1. AGAMOUS TFs have conserved but variably partitioned functions during flower development
In A. thaliana, AG (euAG lineage) is the only gene showing a full C function activity. The ag mutant completely loses male and female organ identity and FMD (Bowman et al., 1989), despite the fact that A. thaliana also possesses two PLE lineage genes, SHP1 and SHP2 (Liljegren et al., 2000). Furthermore, the ectopic expression of AG in the perianth whorls, as observed in the ap2 mutants (Drews et al., 1991) or in transgenic plants ectopically expressing AG from the 35S constitutive promoter (Mizukami & Ma, 1995), is enough to homeotically convert sepals into carpels and petals into stamens, thus suppressing the A function. The ectopic expression of other core eudicot AG lineage genes gave very similar results, not only in A. thaliana but also in, for example, transgenic petunia (Tsuchimoto et al., 1993; Kater et al., 1998; Heijmans et al., 2012b), tobacco (Nicotiana tabacum; Mandel et al., 1992; Kempin et al., 1993) and tomato (Solanum lycopersicum; Pnueli et al., 1994; Giménez et al., 2010). In general, these experiments have shown that euAG and PLE lineage proteins show a very similar ability in promoting reproductive organ development and FMD. In A. thaliana, the SHP genes do not seem to regulate these functions under natural conditions; however, SHP and AG ectopic expression causes the same homeotic conversion of perianth organs as described above (Pinyopich et al., 2003). The gene AP2 is required to suppress the expression of AG in the two perianth whorls (Drews et al., 1991; Krogan et al., 2012), and it also negatively regulates SHP1 and SHP2 in the first whorl (Savidge et al., 1995; Flanagan et al., 1996). In ap2 ag double mutants, the homeotic conversion of sepals into carpels occurs despite the absence of AG activity (Alvarez & Smyth, 1999), which is largely a result of ectopic SHP1/SHP2 activity. This was evidenced by the absence of homeotic conversions in the ap2 ag shp1 shp2 quadruple mutant (Pinyopich et al., 2003). A similar phenotype was seen in crc spt ap2 pi ag pentuple mutants (Alvarez & Smyth, 1999), indicating that SPATULA (SPT) and CRABS CLAW (CRC) regulate, in parallel to AG, distinct features of carpel development. Interestingly, it has been suggested that AG, SHP1 and SHP2 might redundantly promote SPT and CRC expression (Pinyopich et al., 2003; Lee et al., 2005). The ectopic expression of SHP2 largely complemented the ag mutant phenotype, as it restored stamen and carpel identity, FMD and also female fertility, indicating that SHP genes potentially still have C class gene activity (Pinyopich et al., 2003). In wild-type plants, the onset of expression of SHP occurs later than that of AG, being restricted to the two early carpel primordia before their fusion, and later mainly in developing ovules together with STK (Rounsley et al., 1995; Pinyopich et al., 2003; Colombo et al., 2010). SHP genes redundantly regulate, in cooperation with CRC and AINTEGUMENTA (ANT), the fusion of the ovary valve margin and the development of the stylar and stigmatic tissues. However, the unfused valves which develop in crc ant shp1 shp2 quadruple mutants still retain carpel identity (Colombo et al., 2010), further indicating that SHP genes are not contributing to the C function in A. thaliana. SHP genes are also expressed, like AG and probably in an AG-independent manner, in nectaries, which are not affected in the ag mutants (Bowman et al., 1991b; Flanagan et al., 1996; Baum et al., 2001). It is therefore possible that the development of nectaries is specified by these three genes. Although ag shp1 shp2 triple mutants have never been described, Lee et al. (2005) showed that nectary development is abolished in the ap2 pi ag mutant background only when SHP genes are also simultaneously removed, and proposed that, like AG, SHP genes can activate CRC, which is necessary for nectary development (Bowman & Smyth, 1999). Furthermore, SHP genes regulate, redundantly with STK, ovule identity and development. In stk shp1 shp2 triple mutants, ovule integuments lose their identity and are homeotically converted into carpeloid tissues (Pinyopich et al., 2003; Brambilla et al., 2007). However, AG also redundantly contributes to the D function, although protein complexes consisting only of AG and SEP are probably more effective in promoting carpel rather than ovule identity, as evidenced by the stk shp1 shp2 triple mutant phenotype (Western & Haughn, 1999; Pinyopich et al., 2003; Brambilla et al., 2007).
