Multiple interactions amongst floral homeotic - Europe PMC

The EMBO Journal vol.15 no.16 pp.4330-4343, 1996

Multiple interactions amongst floral homeotic MADS box proteins

Brendan Davies1, Marcos Egea-Cortines, Eugenia de Andrade Silva, Heinz Saedler and Hans Sommer2 Department of Molecular Plant Genetics, Max-Planck-Institut fur Zuichtungsforschung, 50829 Koln, Germany tPresent address: Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds LS2 9JT, UK 2Corresponding author

Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism. We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products. Selective heterodimerization is observed between MADS box factors. Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens. In contrast, a third factor, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes expressed at early, intermediate and late stages. We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors may have different criteria for interaction. The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction. These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity. Keywords: flower development/homeotic mutants/MADS box/protein-protein interaction/yeast two-hybrid

Introduction Wild-type Antirrhinum flowers consist of four concentric whorls, each bearing a specific organ type, numbered from the outermost to the innermost. Whorls one and two constitute the perianth and contain sepals and petals respectively. The male and female reproductive organs, stamens and carpels, develop in the third and fourth whorls. In floral homeotic mutants, organs of the flower develop at inappropriate positions. Many of the genes affected in such mutants have been isolated and most of them belong to a structurally related group of genes; the MADS box genes (Schwarz-Sommer et al., 1990). These

'organ identity' MADS box genes include DEFICIENS (DEF) (Sommer et al., 1990), GLOBOSA (GLO) (Trobner et al., 1992) and PLENA (PLE) (Bradley et al., 1993). Recessive mutations at the PLE locus result in the failure of the reproductive organs to develop in whorls three and four and cause perianth organs to develop in their place. Another feature of such mutants is the loss of determinacy, which produces a reiteration of multiple whorls of abnormal flowers within abnormal flowers. Semi-dominant mutations at the same locus can lead to ectopic expression of PLE and hence to ectopic development of reproductive organs in the first and second whorls at the expense of the sepals and petals (Bradley et al., 1993). Mutations at either the GLO or DEF loci lead to homeotic conversion of the second and third whorl organs towards sepals and carpels respectively and result in a failure of organ development inside the third whorl (Trobner et al., 1992). Analogous mutants are also found in other species including Arabidopsis where the equivalent MADS box genes to PLE, DEF and GLO are AGAMOUS (AG) (Yanofsky et al., 1990), APETALA3 (AP3) (Jack et al., 1992) and PISTILLATA (PI) (Goto and Meyerowitz, 1994). Although there is a simple genetic model to explain the phenotypes in mutant and transgenic plants in which the expression patterns of the organ identity genes are altered (Haughn and Somerville, 1988; Coen and Meyerowitz, 1991), the way in which these expression patterns are normally established and how they act to determine the developmental fate of an organ is currently unknown. For example, PLE is required for both carpel development (in the absence of DEF and/or GLO) and stamen development (in the presence of DEF and GLO). In addition to the MADS box genes of known function, there are many for which a function has yet to be determined (Ma et al., 1991; Pnueli et al., 1991; Davies and Schwarz-Sommer, 1994; Yanofsky, 1995). We estimate the total number of MADS box genes in Antirrhinum to be in the range of 25-30 (Shore and Sharrocks, 1995; H.Sommer, unpublished results) and this number is likely to be similar in other dicot plant species. Like the organ identity MADS box genes, these others are developmentally regulated, and show distinct and overlapping spatial and temporal expression patterns (e.g. Flanagan and Ma, 1994; Huang et al., 1995; Yanofsky, 1995). The MADS box itself is a stretch of -60 amino acids which shows extensive homology to part of the DNA binding domains of well characterized transcription factors in yeast (MCM 1, Ammerer, 1990) and mammals (SRF, Norman, et al., 1988). It has been demonstrated that the plant MADS box proteins bind DNA at sequence motifs known as CArG boxes (Schwarz-Sommer et al., 1992; Huang et al., 1993, 1995; Shiraishi et al., 1993) and, at least in the case of one pair, form heterodimers (Schwarz-Sommer et al., 1992; Goto and Meyerowitz, 1994; Zachgo et al., 1995).

