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First published online April 2, 2004; 10.1104/pp.103.036665

Plant Physiology 134:1632-1641 (2004)
© 2004 American Society of Plant Biologists

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DEVELOPMENT AND HORMONE ACTION

Identification and Characterization of Four Chrysanthemum MADS-Box Genes, Belonging to the APETALA1/FRUITFULL and SEPALLATA3 Subfamilies1

Anna V. Shchennikova, Olga A. Shulga, Richard Immink, Konstantin G. Skryabin and Gerco C. Angenent*

Plant Research International, Business Unit Bioscience, Wageningen, The Netherlands (R.I., G.C.A.); and Center Bioengineering, Russian Academy of Sciences, 117312 Moscow, Russia (A.V.S., O.A.S., K.G.S.)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Four full-length MADS-box cDNAs from chrysanthemum, designated Chrysanthemum Dendrathema grandiflorum MADS (CDM) 8, CDM41, CDM111, and CDM44, have been isolated and further functionally characterized. Protein sequence alignment and expression patterns of the corresponding genes suggest that CDM8 and CDM41 belong to the FRUITFULL (FUL) clade, CDM111 is a member of the APETALA1 (AP1) subfamily, and CDM44 is a member of the SEPALLATA3 (SEP3) subfamily of MADS-box transcription factors. Overexpression of CDM111 in Arabidopsis plants resulted in an aberrant phenotype that is reminiscent of the phenotype obtained by ectopic expression of the AP1 gene. In addition, CDM111 was able to partially complement the ap1-1 mutant from Arabidopsis, illustrating that CDM111 is the functional equivalent to AP1. Yeast two- and three-hybrid studies were performed to investigate the potential protein interactions and complexes in which these chrysanthemum MADS-box proteins are involved. Based on these studies, we conclude that CDM44 is most likely the SEP3 functional equivalent, because the CDM44 protein interacts with CDM proteins of the AP1/FUL and AG subfamilies, and as a higher order complex with the heterodimer between the presumed B-type CDM proteins.

The Compositae family has been the subject of many phylogenetic, morphologic, and taxonomic studies (Gronquist, 1955Go; Jeffrey, 1978Go; Stebbins, 1977Go) and the organization of the inflorescence and flowers is particularly attractive to biologists. Nevertheless, molecular genetic information about the control of flower development is restricted to studies with Gerbera hybrida (Yu et al., 1999Go; Kotilainen et al., 1999Go; Kotilainen et al., 2000Go) and Helianthus annuus (Dezar et al., 2003Go). In chrysanthemum, which is also a member of the Compositae family, two types of flowers can be distinguished: marginal ray florets consisting of a single petal and two fused carpels, and disc florets possessing five fused petals and five stamens that are partly fused to the two carpels. In contrast to typical dicot plants such as Arabidopsis and Antirrhinum, clear sepals are absent in chrysanthemum, but instead a pappus is formed, which can be regarded as a rudimentary sepal. After floral initiation inflorescence development goes acropetally. Ray florets are produced first distal to the recently formed involucre, which is composed of bracts belonging to the whole inflorescence. Subsequently, the outermost disc florets are initiated in a centripetal direction until the entire inflorescence is filled (Popham and Chan, 1952Go; Harris et al., 1991Go).

During the last decade enormous progress has been made in the understanding of how floral meristems are formed and how the proper floral organs emerge from this meristem. The identities of the floral meristem and the primordia in the floral whorls are mainly specified by the action of MADS box transcription factors. In Arabidopsis, the meristem identity genes LEAFY (LFY) and APETALA1 (AP1), the latter being a MADS box gene, are both necessary and sufficient for the determination of the floral meristem identity and the concomitant formation of the flowers (Weigel et al., 1992Go; Weigel and Nilsson, 1995Go; Mandel and Yanofsky, 1995Go). In addition, the MADS box gene FRUITFULL (FUL) acts in a redundant manner with AP1 to control inflorescence architecture and floral meristem identity (Ferrándiz et al., 2000bGo). Genes belonging to the AP1/FUL clade have been identified from many other plant species (Theissen et al., 2000Go) and functional analysis studies using gain- or loss-of-function mutants revealed that putative orthologs have slightly diverged over evolution with respect to redundancy and gene function (Huijser et al., 1992Go; Immink et al., 1999Go; Prasad et al., 2001Go).