In conclusion, all four A. thaliana AGAMOUS subfamily genes act redundantly in regulating ovule identity (D function), although the AGL11-like gene STK seems to have unique functions in funiculus development, as this organ is abnormally increased in length in the stk mutant, indicating that STK controls cell division and expansion (Pinyopich et al., 2003). The only C function factor of A. thaliana is AG, because, although SHP proteins fully maintain a similar potential activity, they are not physically present in the meristem and primordia cells at the appropriate time. Their functions are rather restricted to regulate specific tissue types after carpel identity has been established. This is a clear example of subfunctionalization as a result of a change in expression pattern (Airoldi & Davies, 2012).
It is important to note that, because SHP genes are expressed in early carpel primordia, it remains possible that they redundantly regulate carpel identity with AG. In fact, AG seems to be required for SHP expression, and SHP transcripts were dramatically reduced in ag mutant flowers (Savidge et al., 1995). In contrast, it seems that the AGL11 lineage factor STK has no C function activity. Although in A. thaliana 35S::STK transgenic plants sepals were often converted into carpels, these conversions were associated with the ectopic expression of AG, SHP1 and SHP2, whereas petals were only reduced or completely absent, but never converted into stamens (Favaro et al., 2003). The STK-dependent conversion of sepals into carpeloid organs decreased drastically in an ag mutant background. Furthermore, in these 35S::STK ag plants, the ectopic expression of STK seemed not to be able to complement significantly the ag mutant phenotype. The ability of STK to specify carpel identity seemed to be only indirect, largely as a result of the induction of ectopic expression of AG, SHP1 and SHP2 (Favaro et al., 2003).
In addition to A. thaliana, AGAMOUS subfamily members have been functionally characterized in several other species. For instance, in petunia FBP6 and pMADS3 have been intensively studied. FBP6 belongs to the PLE lineage. The fbp6 class C mutant flowers exhibit partial anther to petal conversions (Heijmans et al., 2012b). In the gynoecium, fbp6 flowers have an incomplete fusion of the style and stigma inner tissues and a partial loss of transmitting tissue, and the stigma is transformed into sepal- or leaf-like structures. These defects reduce the fertility of the gynoecium. The silencing of the other class C (euAG) gene pMADS3 by an RNAi approach produced similar but more severe homeotic conversions of stamens, with a reduction in the amount of pollen, whereas the gynoecium developed normally. Plants homozygous for fbp6 carrying the pMADS3-RNAi construct showed a full C function mutant phenotype, with the nearly complete or complete loss of reproductive organ development and the formation of a new flower in the fourth whorl, starting with sepals, and the reiteration of inner flowers was repeated a few more times (Heijmans et al., 2012b; Fig. 1h,i).
