JXB Advance Access originally published online on November 16, 2005
Journal of Experimental Botany 2006 57(1):33-42; doi:10.1093/jxb/erj011
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FOCUS PAPER |
In vivo imaging of MADS-box transcription factor interactions
1Business Unit Bioscience, Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands
2Department of Biochemistry, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands
3Microspectroscopy Centre, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands
* To whom correspondence should be addressed. E-mail: Richard.Immink{at}wur.nl
Received 24 May 2005; Accepted 3 October 2005
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Abstract |
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MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein–protein and protein–DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer–fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.
Key words: FRET-FLIM, in vivo imaging, MADS-box transcription factors
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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MADS-box genes represent a large multigene family in flowering plants and are involved in numerous developmental processes. In angiosperms, many of the genes belonging to this transcription factor family are involved in flower development, most notably in the determination of floral meristem and floral organ identity (Riechmann and Meyerowitz, 1997
; Ferrario et al., 2004a). The complete Arabidopsis genome sequence revealed the existence of over 100 MADS-box genes (De Bodt et al., 2003; Kofuji et al., 2003; Martinez-Castilla and Alvarez-Buylla et al., 2003; Pa
enicová et al., 2003). During the last decade many members of the family have been subject to genetic studies in various plant species, which has led to the robust ‘ABC’ model as the paradigm for flower development in angiosperms. In addition, the functional characterization of a number of MADS-box genes has revealed regulatory roles for other MADS-box genes in flower induction, meristem formation, and fruit development. By contrast to the enormous effort that has been put into this kind of analysis over the last decade, giving rise to detailed knowledge about MADS-box gene functions, virtually nothing is known about the molecular mode of action of the encoded proteins.
Analyses of MADS-box proteins have been mainly restricted to the MIKC type, which has a characteristic modular structure. From the N-terminus to the C-terminus of the protein, four domains can be identified: the MADS-box (M), intervening (I), keratin-like (K), and C-terminal domains (Riechmann and Meyerowitz, 1997). The M-domain is the most conserved among all domains and consists of 
56–58 amino acids. It plays an important role in DNA binding and probably a minor role in dimerization. The I-domain is less conserved, varies in length, and is important for determining the dimerization specificity (Riechmann and Meyerowitz, 1997). The K-domain (80 amino acids) contains several heptad repeats that most likely fold into amphipathic 
-helices, which mediate dimerization (Yang et al., 2003). The C-terminal domain is the least conserved and it has been shown that it is able to act as a transactivation motif for some of the plant MADS-box proteins and, furthermore, it appears to be involved in higher-order complex formation (Egea-Cortines et al., 1999; Honma and Goto, 2001; Yang and Jack, 2004).
The first studies aimed at the elucidation of the molecular mechanisms underlying MADS-box protein functioning, using in vitro DNA binding approaches, revealed that these transcription factors form specific dimers (Schwarz-Sommer et al., 1992; Krizek and Meyerowitz, 1996; Riechmann et al., 1996; West et al., 1998; Egea-Cortines et al., 1999). In addition, the yeast two-hybrid system has been adopted very frequently to obtain information about MADS-box protein–protein interactions. Comprehensive matrix-based screens for petunia and Arabidopsis MADS-box transcription factor interactions have shown that these factors form specific homo- and heterodimers and that these interactions are conserved between different plant species (Immink et al., 2003; De Folter et al., 2005). A further complexity was proposed, based on results obtained with Antirrhinum and Arabidopsis MADS-box proteins in yeast experiments (Egea-Cortines et al., 1999; Honma and Goto, 2001). These experiments revealed that additional MADS-box proteins may bind to a dimer at the C-terminus forming a ternary or quaternary complex. Like dimerization, this complex formation seems to be conserved, because similar complexes could be identified for petunia, Arabidopsis, and chrysanthemum MADS-box proteins using yeast three- and four-hybrid screenings (Favaro et al., 2003; Ferrario et al., 2003; Shchennikova et al., 2004). This ability of MADS-box proteins to form multimeric complexes suggests that they are active in a combinatorial manner. Based on these findings the ‘quartet model’ for MADS-box transcription factor functioning was hypothesized. According to this model, two dimers within a higher-order tetrameric complex recognize two different binding sites in the DNA sequence, which are brought into close proximity by DNA bending. Elaborating on this model, the control of floral organ identity is supposed to be driven by four different tetrameric transcription factor complexes composed of the ‘ABC’–MADS-box proteins (Theißen, 2001; Theißen and Saedler, 2001).