4330 43) Oxford University Press

MADS box protein interactions Table I. Results of the two-hybrid screens Bait

Transformants screened

His+ colonies picked

LacZ+ colonies

Specific interactions

HF7c(BD/DEF) HF7c(BD/GLO) HF7c(BD/PLE)

2.5x 106 3.6x 106 3x 106

175 650 234

14 11 16

4 6 12

If the ability to form heterodimers were to be shared by all the MADS box proteins, hundreds of different MADS box heterodimers could potentially be formed. Heterodimerization of transcription factors is a common feature which has been postulated to enhance their regulatory potential (Lamb and McKnight, 1991). Families of transcription factors which share common dimerization interfaces, such as the bZIP and bHLH motifs, consist of defined sub-groups based on their differential abilities to interact with each other and bind DNA (see Hollenberg et al., 1995, and references therein). The mammalian MADS box-containing genes also form at least two distinct sub-groups based on their heterodimerization specificities, since the MEF2 proteins can form heterodimers with other members of that group (Pollock and Treisman, 1991; Martin et al., 1994), but cannot heterodimerize with SRF (Pollock and Treisman, 1991). Yeast and mammalian MADS box factors also make ternary protein-protein interactions with a variety of unrelated proteins including homeodomain and ETS proteins (reviewed in Shore and Sharrocks, 1995). To understand how the various plant MADS box genes can act in a combinatorial manner to define cell fate during differentiation, it is first necessary to identify the network of protein-protein interactions which exists amongst them and between them and other factors. The yeast two-hybrid system provides a sensitive and specific method to study such interactions (Chien et al., 1991; Fields and Sternglanz, 1994). We have used a yeast twohybrid system to assess the interactions of the products of three of the homeotic organ identity genes of Antirrhinum majus, PLE, GLO and DEF, both by screening a cDNA expression library and by testing directly for interaction between pairs of proteins. The results indicate that specific heterodimerization is a feature of plant MADS box factors and reveal several unsuspected interactions between the organ identity gene products and other MADS box factors. PLE interacts with a range of MADS box proteins, whereas the detected interactions of DEF are limited to heterodimerization with GLO and vice versa. The MADS box proteins which interact with PLE include products of gene classes which are expressed at early, intermediate and late stages of flower development, and represent both previously identified and unidentified members of the family in Antirrhinum. Furthermore, we demonstrate DNA binding of the newly discovered heterodimers and provide evidence that the K box is required for specific heterodimerization between DEF and GLO. The developmental implications for floral organogenesis of limited heterodimerization amongst the MADS box factors are discussed.

Results Two-hybrid screen using a DEFICIENS bait The complete DEF coding sequence, but lacking the initial methionine codon, was cloned into the vector pGBT9 as

described in Materials and methods to form plasmid pBD/ DEF. This plasmid was introduced into the yeast strain HF7c, and the resulting HF7c(BD/DEF) colonies were tested for activation of the HIS3 selectable marker. Since there was no appreciable increase in the ability of the HF7c(BD/DEF) strain to grow on medium lacking histidine as compared with the HF7c strain, we proceeded to introduce the cDNA expression library into HF7c(BD/ DEF). The cDNA expression library was constructed in pGAD424 as described in Materials and methods from mRNA derived from all Antirrhinum floral tissues at different developmental stages. A total of 2.5 X 106 HF7c(BD/DEF) transformants were screened for their ability to grow on medium lacking histidine (see Table I). This initial screen identified 175 His' colonies, which subsequently were tested for activation of the LacZ gene. Fourteen colonies were identified in which both test genes were activated. The activation domain-containing plasmids were rescued from these clones and transformed into the yeast strain SFY526 with and without the plasmid pBD/ DEF. Four of these activation domain plasmids were able to activate the LacZ gene in SFY526 only in the presence of pBD/DEF, and the DNA sequences of the cDNA inserts in these plasmids were completely determined. Remarkably, all four clones corresponded to the GLO gene (see Figure 1), a floral MADS box gene, the protein product of which is known to form heterodimers with DEF. The isolated GLO cDNAs fell into three classes (Figure 1): (i) full coding region and 36 bp of 5' and 4 bp of 3' sequence (clone ydef5O); (ii) coding region truncated at the 5' end by three amino acids and 65 bp of 3' sequence (clone ydef83); and (iii) coding region truncated at the 5' end by three amino acids accompanied by a single amino acid deletion in the MADS box and including 65 bp of 3' sequence (clones ydef33 and ydef48). Since the cDNA library used in the two-hybrid screen was made by random priming, it was to be expected that not all cDNA clones which were rescued would be full length. The regions of the cDNA which are rescued in this functional assay will provide some information about the domains which are required for heterodimerization. The fact that no difference is observed in interactions between the full-length clones and the other two classes indicates that the first three amino acid residues of the GLO coding region (MGR) are not required for heterodimerization with DEF. In addition, in the class of cDNA which contains a single amino acid deletion, most probably arising as a cloning artefact during the cDNA production, the residue which is deleted is a threonine (position 19 in the MADS box) which is highly conserved amongst MADS box genes.