The snapdragon ple-1 class C mutant is not a complete null mutant. Its reproductive organs are replaced by perianth organs and the flower becomes indeterminate, but occasionally still producing small amounts of pollen in the third whorl petaloid organs (Davies et al., 1999). Conversely, mutants in the FARINELLI (FAR, euAG lineage) gene did not show floral homeotic changes (Fig. 1d) and the observed phenotype was only a variable degree of male sterility, attributable to degradation of microspores and tapetal cells. In the ple-1 mutant, the stamens are replaced by unfused petals, still conserving some staminoid appearance, whereas in ple-1 far double mutants these organs are fused and show a more pronounced petaloid morphology. In ple-1, variable sepaloid/petaloid/carpeloid organs were observed in the fourth whorl, inside which a new flower was initiated, composed of mixed sepaloid/petaloid tissue (Fig. 1e). In contrast, the fourth whorl of ple-1 far double mutants consisted of a new tube and corolla of petals followed by further petals in a spiral phyllotaxy (Fig. 1f). These data suggest that, in snapdragon, in contrast to A. thaliana, the C function is mainly regulated by a PLE lineage gene. However, FAR expression decreased consistently in the ple-1 mutant, whereas PLE expression expanded throughout the developing anther and carpel in the far mutants, being more localized in the anther stomium and ovules in the wild type, suggesting that FAR negatively regulates PLE and that in far mutants the expansion of PLE might compensate the loss of FAR activity. Therefore, it is difficult to determine the degree of redundancy and subfunctionalization that exists between the two genes (Davies et al., 1999). Ectopic expression studies in tobacco and snapdragon suggested that FAR has the ability to induce stamen identity, even more so than PLE (Davies et al., 1999; Causier et al., 2005). Furthermore, the ectopic expression of FAR in an A. thaliana ag mutant background restored stamen and carpel identity in the third and fourth whorls, respectively, but not FMD (Airoldi et al., 2010).
Recently, the PLE and euAG-like genes NbSHP and NbAG of Nicotiana benthamiana have also been functionally characterized by virus-induced gene silencing (VIGS; Fourquin & Ferrándiz, 2012). The development of efficient VIGS protocols allowed the characterization of AG lineage genes even in plant taxa not readily transformable, such as the basal eudicots opium poppy (Papaver somniferum; Hands et al., 2011) and California poppy (Eschscholzia californica; Yellina et al., 2010), both belonging to the Papaveraceae family. These experiments led again to typical C mutant phenotypes. Interestingly, a single AG lineage gene has been identified in opium poppy, but encoding two alternative transcripts, PapsAG-1 and PapsAG-2, producing distinct proteins with different lengths of the C-terminal domain downstream of the typical AG motif II. These two proteins retain both functional redundancy and unique functions (Hands et al., 2011).
AGAMOUS subfamily members have also been analysed in monocot species. Functional analysis using loss-of-function mutants have only been reported in the domesticated grasses rice and maize. In rice, OsMADS3 and OsMADS58 belong to the AG lineage. OsMADS3 appears to be more important for stamen identity and stamen development (Yamaguchi et al., 2006). In osmads3 mutants, carpel identity and FMD are just weakly impaired, and only when the osmads3 mutant was combined with the osmads58 mutant was a full C function mutant phenotype observed (Dreni et al., 2011). The osmads3 osmads58 double mutant flowers completely lose FMD and reproductive organ identity, with ectopic lodicules replacing stamens and one or more small palea-like organs developing in place of the pistil (Fig. 1m). Thus, this double mutant largely mimics the A. thaliana ag mutant. In maize, ZMM2 (ZEA MAYS MADS 2; Theissen et al., 1995) and ZAG1 (ZEA AGAMOUS 1; Schmidt et al., 1993) are orthologous to OsMADS3 and OsMADS58, respectively, and they appear to be much more subfunctionalized in regulating stamen development in the tassel and carpel development in the ear, respectively. This is supported by their expression profiles and by the phenotype of the zag1 mutants (Mena et al., 1996; Ambrose et al., 2000). Unfortunately, zmm2 mutant alleles have not yet been reported in the literature. Furthermore, a second OsMADS3 orthologue exists in maize, named ZMM23 (Münster et al., 2002); however, this gene has not yet been functionally characterized.
In conclusion, all these functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C function (see also Notes S3).