The regulatory network of flower organogenesis in Arabidopsis and Anthirrinum majus has been described by the ABC model that was proposed in the early 1990s (Coen and Meyerowitz, 1991Go, Schwarz-Sommer et al., 1990Go). MADS-box genes representing the ABC functions determine in a combinatorial way the identity of the floral organs, from outer to inner, sepals, petals, stamens, and carpels. Additional functions were identified, mainly by studies with petunia (Petunia hybrida). Two redundant MADS-box proteins, floral binding proteins (FBP) 7 and 11 appeared to determine the identity of ovules, which arise from the remaining floral meristem after the carpel primordia are formed (Angenent et al., 1995Go; Colombo et al., 1995Go). Since then these ovule identity genes represent the D-function. A detailed study of genes in Arabidopsis belonging to the AGAMOUS clade, showed that also in Arabidopsis this function is fulfilled by a set of redundant genes (Pinyopich et al, 2003Go). Results of more recent studies, called for an additional extension of the alphabet of floral organ development, introducing a new class of genes referred to as Identity mediating or E-function genes (Egea-Cortines and Davies, 2000Go; Theissen, 2001Go). E-type MADS-box genes were first reported for petunia (FBP2; Angenent et al., 1994Go) and tomato (Lycopersicon esculentum; TM5, Pnueli et al., 1994Go). Knock-down petunia and tomato plants showed very similar phenotypes: homeotic conversion of the organs in whorls two, three, and four into sepalloid organs and loss of determinacy in the center of the flower. A further indication that these factors are essential for floral organ fate came from the analysis of Arabidopsis plants with mutations in the SEPALLATA genes SEP1/SEP2/SEP3, showing the same phenotypic alterations observed in the two Solanaceae species (Pelaz et al., 2000Go).

Plant MADS-box proteins of the type II or MIKC type (Alvarez-Buylla et al., 2000Go; Parenicova et al., 2003Go) are composed of the MADS-box domain located at the N terminus, followed by an intervening region, the K-box involved in protein-protein interactions, and the C terminus, in which more sequence diversity among members is present. The K-box mediates the dimerization between MADS-box proteins, while sequences within the I- and K-regions determine the specificity of interaction. More recently, it became apparent from studies with Antirrhinum MADS-box proteins that even higher-order complexes of at least three MADS-box protein molecules can be assembled (Egea-Cortines et al., 1999Go). Ternary complexes between SQUAMOSA (SQUA) and the dimer of the two B-type proteins DEFICIENS (DEF) and GLOBOSA (GLO) were identified by yeast (Saccharomyces cerevisiae) two- and three-hybrid experiments. Similar higher order complexes were proposed for other MADS-box proteins involved in flower development (Honma and Goto, 2001Go), which recently led to the Quartet model for flower organ specification (Theissen, 2001Go). These complexes add a new dimension to the combinatorial ABCDE model and recent studies with petunia MADS-box proteins show that these interactions are conserved among angiosperm species (Favaro et al., 2002Go; Ferrario et al., 2003Go).

Here we report the identification and partial functional characterization of four novel MADS-box genes from chrysanthemum. Their expression patterns and protein-protein interaction profiles suggest that they are putative orthologs of functionally well-characterized Arabidopsis meristem and organ identity genes. These functional characterizations demonstrate the robustness and conservative nature of plant homeotic genes from higher eudicots with respect to sequence, expression pattern, protein complex formation, and function.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Isolation and Sequence Analysis of Four Chrysanthemum MADS-Box Genes

Full-length cDNA clones from four chrysanthemum MADS-box genes were isolated from a cDNA library constructed from young inflorescences. These clones were designated Chrysanthemum Dendrathema MADS8 (CDM8), CDM41, CDM111, and CDM44. Sequence analysis of CDM8, CDM41, CDM111, and CDM44 revealed open reading frames for putative proteins of 237, 243, 246, and 249 amino acids, respectively.

Figure 1 shows the sequence alignment of the entire CDM protein sequences and other known MADS-box proteins, which indicates that CDM111, CDM41, and CDM8 are new members of the AP1/FUL subfamily, also known as the SQUAMOSA (SQUA) subfamily. CDM44 matches most with members of the SEP3 subfamily, previously known as the AGL2 subfamily (Purugganan et al., 1995Go; Theissen et al., 1996Go).