Two-hybrid screen using a GLOBOSA bait A two-hybrid screen was carried out as described above using a GLO bait (see Materials and methods and Table 4331

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Fig. 1. Schematic diagram showing MADS box factor coding regions which were used as bait and the structure of the cDNAs which were isolated in the two-hybrid screens reported. The boxes represent coding regions and the thin lines denote cDNA regions 5' of the ATG initiation codon and 3' of the stop codon. In all cases except that of DEFH49 (see text), the 5'-non-coding regions which are included in the clones have no in-frame stop codons and can thus be translated as hybrid proteins with the activation domain. The double slashes through the 5' and 3' regions of the DEFH49 cDNA show that these regions are not drawn to scale. The MADS boxes are indicated by dark shading and the K boxes by faint shading. Dotted outlining represents regions of the full-length coding region which were absent from the clone isolated. The point in the GLOBOSA MADS box where a single codon was deleted in two of the isolated clones is indicated by V. The numbers at the right of each of the maps of isolated cDNAs indicate the number of times identical clones were recovered in the screen. The '*' in the case of DEFH49 denotes that this gene was originally identified in a screen for a PLENA-related protein and subsequently also found to interact with PLENA.

I). Six clones were identified which were able to activate the marker genes only in the presence of HF7c(BD/GLO). The DNA sequences of the cDNA inserts in these clones were determined and all corresponded to DEF (see Figure 1). Two distinct clones of DEF were isolated a total of six times. Both clones were intact at the 5' end and included 54 bp of 5'-untranslated leader. However, both were truncated at the 3' end such that their potential to encode the C-terminus of DEF was reduced by 22 amino acids in one case (clones yglo7 and yglo 11) and 76 amino acids in the other (clones yglol8, yglol9, yglo2l and yglo48). It is striking that the larger of the two deletions maps to the C-terminal end of the DEF K box, a region which has been postulated to be involved in proteinprotein interactions amongst the plant MADS box genes. This result confirms previous in vitro DNA binding data which demonstrated that residues C-terminal to the DEF K box are not required for heterodimerization between DEF and GLO (Zachgo et al., 1995). Two-hybrid screen using a PLENA bait A PLE bait was used in a two-hybrid screen as described above (see Materials and methods and Table I). Twelve clones were isolated in which the marker genes were activated only in the presence of the appropriate bait, and the cDNA inserts in these clones were fully sequenced. All except one were MADS box genes. The single nonMADS box gene (clone yple4O) has an unbroken open reading frame with the potential to encode 204 amino acids in-frame with the GAL4 activation domain. The sequence has no obvious structural motifs or similarity to any sequences in the databases. The isolation of fulllength cDNAs for this gene and analysis of its role in floral development is currently in progress. 4332

The MADS box genes fell into three subclasses (see Figure 1), only one of which, SQUAMOSA (SQUA), was previously characterized. SQUA is an early gene, one of the functions of which is to participate in the switch from inflorescence to floral meristem (Huijser et al., 1992). In squamosa mutants, new mutant inflorescences are commonly formed at the position where a flower would normally be expected to develop, and the occasional flower which does develop may be abnormal (Huijser et al., 1992). The other two MADS box genes did not correspond to any of the published Antirrhinum genes and were designated DEFH200 and DEFH72. SQUA was isolated once (clone yple28), three distinct clones of DEFH200 were isolated a total of eight times and DEFH72 was isolated twice (clones yple38 and yple213). The SQUA cDNA has the potential to encode a truncated protein excluding the methionine initiation codon at the N-terminus and 38 amino acids at the C-terminus (see Figure 1 and C-terminus of truncation underlined in Figure 3). Since the cDNAs encoding DEFH200 and DEFH72 which were isolated from the two-hybrid screen all contained truncations, a conventional cDNA library was screened in order to isolate full-length clones. The library was screened using the truncated DEFH200 cDNA as a probe (see Materials and methods). Eleven strongly hybridizing plaques were identified, the cDNA inserts of which were characterized, and the DNA sequences of the longest two were determined by cycle sequencing (see Figure 3). The library was also screened with the almost full-length DEFH72 cDNA, and 10 strongly hybridizing plaques were identified. Two of these cDNAs were sequenced and found to correspond to DEFH72 but to contain additional 5' and 3' sequences. The complete

