Plant Physiol. (1999) 120: 1193-1204

Determination of the Motif Responsible for Interaction between the Rice APETALA1/AGAMOUS-LIKE9 Family Proteins Using a Yeast Two-Hybrid System¹

Yong-Hwan Moon, Hong-Gyu Kang, Ji-Young Jung, Jong-Seong Jeon, Soon-Kee Sung, and Gynheung An^*

Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea

	ABSTRACT

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A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe. With this clone, various MADS box genes that encode for protein-to-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the interaction partners, OsMADS14, was selected for further study. Both genes began expression at early stages of flower development, and their expression was extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/lemmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement.

	INTRODUCTION

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The vegetative-to-reproductive transition is an important event in a plant's development. Upon initiation of the reproductive phase, the development of the floral meristem is initiated by floral meristem identity genes such as LFY (LEAFY) and AP1 (APETALA1) in Arabidopsis (Mandel et al., 1992; Weigel et al., 1992; Hempel et al., 1997). At a later step, the fate of floral organ primordia is specified by three classes of homeotic genes (for review, see Weigel and Meyerowitz, 1994): AP1, AG (AGAMOUS), PI (PISTILATA), and AP3 in Arabidopsis (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994), and SQUA (SQUAMOSA), PLE (PLENA), GLO (GLOBOSA), and DEF (DEFICIENS) in snapdragon (Sommer et al., 1990; Huijser et al., 1992; Tröbner et al., 1992; Bradley et al., 1993). These homeotic genes belong to the MADS-domain protein gene family. MADS box genes encode a family of highly conserved transcription factors that participate in signal transduction and developmental control in plants, animals, yeast, and fungi.

In addition to the three classes of organ identity genes in plants, there are a large number of other MADS box genes whose function is less well defined. In Arabidopsis at least 17 AGL (AG-LIKE) genes have been isolated (Ma et al., 1991; Mandel and Yanofsky, 1995; Rounsley et al., 1995). It was revealed that some of these genes are floral organ specific and appear to be involved in controlling floral organ initiation and development. For example, AGL2 and AGL4 are first expressed very early in flower development (after the floral meristem has emerged from the inflorescence meristem but before any of the organ primordia emerge), suggesting that AGL2 and AGL4 play important roles in the intermediate step between inflorescence initiation and floral organ initiation (Flanagan and Ma, 1994; Savidge et al., 1995). In addition to flower development, several MADS box genes are involved in the control of ovule and seed development, vegetative growth, root development, embryogenesis, or symbiotic induction (Mandel et al., 1994; Angenent et al., 1995; Heard and Dunn, 1995; Flanagan et al., 1996; Perry et al., 1996; Buchner and Boutin, 1998; Zhang and Forde, 1998).

Plant MADS box proteins consist of a MADS box domain, an I region, a K domain, and a C-terminal region. The conserved MADS box domain is required for sequence-specific DNA binding and dimerization (Mizukami et al., 1996; Riechmann et al., 1996a, 1996b; West et al., 1998). The MADS box domains bind to the consensus DNA sequence, the CArG motif (Huang et al., 1993, 1995; Tilly et al., 1998). In AP3 and PI the MADS box and the I region are needed for nuclear localization of the proteins (McGonigle et al., 1996). The K domain is the second conserved region, carrying 65 to 70 amino acid residues and located in the middle of the MADS box proteins. The K domain was named due to its structural resemblance to the coiled-coil domain of keratin, and has been suggested to be involved in protein-to-protein interactions (Ma et al., 1991; Pnueli et al., 1991; Theissen et al., 1995). The C-terminal region is rich in acidic amino acids, which are characteristic of transactivation domains. Using the yeast two-hybrid system, we recently demonstrated that OsMADS16, a rice (Oryza sativa L.) AP3 homolog, contains a transcription activation domain in the C-terminal region of the protein (Moon et al., 1999).

