Research Article

FD, a bZIP Protein Mediating Signals from the Floral Pathway Integrator FT at the Shoot Apex

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Science  12 Aug 2005:
Vol. 309, Issue 5737, pp. 1052-1056
DOI: 10.1126/science.1115983

Abstract

FLOWERING LOCUS T (FT) is a conserved promoter of flowering that acts downstream of various regulatory pathways, including one that mediates photoperiodic induction through CONSTANS (CO), and is expressed in the vasculature of cotyledons and leaves. A bZIP transcription factor, FD, preferentially expressed in the shoot apex is required for FT to promote flowering. FD and FT are interdependent partners through protein interaction and act at the shoot apex to promote floral transition and to initiate floral development through transcriptional activation of a floral meristem identity gene, APETALA1 (AP1). FT may represent a long-distance signal in flowering.

Flowering in Arabidopsis is regulated by several pathways that converge on the transcriptional regulation of the floral pathway integrators FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and LEAFY (LFY) (1). FT is a direct target of CO, a key transcriptional regulator of the photoperiod pathway, and the role of FT as a potent promoter of flowering in response to photoperiods is conserved in Arabidopsis and rice (26). FT is expressed in the phloem tissues of cotyledons and leaves (7, 8) and encodes a 20-kD protein with homology to phosphatidylethanolamine binding protein or Raf kinase inhibitor protein (2, 3). However, the biochemical function of FT and downstream events leading to floral transition and floral morphogenesis at the shoot apex remain unknown.

bZIP protein FD is required for FT function. To understand how signals are mediated from FT to finally cause floral transition and floral morphogenesis, we searched for genes required for FT to promote flowering. Ectopic overexpression of FT by the 35S RNA promoter of cauliflower mosaic virus (35S::FT) causes a precocious-flowering phenotype (2, 3). We screened for suppressors of 35S::FT, because a similar approach with 35S::CO was successful in elucidating the downstream targets of CO, FT and SOC1 (4, 9). In addition to screening mutagenized populations of 35S::FT, we examined known late-flowering mutants (10) for their effect on the 35S::FT phenotype. Through the latter approach, we found that fd-1 is a strong suppressor of 35S::FT (Table 1). In contrast to a strong effect on 35S::FT, fd-1 had only a weak effect on a similar precocious-flowering phenotype of soc1-101D, an activation tagged allele of SOC1 (11) (Table 1), or 35S::LFY (12). These observations suggest that the FD activity is required specifically for the promotion of flowering by FT.

Table 1.

Flowering times of transgenic and mutant plants.

