• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntcellLink to Publisher's site
Plant Cell. Jun 2006; 18(6): 1373–1382.
PMCID: PMC1475495

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in Arabidopsis[W]

Abstract

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30°C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.

INTRODUCTION

The transition from vegetative to reproductive growth is a major developmental switch in the life cycle of plants since it is the key step for the reproductive success of the plant. During the vegetative phase, the shoot apical meristem produces leaf primordia. After perception and processing of several environmental and internal signals, the shoot apical meristem undergoes a change in fate, and an inflorescence meristem is produced. This process is called the floral transition (for reviews, see Komeda, 2004; Putterill et al., 2004; He and Amasino, 2005).

The genetic network controlling the floral transition culminates in the activation of floral meristem identity genes, such as LEAFY (LFY) and APETALA1 (AP1), which subsequently regulate the three classes of floral homeotic genes, A, B, and C (Weigel and Meyerowitz, 1994; Komeda, 2004). These ABC floral homeotic genes function in overlapping domains to specify different floral organ identities. For instance, LFY positively regulates AGAMOUS (AG), the class C gene that is responsible for stamen and carpel identities (Bowman et al., 1991), by binding to sites within the AG control region that is located within the second intron (Busch et al., 1999; Lohmann et al., 2001). LFY, together with AP1, is also required for the activation of the class B gene AP3, which together with PISTILLATA specifies petal and stamen identities (Ng and Yanofsky, 2001; Lamb et al., 2002). AG is regulated by several other factors that all seem to act on the second intron (Sieburth and Meyerowitz, 1997; Deyholos and Sieburth, 2000; Hong et al., 2003; Bao et al., 2004). One of these factors is LEUNIG (LUG), which also regulates the expression of class A and B genes (Liu and Meyerowitz, 1995). LUG encodes a protein with homology to the Tup1 corepressor from yeast and Groucho from Drosophila melanogaster (Conner and Liu, 2000). LUG interacts with SEUSS (SEU) to regulate AG (Sridhar et al., 2004). In contrast with LUG, SEU does not exhibit any direct repressor activity. It is a plant-specific protein that shows sequence similarity to the dimerization domain of the LIM domain binding (Ldb) family of transcriptional coregulators, such as Ldb1 in mouse and Chip in Drosophila (Franks et al., 2002). By analogy with the Ssn6-Tup1 complex (Conlan et al., 1999), it has been suggested that SEU acts as adapter protein between LUG and DNA binding transcription factors (Sridhar et al., 2004).

Here, we report the analysis of two MADS box genes, namely, SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE24 (AGL24), originally isolated and characterized as regulators of the floral transition (Hartmann et al., 2000; Yu et al., 2002; Michaels et al., 2003). Although phylogenetic analysis showed that these genes are closely related (Pařenicová et al., 2003), functional analyses revealed that they have an opposite effect in the control of flowering time. Whereas SVP acts as a repressor of flowering, AGL24 functions as a promoter of flowering. Both genes are expressed in vegetative tissue before the floral transition. SVP exerts its repressive function in a dosage-dependent manner, independently from environmental factors such as daylength or temperature (Hartmann et al., 2000). AGL24 is a dosage-dependent flowering promoter and is gradually activated in the shoot apical meristem during the floral transition (Yu et al., 2002; Michaels et al., 2003). It is one of the key genes in promoting inflorescence fate (Yu et al., 2004), and the meristem identity genes, in particular, AP1 and LFY, downregulate the expression of AGL24 to establish floral meristem identity and subsequently upregulate the ABC floral organ identity genes (Yu et al., 2004).

svp and agl24 single mutants are affected only in the transition to flowering. However, after the transition, their expression has also been detected in the inflorescence meristem and during early stages of flower development (Hartmann et al., 2000; Yu et al., 2002, 2004). This overlap in expression pattern combined with a high level of sequence similarity suggests a possible redundant role after the floral transition. Therefore, we combined these two mutants and studied the effects on flowering time and flower development. The obtained phenotypes showed that for the control of flowering time, svp is epistatic to agl24. Furthermore, flower development was severely affected in this double mutant when grown at 30°C. Both the number and the identity of the floral organs were affected in all floral whorls. In situ expression analysis showed that the homeotic transformations observed in these mutant flowers are possibly due to the deregulation of class B and C identity genes. The observed floral phenotypes are very similar to what has been reported for the lug and seu mutants. Yeast two-, three-, and four-hybrid protein interaction studies showed that a dimer composed of AP1 and SVP, or AP1 and AGL24 can bind the LUG-SEU corepressor. The role of AP1 in these interactions was further supported by genetic experiments that showed that the floral phenotypes in the ap1 agl24 svp triple mutant are significantly enhanced in respect to the agl24 svp double mutant. Our data suggest a role for MADS box factors in the recruitment of the SEU-LUG corepressor complex for the regulation of AG expression during early stages of flower development.