There is also quite extensive information available about the AGAMOUS subfamily members controlling ovule identity. As mentioned in the Introduction, in A. thaliana the four AGAMOUS subfamily genes all redundantly regulate ovule identity. In petunia, based on co-suppression studies, the AGL11-like genes FBP7 and FBP11 were initially reported to be essential in controlling ovule identity (Angenent et al., 1995; Colombo et al., 1995). However, recently it was shown that significant homeotic conversions of ovules into carpeloid structures were only obtained when the fbp7 fbp11 double mutant was combined with the fbp6 class C gene mutant, or with a pMADS3-RNAi silencing construct (Heijmans et al., 2012b). This suggests that a high redundancy between AG and AGL11 lineage genes in regulating ovule identity seems to be maintained within core eudicots. Recently, we reported the functional characterization of the rice AGL11 lineage genes OsMADS13 and OsMADS21 (Dreni et al., 2007, 2011). Despite the fact that this monocot species has two genes in this clade, only OsMADS13 seems to be an ovule identity gene. Nevertheless, preliminary experiments indicated that OsMADS21 is partially able to complement the osmads13 mutant phenotype, when expressed under the control of the OsMADS13 putative promoter. Furthermore, even though the two AGAMOUS homologues OsMADS3 and OsMADS58 are also expressed in developing ovules, the osmads13 single mutant shows strong homeotic ovule to carpeloid organ conversions. This phenotype is similar to those of the A. thaliana stk shp1 shp2 triple mutant and the petunia fbp6 fbp7 fbp11 or pMADS3-RNAi fbp7 fbp11 lines, indicating a different scenario from that in core eudicots. OsMADS13 also regulates, redundantly with OsMADS3 and OsMADS58, the identity of the ovary wall adaxial epidermis, thus retaining some aspects of the C function (Dreni et al., 2011).
Despite the regulation of FMD being originally assigned to AG homologues, our experiments (Dreni et al., 2011) and those of Li et al. (2011a) showed that in rice OsMADS13 strongly contributes to FMD. A redundancy between AG and AGL11 lineage genes in this function was also recently proposed for petunia (Ferrario et al., 2006; Heijmans et al., 2012b), thus suggesting that it might be a common feature of flowers with a central placentation type, while in plants like A. thaliana, with a parietal placentation, the FM terminates in the carpel primordium, and thus those AGAMOUS subfamily genes with ovule-specific expression do not participate in the process of FMD (Colombo et al., 2008).
2. Expression domain of the AGAMOUS subfamily genes
The spatial and temporal expression profile of AGAMOUS subfamily genes is closely consistent with their conserved function and subfunctionalization. The expression of AG lineage genes in the floral meristem after the emergence of the perianth organ primordia, and in both the third and fourth floral whorls, is probably the ancestral expression profile of these genes (Kramer et al., 2004; Zahn et al., 2006), as it is the most frequently observed pattern in basal eudicots (Di Stilio et al., 2005; Zahn et al., 2006; C; Yellina et al., 2010; Hands et al., 2011; Hu et al., 2012), core eudicots and monocots (Notes S4). However, there are clear exceptions to this conserved pattern, as observed in poplar and apple tree (Malus domestica), where a weak expression of AG lineage genes was observed in vegetative tissues (Brunner et al., 2000; van der Linden et al., 2002) Furthermore, in apple the euAG gene MdMADS15 is also clearly expressed in sepals and in the receptacle (van der Linden et al., 2002).
Within the AGL11 lineage, gene expression is usually specific for the ovary placenta, ovule primordium and all the stages of ovule development. The maize gene ZAG2 is also expressed in the central domain of the developing silk (Schmidt et al., 1993). This expression pattern of AGL11-lineage genes is consistent with a role restricted to ovule development. In mature ovules, the expression is often predominant in the integuments. However, there are also exceptions; for instance, the cucumber (Cucumis sativus) gene CUM10 (also named Cucumis AGAMOUS 1, CAG1) is strongly expressed in the stamens, carpels and nectaries of both male and female flowers (Kater et al., 1998; Perl-Treves et al., 1998). Although the cotton (Gossypium barbadense) gene GbAGL1 is mainly expressed in developing ovules, weak expression was also shown in the early petal and stamen primordia (Liu et al., 2010a). In rice, OsMADS21 is expressed in reproductive organs at early stages of their development, later becoming predominantly expressed in the ovule integuments, but its expression level is significantly lower than those of OsMADS3, OsMADS13 and OsMADS58 (Arora et al., 2007; Dreni et al., 2007, 2011). Kramer et al. (2004) hypothesized that the ovule-specific expression of many STK lineage genes has somehow evolved from an ancestral situation in which these genes were broadly expressed in the male and female reproductive organs, which is further supported by the observation that in gymnosperms the AGAMOUS genes are expressed in microsporophylls, megasporophylls, and ovules (reviewed by Kramer et al., 2004; Englund et al., 2011).