Despite the fact that yeast screenings can be performed in a high-throughput manner and offer a first glimpse of dimerization patterns and complex formation, they have many drawbacks, especially when it concerns transcription factors that often contain intrinsic transcriptional activation domains. Because of this, yeast methods give rise to false-positive and false-negative results and, therefore, should be verified by in planta studies (Immink and Angenent, 2002). Moreover, the ability to visualize and follow molecules and events in living cells has become an important aspect in cell biology (Lippincott-Schwartz and Patterson, 2003). Recently, innovative microspectroscopic approaches have been developed in order to combine the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules (Gadella et al., 1999; Hink et al., 2002). Fluorescence resonance energy transfer (FRET)-based methods have become a key for the detection of protein–protein interactions in living cells. The principle of this is that excited-state energy is transferred non-radiatively through space from a donor to an acceptor molecule. This energy transfer takes place only if emission and excitation spectra of the fluorophore pair are overlapping and if the distance between the molecules is very small (within 1 to 10 nm of each other). Hence, protein–protein interactions can be studied by fusing the proteins of interest to two fluorescent molecules with the right characteristics (Gadella et al., 1999; Hink et al., 2002). The combination of cyan and yellow fluorescent proteins (CFP and YFP, respectively) has proven to be the best marriage for in planta FRET studies (Immink et al., 2002; Russinova et al., 2004). FRET can be quantified by observing changes in the fluorescence lifetime of the donor using fluorescence lifetime imaging microscopy (FLIM) (Gadella et al., 1993; Borst et al., 2003). In the case of a protein–protein interaction, FRET will occur and the fluorescence lifetime of the donor molecule will decrease. The advantages of FLIM for the detection of FRET are that it is not dependent on changes in probe concentration, and that it is less sensitive to photobleaching, autofluorescence, and other factors that limit intensity-based steady-state analyses (Chen and Periasamy, 2004).
With respect to MADS-box transcription factors, protein interactions in living cells have only been shown for a few petunia MADS-box proteins by means of FRET-spectral photoimaging microscopy (SPIM) and FRET-FLIM analyses (Immink et al., 2002). The ovule-specific FLORAL BINDING PROTEIN11 (FBP11) appeared to interact specifically with three closely related proteins, FBP2, FBP5, and FBP9, that all belong to the SEPALLATA clade of MADS-box proteins (Ferrario et al., 2003). Recently, another ovule-specific MADS-box gene ABS (Arabidopsis B-sister gene; Becker et al., 2002), formerly known as AGL32 and TT16 (Transparent Testa16; Nesi et al., 2002), has been described. The abs mutant is affected in seed coat pigmentation and probably to some extent in the integrity of the entire inner integument. The petunia FBP24 gene appeared to be very close in sequence to ABS and is expressed specifically in ovules (S De Folter and RGH Immink, unpublished results). Currently, it is not known how the ovule-specific FBP24 protein is acting at the molecular level and to which protein complexes it contributes. Therefore, yeast two- and three-hybrid analyses to study FBP24 protein–protein interactions were performed. Subsequently, FBP24 and its putative interacting partners were tagged with fluorescent proteins and expressed in protoplasts, which allowed the analysis of cellular localization and in planta interactions using FRET-FLIM imaging techniques. The results obtained improve our knowledge about plant MADS-box transcription factor functioning at the molecular level and provide information about their dynamics in living plant cells.
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Materials and methods |
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Plant material
The Petunia hybrida line W115 and cowpea variety Black Eye California were grown under normal greenhouse conditions (14/10 h light/dark, 20 °C for petunia and 28 °C for cowpea).