RESULTS

Flowering Time in the agl24 svp Double Mutant

AGL24 and SVP are two MADS box transcription factors that show significant similarity in primary amino acid sequence (identity is 53%, and similarity is 71%). Despite this similarity, they have opposite functions in the transition to flowering. Mutations in AGL24 confer a dosage-dependent late-flowering phenotype, indicating that AGL24 is a promoter of the floral transition (Yu et al., 2002; Michaels et al., 2003; Table 1). svp mutants show a dosage-dependent early-flowering phenotype, indicating that SVP is a repressor of the floral transition (Hartmann et al., 2000). To investigate whether AGL24 and SVP act in the same pathway, an agl24-2 svp-41 double mutant was created. This double mutant was compared with wild-type and single mutants for differences in flowering time under short-day (SD) (8 h light/16 h dark) conditions. As shown in Figure 1, the svp-41 mutant is early flowering, forming ~14.6 ± 1 rosette leaves in SD conditions. The agl24 mutant is late flowering in respect to the wild type, forming on average 75.0 ± 2.5 rosette leaves in SD conditions. These values are comparable to previously published data (Hartmann et al., 2000; Yu et al., 2002; Michaels et al., 2003). The agl24-2 svp-41 double mutant has the same flowering time as the svp-41 single mutant (14.5 ± 1 rosette leaves in SD conditions) showing that svp is epistatic to agl24.

Table 1.
Effects of svp, agl24, ap1, lug, and seu Mutants on Flower Development
Figure 1.
Flowering Time in the agl24 and svp Single and agl24 svp Double Mutants.

Flower Development in the agl24 svp Double Mutant

Although the svp and agl24 single mutants are only affected in flowering time (Table 1), previously reported expression analyses showed that AGL24 and SVP are also expressed after the floral transition in generative tissues (Hartmann et al., 2000; Yu et al., 2002, 2004; Michaels et al., 2003). These experiments showed that SVP is expressed in the secondary inflorescence meristems but was absent in the primary inflorescence meristem. SVP expression is also detected in the floral primordia until stage 3 when sepal primordia become visible. AGL24 expression in generative tissue is similar to the SVP expression profile. AGL24 overlaps with SVP expression in the secondary inflorescence meristems, although AGL24 mRNAs were also detected in the primary inflorescence meristem. AGL24 is also expressed in the floral meristem, and in later stages of flower development, weak expression is observed in stamens and carpels.

Since there is an overlap in expression in generative tissues, we analyzed the svp agl24 double mutant for defects in flower development (Table 1). Under standard growing conditions (22°C), only the first three flowers of the double mutant were affected. These flowers had a reduced number of organs. In particular, this reduction concerned the second and third floral whorls that produced three petals and five stamens, whereas wild-type Arabidopsis thaliana flowers have four petals and six stamens (Figures 2A and 2B). Furthermore, some flowers showed homeotic conversion of sepals into petaloid organs (Figures 2C and and2D2D).

Figure 2.
Floral Defects in agl24 svp Double and agl24 svp ap1 Triple Mutants.

When double mutant plants were grown at a higher temperature (>30°C), almost all flowers (90%) were severely affected in contrast with wild-type and single mutant plants that showed no floral phenotype. The double mutant flowers exhibited variable floral defects, and all produced a reduced number of organs (Figures 2E to to2I2I).

Frequently (70% of double mutant flowers) sepals were fused and they showed carpelloid features, including stigmatic papillae and multiple ovules developed along the margin of each organ (Figures 2E and and2H).2H). Less frequently we also observed the homeotic conversion of sepals into petaloid (Figure 2F) or staminoid organs (Figures 2F, ,2G,2G, and and2I).2I). In the second whorl, the petals were reduced in number or were completely missing (Figures 2E to to2I).2I). Some plants developed staminoid tissue on top of the petals or stamen-like filaments at the base (Figure 2F). In the third whorl, all flowers had fewer stamens than wild-type flowers, and sometimes they were malformed (Figures 2E to to2I).2I). In the fourth whorl, we sometimes observed defects in carpel fusion (Figures 2E, ,2F,2F, and and2H).2H). Furthermore, in some cases, anther tissue developed on top of these unfused carpels (Figure 2E). A few plants (5 out of the 98 that we analyzed) developed terminal flowers, transforming primary or secondary inflorescence meristems into floral meristems. These terminal flowers were in general composed of only carpels and stamens (Figure 2I).

Scanning electron microscopy analysis of young floral buds (stage 6) of wild-type and agl24 svp double mutant flowers at 30°C confirmed that development is already affected at early stages in the double mutant flower (Figures 3A and 3B). Wild-type stage 6 flowers are enclosed by sepals, whereas the outer whorl organs of mutant flowers are not covering the inner whorls. In the mutant flower shown in Figure 3B, only two sepal primordia and three normal stamen primordia are formed. Furthermore, one stamen primordia is fused with the gynoecium primordia.

Figure 3.
Scanning Electron Microscopy Analysis of Mutant Flowers.

Expression Analysis of Class B and C Genes

The phenotypes described above suggest that in the agl24 svp double mutant, homeotic class B and C genes are deregulated, resulting in their ectopic expression. Therefore, we analyzed by in situ hybridization the expression of AG and AP3 during different stages of Arabidopsis flower development (Figure 4). In wild-type flowers, AP3 expression becomes visible in the floral meristem prior to petal and stamen primordia development (stage 3 flowers), and expression is maintained in petals and stamens during all stages of their development (Figure 4B; Jack et al., 1992). The in situ analysis shows that in the agl24 svp double mutant, AP3 is expressed (starting from stage 3) in all parts of the floral meristem. Subsequently, AP3 expression was detected in all floral primordia and later in all floral organs (Figure 4D).