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  		Figure 1. Comparative analyses of CDM8, CDM41, CDM44, CDM111, Arabidopsis, and petunia MADS-box proteins that belong to the AP1/FUL and SEP3 subfamilies. Conserved AP1, FUL, and SEP motifs in CDM8, CDM41, CDM111, and CDM44 are shown. The double lined box indicates conserved motif of AP1 homologs; solid lined box-conserved motif of FUL homologs; broken lined box-conserved motif of SEP3 homologs (Vandenbussche et al., 2003Go). The MADS-box and K-box domains are underlined.

 
The putative protein product of CDM111 contains the conserved motifs at the C terminus, which is typical for the AP1-like proteins. Similarly, CDM41 and CDM8 share a conserved motif with the FUL protein, while CDM44 shares a conserved motif with the SEP3 protein (Fig. 1, Vandenbussche et al., 2003Go; Litt and Irish, 2003Go). A phylogenetic tree, which is based on MADS-, I-, and K-region sequences, illustrates these relationships between CDM and MADS-box proteins belonging to AP1/FUL and SEP3 subfamilies from different angiosperms (Fig. 2 ).



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  		Figure 2. Phylogenetic tree of CDM8, CDM41, CDM44, CDM111, and their closest MADS-box homolog in other plant species. The phylogenetic analysis was performed with the MADS-, I-, and K-regions of the proteins. The Arabidopsis AP3 protein was used as an outgroup and the chrysanthemum MADS-box genes are underlined. Bootstrap values are indicated.

 

Expression Analysis of CDM8, CDM41, CDM44, and CDM111 in Chrysanthemum Tissues

The expression patterns of the isolated CDM genes were determined by RNA-blot analysis using inflorescences at different developmental stages and floral organs present in mature florets. A schematic presentation of a chrysanthemum inflorescence and separate disc flower is depicted in Figure 3A . Two types of florets can be distinguished in a chrysanthemum inflorescence, ray flowers that lack stamens and the hermaphrodite disc flowers located in the central domain of the inflorescence meristem. The northern blots shown in Figure 3B revealed that all four genes are relatively highly expressed in young inflorescence buds, but are differentially expressed in other tissues of the plant. CDM8 and CDM41 transcripts are also detectable in vegetative tissues such as stems and leaves, which matches with expression patterns observed for the PETUNIA FLOWERING GENE (PFG) MADS- box gene (Immink et al., 1999). Moreover, the expression of CDM41 in the flowers is also reminiscent with that of PFG in petunia flowers with high expression in petals and carpels and absent in stamens. A striking difference in CDM41 expression level was observed between petals from ray and disc flowers, indicating that these petals are clearly distinct from each other. CDM111 transcript levels are high in mature inflorescence bracts and petals of both types of florets, which correspond with expression data from the SQUA-like genes (Huijser et al., 1992Go). CDM44 expression is predominantly in young florets present in small inflorescence buds, but diminishes in the floral organs upon maturity.



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  		Figure 3. Expression patterns of CDM8, CDM41, CDM44, and CDM111 in chrysanthemum. A, A schematic presentation of a chrysanthemum inflorescence meristem (IM) with ray and disc flowers. A disc flower is depicted in detail. B, Northern-blot analysis of CDM8, CDM41, CDM44, and CDM111 in chrysanthemum tissues. R, root; St, stem; L, leaf; I-1, inflorescence (diameter 1–2 mm); I-2, inflorescence (diameter 4–5 mm); B, inflorescence bract; Rp, ray petal; Dp, disc petal; Ds, disc stamen; Dpi, disc pistil; Rpi, ray pistil. In each lane, 10 µg of total RNA was loaded. Equal loading of RNA was demonstrated by staining a representative gel with radiant red before blotting (rRNA).

 
In situ hybridization analysis was performed to determine CDM gene expression at early stages of inflorescence and floret development. Expression of all four genes is detectable in developing ray and disc floret primordia (Fig. 4 ). Only in the case of CDM111 were weak hybridizing signals observed in the inflorescence bracts that cover the floral primordia (Fig. 4A). In developing disc flowers the CDM genes are expressed in petals, stamens, and carpel primordia, although the hybridization signal for the CDM44 gene is weak in developing petals.