It has been demonstrated that the K domain is required for interactions between MADS box proteins (Davies et al., 1996; Fan et al., 1997). In experiments using the yeast two-hybrid system, GLO and DEF, the B class proteins of snapdragon, specifically selected each other as a partner in the protein-to-protein interaction, and the K domain played an important role in the interaction (Davies et al., 1996). Furthermore, in studies using the same system, AG interacted with AGL2, AGL4, AGL6, and AGL9 (Fan et al., 1997). In that study it was demonstrated that the K domain is necessary in the protein-to-protein interaction. These results indicate that MADS box proteins cooperate with other MADS box proteins in a K-domain-mediated interaction to carry out their functions.

Almost all of our knowledge about the interaction between MADS box proteins has been obtained from the two dicots Arabidopsis and snapdragon. Although many MADS box proteins were isolated from monocots, including maize, sorghum, orchid, and rice (Lu et al., 1993; Schmidt et al., 1993; Chung et al., 1995; Kang et al., 1995, 1997; Mena et al., 1995; Montag et al., 1995; Greco et al., 1997; Kang and An, 1997), the interaction between the MADS box proteins has not been elucidated. We previously reported the isolation of OsMADS1, a rice MADS box gene that exhibited the highest homology with AGL2 (Chung et al., 1994). It was demonstrated that ectopic expression of the OsMADS1 gene in homologous and heterologous plants resulted in early flowering, suggesting that the rice MADS gene is involved in flower induction (Chung et al., 1994). In the present study, we report the isolation of a rice AP1/AGL9 family gene, OsMADS6, by screening a young flower cDNA library from rice using OsMADS1 as a probe. With this gene we isolated the protein-to-protein interaction partners by the yeast two-hybrid system. We also investigated the motif responsible for the interaction between MADS box proteins.

	MATERIALS AND METHODS

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Library Screening and Sequence Analysis

Plasmid Construction

Replacement mutants in the K box region of OsMADS6 were generated by PCR as follows: Leu-110 with Ser and Leu-118 with Arg in the KII fragment (OsMADS6-KIIS₁₁₀R₁₁₈); Leu-126 with Arg and Leu-134 with Arg in the KII fragment (OsMADS6-KIIR₁₂₆R₁₃₄); Leu-110 with Ser and Leu-118 with Arg in the KII and KIII fragments (OsMADS6-KIIS₁₁₀R₁₁₈+KIII); Leu-159 with Arg and Leu-166 with Arg in the KII and KIII fragments (OsMADS6-KII+KIIIR₁₅₉R ₁₆₆); and Leu-110 with Ser, Leu-118 with Arg, Leu-159 with Arg, and Leu-166 with Arg of the KII and KIII fragments (OsMADS6-KIIS₁₁₀R₁₁₈ +KIIIR₁₅₉R ₁₆₆). For all constructs, the 5 EcoRI site and the 3 SalI site were introduced by PCR. The PCR profile used was 1 min at 95°C, 1 min at 57°C, and 1.5 min at 72°C for a total of 40 cycles. The PCR products were digested with EcoRI and SalI, ligated to pBDGAL4 or pADGAL4, and transformed into appropriate hosts. The sequences of all inserts were determined to confirm the proper fusion of the constructs.

Yeast Two-Hybrid Screening

Mat

The strain was transformed with 100 µg of plasmid DNA of the HybriZAP (Stratagene) cDNA library, along with 3 mg of salmon-sperm carrier DNA, using the lithium acetate method. The transformants were plated on SD (synthetic dropout) medium lacking Trp, Leu, and His, and containing 1 mM 3-aminotriazole (SD-Trp-Leu-His+3-AT; Kaiser et al., 1994). Approximately 1.4 × 10⁶ transformants were obtained, as estimated based on the number of transformants grown on the SD-Trp-Leu plate. The 59 colonies that grew on the SD-Trp-Leu-His+3-AT plates after 5 d were subsequently transferred onto the filter paper on the SD-Trp-Leu-His plate and incubated for 1 d. The -galactosidase activity was measured by filter assay according to the method of Breeden and Nasmyth (1985). The colonies that turned blue in less than 6 h were selected for isolation of DNA, and were then retransferred into the YRG-2 yeast strain containing pBDGAL4 or pBD/OsMADS6-KC14. Plasmid DNA was recovered from yeast according to the method of Hoffman and Winston (1987) and transformed into Escherichia coli strain XL-1-Blue (Stratagene) by electroporation.

