of CO through direct binding to FT promoter regions (alone or in a complex with another protein). In addition, similarities between the chromatin-binding pattern of GI and that of FT repressors suggests that GI could interfere with FT repressors by regulating their access to the FT promoter or their activity and/or stability.

Fig. 4.
GI binds to FT promoter regions. (A) FT chromatin regions associated with the GFP-GI protein. Plants were harvested 13 h after light onset (ZT13) on day 10 under LD conditions. Relative enrichment in the wild-type sample and in the pGI::GFP-GI/gi-2 sample ...

Involvement of FKF1 in FT Gene Regulation.

Models based on qPCR data predict that FKF1 influences FT expression in a CO-independent way (28). FKF1 functions by forming a complex with GI to regulate the timing of CO gene expression (5). To examine whether FKF1 is also involved in the direct activation of FT gene expression by GI, we performed a series of ChIP experiments with pFKF1::HA-FKF1/fkf1 #24 to examine whether FKF1 can bind to FT promoter regions. These experiments show that FKF1 does bind to FT promoter regions, in a pattern similar to that of GI binding (Fig. 4B).

Next, using yeast two-hybrid assays, we examined interactions between FKF1 and four FT repressors (SVP, FLC, TEM1, and TEM2). We could not detect obvious interactions between FKF1 protein and the FT repressors we assayed (Fig. S6). It is possible that FKF1 has different molecular targets from those that bind to GI. Ultimately, the molecular mechanism by which FKF1 potentially activates FT expression in a CO-independent manner requires further analysis.


We have shown that targeted expression of GI in mesophyll or in vascular bundles rescues the late-flowering phenotype in gi-2 loss-of-function mutants. In our hands, GI activated FT expression in a CO-independent manner under SD conditions. Ectopic expression of GI in mesophyll triggered FT expression in this tissue. In addition, we demonstrated that GI binds directly to SVP, TEM1, and TEM2 in vivo. Finally, our ChIP experiments showed that GI physically associates with FT promoter regions, the same regions that are binding sites for FT repressors SVP, TEM1, and TEM2. Taken together, we suggest a unique model where GI directly regulates FT expression through neutralization of FT repressors by blocking their access to FT promoter regions and/or by effecting their stability and/or activity.

GI Activates FT in a CO-Independent Manner.

Our data showed that GI expression in mesophyll (pLhCB2.1::GI/gi-2) and in vascular tissues (pSUC2::GI/gi-2) can activate FT gene expression without up-regulating CO under SD conditions at both 16 °C and 23 °C (Fig. 2 and Fig. S3).

One explanation for why the strong CO-independent FT induction by GI was detected under SD conditions is that under LD conditions, expression of GI and FKF1 peak at the same time, a sufficient amount of GI–FKF1 complex is formed, CO is up-regulated, and FT is induced. However, under SD conditions, maximal expression of FKF1 does not coincide with maximal GI expression, the amount of GI–FKF1 complex is smaller, and CO is not significantly up-regulated. In contrast, under LD conditions, some CO-independent activation of FT by GI likely occurs, because we detected the physical interaction of GI with the FT promoter in plants grown under LD (Fig. 4A). As additional evidence for our model that GI can induce FT without the involvement of CO, we determined that expression of GI in these tissues rescued the co-101 late-flowering phenotype (Fig. S4). We consider CO-dependent FT activation to be prominent in Arabidopsis, because the effect of ectopic GI expression in co-101 mutant was relatively weak.

GI Expressed in Mesophyll Acts Locally.

We have shown that GI expression in mesophyll (pLhCB2.1::GI/gi-2) rescues the late-flowering phenotype of the gi-2 mutant. GI is more broadly expressed than FT [mesophyll, vascular bundles, root, etc. vs. vascular bundles only (12)]. The fact that mesophyll-targeted expression of GI rescues the gi-2 flowering phenotype could mean that GI induced FT in mesophyll or that GI is transported to vascular bundles to activate FT there. The first possibility seems more likely, mainly because GI expressed in mesophyll alone induced FT expression there (Fig. 3A). Also, GI is a 125-kDa protein. Due to the large size, it is likely that intercellular movement is inefficient (29, 30). In addition, Adrian et al. showed that 35S::CO does not induce FT expression in mesophyll (27). This report supports our model that GI induces FT expression in mesophyll in a CO-independent manner.

Unique Role of GI in the Flowering Pathway.