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  		Figure 4. In situ localization of CDM8, CDM41, CDM44, and CDM111 transcripts in wild-type chrysanthemum inflorescences and developing florets. Longitudinal sections were hybridized with digoxigenin-labeled antisense RNA fragments of CDM111 (A, B), CDM41 (C, D), CDM44 (E, F), and CDM8 (G, H). (A, C, E, G) CDM111, CDM41, CDM44, CDM8 expression in young inflorescences in which floret primordia start to develop. Hardly any signal is detectable in the receptacles (r) or inflorescence bracts (ib). B, D, F, and H, CDM111, CDM41, CDM44, CDM8 expression in young disc florets. p, petals; o, ovary; a, anther; st, style. Bars in A, C, E, and G = 500 µm. Bars in B, D, F, and H = 200 µm.

 
Strikingly, at later developmental stages the expression of CDM8, CDM41, and CDM111 is diminished in stamens (Fig. 3B), while the in situ hybridization experiments reveal that mRNA accumulates in male organs of flowers at early developmental stages. Similar differential expression throughout stamen development has been reported for the Antirrhinum gene SQUA (Huijser et al., 1992Go) and the petunia gene PFG (Immink et al., 1999Go).


Expression of CDM111 in Arabidopsis Complements ap1-1 Mutant Flowers

Sequence homology and expression pattern suggest a possible functional relationship between CDM111 and AP1 from Arabidopsis. To investigate whether the chrysanthemum gene can induce flowering and whether it is able to complement the ap1 mutant phenotype, we expressed CDM111 under the control of the double CaMV 35S promoter in Arabidopsis Columbia and in ap1-1 mutant plants. Fifty Arabidopsis Columbia transgenic plants were generated with the 35S::CDM111 construct from which approximately one-half (21) were affected in flowering time and inflorescence structure. These aberrant primary transformants show early flowering after the production of four to six rosette leaves, curled leaves, and a composite terminal flower, which consists of two or three pistils surrounded by an abnormal number of sepals, petals, and stamens (Fig. 5B ). This early flowering and terminal flower phenotype has been reported for many AP1-like genes when overexpressed in Arabidopsis (Mandel and Yanofsky, 1995Go; Berbel et al., 2001Go), but also ectopic expression of MADS-box genes belonging to the SEP3 clade displays a similar phenotype (Honma and Goto, 2001Go; Ferrario et al., 2003Go). Therefore, we attempted to complement the mutant phenotype of the severe Arabidopsis ap1-1 mutant by overexpression of CDM111, which would confirm the functional equivalency of CDM111 and AP1. Ap1-1 mutant flowers lack petals and new floral buds develop in the axils of the bract-like organs present in the first whorl (Fig. 5C; Bowman et al., 1993Go). Arabidopsis ap1-1 plants (Ler background) were transformed with a 35S::CDM111 construct and the obtained transgenic lines were phenotypically analyzed (Fig. 5, D and E). In 9 plants (out of 19 transformants) sepal and petal formation is partially or completely restored, while axillary flowers are rarely seen. The terminal flower characteristic for the 35S::CDM111 overexpression remains apparent in these restored flowers. Molecular analysis performed by northern hybridization confirmed that in all partly or completely restored plants the CDM111 gene was expressed (data not shown).



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  		Figure 5. Floral phenotypes of 35S::CDM111 lines in wild-type and mutant Arabidopsis plants. A, Mature flower of an Arabidopsis (Col) plant. B, Flower of a 35S::CDM111 line. The terminal flower has multiple pistils and increased numbers of floral organs. C, Flower of an ap1-1 mutant (Ler). Petals are absent and new buds develop in the axils of the first whorl organs (white triangle). D, Inflorescence of a line expressing 35S::CDM111 in the ap1-1 mutant background. The terminal flower (asterisk), which is a hallmark for CDM111 overexpression, is still visible and the petal formation is restored. E, Flower of a 35S::CDM111/ap1-1 line in which petal (arrow) formation is restored.

 

Protein-Protein Interactions between CDM Proteins

MADS-box proteins form specific heterodimers between different members of the MADS-box family. In addition, higher order complexes are formed in yeast and can be analyzed with the GAL4 yeast two- and three-hybrid system (Egea-Cortines et al., 1999Go). The interactions between CDM proteins are presented in Table I, which also includes published data on interactions between homologous MADS-box proteins from other species. Besides the CDM proteins belonging to the AP1/FUL and SEP3 clades as reported in this study, other chrysanthemum MADS-box proteins representing putative flowering, B, and C function MADS-box proteins are included in these interaction studies. The CDM86, CDM115 (CDM19), CDM37, and CDM36 proteins are putative homologs of Arabidopsis MADS-box proteins PI, AP3, AG (Bowman et al., 1989Go), and SOC1 (Lee et al., 2000Go), respectively (Shchennikova et al., 2003Go). Both CDM115 and CDM19 are close homologs of the Arabidopsis AP3 protein.