Plasmid construction
All the clonings were done following the GatewayTM system from Invitrogen (Carlsbad). The complete ORFs of the MADS-box genes were PCR amplified using specific primers yielding entry clones. Vectors containing cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) under control of the CaMV 35S promoter (Immink et al., 2002) were made Gateway-compatible according to the Invitrogen manual. In addition, the coding region of the monomeric red fluorescent protein (mRFP) (Campbell et al., 2002) was cloned in the same vector backbone (pGD120). Finally, expression vectors encoding the various MADS-box transcription factors tagged with a C-terminal fused fluorescent protein were obtained by an LR reaction.
Yeast two- and three-hybrid experiments
Two-hybrid analyses using the CytoTrap and the GAL4 system were performed as described previously (Immink et al., 2003). For this purpose the entire FBP24 coding region was cloned in-frame in the pMYR, pSOSnes, and pADGAL4 and pBDGAL4 vectors. FBP24 was screened against 14 petunia MADS-box proteins in the GAL4 system (FBP2, FBP4, FBP5, FBP9, FBP23, pMADS12, FBP6, FBP7, pMADS3, FBP11, FBP26, FBP29, PFG, FBP24). Selection for interaction was performed using the histidine marker in combination with two different concentrations of 3-amino-triazole (3AT, 1 mM and 5 mM), and by the adenine marker. The three-hybrid experiments were done with a modified yeast two-hybrid GAL4 system as described by Ferrario et al. (2003).
Transient expression in cowpea and petunia protoplasts
Cowpea protoplasts were prepared and transfected according to Shah et al. (2001). Petunia protoplasts were obtained from W115 petunia leaves and transfected as described by Immink et al. (2002). Protoplasts were incubated overnight in protoplast medium at 25 °C in the light for cowpea, and in the dark for petunia, and subsequently imaged for fluorescence.
Localization studies in living cells
The imaging of the fluorescent fusion proteins was done using a confocal laser scanning microscope 510 (Carl Zeiss). Protoplasts were excited by 458 nm and 514 nm Ar laser lines for CFP and YFP, respectively. In addition, a 543 nm He laser line was used to excite mRFP. The pinholes were set at one Airy unit which corresponds to a theoretical thickness (full width at half-maximum) of 1 µm. Images and data analyses were performed with Zeiss LSM510 software (version 3.2).
Fluorescence lifetime-imaging microscopy
For FRET-FLIM analyses, cowpea protoplasts were analysed as described by Russinova et al. (2004), using a Bio-Rad Radiance 2100 MP system (Hercules) in combination with a Nikon TE 300 inverted microscope. Two-photon excitation pulses were generated by a Ti:-Sapphire laser (Coherent Mira) that was pumped by a 5 W Coherent Verdi laser. The excitation light was directly coupled to the microscope and focused to the sample by using a CFI Plan Apochromat 60x water immersion objective lens (N.A. 1.2).
The heterodimer between FBP2 and FBP11 and the combination FBP2 and PFG (Petunia Flowering Gene) were used as positive and negative controls, respectively (Immink et al., 2002).
In this study, a two-photon set-up was used and the donor fluorescence lifetime values were measured pixel by pixel. In all cases, measurements were done for the central part of the nucleus where the fluorescence lifetime is not influenced by autofluorescence from the chloroplasts. For each analysis at least 10 representative cells were measured, expressing either a single CFP-labelled MADS-box protein, or a combination of a CFP- and a YFP-labelled protein.
Images with a frame size of 64x64 pixels were acquired using the Becker and Hick1 SPC 830 module, and for the data analysis, the SPCimage 2.8 software was used.
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Results |
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Yeast two- and three-hybrid analyses
To get a first impression about putative FBP24 interaction partners and to select candidates for in vivo studies, yeast two- and three-hybrid analyses were performed. Initially, FBP24 was tested for dimerization with the 23 known petunia MADS-box proteins (Immink et al., 2003
) in the CytoTrap two-hybrid system. Remarkably, none of the couples tested resulted in growth of the yeast at 37 °C, suggesting that a putative FBP24 heterodimerization partner is not present in the collection. Alternatively, FBP24 is able to interact weakly with one of the known MADS-box factors, but it just cannot be detected by the yeast CytoTrap system, due to the relative high assay temperature in this system. Therefore, FBP24 dimerization was tested in the yeast two-hybrid GAL4 system at room temperature. This analysis revealed that FBP24 interacts specifically with FBP2 and FBP4 and is neither able to dimerize in yeast with the ovule-specific and very closely related FBP7 and FBP11 D-type proteins (Angenent et al., 1995) nor the putative C-type proteins FBP6 and pMADS3 (Kater et al., 1998).