GenotypeView inline No. of rosette leavesView inline No. of cauline leavesView inlineN
Experiment 1, LDs
Wild type (Col) 14.2 ± 2.2 (11-18) 3.0 ± 0.7 (2-4) 25
fd-1 23.1 ± 3.1 (19-29) 6.6 ± 1.2 (4-8) 14
35S::FT (YK#1-5C) 2.8 ± 0.4 (2-3) 1.0 ± 0.4 (0-2) 25
35S::FT (YK#1-5C); fd-1 18.4 ± 2.7 (14-24) 5.4 ± 1.2 (4-8) 20
soc1-101D View inline 1.9 ± 0.3 (1-2)View inline 2.1 ± 0.3 (2-3) 15
soc1-101D; fd-1 View inline 2.0 ± 0 (2) 1.9 ± 0.3 (1-2) 21
Wild type (Ler) 7.2 ± 1.1 (6-9) 2.4 ± 0.8 (1-4) 22
fd-1 12.3 ± 0.7 (11-15) 4.1 ± 0.7 (3-5) 43
35S::FT (YK#1-5L) 2.0 ± 0 (2) 1.2 ± 0.4 (1-2) 40
35S::FT (YK#1-5L); fd-1 4.0 ± 0 (4) 2.4 ± 0.8 (1-4) 24
Experiment 2, SDs
Wild type (Col), 45.3 ± 5.1 (38-54) 10.4 ± 2.0 (7-15) 17
fd-1 51.0 ± 5.8 (43-61) 9.8 ± 1.7 (7-13) 15
35S::FT (YK#1-5C) 2.3 ± 0.5 (2-3) 1.0 ± 0.5 (0-2) 25
35S::FT (YK#1-5C); fd-1 18.2 ± 4.3 (11-26) 4.4 ± 1.1 (3-7) 19
Experiment 3, LDs
Wild type (Ler) 5.3 ± 0.5 (5-6) 1.7 ± 0.5 (1-2) 14
ft-3 View inline 11.2 ± 0.8 (10-12) 2.8 ± 0.4 (2-3) 10
FD::FT (YD#2-1); ft-3 3.5 ± 0.5 (3-4) 0.5 ± 0.5 (0-1) 14
PDF1::FT (MA#4-12); ft-3 2.1 ± 0.4 (2-3) 1.0 ± 0.5 (0-2) 35
SULTR2;1::FT (YD#1-1); ft-3 3.0 ± 0 (3) 0.7 ± 0.5 (0-1) 15
IAA14::FT (YD#4-3); ft-3View inline 10.7 ± 0.8 (9-12) 2.4 ± 0.5 (2-3) 20
Experiment 4, LDs
Wild type (Col) 10.0 ± 0.7 (9-12) 2.4 ± 0.6 (1-3) 21
fd-1 View inline 21.1 ± 2.0 (18-24) 5.5 ± 0.5 (5-6) 22
FD::FD (MN#7-1); fd-1 8.4 ± 0.5 (8-9) 2.9 ± 0.5 (2-4) 21
SULTR2;1::FD (MN#4-2); fd-1View inline 20.7 ± 1.6 (18-24) 5.0 ± 0.7 (4-6) 23
Experiment 5, LDs
Wild type (Ler) 5.3 ± 0.5 (5-6) 1.8 ± 0.4 (1-2) 15
ft-3 13.7 ± 0.7 (12-15) 3.3 ± 0.7 (2-5) 15
ft-3; fd-1 17.8 ± 0.8 (16-19) 5.3 ± 0.4 (5-6) 16
FD::FT (YD#2-1); ft-3 2.9 ± 0.6 (2-4) 0.8 ± 0.4 (0-1) 15
FD::FT (YD#2-1); ft-3; fd-1 5.9 ± 0.6 (5-7) 1.8 ± 0.6 (1-3) 15
PDF1::FT (MA#4-12); ft-3 2.0 ± 0 (2) 1.3 ± 0.5 (1-2) 18
PDF1::FT (MA#4-12); ft-3; fd-1 6.4 ± 0.6 (5-7) 2.5 ± 0.5 (2-3) 20
SULTR2;1::FT (YD#1-1); ft-3 2.7 ± 0.5 (2-3) 1.2 ± 0.4 (1-2) 13
SULTR2;1::FT (YD#1-1); ft-3; fd-1 4.1 ± 0.3 (4-5) 2.0 ± 0 (2) 13
Experiment 6, LDs
Wild type (Ler), -Dex 6.8 ± 0.9 (5-8) 3.0 ± 0.6 (2-4) 22
Wild type (Ler), +DexView inline 6.4 ± 0.9 (5-8) 2.9 ± 0.7 (2-4) 24
ft-3, -Dex 14.1 ± 2.5 (11-19) 4.9 ± 1.0 (4-7) 12
ft-3, +DexView inline 12.7 ± 1.8 (10-15) 4.2 ± 0.6 (3-5) 11
35S::FT:GR (YD#9-a); ft-3, -Dex 8.8 ± 1.7 (6-12) 3.7 ± 0.6 (3-5) 24
35S::FT:GR (YD#9-a); ft-3, +Dex 3.3 ± 0.7 (2-4) 2.1 ± 0.4 (1-3) 24
  • View inline* Genetic background: Col, Columbia; Ler, Landsberg er. Plants in each experiment were grown under LDs or SDs as indicated. +Dex indicates Dex treatment (14).

  • View inline Indicators of flowering time (10) and shown as average ± SD (range). Statistical tests were done on the number of rosette leaves.

  • View inline No statistically significant difference among indicated genotypes in each experiment (Student's t test, P > 0.1). No symbol means that there was a statistically significant difference (P < 0.001) among the genotypes or conditions compared in each experiment.

  • View inline§ Includes two plants with elongated internodes.

  • View inline Indicates no statistically significant difference from the above condition (P > 0.06).