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  		Table I. Two-hybrid and three-hybrid interactions of CDM proteins in the yeast GAL4 system

 
The two-hybrid analysis revealed that only CDM44 displays auto-activation of the yeast reporter gene in the absence of a bait containing the GAL4 activation domain. CDM8, CDM41, and CDM111 do not form homodimers or heterodimers with each other, but they all interact with CDM44 at room temperature. Also, the putative AG homolog CDM37 interacts weakly with CDM44, which is related to the Arabidopsis SEP3 protein.

It is known from studies with Arabidopsis, Antirrhinum, and petunia class B proteins that they form heterodimers between each other and specific ternary complexes with proteins representing the A, C, and E homeotic functions (Egea-Cortines et al., 1999Go; Honma and Goto, 2001Go; Ferrario et al., 2003Go). Two-hybrid analysis revealed that CDM86 interacts with both CDM115 and CDM19 in a similar way as the B proteins PI-AP3, GLO-DEF, and FBP1-pMADS1 for Arabidopsis, Antirrhinum, and petunia, respectively. The chrysanthemum B proteins were not able to form homodimers in a two-hybrid experiment, neither did they interact with the putative C-class protein CDM37 (results not shown). Three-hybrid studies with the CDM proteins were performed, which may give clues on the conservation of MADS-box protein complexes (Table IB). CDM44 forms ternary complexes with B-type heterodimers CDM86-CDM115 and CDM86-CDM19 at both room temperature and 30°C, which is in agreement with interactions observed between the E-type protein and a B-dimer in other species. Also the petunia FBP2 protein is able to interact with both chrysanthemum B-type heterodimers CDM86-CDM19 and CDM86-CDM115. In contrast, CDM8, CDM41, and CDM111 form a ternary complex with CDM86-CDM115 only and not with the heterodimer combination CDM86/CDM19. This indicates that the presumed paralogs CDM115 and CDM19, being AP3 homologs, act differently in ternary complex formation. Also in three-hybrid studies with the C-type protein CDM37 and the various B-heterodimers, CDM115 and CDM19 appeared to be different (compare interactions 21 and 22, Table IB). A similar observation was made for paralogous E- and B-class proteins from petunia, revealing that only specific combinations of E and B proteins are allowed (Ferrario et al., 2003Go).


   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
Duplication of MADS-box genes is a common phenomenon and has led to innovations in floral morphogenesis due to diversification of function of the duplicated genes (Shepard and Purugganan, 2002Go; Kramer et al., 2003Go). In the AP1/FUL lineage two major clades, represented by AP1 (AP1 lineage) and FUL (paleoAP1 lineage), have evolved. FUL possesses a C-terminal conserved motif LPAWMLRPTTTNE, whereas all AP1-like sequences share another C-terminal conserved motif NCNLGCFAA, which includes the C-terminal prenylation signal CFAA (Rodriguez-Concepcion et al., 1999Go; Vandenbussche et al., 2003Go; Litt and Irish, 2003Go). These changes in amino acid sequence have been explained by a frame-shift mutation in an ancestral AP1/FUL-like gene (VandenBussche et al., 2003Go; Litt and Irish, 2003Go) and are responsible for gene-specific functions (Lamb and Irish, 2003Go). Although the AP1 and FUL genes still possess partial overlapping function in the floral meristem determination process, they fulfill distinct functions in the flower, where AP1 represents the A function for sepal and petal identity (Mandel et al., 1992Go) and FUL is essential for proper silique valve development (Ferrandiz et al., 2000aGo).