Figure 4.
Class B and C Gene Expression in Wild-Type and agl24 svp Double Mutant Flowers.

AG is the class C gene of Arabidopsis and is expressed in the inner part of the floral meristem where stamen and carpel primordia develop (Bowman et al., 1989; Yanofsky et al., 1990; Drews et al., 1991). During flower development, AG expression is restricted to whorls 3 and 4 (Figure 4A). In the agl24 svp double mutant, AG mRNAs were already detected in the inflorescence and floral meristems starting from stage 1, indicating precautious AG expression. In later stages, AG remains expressed in all floral organs.

This expression analysis showed that in the double mutant, AG is expressed earlier during flower development, and both AG and AP3 are not restricted to specific floral whorls. Furthermore, the expression of these genes is often less uniform than observed in wild-type whorl 2 and 3 organs, since often we see expression concentrated in patches (Figures 4E and and4F).4F). This misexpression of class B and C genes reflects the homeotic transformations of floral organs as observed in the flowers of this double mutant.

LUG and SEU Expression Analysis

The floral phenotypes of the agl24 svp double mutant described above are strikingly similar to those observed in the lug and seu single and double mutants (Liu and Meyerowitz, 1995; Franks et al., 2002). In these mutants, the observed homeotic transformation of floral organ identity is due to precocious and ectopic class B and C gene expression. To investigate whether the expression of LUG and/or SEU was affected in the agl24 svp double mutant, RT-PCR analysis was performed on inflorescence RNA extracted from wild-type, svp, and agl24 single and double mutants grown at 30°C. This analysis revealed that the expression levels in wild-type and mutant plants are comparable, which indicates that the observed phenotype in the agl24 svp double mutant is not due to the silencing of LUG and/or SEU (Figure 5).

Figure 5.
Expression of LUG and SEU Is Not Changed in the agl24 svp Double Mutant.

Protein Interaction Analysis

Sridhar et al. (2004) have recently shown that the molecular basis for the similar mutant phenotype of lug and seu single mutants is based on the physical interaction between the LUG and SEU proteins in forming a corepressor complex. In this complex, LUG functions as the repressor of AG that acts via the second regulatory intron (Sieburth and Meyerowitz, 1997; Deyholos and Sieburth, 2000), whereas SEU does not seem to have any inherent function in repressing transcription. SEU is thought to function as an adapter protein bridging the interaction between the repressor LUG and specific DNA binding transcription factors.

Since there are two MADS box transcription factor binding sites (CArG boxes) in the regulatory intron of AG (Deyholos and Sieburth, 2000; Hong et al., 2003), we investigated whether SEU could directly interact with AGL24 and SVP. These interactions would explain how the LUG-SEU repressor complex is recruited to the DNA and by that the observed phenotypes in the agl24 svp double mutant.

To investigate this, yeast two-hybrid assays were performed using SVP, AGL24, SEU, and LUG since a direct interaction between LUG and the MADS box proteins could not be excluded. The coding part of the cDNAs encoding these proteins were fused to the activation domain (AD) and binding domain (BD) and tested for interaction (Table 2). SEU could not be tested in the BD vector since this protein has strong autoactivation activity. In this assay, we observed that LUG forms a strong interaction with SEU (growth on adenine selection and on His selection media with 5 mM 3AT), but neither SEU nor LUG showed an interaction with AGL24 and SVP.

Table 2.
Interactions among MADS Box, LUG, and SEU Proteins in Yeast

Recently, Sridhar et al. (2004) indicated as unpublished data that SEU interacts with AP1 and SEPALLATA3 (SEP3). Interestingly, both SVP and AGL24 have been shown to interact with AP1 and SEP3 (Pelaz et al., 2001; de Folter et al., 2005), suggesting that SEU could interact with a complex composed of SVP or AGL24 and SEP3-AP1. Furthermore, Bowman et al. (1993) showed that strong ap1 alleles develop carpels instead of sepals when these mutants were grown at high temperatures (25 to 30°C). This phenotype is similar to our agl24 svp double mutant; therefore, we used two-hybrid assays to confirm that AP1 and SEP3 interact with SEU. However, all these three proteins show high autoactivation activity when used as bait in the BD vector, making it impossible to test these interactions. To overcome this problem, we tested a deletion construct encoding 1 to 196 of AP1 (AP1Δ1), eliminating the transactivating terminus (Pelaz et al., 2001). This experiment showed that with the AP1Δ1 protein, no interaction with SEU was observed (Table 2), although this might be due to the loss of a part of the C terminus. An explanation for these results could be that either full-length AP1 can interact with SEU or a MADS box dimer composed of, for instance, AP1-AGL24, or AP1-SVP forms the surface for SEU interaction. To test whether at least the dimer is able to interact (which does not exclude the possibility that AP1 is establishing the interaction by itself), we performed yeast three-hybrid assays by fusing the full-length AP1 protein with the nuclear localization signal of the TFT vector (Egea-Cortines et al., 1999). Clear growth of single colonies was observed when the three proteins (SVP/AGL24, AP1, and SEU) were expressed, whereas all controls were clearly negative, showing that the AP1-AGL24 and AP1-SVP dimers can bind to SEU. However, when low concentrations of 3AT were added, no growth was observed, indicating that the interaction is weak (Table 2). To verify this weak interaction, we also performed semiquantitative lacZ assays using yeast strain SFY526, which has a different genetic background (see Supplemental Table 1 online). These assays confirmed the interaction between SEU and the AGL24-AP1 and SVP-AP1 dimers.

