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. 2014 Feb 14;26(2):552–564. doi: 10.1105/tpc.113.115220

Structural Features Determining Flower-Promoting Activity of Arabidopsis FLOWERING LOCUS T[W],[OPEN]

William Wing Ho Ho 1, Detlef Weigel 1,1
PMCID: PMC3967025  PMID: 24532592

FT, also known as florigen, activates flowering, while the closely related TFL1 represses flowering. By mutating most FT residues, the authors identified a comprehensive set of mutations that convert FT into a TFL1 mimic. These mutations are all predicted to change the surface charge, pointing to important differences in FT and TFL1 interactions with other proteins.

Abstract

In Arabidopsis thaliana, the genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) have antagonistic roles in regulating the onset of flowering: FT activates and TFL1 represses flowering. Both encode small, closely related transcription cofactors of ∼175 amino acids. Previous studies identified a potential ligand binding residue as well as a divergent external loop as critical for the differences in activity of FT and TFL1, but the mechanisms for the differential action of FT and TFL1 remain unclear. Here, we took an unbiased approach to probe the importance of residues throughout FT protein, testing the effects of hundreds of mutations in vivo. FT is surprisingly robust to a wide range of mutations, even in highly conserved residues. However, specific mutations in at least four different residues are sufficient to convert FT into a complete TFL1 mimic, even when expressed from TFL1 regulatory sequences. Modeling the effects of these mutations on the surface charge of FT protein suggests that the affected residues regulate the docking of an unknown ligand. These residues do not seem to alter the interaction with FD or 14-3-3 proteins, known FT interactors. Potential candidates for differential mediators of FT and TFL1 activities belong to the TCP (for TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family of transcription factors.

INTRODUCTION

The onset of flowering is a crucial event in the plant life cycle. A multitude of external and endogenous signals are integrated on the promoters of a small set of genes, among which homologs of FLOWERING LOCUS T (FT) play a particularly important and highly conserved role (Kardailsky et al., 1999; Kobayashi et al., 1999; Kojima et al., 2002; Lifschitz et al., 2006; Kobayashi and Weigel, 2007; Turck et al., 2008; Hecht et al., 2011). While FT RNA is produced exclusively in leaves, the protein acts primarily at the shoot apex, apparently by associating with the FD transcription factor to directly activate downstream genes (Abe et al., 2005; Wigge et al., 2005). A large body of circumstantial evidence suggests that FT protein moves from the site of its production, the leaves, through the plant to the site of its action, the shoot apex (Lifschitz et al., 2006; Corbesier et al., 2007; Jaeger and Wigge, 2007; Lin et al., 2007; Mathieu et al., 2007; Tamaki et al., 2007; Notaguchi et al., 2008). FT thus qualifies as an essential component of the mobile flower-promoting signal, florigen (Kobayashi and Weigel, 2007; Turck et al., 2008). FT encodes a small protein of only 175 amino acids, and its mobility is thus not surprising. However, recent work indicates that FT trafficking is regulated (Liu et al., 2012).

In Arabidopsis thaliana, FT belongs to a small gene family that includes TERMINAL FLOWER1 (TFL1), an inhibitor of flowering (Bradley et al., 1997; Ohshima et al., 1997). The structures of FT, TFL1, and the TFL1 orthologous protein CENTRORADIALIS from snapdragon (Antirrhinum majus) have all been solved (Banfield and Brady, 2000; Ahn et al., 2006). As expected from the primary sequence similarity, the fold is similar to that of the phosphatidylethanolamine binding protein (PEBP) family from animals, with some notable differences in and around the anion binding pocket of the animal proteins (Banfield et al., 1998; Serre et al., 1998). In plants, there is no evidence for in vivo binding of phospholipids or other anions.

There are only 39 nonconservative substitutions that distinguish FT and TFL1, yet their in vivo activities are opposite, even when expressed from the same promoter. This intriguing observation has led several groups to investigate the sequence differences underlying antagonistic function. An early report showed that a Y85H substitution conferred partial TFL1-like activity on FT, while the converse change, H88Y, conferred weak FT-like activity on TFL1 (Hanzawa et al., 2005). Subsequent experiments demonstrated that an external loop together with the adjacent segment is largely sufficient to convert TFL1 into an FT-like activator. The effects are not entirely symmetrical, as both the external loop and the adjacent segment on their own can convert FT into a TFL1-like repressor (Ahn et al., 2006). Notably, the external loop is not the most different portion of the FT and TFL1 proteins, but it evolves very differently in the two proteins: While it is almost completely conserved in FT, it appears to be under relaxed selection in TFL1 (Ahn et al., 2006). Furthermore, a residue in the external loop of TFL1 interacts with the Y85 residue required for TFL1 function, while there is no equivalent interaction in FT (Hanzawa et al., 2005; Ahn et al., 2006). This difference has been exploited during natural evolution in sugar beet (Beta vulgaris ssp vulgaris), where mutations in the loop have imparted flower-repressing activity of FT paralogs (Pin et al., 2010).