Here we report the isolation and partial functional characterization of four chrysanthemum MADS-box (CDM) genes. Alignment of their sequences and identification of specific conserved motifs at the C termini of the CDM proteins suggest that CDM111, CDM8, and CDM41 are members of the AP1/FUL subfamily, and that CDM44 belongs to the SEP3 subfamily. A further division within the AP1/FUL subfamily can be made with CDM111 as the AP1 equivalent and CDM8 and CDM41 as members of the FUL clade. The expression pattern of CDM8, which is predominantly during the early stages of inflorescence development, suggests a role in meristem identity, similar to the early functions represented by FUL in Arabidopsis (Ferrandiz et al., 2000aGo) and PFG in petunia (Immink et al., 1999Go). CDM41, however, is expressed in vegetative tissues and throughout flower development, which is reminiscent of the expression pattern of PFG, but is also in line with the late function of FUL in carpel and fruit development (Gu et al., 1998Go). These observations support the hypothesis that CDM41 and CDM8 have arisen by gene duplication, followed by modification of their expression patterns. This may have led to diversification of their functions in chrysanthemum, while in Arabidopsis these early and late functions are still retained by a single gene, FUL. Although this separation of functions of duplicated genes is a general phenomenon that drives evolutionary changes, it awaits further functional characterization of CDM8 and CDM41 to determine whether it is also the case for these chrysanthemum genes.


CDM111 is the Functional Homolog of AP1

AP1 is an early-acting gene in Arabidopsis and is functioning together with FUL and CAL in a redundant way to control inflorescence architecture (Ferrandiz et al., 2000bGo). Moreover, AP1 specifies the identity of sepals and petals, thereby representing the A function in Arabidopsis (Mandel et al., 1992Go). The expression pattern of CDM111 is very similar to that described for AP1 and for its ortholog in Antirrhinum SQUA (Bowman et al., 1989Go; Gustafson-Brown et al., 1994Go; Huijser et al., 1992Go), although CDM111 is expressed at earlier stages in bracts and inflorescence meristems, while AP1 fails to show expression in these tissues. Furthermore, expression of CDM111 in Arabidopsis plants caused a similar early flowering phenotype as was observed with ectopic expression of AP1 (Mandel and Yanofsky, 1995Go). The strongest available evidence so far that demonstrated the functional equivalency of CDM111 and AP1 came from the complementation studies that revealed a nearly complete rescue of the floral organ defects in the strong ap1-1 mutant.


CDM Protein Complexes

Biochemical and yeast two-hybrid studies have shown that MADS-box proteins form specific homo- and heterodimers, which are assembled to higher order complexes (for review, see Egea-Cortines and Davies, 2000Go). Based on the results of these studies, a Quartet model for MADS-box transcription factor functioning was proposed in which the identity of each floral organ is controlled by a set of four MADS-box proteins (Theissen and Saedler, 2001Go). A central factor in these complexes is the E-class protein, represented by SEP1/SEP2/SEP3 in Arabidopsis (Honma and Goto, 2001Go) and FBP2/FBP5 in petunia (Ferrario et al., 2003Go). In chrysanthemum the CDM44 gene is the closest homolog, which exhibited a similar expression pattern when compared to SEP3 (Mandel and Yanofsky, 1998Go) and FBP2 (Angenent et al., 1992Go), although CDM44 transcript levels are reduced in mature flowers. In our yeast interaction studies we demonstrated heterodimer formation between CDM44-CDM111 and CDM44-CDM37, which is in agreement with observed interactions between SEP3-AP1 and SEP3-AG, respectively (Honma and Goto, 2001Go; Table I). Interestingly, CDM44 also interacts with the FUL-like proteins CDM8 and CDM41 and furthermore, these FUL homologs appear to form ternary complexes with the presumed B-type heterodimer CDM115-CDM86 in yeast. Similar higher-order interactions were observed between the petunia B-type heterodimer pMADS1-FBP1 and the FUL-like protein FBP26 (R. Immink and G.C. Angenent, unpublished data), but ternary complex formation of the homologous Arabidopsis proteins has not been revealed yet. These observations indicate that FUL-like proteins are still able to interact with the class B and E floral MADS-box proteins in a similar way as proteins belonging to the AP1-clade (AP1 and CDM111). Whether these complexes are also formed in planta and have a biological function needs further investigation.

Another surprising result was obtained with our two-hybrid experiments between the putative E protein CDM44 and the presumed B-type proteins CDM86 or CDM19 protein. This interaction has never been observed in other dicot species in which class B proteins exclusively form heterodimers with each other. Homodimers composed of a single B protein type have been reported for the gymnosperm Gnetum gnemon and the monocot Lilium longiflorum (Winter et al., 2002Go). The authors suggest that homodimerization is the ancestral state within the clade of B proteins and that facultative heterodimerization as observed in lily is an intermediate state toward the obligate heterodimerization typical of the higher eudicot. An explanation for the observed CDM44-CDM86/CDM19 interaction might be that instead of a heterodimer a ternary complex is scored between a homodimer of the B proteins and the E protein CDM44. Homodimers CDM86/CDM86 and CDM19/CDM19 were not observed in two-hybrid experiments, but it may well be that the CDM44 protein stabilizes the formation of homodimers in a ternary complex. Obviously, this needs further investigation, but it points to differences in the way Angiosperm species have evolved their regulatory mechanism underlying the control of flower development. Despite these subtle divergences in protein interactions the overall homeotic functions are well conserved among flowering plants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Plant Material and Transformation