To have the ultimate evidence that the LUG-SEU corepressor can be recruited by a MADS box dimer, we developed a yeast four-hybrid assay in which we fused SVP to the BD domain, LUG to the AD domain, and both AP1 (in the TFT vector) and SEU (in the pRED vector) to a nuclear localization signal. Only when the four proteins were simultaneously expressed was growth on selective media observed, whereas all controls were clearly negative (Figure 6, Table 2), indicating that the four proteins indeed form a complex. These experiments support the idea that SEU mediates the interaction between the repressor protein LUG and the DNA binding MADS box dimers AP1-SVP and AP1-AGL24.

Figure 6.
Interactions among the SVP, AP1, SEU, and LUG Proteins in Yeast.

Analysis of the ap1 agl24 svp Triple Mutant

To reveal genetic evidence that AP1, together with AGL24 and SVP, is involved in the recruitment of the LUG-SEU corepressor complex, we combined the svp agl24 mutant with the weak ap1-12 allele (Figure 2J). Under standard growing conditions, the ap1-12 mutant forms additional flowers that arise in the axils of the first-whorl sepals (Table 1). Furthermore, the number of petals is reduced in this mutant. To obtain information about the mutation that causes this phenotype, we sequenced the AP1 cDNA, which showed that the ap1-12 allele contains a nonsense mutation due to a single base pair change 547 bp downstream of the ATG.

In the agl24 svp double mutant, we only observed severe phenotypes similar to the lug mutant when plants were grown at 30°C (Figures 2E to to2I).2I). Interestingly, in the ap1 agl24 svp triple mutant, severe lug-type phenotypes were observed under normal growing conditions (22°C) (Figures 2K to to2M).2M). Whereas none of the segregants (single or double mutant combinations) showed these kind of severe phenotypes, scanning electron microscopy analysis shows clearly that the whorl 1 organs are converted into carpelloid structures on which ovules and stigmatic tissue develops (Figures 3E and and3F)3F) or into chimeric organs composed of sepal and stamen tissues, as previously observed in the lug mutant (Figures 3C and and3D).3D). These phenotypes make clear that combining a mild ap1 allele with the agl24 svp double mutant significantly enhances the phenotype. These data suggest that AP1, together with SVP and AGL24, indeed regulates AG.

DISCUSSION

The agl24 svp Double Mutant Phenocopies lug and seu Mutant Phenotypes

Functional redundancy between homologous MADS box genes seems to be a common feature (Pařenicová et al., 2003). The phylogenetic analysis of all Arabidopsis MADS box factors is a helpful tool to predict these redundancies. This analysis grouped the MADS box factors SVP and AGL24 closely together, indicating that the AGL24 and SVP genes are the result of a gene duplication event and therefore might have redundant functions. Surprisingly, the single mutant phenotypes indicated the contrary, since SVP and AGL24 have opposite functions in the control of flowering time. In this study, we combined the two mutants and carefully analyzed the agl24 svp double mutant plants (Table 1). Concerning flowering time, the agl24 svp double mutant flowers as early as the svp single mutant, indicating that SVP is epistatic to AGL24. Analysis of the flowers of this double mutant showed that AGL24 and SVP have indeed a redundant function as predicted by phylogenetic analysis. The observed phenotype was mild under standard growing conditions (22°C) but was significantly enhanced when the plants were grown at 30°C, indicating a temperature-sensitive effect. The phenotypes observed at 30°C were very similar to those reported for the lug and seu mutants (Liu and Meyerowitz, 1995; Conner and Liu, 2000; Franks et al., 2002; Sridhar et al., 2004). Typical for these mutants are homeotic conversion of the sepals into staminoid and carpelloid structures, and petals are staminoid or absent. In situ hybridization analysis of agl24 svp double mutant flowers showed that the class C gene AG is expressed earlier and that both AG and the class B gene AP3 are not restricted to specific floral whorls. This misexpression is also observed in the lug and seu mutants and explains the observed phenotypes. Another similarity between these mutants is that the misexpressed genes are often expressed in patches, which probably results in the observed mosaic floral organs with various identities.