MADS box protein interactions

cDNA sequences of DEFH200 and DEFH72 have been submitted to the EMBL database with accession Nos X95469 and X95468 respectively. The two DEFH72 clones which were isolated from the two-hybrid screen are identical and contain the complete coding region (with the exception of the first three amino acids) and an additional 20 bp of 3'-untranslated sequence (Figure 1). The DEFH200 cDNAs were all truncated at the C-terminal end from which 80 amino acids (clones yplel2, yplel3, yple69, yple200, yple22 and yple217) or 81 amino acids (clones yple 11 and yple 162) were deleted (Figure 1 and C-terminus of truncations underlined in Figure 3). All the DEFH200 clones isolated from the twohybrid screen were intact at the 5' end of the cDNA and included 3 bp (yple22 and yple217) or 9 bp (clones yple 11, yple12, yplel3, yple69, yplel62, yple200) of 5' sequence. Again, the truncated clones rescued from the two-hybrid screen demonstrate that, at least in the case of the PLEDEFH200 interaction, residues C-terminal to the K box of DEFH200 are not required for heterodimerization.

Isolation of DEFH49, an additional PLENA interaction partner During a similar two-hybrid screen for factors capable of interaction with DEFH1, a MADS box protein which is highly related to PLE (B.Davies and H.Sommer, unpublished), we identified one clone which was strongly positive when cross-tested for interaction with PLE. Sequence analysis of this clone revealed that it corresponded to a MADS box gene, DEFH49, which we had identified previously by its homology to DEF (Davies and Schwarz-Sommer, 1994). The DEFH49 clone which was isolated from the two-hybrid screen contained 232 bp of 5'- and 251 bp of 3'-untranslated cDNA sequences (see Figure 1), which contain stop codons in all reading frames, making it unlikely that it could be translated as a fusion protein with the transcription activation domain of GAL4. One explanation which would account for this anomaly is that DEFH49 itself contains an activation domain which is capable of acting in the yeast system. This was confirmed when the coding region of DEFH49 subsequently was cloned into the vector pGBT9, forming the plasmid pBD/ H49, and expressed as a fusion with the GAL4 DNA binding domain. This fusion strongly activated the marker genes in both yeast strains HF7c and SFY526 (data not shown). It will be interesting to establish whether the transcriptional activation observed in yeast for DEFH49, but not for PLENA, DEF or GLO, has any relevance in plants. The DEFH49 cDNA sequence has been submitted to the EMBL database with accession No. X95467. DNA binding of newly identified heterodimers To establish whether the heterodimers between PLE and DEFH200, DEFH72, SQUA and DEFH49 were capable of DNA binding, various combinations of proteins were co-translated and used in gel mobility shift assays (Figure 2). DNA binding was observed only when certain combinations of proteins were produced by co-translation. GLO or PLE alone did not bind DNA and neither did GLO or DEF together with DEFH200 (Figure 2). No DNA binding was observed with DEFH200 or DEFH72 alone (not shown). As previously described, DEF and GLO bound DNA when produced by co-translation (Schwarz-Sommer

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Fig. 2. DNA binding of interacting proteins. The proteins are indicated above each lane and were produced by translation, co-translation or, in one case, by mixing individually translated products (DEF+GLO). The labelled DNA probe was the CArGI box from the promoter of DEF (Trobner et al., 1992). PLEm is a modified PLENA containing 11 additional amino acids (EQKLISEEDLN) at the carboxy-terminal end. The lanes labelled 'LYSATE' and 'NO LYSATE' contain binding reactions carried out with untreated lysate (no added template) or no lysate. The faint smudge visible in the PLE lane results from the inclusion of [35S]methionine in the translation of PLE alone to allow protein production to be assayed. The PLE-SQUA complex cannot be distinguished from a SQUA-SQUA complex.

et al., 1992) or when separately translated and subsequently mixed (Figure 2, DEF/GLO and DEF + GLO). DNA binding was observed when PLE was co-translated with DEFH200, DEFH72, SQUA or DEFH49. Co-translation and DNA binding of combinations of truncations of PLE and DEFH49 have also been carried out and confirm that PLE and DEFH49 interact (not shown). In the case of SQUA, it is not possible to distinguish between binding by SQUA alone and a PLE-SQUA complex. Further

truncation experiments will be required to demonstrate DNA binding by PLE-SQUA conclusively.

Comparison of DEFH200, DEFH72 and DEFH49 sequences All the MADS box factors which were isolated based on their ability to heterodimerize with PLE are related (Figure 3). In fact, SQUA, DEFH200, DEFH72 and DEFH49 all fall within one of three major groups of MADS box factors (AP1/AGL9) (Purugganan et al., 1995). However, DEFH200, DEFH72 and DEFH49 are more related to each other than to SQUA and belong to a separate sub-group within the AP1/AGL9 group. DEFH200 and DEFH72 are very closely related, with ~90% amino acid 4333

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