Chrysanthemum (Dendrathema grandiflorum cv Parliament) plants, which are strongly determinate short-day plants, were grown under standard greenhouse conditions and then transferred for 8 weeks for short day conditions (day/night–8/16 h) for flower initiation.

For functional analysis of CDM genes Arabidopsis plants (Columbia and Landsberg erecta ap1-1) were used. The CDM111 overexpression construct was made by cloning of a full-length cDNA clone of CDM111 in sense orientation into the pBin19 plasmid (Bevan, 1984Go) under the control of the double 35S cauliflower mosaic virus (CaMV) promoter. Plants (ecotype Col) were grown under standard greenhouse conditions and transformed according to Desfeux et al. (2000)Go.


Screening of a cDNA Library

A cDNA library was constructed from purified poly(A)+ RNA of young chrysanthemum buds (3–5 mm in diameter), using the HybriZAP-2.1 Two-Hybrid cDNA Synthesis kit (Stratagene, La Jolla, CA). Approximately 100,000 plaques were screened with [32P]dATP-labeled MADS-box domain fragment of HAM75 (Helianthus annuus MADS75, O.A. Shulga and G.C. Angenent, unpublished data). Filter hybridizations were performed under low-stringency conditions; 55°C hybridization and washing at 55°C twice in 2x SSC, 0.1% SDS (Angenent et al., 1993Go). cDNA insertions from positive clones were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Stratagene). Sequence data were analyzed with the BLAST search program (Altschul et al., 1997Go) at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). All isolated MADS-box genes were aligned with known MADS-box genes from other plants using the ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX) and Tree-View programs (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).


Northern-Blot Analysis and In Situ Hybridization

For northern-blot analysis total plant RNA was denatured and fractionated in a 1.2% agarose gel, blotted onto Hybond Plus membrane, hybridized to a CDM probe lacking the conserved MADS-box domain at 65°C, and washed at 65°C (twice in 2x SSC, 0.1% SDS, and 2 times 0.2x SSC, 0.1% SDS; Feinberg and Vogelstein, 1984Go; Angenent et al., 1992Go). Synthesis of antisense and sense CDM probes for in situ hybridizations (3'-terminal 300–500-bp fragments) was performed according to Angenent et al. (1995)Go. Chrysanthemum bud fixation, embedding in paraffin, section preparation, in situ hybridization, and immuno-staining were performed as described by Cañas et al. (1994)Go.


Analysis of Protein-Protein Interactions with the Yeast Two-Hybrid GAL4 System

Two- and three-hybrid analyses were performed according to HybriZAP-2.1 Two-Hybrid cDNA Synthesis kit protocol (Stratagene) at room temperature and 30°C. The bait and prey constructs were generated by cloning full-length cDNAs of the CDM genes into pAD-GAL4 and pBD-GAL4-Cam vectors (Stratagene). For three-hybrid GAL4 analysis (Egea-Cortines et al., 1999Go) the CDM86 coding sequence was cloned into pRED-NLSa vector (Ferrario et al., 2003Go) in frame with a nuclear localization signal. The two- and three- hybrid experiments were carried out as previously described (Immink et al., 2003Go; Ferrario et al., 2003Go).

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY173056 (CDM8), AY173055 (CDM41), AY173054 (CDM111), AY173057 (CDM44), AY173065 (CDM36), AY173061 (CDM86), AY173064 (CDM19), AY173060 (CDM115), and AY173059 (CDM37).


   ACKNOWLEDGMENTS
 
We thank Dr. S. Dolgov and Annemarie Meijer, who kindly provided us the chrysanthemum plant material and yeast vector pRED-NLSa, respectively.

Received November 24, 2003; returned for revision January 28, 2004; accepted February 3, 2004.


   FOOTNOTES
 
1 This work was supported by the NWO Dutch-Russian Research Cooperation program. Back

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036665.

* Corresponding author; e-mail gerco.angenent{at}wur.nl; fax: 31–317–423110.


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