  • The FD gene was identified with AtbZIP14 (At4g35900) (13) by a map-based approach (figs. S1 and S2 and table S1). In seedlings, FD expression was observed mainly in the shoot apex (Fig. 1, A to E and J), did not show distinct circadian oscillation, and was not affected by photoperiods and CO activity (fig. S3). Under both short days (SDs) and long days (LDs), FD mRNA levels increased with time after germination (fig. S3). To examine the subcellular distribution of FD protein, we made a construct to express enhanced green fluorescent protein–FD (EGFP:FD) fusion protein by the FD promoter (FD::EGFP:FD) (14). That the fusion protein is functional was confirmed by the rescue of fd-1 (table S2). EGFP:FD was localized in the nucleus in cells of the shoot apex (Fig. 1, F and G). Similarly, nuclear localization of functional enhanced yellow fluorescent protein–FD (EYFP:FD) fusion protein (table S2) was observed in cells of the shoot apex and other tissues in 35S::EYFP:FD seedlings with various genetic backgrounds (fig. S4) (14), suggesting a constitutive nuclear localization.

    Fig. 1.

    Expression of FD and subcellular distribution of EGFP:FD and FT:EGFP. (A and B) Expression of FD in shoot apex of wild-type seedlings grown under LDs (16 hours light) for 6 (A) and 7 (B) days. (C to E) GUS staining of gFD::GUS seedlings grown under LDs for 6 (C and D) and 7 (E) days. (C) A whole seedling. The arrow indicates a root tip, which is enlarged in the inset. (D) Shoot apical region. (E) Longitudinal section of shoot apex. (F to I) Distribution of functional EGFP:FD fusion protein (F and G) and functional FT:EGFP fusion protein (H and I) expressed in shoot apex by FD promoter. (G) and (I) are enlargements showing subcellular distribution. Arrowheads in (I) indicate nuclei. (J to M) Expression of FD (J and L) and AP1 (K and M) in shoot apex of wild-type plants at floral transition on day 10 (J and K) and early inflorescence stage on day 15 (L and M) under LDs. Arrowheads in (L) and (M) indicate floral meristems at stage 1. Scale bars: 50 μm in (F), (H), (L), and (M); 20 μmin(A), (B), (E), (J), and (K); and 10 μm in (G) and (I).

    To gain clues to the molecular basis of the requirement of FD for FT function, we investigated protein interactions. FT interacted with FD in yeast cells (Fig. 2A) and in vitro (Fig. 2C). In contrast, FD showed very weak interaction in yeast cells with TERMINAL FLOWER 1 (TFL1), the FT-related protein with an antagonistic role in the regulation of flowering (2, 3) (Fig. 2A and fig. S5). Because the subcellular distribution of the FT protein remains unknown, we examined the distribution of functional FT:EGFP fusion protein (table S2) expressed in the shoot apex of Arabidopsis by the FD promoter or in leaf epidermal cells of Nicotiana benthamiana by the 35S promoter (14). In both cases, FT:EGFP was observed in the nucleus and cytoplasm (Fig. 1, H and I, and fig. S4). That FT is able to function in the nucleus was supported by observations that FT protein fused to a glucocorticoid receptor (GR) expressed by the 35S promoter (35S::FT:GR) (14) promoted flowering on dexamethasone (Dex) treatment (Table 1). These findings suggest that FD and FT proteins coexist in the nucleus. We further analyzed the interaction of FT and FD proteins in plant cells using bimolecular fluorescent complementation (BiFC) (15). In tobacco leaf epidermal cells coexpressing the N-terminal half of EYFP fused to FD (YN-FD) and the C-terminal half of EYFP fused to FT (YC-FT) (14), YFP fluorescence was observed in the nucleus (Fig. 2D). These findings show that protein interaction is the basis of the dependence of FT on FD.

    Fig. 2.

    Protein interaction. (A) Yeast two-hybrid assay of interaction among FD, FDP, FT, and TFL1. FDP, FD PARALOG (AtbZIP27). (B) Yeast two-hybrid assay of interaction between FT and C-terminal mutants of FD. C-terminal sequences of wild-type (WT) FD and mutant (ΔC, T282A, T282S) FD proteins including a possible CDPK site (blue). The asterisk on the WT sequence indicates a threonine (T) residue expected to be phosphorylated. T282A (substitution with an alanine), but not T282S (substitution with a serine), abolishes the CDPK site. None of the mutants affected interaction with wild-type FD. (C) Invitro pull-down assay of interaction between FT and FD (14). “10% input” indicates 10% of 35S-labeled FD subjected to pull-down by GST or GST-FT. Arrows indicate labeled FD. (D) BiFC analysis of interaction between FT and FD in N. benthamiana leaf epidermis (14). BF, blight field image; YFP, YFP fluorescence; PI, propidium iodide fluorescence (nuclei); Merge, merge of YFP and PI; YN-FD, expression of YN-FD alone; YC-FT, expression of YC-FT alone; YN-FD & YC-FT, coexpression of YN-FD and YC-FT. At the bottom are higher magnification images of a nucleus of a cell coexpressing YN-FD and YC-FT. Not all nuclei were stained with PI. Scale bars: 100 μm (upper three rows) and 10 μm (bottom row).