Taking into account that for some MADS-box proteins higher-order complexes have been identified, it was wondered whether FBP24 may interact with the ovule-specific D-type proteins in a higher-order complex. To test this ability, a yeast three-hybrid analysis was performed. In this screen the FBP2 protein lacking the C-terminal domain (FBP2
C) was used, because FBP2 contains an intrinsic transcriptional activation domain in this region (Ferrario et al., 2003). Although, the interactions detected were very weak and could be detected at room temperature and low concentrations of 3AT only, the combinations FBP24–FBP11–FBP2 and FBP24–FBP7–FBP2 clearly gave growth of yeast in comparison to the controls (Table 1).
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Localization of MADS-box proteins in living plant cells
In order to analyse the various MADS-box proteins in vivo, the proteins were labelled with CFP, YFP, and the monomeric mRFP at their C-termini. It is known that tagging proteins with fluorescent groups may disturb their physical properties, which may affect the localization and accumulation of the proteins. However, analyses of the MADS-box protein APETALA1 (AP1) labelled with GFP at its C-terminus revealed an active protein that was able to rescue the ap1 mutant. On the other hand, the N-terminal GFP:AP1 fusion appeared to be non-functional and its subcellular localization was abnormal, being mostly cytoplasmic (Wu et al., 2003
). Also C-terminal fusions with Arabidopsis proteins FRUITFULL and AGAMOUS do not affect the biological activity of these MADS proteins (GC Angenent and SL Urbanus, unpublished results). Considering these data and the results from Immink et al. (2002), C-terminal fusions were generated.
Subsequently, the fusion products obtained were transiently expressed in both petunia and cowpea leaf protoplasts. Because similar localizations were obtained in petunia (not shown) and cowpea protoplasts, and cowpea protoplasts are more amenable for transfections than petunia protoplasts, all further experiments were done with cowpea cells. Initially, localization experiments were performed with the single proteins, FBP11, FBP2, and FBP24 (Fig. 1A–C). The proteins FBP2 and FBP24 appear to be localized in the nucleus, whereas FBP11 remains in the cytoplasm. Most likely, the cytoplasmic localization of FBP11 is not due to incorrect folding or inactivity of this protein, because the protein is still able to interact with its heterodimerization partners FBP2, FBP5, and FBP9, and it is transported into the nucleus upon the formation of these heterodimers (Immink et al., 2002). Based on this, it is hypothesized that dimerization is a prerequisite for MADS-box proteins to move into the nucleus, and hence the cytoplasmic localization of FBP11 can be explained by the inability of this protein to homodimerize. Surprisingly, both FBP24 and FBP2, for which no homodimerization could be detected by the yeast two-hybrid experiments, were localized in the nucleus. FRET-FLIM analyses performed in the past for FBP2, revealed that this protein is able to homodimerize in protoplasts and, hence, are transported into the nucleus (Immink et al., 2002).
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In the next step, cells co-transfected with two labelled proteins, for which either dimerization or no interaction could be detected in yeast two-hybrid experiments, were analysed. Nuclear co-localization has already been described for the partners FBP2 and FBP11 (Immink et al., 2002
). The combination FBP2 and FBP24 appeared to result in nuclear co-localization as well (Fig. 1D–G). Surprisingly, both proteins FBP11 and FBP24 were present in the nucleus in the double-transfected cells (Fig. 1H–K), while the single FBP11 protein was localized in the cytoplasm (Fig. 1A). Taking into account the hypothesis that dimerization is essential for transport into the nucleus (Immink et al., 2002), their co-localization suggests heterodimerization. Finally, all three proteins were imaged simultaneously by transient expression of FBP11, FBP2, and FBP24, labelled with different fluorescent molecules. In this case, all three proteins were present in the nucleus (Fig. 1L–P).