We used an unbiased approach in which we examined hundreds of mutations to map residues required for FT flower-promoting activity. We show that altering surface charge can convert FT into a TFL1-like repressor. Based on these observations, we propose a model for docking of an unknown ligand. This ligand does not appear to be the FD transcription factor (Abe et al., 2005; Wigge et al., 2005) or the 14-3-3 proteins that link FD and FT or TFL1 (Pnueli et al., 2001; Taoka et al., 2011). Instead, members of the TCP family of transcription factors are candidates for factors that mediate differential activity of FT and TFL1 (Cubas et al., 1999; Mimida et al., 2011).

RESULTS

Assay System and Random Mutagenesis

The FT protein sequence is highly conserved among flowering plants (Supplemental Figure 1). To identify residues with strong effects on FT activity, we chose to overexpress random point mutations of the FT cDNA. Overexpression of wild-type FT leads to very early flowering and the formation of terminal flowers, with transformants having a narrow distribution of flowering times (Kardailsky et al., 1999; Kobayashi et al., 1999). The absence of accelerated flowering in transformants therefore indicates mutations that inactivate FT.

We used PCR to generate random mutants of FT. After evaluating the PCR-based mutagenesis conditions (Supplemental Figure 2), we sequenced 1241 clones of the final FT mutagenesis, of which 1100 had a complete FT insert. Among these, 9% had no mutation and 31% had only synonymous substitutions. The remainder, 60%, was almost equally divided between clones with one or with more than one nonsynonymous mutation.

The products from the mutagenesis reaction were cloned into an Escherichia coli plasmid vector, plasmid DNA was prepared from a pool of ∼36,000 bacterial colonies, and the inserts were excised and subcloned en masse into a plant transformation vector between a cauliflower mosaic virus 35S (CaMV35S) promoter and yellow fluorescent protein (YFP [mCitrine]) sequences. The purpose of fusing the FT open reading frame to that of YFP was to avoid false negatives in the overexpression assay due to introduction of premature stop codons. Agrobacterium tumefaciens transformants were pooled and used to infect wild-type Arabidopsis plants.

The general strategy for assessing FT activity of mutants is delineated in Figure 1. Because the effects of FT overexpression are strongest in short days, we first screened 3320 primary transformants in this condition. There was no overlap in the flowering times of the positive (overexpressing FT-YFP) and negative (empty vector) controls (Figure 2A). Two hundred and fifty-two negative control plants transformed with an empty vector flowered with 17 leaves or more (average, 35), and of 401 positive controls transformed with an unmutagenized 35S:FT-YFP construct, all flowered with fewer than 13 leaves, and 398 with fewer than nine leaves.

Figure 1.

Figure 1.

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Random Mutagenesis Assay Strategy.

Around 36,000 randomly mutated FT cDNA copies were generated by PCR. These were cloned en masse into a plant transformation vector for overexpression in wild-type plants as YFP fusions. Screening of 3320 primary transformants in short days identified 52 late-flowering, YFP-positive lines with reduced FT activity, representing 33 unique mutants. The progeny of these 33 lines was reevaluated in long days to distinguish mutations that inactivated FT (14 lines) from those that introduced neomorphic activity and delayed rather than accelerated flowering when overexpressed (19 lines). See Supplemental Figure 3 for fluorescence levels.

Figure 2.

Figure 2.

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Flowering Times of T1 and T2 Lines.

(A) Flowering times of T1 transformants and controls in short days. Light green and pink indicate distribution of positive and negative controls. The two cohorts on the right are a subset of the 35S:mFT-YFP population shown in the middle. Note that many plants with barely detectable fluorescence also flowered very early but were not considered further (see Figure 1 and Supplemental Figure 3).

(B) Flowering time ranges of selected T2 lines in long days. Ochre indicates range between negative control and ft-10. Red type indicates residues subsequently investigated in detail. Lines in dark gray are statistically significantly later than ft-10 in a two-tailed Student’s t test with Bonferroni correction. Error bars indicate sd. See also Supplemental Table 1.

Given the fraction of nonsynonymous mutations determined by sequencing, at least 40% of all transformants were expected to flower similarly early as the positive control. Indeed, the majority of T1 plants flowered with fewer than nine leaves, as seen in 99.3% of positive controls. These were assumed to be early-flowering and to have retained substantial FT activity, even though some had only barely detectable YFP fluorescence (Figure 2A; Supplemental Figure 3).

Only a minority of T1 plants, <4%, flowered in the same range as the negative controls, with 17 leaves or more and were classified as not early flowering. Fifty-two T1 plants with strong or intermediate fluorescence, representing 33 unique mutations, fell into this class. To distinguish mutations that inactivated FT activity from those that interfered with normal FT function in a dominant-negative manner or that had gained neomorphic activity, the T2 progeny from these transformants was evaluated in long days (Figure 2B; Supplemental Table 1), where FT function is required for the acceleration of flowering (Koornneef et al., 1991). The transgene in 14 of these T2 lines had apparently lost FT activity, and plants flowered with fewer than 13 leaves on average, while 19 transgenes had dominant-negative or neomorphic activity and caused plants to flower with on average 24 or more leaves, with some flowering even later than ft-10, a null allele (Yoo et al., 2005).