3. AGAMOUS subfamily protein–protein interactions
Genetic and molecular studies in A. thaliana revealed that floral organ identity MADS-domain factors are dependent for their function on the interaction with SEP (E function) MADS-domain proteins. SEP proteins are thought to act as a ‘bridge’ allowing the formation of higher order complexes and to add transcriptional activator activity to these complexes (Honma & Goto, 2001; Pelaz et al., 2001). Based on this knowledge, the ABCDE model was translated into the ‘quartet model’ of MADS-domain proteins (Theissen, 2001; Theissen & Saedler, 2001). The model predicts that a dimer composed of two MADS-domain proteins binds a specific target sequence, named the CArG box (CCA/T6GG; reviewed by Kaufmann et al., 2005), in a promoter region and that another MADS-dimer binds another CArG box. Subsequently, these two dimers interact to form a tetramer (‘quartet’) and loop the DNA that lies between the two CArG boxes (Melzer et al., 2009; Melzer & Theissen, 2009; Smaczniak et al., 2012b; Notes S5).
AGAMOUS subfamily proteins efficiently dimerize with SEP proteins. According to the quartet model, in the third floral whorl of A. thaliana, the B function heterodimer APETALA3-PISTILLATA (AP3-PI) interacts with the AG-SEP heterodimer to form the functional stamen identity tetrameric complex (Fig. 3). In the fourth innermost whorl, the interaction of two AG-SEP heterodimers forms the FMD and carpel identity complex. Ovule identity complexes are established by complexes of SEP, STK and/or AG and SHP proteins (Favaro et al., 2003; Fig. 3). Very recently, Mendes et al. (2013) provided in vitro and in vivo evidence for the existence of SEP3-STK quartets and for the importance of these tetramers in the regulation of the expression of target genes during ovule and seed development.
Figure 3. Conserved MADS-box tetrameric complexes regulate stamen (yellow), carpel (green) and ovule (purple) identity in Arabidopsis thaliana (left) and rice (right). SEPALLATA (SEP) proteins are shown in black, AP3/PI in blue and AGAMOUS in red. In rice, the names of MADS-domain factors are abbreviated and only the number is shown. In rice, the AGL6-like factor OsMADS6 is not present in anthers but seems redundant with SEP proteins in the fourth whorl. However, as the single osmads6 mutant already shows obvious phenotypes, it could also form alternative complexes and have specific functions. The floral meristem determinacy (FMD) is regulated by the carpel identity complexes and in rice probably also by the ovule identity complex.
Download figure to PowerPoint
Based on the high functional conservation of the AGAMOUS subfamily members in many species and the fact that they show similar interaction patterns (Davies et al., 1996; Ferrario et al., 2003; Vandenbussche et al., 2003), analogous tetrameric complexes are predicted to be formed, at least within core eudicots. The complexes, however, do not seem to contain only MADS-domain proteins. For instance, in A. thaliana, molecular and genetic evidence suggests that AG-SEP complexes interact with the homeodomain TF BELL1 (BEL1) to regulate ovule development, in particular to repress WUSCHEL in the chalaza to control outer integument development (Brambilla et al., 2007).