The LUG and SEU proteins are considered to form a corepressor complex that prevents AG expression in the outer two whorls during flower development (Conner and Liu, 2000; Franks et al., 2002; Sridhar et al., 2004). Combining lug and ag mutants showed that the ectopic B activity was strongly reduced in this double mutant, indicating that ectopic AG activity in the lug single mutant induces misexpression of class B genes (Liu and Meyerowitz, 1995). Besides AG, LUG seems to regulate several other target genes since defects like narrow leaves, sepals, and petals, split stigma, and abnormal carpels and ovules observed in lug mutants are independent of AG (Conner and Liu, 2000). Interestingly, these phenotypic effects independent of AG ectopic expression are not observed in the agl24 svp double mutants. We rarely observed partially fused carpels, but this is likely due to the formation of staminoid tissue. This probably indicates that the AGL24 and SVP genes are mainly involved in AG repression and not in other processes in which SEU and LUG are involved.

The repression of AG by SEU and LUG seems to act early in flower development, like has been observed for AP2, whereas the repressive function of the polycomb group gene CURLY LEAF (CLF) is later in development, which is also reflected by the milder phenotypes of clf mutants (Goodrich et al., 1997). SVP and AGL24 are both expressed during early stages of flower development (SVP until stage 3; sepal primordia visible as shown in Hartmann et al., 2000). The severe phenotypic effect observed in the agl24 svp double mutant suggests a repressive role for these MADS box factors also during later stages of development (at least until around stage 5). An explanation for this might be that after stage 3, the expression of SVP is too low to be detected by in situ hybridization or that this MADS box protein is stable enough to control AG expression during subsequent stages of flower development. A more likely explanation might be that due to the absence of AGL24 and SVP at very early stages of flower development, there is a precocious accumulation of AG and other floral identity factors that could deregulate negative and positive feedback loops that control flower development, causing effects on later stages of flower development (de Folter et al., 2005; Gómez-Mena et al., 2005).

MADS Box Dimer Interacts in Yeast with the SEU-LUG Corepressor

LUG encodes seven WD repeats, a LUFS motif, and two Q-rich regions (Conner and Liu, 2000). SEU interacts directly with the LUFS domain of LUG, although for the interaction with LUG, the entire SEU protein is needed (Sridhar et al., 2004). LUG is similar in motif structure to the yeast corepressor Tup1, and SEU encodes a plant-specific regulatory protein with sequence similarity to Ssn6. In yeast, Ssn6 functions as an adaptor protein bridging the interaction between Tup1 and specific DNA binding transcription factors (Smith and Johnson, 2000). Recent results of Pfluger and Zambryski (2004) suggest that SEU might have a similar function. They showed that SEU physically interacts with ETTIN, a transcription factor belonging to the auxin response factor family, probably bridging the interaction with other regulatory molecules to modulate transcription of auxin response genes. SEU seems to function in a similar way to bridge the interaction between LUG and DNA binding factors. Genetic studies have shown that intragenic regions of AG are essential for the regulation by LUG (Sieburth and Meyerowitz, 1997; Deyholos and Sieburth, 2000). Candidate factors that could recruit the LUG-SEU corepressor complex to DNA are BELLRINGER (BLR) and MADS box proteins, the latter because of the presence of two CArG boxes in the AG regulatory intron. BLR has been shown to act as a repressor of AG and directly binds to the AG intron. Furthermore, blr shows synergistic genetic interactions with lug and seu, which makes it a perfect candidate (Bao et al., 2004). We assayed interactions between BLR and SEU or LUG in yeast, but we did not observe direct interactions between these proteins (data not shown).

The yeast two-hybrid assays using AGL24 and SVP also showed that these MADS box factors do not directly recruit SEU or LUG to the DNA. Since AP1 was suggested to interact with SEU (Sridhar et al., 2004) and because AP1 also interacts with SVP and AGL24 (de Folter et al., 2005), we also tested AP1 for interactions with SEU and LUG. These assays showed, using a truncated version of AP1 (AP1Δ1) due to autoactivation of this protein, that AP1Δ1 does not interact with SEU or LUG. Subsequently, we tested the MADS box dimers AP1-AGL24 and AP1-SVP for interactions with the SEU-LUG corepressor by yeast three- and four-hybrid assays. These experiments revealed that MADS box dimers (AP1-AGL24 and AP1-SVP) interact weakly in yeast with the corepressor, which supports the idea that SEU mediates the interaction between the repressor protein LUG and the DNA binding MADS box dimers AP1-SVP and AP1-AGL24. Future experiments will have to reveal whether these MADS box corepressor complexes indeed directly bind to the CArG boxes that are located in the AG regulatory intron.

To reveal genetic evidence that AP1, together with AGL24 and SVP, is involved in the recruitment of the LUG-SEU corepressor complex, we combined the svp agl24 mutant with the weak ap1-12 mutant (Table 1). Interestingly, in the agl24 svp double mutant, we only observed severe phenotypes similar to the lug mutant when plants were grown at 30°C, whereas in the ap1 agl24 svp triple mutant, severe lug-type phenotypes were observed under normal growing conditions. These data suggest that AP1 indeed recruits, together with SVP and AGL24, the SEU-LUG corepressor complex for the regulation of AG. This is further supported by the fact that the ap1-1 mutant enhances floral homeotic transformation and AG misexpression in the lug-1 ap1-1 double mutant (Liu and Meyerowitz, 1995).