The FT coding regions from the mutated transgenes in all three categories (wild type, loss of function, and gain of function) were amplified by PCR, and the products were individually sequenced. Out of the 175 codons, 472 unique substitutions in 166 codons were found (Supplemental Figure 4). One hundred and fifty mutants had nonsynonymous mutations in more than one codon. In the 245 mutants with retained FT activity, 155 codons were affected, in the 14 mutants with reduced FT function, 11 codons were affected, and in the 19 mutants with anti- or neomorphic activity, 12 codons were affected (Supplemental Figure 4). In some cases, which are discussed in more detail below, different substitutions at the same codon differed in their effects on FT activity.

Targeted Mutagenesis

Twelve of fifteen unique anti- or neomorphic mutations were found at or close to the potential ligand binding pocket and the adjacent domains encoded by segments B and C of the fourth exon (Ahn et al., 2006). The Adaptive Poisson-Boltzmann Solver software (Baker et al., 2001) predicted this surface to have mostly negative charge (Supplemental Figure 5). Intriguingly, at least 11 of the anti- or neomorphic activity mutations (Y85H, E109K, Q127R, L128H, W138R, Q140H, Q140K, E146K, N152K, L155R, and R173E) changed the charge of the corresponding residues, suggesting that they might also affect overall surface charge of FT. Therefore, we modeled such changes by mutating in silico 170 of the 175 residues of FT. Point mutations predicted to change the surface charge were then engineered into the FT open reading frame by site-directed mutagenesis. In this series of experiments, we did not use the YFP tag, to avoid any potential confounding effects due to the more than doubling in size of FT-YFP relative to native FT. In addition, we used site-directed mutagenesis to confirm the effects of mutations in 108 residues that are highly conserved in FT homologs (Supplemental Figure 1). Altogether, 228 mutations at 122 codons were introduced into wild-type plants. Some mutations at conserved positions did not affect the ability of FT to induce early flowering but reduced the extent to which terminal flowers were formed, while others had no detectable effects on FT function at all (Supplemental Table 2). Only a relatively small number, mutations at 17 out of 108 highly conserved residues, reduced both the ability of FT to accelerate flowering in short days and to convert the indeterminate shoot meristem into a determinate floral meristem. Because plants transformed with these variants did not have any of the features seen in TFL1 overexpressors (Ratcliffe et al., 1998), we infer that these were loss-of-function mutations.

Mutations Near the Entrance of the Potential Ligand Binding Pocket

The entrance of the potential ligand binding pocket is important for FT- or TFL1-like activity, as a Y85H substitution partially confers TFL1 properties onto FT (Hanzawa et al., 2005). Both Tyr-85 in FT and the corresponding His-88 in TFL1 form hydrogen bonds with the orthologous residues Glu-109 and Glu-112, but only His-88 forms a second hydrogen bond with Asp-144 in TFL1, while Tyr-85 and Gln-140 (which corresponds to Asp-144) in FT do not (Ahn et al., 2006) (Figure 3). Importantly, both pairs of residues, Y85/Q140 and H88/D144, are diagnostic for FT- and TFL1-like proteins (with the exception of Brassica TFL1 proteins, which have an Arg instead of a His at position 88 (Mimida et al., 1999). The importance of hydrogen bonds was confirmed by an Y85A substitution (Figure 3), which largely inactivated FT (Supplemental Table 2). We also examined a Y85W substitution; similar to the side groups of Tyr-85 and His-88, the indole side group of Trp can support π-π stacking, but it should form a hydrogen bond only with Glu-109 (Figure 3). In contrast with Y85A, Y85W had partial FT activity, supporting the hypothesis that the intramolecular bond with Glu-109 is important for FT function (Supplemental Table 2). We note that with regard to the Y85H mutation, the 2.9-Å hydrogen bond between His-88 and Asp-144 in TFL1 (Ahn et al., 2006) would be replaced by a weaker interaction between the equivalent His-85 and Gln-140 in FT, with a predicted length of more than 3.5 Å (Figure 3). This may explain the relatively weak TFL1-like activity of Y85H.

Figure 3.

Figure 3.

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Intramolecular Hydrogen Bonds.

Effects of Tyr-85 mutations are shown; wild-type FT is at the far left, and wild-type TFL1 is at the far right. Hydrogen bonds are shown as dashed lines. Hot pink indicates FT residues, cyan TFL1 residues, and green other substitutions.

Among the newly identified residues that could be mutated to confer TFL1-like properties, we decided to focus on six positions that are invariant or almost invariant among FT homologs. The first two are Glu-109, which shows similar interactions in FT and TFL1, and Gln-140, which does not. The surface surrounding these two residues is strongly negative in charge in FT, but positive in TFL1 (Figure 4). Notably, even though Glu-109 in FT corresponds to Glu-112 in TFL1, charge reversal through a E109K mutation caused FT to behave like TFL1 (Figure 5). Similar effects were seen when hydrophobic side chains were introduced (E109A and E109M), predicted to cause a localized expansion of positive charge (Figure 4). By contrast, E109D, which preserves the negative charge, did not affect FT activity. Compared with Glu-109, Gln-140 tolerates both negative and neutral substitutions (Q140D and Q140A) but not positive ones (Q140K and Q140R) (Figures 4 and 5; Supplemental Table 3). Furthermore, charge reversion deep inside the ligand binding pocket (such as D71N) did not convert FT into a TFL1-like molecule (Supplemental Table 2). This is reminiscent of the observation that binding of the human PEBP homolog Raf kinase inhibitor (RKIP) to Raf-1 relies on residues near but not inside the ligand binding pocket (Tavel et al., 2012).