Isolation of the 5 Region of the OsMADS14 cDNA

DNA and RNA Blot Analyses

Total RNA was isolated (TRI reagent, Molecular Research Center, Cincinnati) from young flowers with a panicle size of 1 to 5 cm, flowers at the early vacuolated pollen stage, flowers at the late vacuolated pollen stage, leaves, roots, lodicules, sterile lemmas, paleas/lemmas, stamens, and carpels. Total RNA (20 µg) was fractionated on a 1.3% (w/v) agarose gel as described previously (Sambrook et al., 1989). After RNA transfer onto a nylon membrane, the blots were prehybridized, hybridized, and washed according to the method of Moon et al. (1999).

The OsMADS6-specific probe (296-bp fragment between nucleotides 748 and 1043) and the OsMADS14-specific probe (626-bp fragment between nucleotides 730 and 1355) were labeled by the random priming method (Amersham).

Quantitative Assay of -Galactosidase Activity

Construction of Phylogenetic Trees

	RESULTS

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Isolation of a Rice cDNA Clone Encoding a MADS Box Protein

View larger version (38K): [in this window] [in a new window]   Figure 1. Deduced amino acid sequence comparisons of MADS box proteins. A, Alignment of the amino acid sequences of OsMADS6, ZAG3, ZAG5, and AGL6. The two conserved motifs of the C region are indicated in bold. B, Alignment of the amino acid sequences of OsMADS14 and ZAP1, an AP1 homolog of maize. The MADS box regions are underlined and the K domains are double underlined. Asterisks indicate identical amino acid residues. Dashes indicate gaps introduced to maximize alignments.

The OsMADS6 protein contains two variable regions: the I region between the MADS and K boxes and the C-terminal region downstream of the K box (Purugganan et al., 1995). The C-terminal region of OsMADS6 has two short motifs, EPTLQIG and AENNFMLGWVL, which are partially conserved in ZAG3, ZAG5, AGL2, AGL4, and AGL9 (Fig. 1A). Based on amino acid sequence similarity of the entire coding region, OsMADS6 can be grouped into the AP1/AGL9 family (Purugganan et al., 1995), which includes AGL2 and AGL4 of Arabidopsis (Ma et al., 1991), ZAG3 and ZAG5, the maize homolog of AG (Mena et al., 1995), floral binding protein 2 (FBP2) of petunia (Angenent et al., 1994), TM5 of tomato (Pnueli et al., 1994), and OsMADS1, OsMADS5, OsMADS7, and OsMADS8 of rice (Chung et al., 1994; Kang and An, 1997; Kang et al., 1997). Among these genes, OsMADS6 was the most homologous to ZAG3 (84%) and ZAG5 (82%).

Two-Hybrid Screening

We therefore proceeded to introduce the cDNA expression library constructed from the mRNA of young rice panicles into the YRG-2 yeast strain containing pBD/OsMADS6-KC14. A total of 1.4 × 10⁶ transformants was screened for their ability to grow on a medium lacking His. This initial screening identified 59 colonies, which were subsequently tested for activation of the LacZ gene. These experiments resulted in the identification of 45 colonies that activated both HIS3 and LacZ. Plasmid DNAs were prepared from these colonies and retransferred into the YRG-2 strain to confirm whether the activation is indeed due to the presence of the fusion protein. We observed that 39 plasmids were able to activate the LacZ gene only in the presence of pBD/OsMADS6-KC14. Sequence determination of these clones revealed that 38 plasmids contained an ORF that exhibited a significant homology to MADS box proteins (Table I). The remaining plasmid had some homology to MADS genes, but was significantly different from typical plant MADS box genes. This clone was not studied further.