Interestingly, mutant alleles of AP1, BLR, or a combination of SVP and AGL24, which all encode putative partners of a complex that recruits the SEU-LUG repressor to regulate AG, cause a temperature-sensitive phenotypic effect (Bowman et al., 1993; Bao et al., 2004). It seems that when one of the components of this complex is missing, the remaining factors can still recruit the repressor complex. However, at higher temperatures, the incomplete complex might get unstable, and AG repression is lost. In the ap1 agl24 svp triple mutant, the absence of three essential factors can probably not be compensated; therefore, AG repression is also lost at normal growing temperatures.

Molecular Mechanism underlying the ABC Model

The ABC model of flower development explains how three classes of genes control sepal, petal, stamen, and carpel identity and predicts that class A genes control sepal and petal identity (Coen and Meyerowitz, 1991). Furthermore, the model indicates that class A and C genes are mutually antagonistic, which means that class A genes prevent C expression in the outer two whorls and vice versa (Drews et al., 1991). In Arabidopsis, two class A genes have been identified, which are AP1 and AP2. Typically, the ap1 mutant forms bracts in stead of sepals, and petals mostly do not develop (Irish and Sussex, 1990). Furthermore, in the axil of the first-whorl organs, a new ap1 flower develops. The ap2 mutant also rarely develops petals, but in this mutant, sepals are transformed into carpelloid structures due to ectopic AG expression. These phenotypic observations resulted in the generally accepted idea that AP2 is the key player in preventing AG expression in whorls 1 and 2 (Kunst et al., 1989). Liu and Meyerowitz (1995) suggested that AP1 is likely a redundant repressor of AG since ap1-1 enhanced floral homeotic transformations in lug-1 ap1-1 and ap1-1 ap2-1 double mutants. Our results confirm this role of AP1 in the control of AG expression and show that AP1 is redundant for this function with SVP and AGL24.

Interestingly, in ap2 mutants, AG deregulation seems to start from around stage 3 of flower development (Drews et al., 1991). In the agl24 svp double mutant, AG was deregulated at earlier stages (1 to 3). This suggests that AP1, AGL24, and SVP are involved in AG repression in the first stages of flower development, whereas AP2 seems to act later, restricting AG expression to the inner two whorls.

The data presented here show that MADS box factors play different roles in the developmental pathway that finally leads to plant reproduction. For instance, in the vegetative phase, high levels of SVP expression repress the transition to flowering (Hartmann et al., 2000); however, when its expression reduces and AGL24 expression increases, the floral transition is promoted and then AGL24 promotes inflorescence identity (Yu et al., 2002; Michaels et al., 2003). To establish floral meristem identity, AGL24 is repressed by AP1 (Yu et al., 2004). Subsequently, all three factors have a function in the repression of AG in the floral meristem. The diversity in function of these MADS box factors is probably obtained by making different protein–protein interactions. This all illustrates the complexity of the regulation of developmental processes and how transcription factors are recycled for different functions.

METHODS

Plant Material and Growth Conditions

The plants were grown at 22 or 30°C under SD (8 h light/16 h dark) or long-day conditions (16 h light/8 h dark). The agl24-2 and svp-41 Arabidopsis thaliana mutants (ecotype Columbia) have been kindly given by R.M. Amasino and P. Huijser, respectively. The agl24-2 allele is an En transposon line, and genotyping of the alleles was performed as described previously (Michaels et al., 2003). In the svp-41 mutant, a 2-bp deletion causes a frame shift (Hartmann et al., 2000). Genotyping of SVP alleles was performed by PCR using the gene-specific oligonucleotides 198 and 199 for the wild type (5′-GACCCACTAGTTATCAGCTCAG-3′ and 5′-AAGTTATGGCTCTCTAGGAC-3′) and oligonucleotide 200 designed on the mutation (5′-AAGTTATGGCTCTCTAGGTT-3′). Seeds from the ap1-12 mutant in Columbia were obtained from the Nottingham Arabidopsis Stock Centre.

Expression Analysis

For the in situ hybridization, Arabidopsis flowers were fixed and embedded in paraffin as described previously (Lopez-Dee et al., 1999). Digoxigenin-labeled gene-specific antisense RNA probes were generated by in vitro transcription following the instructions of the in vitro transcription kit (Roche). Hybridization and immunological detection were performed as described previously (Lopez-Dee et al., 1999).

Total RNA was extracted from Arabidopsis tissues using the SV total RNA isolation system (Promega). RT-PCR reactions were performed as described previously (Lago et al., 2004) using primers AtP536 and AtP537 for SEU (5′-GAAGACTTTTGATACCGCAGG-3′ and 5′-TGCAGATGAAGGGCCTGTTCTC-3′) and AtP531 and AtP532 for LUG (5′-CTTAAGTTAAAGATGGCTCTG-3′ and 5′-TCAACATTGTCGTCAAGTGATCC-3′).