FRET-FLIM analyses reveal differences between dimers
Although subcellular co-localization may suggest dimerization and complex formation in living cells, evidence for physical interaction between proteins can only be obtained after application of an appropriate methodology. Therefore, FRET-FLIM analyses were used to detect homodimerization and heterodimerization of the ovule-specific MADS-box transcription factors described above. To calculate the fluorescence lifetime of CFP in the absence of the YFP acceptor, the single proteins FBP2 and FBP24 labelled with the donor molecule CFP were used for protoplast transfections. The fluorescence lifetime values obtained and their distribution over the nucleus were used as a reference for values obtained with the various double transfections. Cells transfected with either FBP2–CFP or FBP24–CFP show a limited variation in fluorescence lifetime values, with an average around 2.45 ns (Fig. 2A–F). The fluorescence lifetime for the negative control, the combination of FBP2 and PFG, appeared to be in the same range; however, the variation in lifetime values for different cells is slightly larger (Fig. 2G–I). For the positive control (FBP2–FBP11), the fluorescence lifetime drops to about 1.9–2.0 ns on average, which can be measured throughout the nucleus (Fig. 2J–L).
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Subsequently, the combination FBP2–CFP and FBP24–YFP was analysed. For this combination, heterodimerization was detected in yeast and co-localization of the proteins was observed in living plant cells (Fig. 1D–G). The FLIM data depicted in Fig. 2M–O show that this combination gives a strong reduction in fluorescence lifetime, demonstrating that these proteins interact in living plant cells. The reciprocal combination (FBP24–CFP and FBP2–YFP) has been tested as well and gave the same result (data not shown). Surprisingly, in the case of the combination FBP11–FBP24 a distribution of different fluorescence lifetime values over the nucleus was observed (Fig. 2P–R), suggesting that there are subnuclear regions with and without interaction between the two proteins. Finally, cells transfected with both FBP24–CFP and FBP24–YFP were analysed, in order to determine whether this protein is able to homodimerize. Interestingly, the same variation of fluorescence lifetime values distributed over the nucleus was found as described for FBP11–FBP24 (Fig. 2S–U).
Competition and higher-order complex formation
It has been proposed that MADS-box proteins are active as multimeric complexes, such as ternary or quaternary complexes (Egea-Cortines et al., 1999). However, if and how these complexes are formed in a plant cell is unknown. The final constitution of the complexes formed is dependent on various characteristics of the proteins and of the processes that play a role, like protein concentration, protein subcellular localization, interaction affinity, the stability of interactions, and the dynamics of complex formation. In turn, all these factors will influence competition between individual proteins for dimerization, and competition for higher-order complex formation between dimers and/or monomers. The present FRET-FLIM analyses clearly revealed differences between dimers with respect to distribution of lifetime values over the nucleus, most likely directed by the above-mentioned processes and protein characteristics. Some combinations display a narrow distribution of lifetime values that are homogenously spread, while others display a wide distribution, with a clearly detectable FRET signal in subnuclear regions only. To get a possible explanation for this difference in distribution, and to get an insight into higher-order complex formation in planta, FLIM studies were done using a non-labelled third factor (FBP2) in combination with FBP11–CFP and FBP24–YFP. This experiment revealed a reduction in fluorescence lifetime, with a more uniform distribution over the nucleus for the triple combination (Fig. 2V–Y versus Fig. 2P–Q). Thus, addition of FBP2 resulted in the detection of a strong FRET signal all over the nucleus for the combinations FBP11–CFP and FBP24–YFP while, without FBP2 the FRET signal was observed in subnuclear spots only.