Figure 4.

Figure 4.

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Predicted Effects of Point Mutations on Surface Electrostatic Potential of FT.

Residues tested in each structure are highlighted in yellow and the external loop and FT sequence signature from segment 4C (Ahn et al., 2006) in purple in the ribbon representation on the far left. The ligand binding pocket is indicated by an asterisk where visible. TFL1 is shown for comparison on the right. FT variants with FT-like activity are labeled red, those with TFL1-like activity blue, and reduced- or loss-of-function variants in black.

Figure 5.

Figure 5.

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Effects of Side Chain Properties at Critical Positions on FT Activity.

Flowering time ranges of T1 transformants overexpressing FT variants and controls in short days. Far left, controls; negative control is empty vector with CaMV35S promoter. Middle, charge-dependent positions; right, charge-independent positions. T1 plants were grown in short days. The mutants indicated in ochre were not significantly earlier than 35S:TFL1 using two-tailed Student’s t test with Bonferroni correction. Error bars indicate ± sd.

Mutations Distant from the Potential Ligand Binding Pocket

The next two residues we investigated in detail were Leu-128 and Asn-152, which are positioned in an external loop and an adjacent α-helix. The external loop including Leu-128 is under strong purifying selection and almost invariant in FT proteins, but it is under relaxed selection and very variable in TFL1 proteins (Ahn et al., 2006). The only tolerated mutation was a very conservative substitution, L128I, while introduction of a charged Lys, as in TFL1 (L128K), bestowed weak TFL1-like activity on the mutant protein (Supplemental Table 3). Different from the external loops, the adjacent α-helices are very similar in FT and TFL1. However, although Asn-152 is strongly conserved in FT, but not TFL1 orthologs, mutations to negative (N152D), neutral (N152A), hydrophilic (N152S), or aromatic (N152Y) side chains were all largely tolerated. The exception was N152K, which in contrast with the other mutations strongly affected surface charge and completely converted FT into a TFL1-like floral repressor (Figures 4 and 5).

A beautiful example of Darwinian selection having changed a floral activator in the FT/TFL1 family into a floral repressor comes from sugar beet, where floral repressing activity of one of the two FT orthologs, Bv-FT1, is associated with three mutations in the external loop corresponding to Y134N, G137Q, and W138Q in Arabidopsis FT. Simultaneous mutation of the three residues converts Bv-FT1 into a floral activator, while the opposite mutations in the activator Bv-FT2 impart floral repressive activity (Pin et al., 2010), in line with our previous demonstration of the external loop as being essential for discriminating between FT- and TFL-like activities (Ahn et al., 2006). Based on heterologous overexpression in Arabidopsis, it has been suggested that only a Y134N/W138Q double mutation can turn Bv-FT2 into a floral repressor (Klintenäs et al., 2012).

To dissect the contributions made by individual residues in the external loop, we introduced mutations at all three positions. We found that neither G137A, G137W, G137E (which is similar to G137Q), nor G137R conferred repressive activity on Arabidopsis FT, while Y134A and Y134K as well as W138E, W138S, and W138A do (Supplemental Table 3). That mutating either Tyr-134 or Trp-138 was sufficient for converting FT into a TFL1-like molecule is in agreement with the hypothesis that the external loop functions as a molecular interface with other proteins only in FT, but not in TFL1 (Ahn et al., 2006). However, unlike the Glu-109 and Gln-140 residues near the ligand binding pocket or Leu-128 and Asn-152 in and next to the external loop, the activities of Tyr-134 or Trp-138 were independent of the surface charge change (Figure 5; Supplemental Figure 6). They share with Y85 the characteristic of an aromatic side chain, and all three are close in the FT structure. Different from Tyr-85, which is embedded in the binding pocket entrance, the side groups of Tyr-134 and Trp-138 are protruding and easily accessible, suggesting roles in mediating interactions with other molecules.

Finally, to confirm that the TFL1-like mutants discussed above, E109K, Y134K, W138E, Q140K, and N152K, had full TFL1-like activity, we expressed them in a TFL1 genomic context with 2.2-kb upstream and 4.6-kb downstream sequences. Even though it has not been shown directly that these sequences recapitulate the native TFL1 expression domain, they are sufficient for phenotypic complementation of tfl1 mutants without inducing gain-of-function phenotypes (Sohn et al., 2007; Kaufmann et al., 2010). Inactivation of FT and its paralog TWIN SISTER OF FT (TSF) greatly delays flowering but does not affect flower and shoot morphology, as overexpression of TFL1 does (Figure 6A), allowing differentiation between antimorphic, dominant-negative FT mutants and neomorphic FT mutants with TFL1-like activity. All five mutants behaved very differently from normal or loss-of-function variants of FT expressed from TFL1 regulatory sequences and complemented the tfl1-1 mutant defects (Figure 6B; Supplemental Table 4), confirming that TFL1-like activity did not require constitutive overexpression.