Yeast Two-, Three-, and Four-Hybrid Assays

The two-, three-, and four-hybrid assays were performed in the yeast strains PJ69-4A and SFY526 as described previously (Davies et al., 1996; James et al., 1996). pBD, pAD, pTFT1, and pRED vector constructs were selected on YSD media lacking Leu, Trp, adenine, and uracil, respectively. Three-hybrid interactions were assayed on selective YSD media lacking Leu, Trp, adenine, and His supplemented with different concentrations of 3AT (1, 3, or 5 mM). Four-hybrid interactions were assayed on selective YSD media lacking Leu, Trp, adenine, uracil, and His supplemented with different concentrations of 3AT (1, 3, or 5 mM). Genes used for the yeast two-, three-, and four-hybrid assays were cloned in the Gateway vector GAL4 system (pDEST32 for BD and pDEST22 for AD) passing through pDONOR201 (Life Technologies) as described by de Folter et al. (2005). The coding sequences of LUG and SEU were amplified using primers AtP524 and AtP525 for LUG (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTCAGACCAACTGG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTATAGTTTTCACTTCCACAG-3′) and AtP526 and AtP527 for SEU (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTACCATCAGAGCCGCCTAATCC-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTCATTTCACGCGTTCC-3′).

Scanning Electron Microscopy

Samples were prepared and analyzed as described previously (Favaro et al., 2003).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At2g22540 (SVP), At4g24540 (AGL24), At1g69120 (AP1), At1g43850 (SEU), and At4g32551 (LUG).

Supplemental Data

The following material is available in the online version of this article.

  • Supplemental Table 1. Semiquantitative Yeast Three-Hybrid Assays.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Rick M. Amasino for providing the agl24 mutant, Peter Huijser for providing the svp mutant, and Soraya Pelaz for ΔAP1 in the BD vector. Scanning electron microscopy analysis was conducted at the Centro Interdipartimentale Microscopia Avanzata, an advanced microscopy laboratory established by the University of Milan. This research was supported by the Fondo per gli Investimenti della Ricerca di Base 2002 and the Ministero dell'Istruzione, dell'Università e della Ricerca Cofinanziamento 2003.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Martin M. Kater (ti.iminu@retak.nitram).

[W]Online version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.106.041798.