    Floral meristem identity genes are regulatory targets of FT and FD. We next tried to identify the regulatory targets of FT and FD in the shoot apex that cause floral transition and morphogenesis. We obtained clues from analysis of ft; lfy and fd; lfy double mutants. A previous work showed that ft; lfy greatly reduced mRNA levels of AP1 and caused severe defects in floral meristem specification (16). These observations led to the suggestion that FT and LFY play redundant roles in up-regulation of AP1 and floral fate specification (1, 16). In agreement with a previous report, ft; lfy greatly reduced the amount and region of AP1 expression in the shoot apex and caused severe defects in floral development (Fig. 3, A to D, and fig. S6). fd; lfy plants had an inflorescence phenotype indistinguishable from that of ft; lfy and displayed a severely reduced amount and spatial extent of AP1 expression (Fig. 3, A to D, and fig. S6). These observations suggest that FT and FD together are involved in the regulation of AP1 redundantly with LFY.

    Fig. 3.

    FT-dependent activation of AP1 by FD. (A) Floral defect in lfy-26, fd-1; lfy-26, and ft-2; lfy-26. Lateral structures on the primary inflorescence that would be single flowers in the wild-type plant. Inset shows a flower formed later in lfy-26. Scale bars: 2 mm and 1 mm (inset). (B) Rescue of floral defect in ft-2; lfy-26 by tissue-specific expression of FT. Young primary inflorescences and flowers of ft-2; lfy-26 with indicated FT constructs. Arrowheads indicate a terminal flower. Scale bars, 2 mm and 1 mm (inset). (C) AP1 and FUL expression in shoot apex of various genotypes at two different stages. SOC1 and ACT2 were amplified for reference. (D) AP1 expression in young inflorescence apex. Scale bar: 10 μm. (E to J) AP1 and FUL expression in 35S::FD and wild-type (WT) seedlings. SOC1, EF-1, and ACT2 were amplified for reference. Whole seedlings (E to H and J) or cotyledons (I) were harvested for RNA extraction at the indicated Zeitgeber time (ZT) points. (E) Seven-day-old seedlings under LDs. (F) Seven-day-old 35S::FD in FT+ and ft-3 background and wild type under LDs. (G) Seven-day-old seedlings under LDs and SDs (8 hours light). (H) 35S::FD seedlings grown for 6 days under SDs and subjected to day-length extension from 8 to 16 hours on day 7. (I) Vascular- and mesophyll-rich fractions from cotyledons of 10-day-old seedlings under LDs (14). SULTR1; 3 is a vascular marker and RbcS is preferentially expressed in mesophylls. (J) Seven-day-old seedlings of wild-type and fd-1 with 35S::FD or 35S::FDT282A or without a transgene (–) under LDs.

    Consistent with a role of FD in the regulation of AP1, ectopic expression of AP1 was observed in 35S::FD seedlings (Fig. 3E). Ectopic induction of AP1 expression by FD was abolished by ft mutation (Fig. 3F) or under SDs, which reduce FT expression (2, 3, 17, 18) (Fig. 3G). Upon a shift from SDs to LDs, which induces FT expression (8, 18), AP1 expression was induced (Fig. 3H). In young seedlings, FT is expressed mainly in the vasculature of cotyledons, and little is detected in mesophyll cells (7, 8). In 35S::FD seedlings, AP1 expression was detected in the vascular-rich fraction but not in the mesophyll-rich fraction from cotyledons (Fig. 3I). These observations suggest that activation of AP1 expression by FD requires the FT function. Finally, in the shoot apex around the stage of floral transition and in the young inflorescence apex, the region of AP1 expression was within the expression domain of FD (Fig. 1, J to M). Thus, AP1 seems to be a regulatory target of FD, which requires the FT activity through protein interaction. In support of this conclusion, several potential bZIP protein binding motifs were found in a 1.7-kb AP1 promoter (19) (fig. S7).