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Discussion |
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During the last decade, many studies have been performed to identify the genes involved in the regulation of important steps in plant development. Transcription factors belonging to the MADS-box family appeared to play pivotal roles in these processes and can be considered as the main regulators of plant development. Nevertheless, little is known about their behaviour in plant cells at the molecular level and the dynamic process of gene regulation in the nucleus. It has been hypothesized that the MADS-box proteins form specific dimers, which are further assembled into tetrameric complexes (reviewed in Theißen and Saedler, 2001
). Intriguing questions remain whether these complexes are actually formed and how stable these complexes are. In this study, an in vivo approach has been followed to investigate the dynamics of MADS-box transcription factor interactions in a plant cell environment. For this purpose, the petunia MADS-box proteins FBP11, FBP2, and FBP24 that are supposed to be involved in ovule development were selected as the object, and studied upon transient expression in protoplasts. Transient assays should always be performed with the proper controls and be analysed with caution, because the proteins are expressed at relatively high levels. The fact that, in this study, for some MADS-box transcription factor combinations, strong FRET signals were obtained, whereas for other combinations of MADS-box proteins, with similar expression levels and localization patterns, no FRET signal was detected, indicates that interactions can be detected in a specific and sensitive manner and that the approach followed is suitable for the initial analysis of MADS-box transcription factor complex formation in living plant cells.
Initially, yeast two- and three-hybrid experiments were performed to get the first indication about FBP24 complex formation. Surprisingly, no dimerization partner could be detected for FBP24 in the yeast CytoTrap two-hybrid system. In this system the selection for protein–protein interactions is based on the Ras signal transduction cascade and, due to this, the temperature-sensitive yeast strain is able to grow at a relative high temperature of 37 °C upon a protein–protein interaction (Aronheim et al., 1997). A temperature-dependent interaction has been reported for the class B proteins PISTILLATA and APETALA3 from Arabidopsis (Kohalmi et al., 1996), which can be stabilized by the presence of additional MADS-box factors. FBP24 has been designated as a ‘B-sister’ gene (Becker et al., 2002), based on its evolutionary relationship with the class B proteins. The authors' yeast two-hybrid results also point to weak and temperature-sensitive interactions between FBP24 and other MADS-box proteins.
It has been hypothesized that dimerization is a prerequisite for nuclear localization of plant MADS-box transcription factors (Immink et al., 2002; Ferrario et al., 2004b). In line with this, FBP11 that is not able to homodimerize is localized in the cytoplasm, whereas FBP2 molecules form homodimers and are subsequently transported to the nucleus. Despite the fact that FBP24 did not show homodimerization in yeast, it appeared to be localized in the nucleus of plant cells. FRET-FLIM analyses in living plant cells demonstrated, however, that, by contrast to the yeast two-hybrid results, homodimerization could be detected for this protein. Like FBP24, homodimerization of FBP2, FBP5, and FBP9 (Immink et al., 2002) could only be detected in planta and not by a traditional yeast two-hybrid system. A possible explanation for this discrepancy could be that plant MADS-box transcription factor homodimers are, in general, less stable than heterodimers and can be detected only by very sensitive methods. The fact that only five homodimers have been identified in a large-scale yeast two-hybrid screening with over 100 Arabidopsis MADS-box transcription factors (De Folter et al., 2005) supports this observation.
For the putative FBP2–FBP24 heterodimer the yeast result was confirmed by the in planta analysis. A relative low fluorescence lifetime with little variation was observed for this combination, suggesting the formation of a ‘stable’ heterodimer. On the other hand, the proteins FBP11 and FBP24 that are not interacting in yeast give a FRET signal in subnuclear regions in plant cells. A similar kind of fluorescence lifetime distribution over the nucleus, with regions with relatively low fluorescence lifetime values and regions with high fluorescence lifetime values, was observed for the FBP24 homodimer. This kind of fluorescence lifetime value distributions were always found for these two specific combinations in successively performed experiments, but were never obtained for the other dimers tested. The stronger FRET signal in the subnuclear spots is not due to a higher concentration of the fluorescence proteins at these places, because fluorescence intensity measurements showed a more or less equal distribution of signal over the nucleus. Based on this, and the fact that most interactions are dynamic and proteins can associate and dissociate during time, it is hypothesized that the transient and dynamic interaction between FBP11 and FBP24 is stabilized in the specific subnuclear spots. However, at the moment, it is not clear what these subnuclear regions represent. A number of studies indicate that homo- and/or heterodimerization facilitate the binding to specific DNA sequences (Pellegrini et al., 1995; Shore and Sharrocks, 1995) and that higher-order complex formation of MADS-box transcription factors is stabilized by specific DNA binding (Egea-Cortines et al., 1999). It might be possible that the subnuclear regions represent places where the chromatin is available for transcription and to which the transcription factors are recruited, resulting in a more stable association of the transcription factor proteins around and at these sites.