References

  • Bao, X., Franks, R.G., Levin, J.Z., and Liu, Z. (2004). Repression of AGAMOUS by BELLRINGER in floral and inflorescence meristems. Plant Cell 16 1478–1489. [PMC free article] [PubMed]
  • Bowman, J.L., Alvarez, J., Weigel, D., Meyerowitz, E.M., and Smyth, D.R. (1993). Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes. Development 119 721–743.
  • Bowman, J.L., Smyth, D.R., and Meyerowitz, E.M. (1989). Genes directing flower development in Arabidopsis. Plant Cell 1 37–52. [PMC free article] [PubMed]
  • Bowman, J.L., Smyth, D.R., and Meyerowitz, E.M. (1991). Genetic interactions among floral homeotic genes of Arabidopsis. Development 112 1–20. [PubMed]
  • Busch, M.A., Bomblies, K., and Weigel, D. (1999). Activation of a floral homeotic gene in Arabidopsis. Science 285 585–587. [PubMed]
  • Coen, E.S., and Meyerowitz, E.M. (1991). The war of the whorls: Genetic interactions controlling flower development. Nature 353 31–37. [PubMed]
  • Conlan, R.S., Gounalaki, N., Hatzis, P., and Tzamarias, D. (1999). The Tup1-Cyc8 protein complex can shift from a transcriptional co-repressor to a transcriptional co-activator. J. Biol. Chem. 274 205–210. [PubMed]
  • Conner, J., and Liu, Z. (2000). LEUNIG, a putative transcriptional co-repressor that regulates AGAMOUS expression during flower development. Proc. Natl. Acad. Sci. USA 97 12902–12907. [PMC free article] [PubMed]
  • Davies, B., Egea-Cortines, M., de Andrade Silva, E., Saedler, H., and Sommer, H. (1996). Multiple interactions amongst floral homeotic MADS-box proteins. EMBO J. 15 4330–4343. [PMC free article] [PubMed]
  • de Folter, S., Immink, R.G., Kieffer, M., Parenicova, L., Henz, S.R., Weigel, D., Busscher, M., Kooiker, M., Colombo, L., Kater, M.M., Davies, B., and Angenent, G.C. (2005). Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell 17 1424–1433. [PMC free article] [PubMed]
  • Deyholos, M.K., and Sieburth, L.E. (2000). Separable whorl-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron. Plant Cell 12 1799–1810. [PMC free article] [PubMed]
  • Drews, G.N., Bowman, J.L., and Meyerowitz, E.M. (1991). Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 65 991–1002. [PubMed]
  • Egea-Cortines, M., Saedler, H., and Sommer, H. (1999). Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J. 18 5370–5379. [PMC free article] [PubMed]
  • Favaro, R., Pinyopich, A., Battaglia, R., Kooiker, M., Borghi, L., Ditta, G., Yanofsky, M.F., Kater, M.M., and Colombo, L. (2003). MADS-box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell 15 2603–2611. [PMC free article] [PubMed]
  • Franks, R.G., Wang, C., Levin, J.Z., and Liu, Z. (2002). SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development 129 253–263. [PubMed]
  • Gómez-Mena, C., de Folter, S., Costa, M.M., Angenent, G.C., and Sablowski, R. (2005). Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development 132 429–438. [PubMed]
  • Goodrich, J., Puangsomlee, P., Martin, M., Long, D., Meyerowitz, E.M., and Coupland, G. (1997). A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386 44–51. [PubMed]
  • Hartmann, U., Hohmann, S., Nettesheim, K., Wisman, E., Saedler, H., and Huijser, P. (2000). Molecular cloning of SVP: A negative regulator of the floral transition in Arabidopsis. Plant J. 21 351–360. [PubMed]
  • He, Y., and Amasino, R.M. (2005). Role of chromatin modification in flowering-time control. Trends Plant Sci. 10 30–35. [PubMed]
  • Hong, R.L., Hamaguchi, L., Busch, M.A., and Weigel, D. (2003). Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. Plant Cell 15 1296–1309. [PMC free article] [PubMed]
  • Irish, V.F., and Sussex, I.M. (1990). Function of the apetala-1 gene during Arabidopsis floral development. Plant Cell 2 741–753. [PMC free article] [PubMed]
  • Jack, T., Brockman, L.L., and Meyerowitz, E.M. (1992). The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68 683–697. [PubMed]
  • James, P., Halladay, J., and Craig, E.A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144 1425–1436. [PMC free article] [PubMed]
  • Komeda, Y. (2004). Genetic regulation of time to flower in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55 521–535. [PubMed]
  • Kunst, L., Klenz, J.E., Martinez-Zapater, J., and Haughn, G.W. (1989). AP2 gene determines the identity of perianth organs in flowers of Arabidopsis thaliana. Plant Cell 1 1195–1208. [PMC free article] [PubMed]
  • Lago, C., Clerici, E., Mizzi, L., Colombo, L., and Kater, M.M. (2004). TBP-associated factors in Arabidopsis. Gene 342 231–241. [PubMed]
  • Lamb, R.S., Hill, T.A., Tan, Q.K., and Irish, V.F. (2002). Regulation of APETALA3 floral homeotic gene expression by meristem identity genes. Development 129 2079–2086. [PubMed]
  • Liu, Z., and Meyerowitz, E.M. (1995). LEUNIG regulates AGAMOUS expression in Arabidopsis flowers. Development 121 975–991. [PubMed]
  • Lohmann, J.U., Hong, R., Hobe, M., Busch, M.A., Parcy, F., Simon, R., and Weigel, D. (2001). A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell 105 793–803. [PubMed]
  • Lopez-Dee, Z.P., Wittich, P., Pe, E.M., Rigola, D., Del Buono, I., Gorla, M.S., Kater, M.M., and Colombo, L. (1999). OsMADS13, a novel rice MADS-box gene expressed during ovule development. Dev. Genet. 25 237–244. [PubMed]
  • Ng, M., and Yanofsky, M.F. (2001). Activation of the Arabidopsis B Cless Homeotic Genes by APETALA1. Plant Cell 13 739–753. [PMC free article] [PubMed]
  • Michaels, S.D., Ditta, G., Gustafson-Brown, C., Pelaz, S., Yanofsky, M., and Amasino, R.M. (2003). AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 33 867–874. [PubMed]
  • Pařenicová, L., de Folter, S., Kieffer, M., Horner, D.S., Favalli, C., Busscher, J., Cook, H.E., Ingram, R.M., Kater, M.M., Davies, B., Angenent, G.C., and Colombo, L. (2003). Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: New openings to the MADS world. Plant Cell 15 1538–1551. [PMC free article] [PubMed]
  • Pelaz, S., Gustafson-Brown, C., Kohalmi, S.E., Crosby, W.L., and Yanofsky, M.F. (2001). APETALA1 and SEPALLATA3 interact to promote flower development. Plant J. 26 385–394. [PubMed]
  • Pfluger, J., and Zambryski, P. (2004). The role of SEUSS in auxin response and floral organ patterning. Development 131 4697–4707. [PubMed]
  • Putterill, J., Laurie, R., and Macknight, R. (2004). It's time to flower: The genetic control of flowering time. Bioessays 26 363–373. [PubMed]
  • Sieburth, L.E., and Meyerowitz, E.M. (1997). Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9 355–365. [PMC free article] [PubMed]
  • Smith, R.L., and Johnson, A.D. (2000). Turning genes off by Ssn6-Tup1: A conserved system of transcriptional repression in eukaryotes. Trends Biochem. Sci. 25 325–330. [PubMed]
  • Sridhar, V.V., Surendrarao, A., Gonzalez, D., Conlan, R.S., and Liu, Z. (2004). Transcriptional repression of target genes by LEUNIG and SEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proc. Natl. Acad. Sci. USA 101 11494–11499. [PMC free article] [PubMed]
  • Weigel, D., and Meyerowitz, E.M. (1994). The ABCs of floral homeotic genes. Cell 78 203–209. [PubMed]
  • Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A., and Meyerowitz, E.M. (1990). The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346 35–40. [PubMed]
  • Yu, H., Ito, T., Wellmer, F., and Meyerowitz, E.M. (2004). Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development. Nat. Genet. 36 157–161. [PubMed]
  • Yu, H., Xu, Y., Tan, E.L., and Kumar, P.P. (2002). AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals. Proc. Natl. Acad. Sci. USA 99 16336–16341. [PMC free article] [PubMed]

Articles from The Plant Cell are provided here courtesy of American Society of Plant Biologists
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...