FT expression is regulated by multiple factors. One mode of activation involves CO expression in vascular bundles (13, 31). On the other hand, FT expression is repressed by SVP, FLC, TEM1, and TEM2 in both vascular bundles and mesophyll (1517). We found that GI interacts with SVP, TEM1, and TEM2 in vivo. In addition, our ChIP experiments showed that GI associates with FT promoter regions, where FT repressors bind (1517). CO-independent activation of FT was inferred in previous studies (7, 10). Kim et al. presented a model of multiple parallel pathways within the COFT pathway. Mizoguchi et al. have also suggested that GI may act in a CO-independent fashion. As a function of GI in a CO-independent pathway, Jung et al. showed that GI regulates miR172 levels, presumably through its processing pathway (8). miR172 has multiple targets such as TOE1 that repress FT in vascular bundles. Our findings suggest that GI is involved in FT regulation in a more direct way, such as regulating the stability of FT repressors or their accessibility to FT promoter regions. This is a unique function of GI in the circuits regulating flowering time.

GI in Mesophyll Might Modulate Environmental and Developmental Signals.

In this series of experiments, we show that GI interacts with FT repressors such as SVP, TEM1, and TEM2, which are normally expressed in both mesophyll and vascular tissues (Fig. 3 B and C). In addition, GI associates with the FT promoter regions where SVP, TEM1, and TEM2 bind (Fig. 4A) (15, 17). These data support the hypothesis that GI activates FT in mesophyll. Expression of SVP is influenced by temperature (15). TEM1 and TEM2 expression modulates FT expression during development (17). Based on the interactions between GI and those proteins and their binding to overlapping regions of the FT promoter, we speculate that GI may interact with FT repressors to transduce temperature and/or developmental signaling.

GI Expression in Vascular Bundles Is Most Effective in Promoting Flowering.

We have shown that GI expression in mesophyll or vascular tissues can rescue the late-flowering phenotype of a gi-2 mutant. Under SD conditions, vascular expression of GI (pSUC2::GI/gi-2) rescued the gi-2 flowering phenotype more efficiently than mesophyll expression (pLhCB2.1::GI/gi-2). These data indicate that FT expression is regulated differently in mesophyll and vascular tissues. Alternatively, it is possible that FT protein is less stable in mesophyll or that transport of FT from mesophyll to vascular tissues somehow decreases its effect on the induction of flowering. It is also possible that GI moves to the vascular bundles. The size of GI reduces the efficiency of activating flowering. Efficiency of the internal transport of proteins such as FT and GI within the plant and/or the stability of the proteins in mesophyll needs to be examined to explain why the expression of GI in mesophyll is less effective at triggering flowering than expressing it in the vasculature.

Materials and Methods

Plant Materials and Growth Conditions.

Details on the construction of the binary vectors, plant lines, and plant growth conditions are provided in SI Materials and Methods. Primer sets used in the construction are listed in Table S1.

RNA Preparation and Quantitative PCR.

Whole plants were collected every 4 h. RNA isolation, cDNA preparation, and qPCR techniques are described in SI Materials and Methods. Each PCR was repeated atleast three times using biologically independent samples.

GUS Staining.

Details on the construction of pFT::GUS construct and experimental procedures are described in SI Materials and Methods. Primer pairs used to clone FT promoter are listed in Table S1.

Coimmunoprecipitation Experiments.

Details on the constructions and antibodies used in this study are included in SI Materials and Methods. A list of primer sequences used in construction are found in Table S1.

ChIP Assays.

ChIP assays were conducted as described previously (5). Anti-GFP antibody (A11122; Invitrogen) and anti-HA antibody (3F10; Roche) were used for immunoprecipitation. The primers used for amplification were described previously (15).

Supplementary Material

Supporting Information:


We thank E. Hamilton, E. Kolmos, D. Nagel, J. Nelson, D. A. Nusinow, and J. Pruneda-Paz for critical review of the manuscript. We thank J. Putterill for providing plasmids (35S::HA-GI) and plant line (35S::HA-GI/gi-2), M. Endo for cloning the tissue-specific promoters, and L. Knutstad and M. Nishi for technical assistance. This work was supported by National Institutes of Health Grants R01 GM67837 and GM50006 (to S.A.K.). M.S. was supported by a Japan Society for the Promotion of Science fellowship.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106771108/-/DCSupplemental.