To get an insight into the interaction dynamics, and eventually into higher-order complex formation of MADS-box proteins in planta, a triple transfection experiment was performed in this study. Information about the stability of dimeric MADS-box protein interactions and the influence of additional factors is limited to yeast experiments, and is completely lacking for in planta interactions. Based on differences in FRET signal that were obtained for the individual dimers (FBP2–FBP2, FBP24–FBP24, FBP2–FBP11, FBP2–FBP24, and FBP11–FBP24), it was assumed that competition for dimerization between the individual proteins will occur when more than two factors are co-expressed. When competition for dimerization is the only aspect that plays a role, addition of non-labelled FBP2 to the combination FBP11–CFP+FBP24–YFP will result in less or no dimerization between FBP11 and FBP24 and, hence, an increase in the fluorescence lifetime. However, the fluorescence lifetime was decreased for this specific triple combination demonstrating that FBP11 and FBP24 are still in one and the same complex. This observation and the results of the yeast three-hybrid experiments, suggest that higher-order complex formation plays a role. Most likely, all three proteins are present in one and the same complex and FBP2 is having a stabilizing effect on this complex. Considering these results, some hypotheses can be drawn about complex formation that probably occurs in vivo for the ovule-specific MADS-box transcription factors (Fig. 3). For example, a ternary complex might be formed between the monomers FBP24, FBP11, and FBP2. However, both FBP24 and FBP2 are able to homodimerize and, hence, a quaternary complex involving a heterodimer in combination with either a FBP2 or FBP24 homodimer could theoretically be formed. Nevertheless, the yeast and FRET-FLIM analyses suggest that these homodimers are less stable than the FBP2–FBP11 and FBP2–FBP24 heterodimers. Taking this into account, it is hypothesized that it is more likely that in vivo a quaternary complex is formed by the two very stable dimers FBP24–FBP2 and FBP2–FBP11. A putative quaternary complex like this would fit perfectly in the proposed ‘quartet model’ of MADS-box transcription factor functioning (Egea-Cortines et al., 1999; Honma and Goto, 2001; Theißen and Saedler, 2001).
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In conclusion, this report has demonstrated that the in vivo FRET-FLIM analyses provide more detailed and spatial information about protein–protein interactions than the yeast systems. The experiments performed here give a first glimpse into the behaviour of MADS-box transcription factors in plant cells. Certainly, more analyses are required to get final proof of higher-order complex formation between plant MADS-box transcription factors and to understand the exact stochiometry of these kinds of complexes in the plant tissue where they are active. The question remains: what is the biological function of the complex involving FBP2, FBP11, and FBP24? As mentioned before, FBP24 has a high sequence similarity with ABS from Arabidopsis, which is supposed to play a role in seed coat pigmentation and probably is essential for the formation or maintenance of the endothelial cells (Nesi et al., 2002
). Probably, the petunia homologue FBP24 is required for late ovule development as well, in combination with FBP2 and the ovule-specific protein FBP11. This suggests that FBP11 plays a dual role in ovule development, being involved in the initiation of ovules (Colombo et al., 1995, 1997) and in late ovule development. The higher-order complex identified between FBP11, FBP2, and FBP6 (RGH Immink and GC Angenent, unpublished results) and their Arabidopsis orthologues SEEDSTICK (STK), SEPALATA3 (SEP3), and AGAMOUS (AG) (Favaro et al., 2003), respectively, are supposed to be involved in early ovule function, while the complex between FBP11, FBP2, and FBP24 identified in this study, might be responsible for a late ovule function. |
Acknowledgements |
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We are very grateful to Roger Tsien who kindly provided mRFP, to Stefan de Folter for helpful comments and suggestions on this manuscript, Ronny Joosen for help with the statistical analyses and graphs, and Mark Hink for microscopy analyses. This work has been financially supported by CAPES-Brazil (BEX 0970/01-8), the Netherlands Proteomics Centre (NPC), and a NWO-VENI grant to RGHI.
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References |
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