In contrast to the AGAMOUS subfamily, the SEP subfamily is only found in angiosperms, and is probably closely related to the AGL6 subfamily, which in contrast is found in all seed plants (Zahn et al., 2005). The involvement of SEP in the floral meristem and floral organ identity complexes suggests that the rise of this subfamily represented a crucial step in the evolution of the flower (Zahn et al., 2005). The four SEP factors in A. thaliana are SEP1, SEP2 and SEP4 (AGL2/3/4 or LOFSEP group) and SEP3 (AGL9 or SEP3 group; Malcomber & Kellogg, 2005; Zahn et al., 2005). Despite the fact that these four factors are highly redundant in specifying the floral state, it is evident that SEP3 is the most important and that it forms the most effective B, C and D function complexes (Honma & Goto, 2001; Favaro et al., 2003; Ditta et al., 2004). In rice, OsMADS3 and OsMADS13 seem to interact mainly with the two SEP3 orthologues OsMADS24 and OsMADS45 (Favaro et al., 2002; Cooper et al., 2003; they are allelic to OsMADS8 and OsMADS7, respectively; Kang et al., 1997), rather than with LOFSEP orthologues (reviewed by Dreni et al., 2013). This suggests that SEP3-like factors might be the most important direct partners of AGAMOUS factors in all flowering plants. Besides OsMADS24 and OsMADS45, a third strong interactor with OsMADS13 is OsMADS6 (Favaro et al., 2002), which belongs to the AGL6 subfamily; for more information about this, see Notes S6.
In 1995, Sieburth et al. showed that in A. thaliana the functions of AG in the specification of stamen identity, carpel identity and FMD are genetically separable, as they depend on specific parts of the K-box or specific amino acid residues within it. The most likely explanation for this finding is that different parts of the K-box are required for specific protein–protein interactions. Thus, particular mutations could specifically affect the formation or the functionality of just one complex.
Interestingly, the ectopic expression of FAR (35S::FAR) in both snapdragon and A. thaliana mostly results in the homeotic conversion of petals into stamens, but sepals do not turn into carpeloid organs like they do when other AG lineage genes are used (Causier et al., 2005). Airoldi et al. (2010) were able to elucidate the molecular mechanism behind this peculiar behaviour of FAR through the heterologous expression of artificial variants in A. thaliana. These experiments revealed that the inability of FAR to induce carpel identity in the first whorl is caused by the presence of an additional glutamine in the K3 helix of the protein. This additional amino acid limits FAR to interact in A. thaliana only with SEP3, which is not expressed in the first whorl at early developmental stages. The result of this is that the ectopic expression of FAR cannot lead to the formation of carpel identity complexes in the first whorl of A. thaliana, as a consequence of the absence of the only SEP partner of FAR. However, the ectopic expression of FAR in an ag mutant background was able to restore both stamen identity in the third whorl and carpel identity in the fourth whorl (but not FMD), because of the concomitant presence of SEP3.
Conversely, a variation in the Medicago truncatula SHP homologue protein, MtruSHP, strengthened its interaction with SEP3 in yeast and was associated with the coiled fruit formation observed in this and related species. Other Medicago species having the threonine residue develop uncoiled pods (Fourquin et al., 2013). This threonine-to-alanine variation is located in the C terminus immediately upstream of the conserved AG motif I that was identified by Kramer et al. (2004). Therefore, it is possible that this conserved motif has an important function in mediating protein–protein interactions.
The data as described in this paragraph suggest that MIKCC MADS-box factors establish interactions to form higher order complexes, which determine the function of the individual proteins. These data therefore indicate that proteins forming evolutionarily conserved complexes must have constraints in the modification of their sequence as they need to ‘coevolve’, because a mutation in one of the proteins that impairs their interaction, or the activity of the complex, will be negatively selected, when it is not masked by redundancy or compensated by some other change in the partner protein. These observations also provide a reason why duplicated copies of MADS-box genes are frequently observed in plant genomes. The presence of redundant homologous gene copies removes their functional constraint, leaving one or both of them free to sub- or neofunctionalize (Airoldi & Davies, 2012). Furthermore, the ‘balance hypothesis’ suggests that duplicated copies of transcription factor-encoding genes, obtained by WGDs as often occurred in plants, are maintained to prevent imbalance in the concentration of the subcomponents of these protein complexes (Maere et al., 2005). This is probably also the case for the AGAMOUS subfamily members, where concentrations of the AGAMOUS subfamily proteins and their SEP partners are probably important (Brambilla et al., 2007).