Figure 6.

Figure 6.

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FT Mutants with TFL1-Like Activity.

(A) Comparison of inflorescence tips of plants overexpressing TFL1, FT, or FT mutants with TFL1-like activity. ft-10 tsf-1 is shown for comparison on the far right. Asterisks indicate leafy shoots typical for 35S:TFL1 plants (Ratcliffe et al., 1998). Arrow points to terminal flower in 35S:FT.

(B) Complementation of tfl1-1 mutant with constructs using TFL1 genomic sequences. The bottom panel shows top views of inflorescences.

Correlation of TCP Interaction with FT Surface Charge

Several of the mutations that endow FT mutants with TFL1-like activity, especially E109K and Q140K, were predicted to drastically alter the charge of a single surface near the potential ligand binding pocket. So far, two types of proteins that interact with both FT and TFL1 orthologs have been described in detail: 14-3-3/GENERAL REGULATORY FACTOR (GRF) proteins and the FD transcription factor (Pnueli et al., 2001; Abe et al., 2005; Wigge et al., 2005). Based on studies with a small FT peptide, it has been suggested that the interaction of FT and TFL1 with FD is thought to be indirect, mediated by the 14-3-3 adaptor protein, with yeast 14-3-3 being able to substitute for the plant 14-3-3 in yeast two-hybrid assays (Taoka et al., 2011). We tested protein interactions in quantitative yeast two-hybrid assays and in qualitative bimolecular luminescence complementation assays after transient expression in Nicotiana benthamiana. As expected, FT, TFL1, the TFL-1–like FT mutant Q140K as well as the FT mutant Q140D, which is predicted to have the same surface charge as native FT, all interacted similarly well with several 14-3-3/GRF proteins and with FD (Figures 7 and 8; Supplemental Figure 7).

Figure 7.

Figure 7.

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Yeast Two-Hybrid Interaction Assays.

Results from six independent replicates. The darker gray line is for comparison to GRF results. Symbols (‡) indicate significant differences between vertical comparisons using two-tailed Student’s t test with Bonferroni correction at P < 0.001. Error bars indicate ±sd.

Figure 8.

Figure 8.

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Bimolecular Luciferase Complementation Assays.

Young but fully expanded 4-week-old leaves were strictly chosen for infiltration. Experiments were repeated twice. Complete set of assay results is shown in Supplemental Figure 7.

Recently, a diverse set of TCP proteins, from both miR319-targeted and non-miR319-targeted clades (Martín-Trillo and Cubas, 2010), has been shown to interact with FT (Mimida et al., 2011; Niwa et al., 2013). At least one, TCP18/BRANCHED1 (BRC1), interacts specifically with FT but not TFL1 (Niwa et al., 2013). Furthermore, several of these affect flowering time (Efroni et al., 2008; Schommer et al., 2008; Niwa et al., 2013) (Supplemental Table 5). TCPs are thus candidates for discriminating between FT and TFL1 proteins based on their surface charge. We tested all 24 TCP proteins encoded in the Arabidopsis genome for interaction with FT in yeast two-hybrid assays; nine TCP proteins from class I and one from class II interacted with FT at a level that was comparable to that of FD and the majority of 14-3-3/GRF proteins (Figure 7). In seven cases, the interactions with TFL1 were significantly weaker, and a similar trend was seen with the TFL1 mimic FT (Q140K), but not with the Q140D mutant, which retains FT activity. By contrast, no statistically significant differences were seen for the interaction with GRF proteins or FD. The same trends were observed in qualitative assays using bimolecular luminescence complementation assays in N. benthamiana (Figure 8; Supplemental Figure 7).

Effect of Expression Domain on Mutant FT Activity

FT protein likely travels from its site expression in phloem companion cells via the sieve tubes to the shoot apex, where it forms an active complex with the FD transcription factor (Takada and Goto, 2003; Abe et al., 2005; Wigge et al., 2005; Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007; Notaguchi et al., 2008). Because the export of FT from the phloem companion cells has recently been shown to be regulated (Liu et al., 2012), we wanted to ascertain whether any of the mutations were likely to specifically affect FT mobility. To this end, we expressed the 228 variants from the targeted mutagenesis collection under the companion cell-specific SUCROSE-PROTON SYMPORTER2 (SUC2) promoter and shoot apical meristem–specific FD promoter and compared the effects with those resulting from constitutive expression from the CaMV35S promoter (Odell et al., 1985; Imlau et al., 1999; Mathieu et al., 2007). While in the vast majority there was very good correlation between the three promoters, two mutations stood out: D17K and V18A, where the SUC2 promoter driven mutants seemed to be much less effective than the FD or CaMV35S promoter–driven mutants (Supplemental Figure 8 and Supplemental Table 6). This was reminiscent of what has been observed for FT variants with limited mobility due to the addition of large protein tags (Jaeger and Wigge, 2007; Mathieu et al., 2007; Notaguchi et al., 2008). The two adjacent conserved residues Asp-17 and Val-18 are thus good candidates for interaction with proteins such as FT-INTERACTING PROTEIN, which regulates FT export from the phloem (Liu et al., 2012). In addition, we also found five mutants where the FD promoter construct was less effective than the CaMV35S or SUC2 promoter constructs (N39A, N39D, R44F, G57H, G58E, and D60H; Supplemental Table 6). As there was a general tendency for the FD promoter constructs to have weaker effects, these five mutants are more difficult to interpret.