    Because AP1 expression was observed only in a subset of the expression domain of FD, there should be factors that restrict AP1 expression to nascent lateral meristems. TFL1, which has a role antagonistic to FT (2, 3), is likely to be responsible for suppressing AP1 expression in the shoot apical meristem proper (20, 21). That the loss of AP1 alone does not affect the precocious-flowering phenotype of 35S::FT (3) suggests that other regulatory targets of FD contribute to the promotion of floral transition. FRUITFULL (FUL) and CAULIFLOWER (CAL), which act redundantly with AP1 to promote flowering (22), are candidates for such targets (Fig. 3, C and E to J, and fig. S8).

    How FT regulates FD activity is another important question. Constitutive nuclear localization of FD and the presence of FT in the nucleus suggest the regulation of FD activity in the nucleus. Whether FD and FT form a stable transcriptional complex or interact only transiently remains to be investigated. FD protein has a potential phosphorylation site for Ca2+-dependent protein kinases (CDPKs) at the C terminus (Fig. 2B and fig. S2). Deletion or mutation of this site abolished interaction with FT (Fig. 2B), ectopic induction of AP1 expression (Fig. 3J), and the ability to complement fd-1 (fig. S9), although nuclear localization was not affected (fig. S4). These findings suggest the importance of phosphorylation of FD in the interaction with FT and in its functional regulation.

    Mutual dependence and site of action of FT and FD. The mutual dependence of FT and FD, as shown above, is further supported by the observation that flowering of 35S::EYFP:FD plants was delayed under SDs, which reduce FT expression (table S3). Furthermore, the enhanced phenotype in 35S::FT; 35S::FD (tables S3 and S4) indicates that FT and FD are mutually limiting for the combined activity of FT and FD. These raise the question of the site(s) of action of FT and FD. In seedlings, FT is expressed in the vasculature of cotyledons, but not in the shoot apex (7) (fig. S10), whereas FD is expressed in the shoot apex but not in cotyledons and leaves (Fig. 1, A to E, and fig. S10). As expected, restoration of the FT function in the vasculature through expression by SULTR2; 1::FT (14) could rescue the late-flowering phenotype of ft (Table 1 and table S5). Restoration of the FT function in the shoot apex of ft, either in the whole region by FD::FT or in the outermost cell layer (L1) by PDF1::FT (14), also rescued the late-flowering phenotype (Table 1 and table S5). These findings agree well with those of a previous report that the late-flowering phenotype of co is suppressed by similar constructs for FT misexpression (23). We further observed that FD::FT, PDF1::FT, and SULTR2; 1::FT rescued delayed flowering, reduced AP1 expression, and severe floral defect in ft; lfy (Fig. 3, B and C, and fig. S6). By contrast, restoration of FT in root vasculature by IAA14::FT (14) failed to rescue ft (Table 1). These observations indicate that ectopically expressed FT in the shoot apex can exert an effect on flowering. FT expressed in the shoot apex requires the FD function, because fd-1 attenuated the rescued phenotype (Table 1 and table S5). In contrast, FD rescued the late-flowering phenotype of fd through expression in the whole shoot apex (by FD::FD), but not through ectopic expression in leaf vasculature (by SULTR2; 1::FD), where FT is expressed (Table 1). Therefore, FD acts in the shoot apex and seems to be required in all cell layers. These findings, together with observations that protein interaction is the basis for interdependence between FT and FD, suggest that the shoot apex is the site of the FT and FD action.

    FT and the long-distance signal in flowering. It has long been believed that a long-distance signal, named florigen (24), is generated in leaves upon exposure to inductive photoperiods, is transported to the shoot apex, and acts there to promote flowering (25). However, the nature of the signal has remained elusive (25). Our present work supports an emerging hypothesis (7, 8, 23) that the FT products represent a part of the long-distance signal(s) generated in cotyledons and leaves (mainly in the phloem tissues) and act at the shoot apex to promote floral transition and to initiate floral development (fig. S11).

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/309/5737/1052/DC1

    Materials and Methods

    SOM Text

    Tables S1 to S5

    Figs. S1 to S11

    References

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