1. Imaizumi T, Kay SA. Photoperiodic control of flowering: Not only by coincidence. Trends Plant Sci. 2006;11:550–558. [PubMed]
2. Corbesier L, Coupland G. The quest for florigen: A review of recent progress. J Exp Bot. 2006;57:3395–3403. [PubMed]
3. Valverde F, et al. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science. 2004;303:1003–1006. [PubMed]
4. Yanovsky MJ, Kay SA. Molecular basis of seasonal time measurement in Arabidopsis. Nature. 2002;419:308–312. [PubMed]
5. Sawa M, Nusinow DA, Kay SA, Imaizumi T. FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis. Science. 2007;318:261–265. [PMC free article] [PubMed]
6. Sawa M, Kay SA, Imaizumi T. Photoperiodic flowering occurs under internal and external coincidence. Plant Signal Behav. 2008;3:269–271. [PMC free article] [PubMed]
7. Mizoguchi T, et al. Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis. Plant Cell. 2005;17:2255–2270. [PMC free article] [PubMed]
8. Jung JH, et al. The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering independent of CONSTANS in Arabidopsis. Plant Cell. 2007;19:2736–2748. [PMC free article] [PubMed]
9. Mathieu J, Yant LJ, Mürdter F, Küttner F, Schmid M. Repression of flowering by the miR172 target SMZ. PLoS Biol. 2009;7:e1000148. [PMC free article] [PubMed]
10. Kim WY, Hicks KA, Somers DE. Independent roles for EARLY FLOWERING 3 and ZEITLUPE in the control of circadian timing, hypocotyl length, and flowering time. Plant Physiol. 2005;139:1557–1569. [PMC free article] [PubMed]
11. McDonald MS. Photobiology of Higher Plants. New York: Wiley; 2003.
12. Takada S, Goto K. TERMINAL FLOWER2, an Arabidopsis homolog of HETEROCHROMATIN PROTEIN1, counteracts the activation of FLOWERING LOCUS T by CONSTANS in the vascular tissues of leaves to regulate flowering time. Plant Cell. 2003;15:2856–2865. [PMC free article] [PubMed]
13. An H, et al. CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development. 2004;131:3615–3626. [PubMed]
14. Fornara F, et al. Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response. Dev Cell. 2009;17:75–86. [PubMed]
15. Lee JH, et al. Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes Dev. 2007;21:397–402. [PMC free article] [PubMed]
16. Li D, et al. A repressor complex governs the integration of flowering signals in Arabidopsis. Dev Cell. 2008;15:110–120. [PubMed]
17. Castillejo C, Pelaz S. The balance between CONSTANS and TEMPRANILLO activities determines FT expression to trigger flowering. Curr Biol. 2008;18:1338–1343. [PubMed]
18. Winter D, et al. An “electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS One. 2007;2:e718. [PMC free article] [PubMed]
19. Sawchuk MG, Donner TJ, Head P, Scarpella E. Unique and overlapping expression patterns among members of photosynthesis-associated nuclear gene families in Arabidopsis. Plant Physiol. 2008;148:1908–1924. [PMC free article] [PubMed]
20. Susek RE, Ausubel FM, Chory J. Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development. Cell. 1993;74:787–799. [PubMed]
21. Truernit E, Sauer N. The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2. Planta. 1995;196:564–570. [PubMed]
22. Yoshimoto N, Inoue E, Saito K, Yamaya T, Takahashi H. Phloem-localizing sulfate transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis. Plant Physiol. 2003;131:1511–1517. [PMC free article] [PubMed]
23. Hoecker U, Quail PH. The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis. J Biol Chem. 2001;276:38173–38178. [PubMed]
24. Long JA, Moan EI, Medford JI, Barton MK. A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature. 1996;379:66–69. [PubMed]
25. Chen F, et al. Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole. Plant Physiol. 2004;135:1956–1966. [PMC free article] [PubMed]
26. Suárez-López P, et al. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature. 2001;410:1116–1120. [PubMed]
27. Adrian J, et al. cis-regulatory elements and chromatin state coordinately control temporal and spatial expression of FLOWERING LOCUS T in Arabidopsis. Plant Cell. 2010;22:1425–1440. [PMC free article] [PubMed]
28. Salazar JD, et al. Prediction of photoperiodic regulators from quantitative gene circuit models. Cell. 2009;139:1170–1179. [PubMed]
29. Balachandran S, Xiang Y, Schobert C, Thompson GA, Lucas WJ. Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata. Proc Natl Acad Sci USA. 1997;94:14150–14155. [PMC free article] [PubMed]
30. Kempers R, van Bel AJE. Symplasmic connections between sieve element and companion cell in the stem phloem of Vicia faba L. have a molecular exclusion limit of at least 10 kDa. Planta. 1997;201:195–201.
31. Putterill J, Robson F, Lee K, Simon R, Coupland G. The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell. 1995;80:847–857. [PubMed]

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