DISCUSSION

The closely related FT and TFL1 proteins have opposite functions, with FT strongly promoting flowering and TFL1 repressing flowering as well as suppressing floral identity (Shannon and Meeks-Wagner, 1991; Bradley et al., 1997; Ohshima et al., 1997; Ratcliffe et al., 1998; Kardailsky et al., 1999; Kobayashi et al., 1999). Previous work identified individual residues that reduce the floral promoting activity of FT as well as larger segments that impart floral repressing activity on FT, with only the larger changes converting FT completely into a TFL1-like molecule (Hanzawa et al., 2005; Ahn et al., 2006; Pin et al., 2010). Here, we demonstrated that despite 39 nonconservative amino acid differences between FT and TFL1, specific mutations in each of four critical residues, Glu-109, Trp-138, Gln-140, and Asn-152, can turn FT into a TFL1-like floral repressor. The relevant mutations at all four positions have strong effects on protein surface charge. It is striking that the effect of the E109K mutation could not have been predicted based on a sequence comparison between FT and TFL1, as the orthologous position in TFL1 is also occupied by a Glu.

It has been noted before that the conformation of the potential ligand binding pocket does not readily explain differences in FT and TFL1 activity (Ahn et al., 2006) but that the external loop, which is critical for FT and TFL1 function, could be involved in regulating access of a potential ligand to the binding pocket. The surfaces adjacent to the ligand binding pocket as well as the external loop have the same negative charge as the ligand binding pocket only in FT, suggesting that FT, but not TFL1, would support access by a positively charged coactivator. The two aromatic residues Tyr-134 and Trp-138 found only on FT may further increase selectivity for a coactivator with corresponding aromatic side groups that can engage in π-π stacking (Figure 9). This repulsion/lock model thus makes explicit predictions about the molecular details of FT and TFL1 interaction with so far unknown coregulators. Whether TFL1 is merely unable to effectively bind this coactivator, as proposed before (Ahn et al., 2006), or actively recruits a corepressor remains unclear.

Figure 9.

Figure 9.

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Repulsion/Lock Models.

FT binds a coactivator, while TFL1 may either merely exclude a coactivator (middle) or attract a corepressor (bottom). Blue color represents positively charged, while red and pink represent strongly and more weakly negatively charged regions. Gray arrows indicate electrostatic interactions. π-π represents stacking between aromatic groups. AR, aromatic side chain.

There has been conflicting evidence whether FD protein interacts similarly well with FT and TFL1 (Abe et al., 2005; Wigge et al., 2005; Hanano and Goto, 2011). It has recently been shown that FD is genetically required for the effects of TFL1 overexpression (Jaeger et al., 2013). This may explain why plants that lack activity of both FD and its paralog FDP flower substantially earlier than mutants in which FT and its close paralog TSF are inactivated (Yoo et al., 2005; Jang et al., 2009; Jaeger et al., 2013), since ft tfl1 double mutants flower at an intermediate time relative to the ft and tfl1 single mutants (Hanzawa et al., 2005; Ahn et al., 2006). Thus, FD and FDP are unlikely candidates for discriminating between FT and TFL1 effects, in agreement with our observation that changing the surface charges of FT does not have a major impact on interaction with FD.

It is possible that FT directly recruits the basic transcription activation machinery through interaction with a positively charged subunit, similar to fusions of FT or TFL1, which have hyper- and antimorphic activity, respectively (Wigge et al., 2005; Hanano and Goto, 2011). Alternatively, their opposite activities might be mediated by differential recruitment of specific transcription factors. However, increasing evidence supports recruitment of bridging molecules, such as TCP proteins (Kosugi and Ohashi, 1997; Cubas et al., 1999). It has been reported that TCP18/BRC1 interacts in yeast with FT and TSF, but not with TFL1, and genetic studies have shown that BRC1 functions apparently as an inhibitor of FT/TSF activity in lateral shoots (Niwa et al., 2013). We have found additional TCPs that interact more strongly with FT than with TFL1 in both yeast and in transient plant assays, with most of them belonging to class I. Flowering defects have been described for class II TCPs, although the molecular targets are unknown (Palatnik et al., 2007; Efroni et al., 2008; Koyama et al., 2010; Niwa et al., 2013). Common to both class I and class I TCPs are effects on cell proliferation (Martín-Trillo and Cubas, 2010), and there is evidence for FT and TFL1 being able to affect more general growth processes (Pnueli et al., 1998, 2001; Lifschitz et al., 2006; Kumar et al., 2012). Further experiments are required both for identifying shared genomic targets of FT, TFL1 and TCPs, and for substantiating the generality of FT/TFL1 interactions with TCPs in vivo.

In conclusion, we presented evidence for surface charge being a critical determinant of FT and TFL1 function, likely affecting the recruitment of transcriptional coactivators and corepressors. In addition, our work confirms the power of large-scale random mutagenesis experiments coupled with functional tests in transgenic plants for the identification of structural activity determinants (Mateos et al., 2010).

METHODS

Plant Material and Growth Conditions

The wild type was Arabidopsis thaliana Columbia-0. T-DNA insertion lines were obtained from the Nottingham Arabidopsis Stock Center unless otherwise specified. tcp5 tcp13 tcp17 and tcp2 tcp4 tcp10 triple mutants have been described (Efroni et al., 2008; Schommer et al., 2008). Plants were cultivated under a 1:2 mixture of cool white and warm fluorescent light, with a fluence rate of 125 to 175 µmol m−2 s−1. Plants were grown at 65% humidity under long (16 h of light) or short days (8 h of light) at 23°C. Transgenic Arabidopsis plants were generated with the floral dip method (Clough and Bent, 1998). Transgenic seedlings were selected on soil with 0.05% glufosinate (Basta).

Plasmid Construction

To construct 35S:HA:mFT:(GS)4:YFP:rbcs for random mutagenesis, a mCitrine version of YFP (Griesbeck et al., 2001) preceded by a multicloning site (MCS) was cloned into pGEM-T Easy (Promega) (pWH26). The CaMV35S promoter and rbcs terminator were amplified and inserted into ApaI-SacII and SpeI-PstI sites of pWH26, and the resulting gene cassettes were shuttled into pFK202, a derivative of pGREEN-IIS (Hellens et al., 2000), yielding pWH31. PCR was used to insert sequences coding for an HA tag at the N terminus and a (Gly-Ser)4 linker at the C terminus of FT protein. This fragment was cloned into pGEM-T Easy (pWH25). Using primers in the HA and GS4 sequences, PCR-mediated random mutagenesis was performed with the GeneMorph II random mutagenesis kit (Clontech). PCR products were digested and ligated into pWH31 linearized with HindIII-XhoI (pWH32). pWH31 was used as empty vector control, while the positive control was made in the same way as pWH32 but using Pfu DNA Pol (Fermentas) for FT amplification (pWH33).

For site-directed mutagenesis of the central portion of FT, overlap extension PCR was performed (Ho et al., 1989). For site-directed mutagenesis of the termini, single mutagenic primers were used. The PCR products were cloned into entry vector pJLBlueF and recombined into pGREEN-IIS with CaMV35S, SUC2, or FD promoters (Mathieu et al., 2007) using LR Clonase II (Invitrogen).

For tfl1-1 complementation, the SUC2 promoter in an pGREEN-IIS destination vector (de Felippes et al., 2011) was released by XhoI-PstI excision and replaced by 2.2-kb upstream sequences of TFL1 (pWH1352). The 4.6-kb downstream sequences essential for TFL1 expression (Kaufmann et al., 2010) and a MCS (PstI-NotI-SmaI-SalI-NcoI-BamHI) were inserted into SpeI-SacI and PstI/BamHI sites of pWH1352 (pWH1354). Sequences of FT, mFT, and TFL1 from start to stop codon were inserted into NotI-NcoI and PstI-SpeI sites of pWH1354.

To facilitate subsequent cloning, pairs of new entry and destination vectors were made for yeast growth and quantitative ortho-nitrophenyl-β-galactoside assays. For the entry vector, an ApaI site was removed from a self-ligated pCR8/GW/TOPO vector (Invitrogen) by treatment of S1 nuclease (Fermentas) (pEN1). A linker with MCS (EcoRI overhang-2 bp-NotI-NcoI-KpnI-HindIII-SmaI-BglII-PstI-ApaI-SalI-SpeI-1 bp-EcoRI overhang) was ligated to pEN1 linearized by EcoRI. The entry vector with forward-oriented MCS was named pEN2F. For destination vectors, MCS of pGBKT7:GAL4_DBD and pGADT7:GAL4_AD (Clontech) were replaced by Gateway cloning sites (attR:ccdB-CmR) flanked by NdeI-NotI (pWH1016) and NdeI-XhoI (pWH1017) sites. cDNA was synthesized from wild-type RNA using the SuperScript II cDNA synthesis kit (Invitrogen). GRF1-15 and TCP1-24 (except TCP9, 11, and 21) were amplified from the cDNA and cloned into pEN2F using NotI-XhoI:SalI sites. TCP9, 11, and 21 were cloned into pEN2F using NotI-SpeI sites. FT, mFT, FD, and TFL1 were cloned into pEN2F using HindIII-XhoI sites. Entry clones of GRF1-15, TCP1-24, and FD were recombined to pWH1017 and transformed to yeast strain AH109. FT, Q140K, Q140D, and TFL1 were recombined into pWH1016 and transformed into yeast strain Y187 for mating (Clontech).

For the bimolecular luciferase complementation assays, destination vectors were constructed by replacing the MCS of JW771 (35S:LUCn) and JW772 (35S:LUCc) (Gou et al., 2011) with Gateway cloning sites (attR:ccdB-CmR) flanked by KpnI-SalI (pWH1354) and KpnI-PstI (pWH1353) sites. FT, TFL1, Q140K, and Q140D were cloned into pEN2F using HindIII-XhoI sites. Entry clones of GRF1-15, TCP1-24, and FD (from the yeast two-hybrid set) were recombined into pWH1353, while FT, Q140K, Q140D, and TFL1 were recombined into pWH1354. Expression clones were transformed to Agrobacterium tumefaciens GV3101 for infiltration of Nicotiana benthamiana.

Primers used for vector construction are shown in Supplemental Table 7.

Fluorescence Measurement

A Leica MZ FLIII stereomicroscope was calibrated each time using leaves of transgenic plants transformed with pWH33 (positive control) and pWH31 (negative control) to define maximal as well as background fluorescence. Rosette leaves (at four- to six-leaves stage) from each plant were freshly excised in dim light. Transgenic plants were examined for the expression of YFP using the stereomicroscope fitted with wide- and band-pass YFP filters and an AxioCam HRc digital camera (Carl Zeiss) with AxioVison software (version 3.1).

In Silico FT Mutagenesis and Electrostatic Potential Calculation

Three-dimensional structures of wild-type FT (entry 1WKP) and TFL1 (entry 1WKO) were obtained from the RCSB Protein Data Bank and were visualized by PyMOL molecular graphics system (version 1.5.0.4; Schrödinger) (Ahn et al., 2006). During the in silico mutagenesis, first the backbone-dependent rotamer representing the highest frequencies of occurrence in proteins was adopted, unless otherwise specified. For the electrostatic potential calculation, the Adaptive Poisson-Boltzmann Solver plug-in was applied (Baker et al., 2001). Before calculation, any water and solvent molecules that disturb the native potential of proteins found in the visualization were removed. PDB2PQR and default grid settings were applied for the calculations.

Yeast Two-Hybrid Assays

β-Galactosidase activity was quantified using ortho-nitrophenyl-β-galactoside assays as described in the Yeast Protocols Handbook (Clontech), with at least six replicates.

Bimolecular Luciferase Complementation Assays

N. benthamiana was transiently transformed as described (Voinnet et al., 2003), and samples were analyzed 4 d after inoculation (Gou et al., 2011). Before measurement, leaves only infiltrated with luciferin (negative control) and leaves infiltrated with a 35S:LUC construct and luciferin (positive control) were used for calibrating the Orca 2-BT cooled charge-coupled device camera (Hamamatsu Photonics). At least three replicates were performed on independent plants for each assay.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: FT (At1g65480), TFL1 (At5g03840), FD (At4g35900), GRF1 (At4g09000), GRF2 (At1g78300), GRF3 (At5g38480), GRF4 (At1g35160), GRF5 (At5g16050), GRF6 (At5g10450), GRF7 (At3g02520), GRF8 (At5g65430), GRF9 (At2g42590), GRF10 (At1g22300), GRF11 (At1g34760), GRF12 (At1g26480), GRF13 (At1g78220), GRF14 (At1g22290), GRF15 (At2g10450), TCP1 (At1g67260), TCP2 (At4g18390), TCP3 (At1g53230), TCP4 (At3g15030), TCP5 (At5g60970), TCP6 (At5g41030), TCP7 (At5g23280), TCP8 (At1g58100), TCP9 (At2g45680), TCP10 (At2g31070), TCP11 (At2g37000), TCP12 (At1g68800), TCP13 (At3g02150), TCP14 (At3g47620), TCP15 (At1g69690), TCP16 (At3g45150), TCP17 (At5g08070), TCP18 (At3g18550), TCP19 (At5g51910), TCP20 (At3g27010), TCP21 (At5g08330), TCP22 (At1g72010), TCP23 (At1g35560), and TCP24 (At1g30210).

Supplemental Data

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Supplementary Material

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Acknowledgments

We thank Pauline Ip and Rebecca Schwab for support, Robert Leo Brady for communicating unpublished findings, Markus Schmid for the mCitrine clone, Yuval Eshed for seeds, and Yasushi Kobayashi for FT variant P75L. We also thank Michael Christie, Yasushi Kobayashi, Roosa Laitinen, Sascha Laubinger, Ignacio Rubio, Markus Schmid, Fritz Schöffl, Rebecca Schwab, and Jiawei Wang for advice and comments on the article. W.W.H.H. was supported by a PhD scholarship from the Croucher Foundation. This work was supported by the Max Planck Society.

AUTHOR CONTRIBUTIONS

W.W.H.H. and D.W. designed research. W.W.H.H. performed research. W.W.H.H. and D.W. analyzed data. W.W.H.H. and D.W. wrote the article.

Glossary

CaMV35S

cauliflower mosaic virus 35S

MCS

multicloning site

Footnotes

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