Eleven of the clones encoded for previously identified MADS box proteins; of these, six clones belong to OsMADS1 (Chung et al., 1994), two to OsMADS5 (Kang and An, 1997), and three to OsMADS7 (Kang et al., 1997). The remaining plasmids encode for MADS box proteins not previously reported. Twelve of these were partial clones of an identical MADS box gene, although the 5 ends were different from each other (Fig. 2). This gene was designated OsMADS14. Thirteen clones encoded for an identical protein of another MADS box protein. The gene for these clones was designated OsMADS15. Both the OsMADS14 and OsMADS15 proteins were highly homologous to ZAP1. Among the remaining two clones, one clone, OsMADS17, showed a high similarity with ZAG3 and the last clone, OsMADS18, was the most homologous to ZAP1.

View larger version (50K): [in this window] [in a new window]   Figure 2. The nucleotide sequence and the deduced amino acid sequence of OsMADS14. The MADS box region is underlined, and the K domain is double underlined. The 10 repeats of the GGA sequence in the 5-UTR are indicated in bold type. The primer sequence used in isolation of the 5 region of the gene is underlined. Arrowheads and numbers below the amino acid sequence indicate positions of the first amino acid of the fusion proteins selected by the yeast two-hybrid screening and the number of selected clones with the same first amino acid, respectively. The XhoI site used for generation of the gene-specific probe (accession no. AF058697) is indicated in bold type.

Isolation of the OsMADS14 cDNA Clone Containing an Entire ORF

The 5 UTR of OsMADS14 cDNA contains 10 repeats of the GGA sequence (Fig. 2), and such repeat sequences were previously observed from other rice MADS-box genes (Chung et al., 1994; Kang and An, 1997; Kang et al., 1997). The OsMADS14 protein contains a MADS box domain which consists of 56 conserved amino acids present in the N-terminal region of all of the MADS transcription factors (Figs. 1B and 2). The K box domain, a region considered to participate in the protein-to-protein interaction, is also present between amino acid residues 91 and 158. Amino acid sequence comparison revealed that OsMADS14 was 72.4% homologous to ZAP1, an AP1 homolog of maize.

Expression Patterns of OsMADS6 and OsMADS14

View larger version (55K): [in this window] [in a new window]   Figure 3. Genomic DNA blot analysis and RNA blot analysis of OsMADS6 and OsMADS14. A, Southern blot analysis of OsMADS6 and OsMADS14. The rice genomic DNA was digested with EcoRI (E), HindIII (H), or PstI (P). The numbers indicate the size (in kb) of the DNA markers. B, RNA blot analysis of OsMADS6 and OsMADS14. Ethidium bromide staining of 25S and 17S rRNAs demonstrated equal amounts of RNA loading (data not shown). Lane L, Leaves; lane R, roots; lane Sl, sterile lemmas; lane P, paleas/lemmas; lane Lo, lodicules; lane S, stamens; lane C, carpels; lane 1, young flowers with a panicle size of 1 to 5 cm; lane 2, flowers at the early vacuolated pollen stage; lane 3, flowers at the late vacuolated pollen stage.

RNA blot analyses were conducted using the gene-specific probes. The results showed that the OsMADS6 transcript was detectable primarily in lodicules and also weakly in sterile lemmas and carpels of flowers at the late vacuolated pollen stage (Fig. 3B). However, the transcript was not detectable in stamens, paleas/lemmas, and vegetative organs. The spatial expression pattern of OsMADS14 was different from that of OsMADS6. Transcripts of this clone were detectable primarily in sterile lemmas and also weakly in paleas/lemmas, stamens, and carpels (Fig. 3B). However, the OsMADS14 transcripts were not detected in lodicules and vegetative organs. During flower development the OsMADS6 and OsMADS14 genes were expressed at an early stage, and their expression was extended into later stages of flower development (Fig. 3B).

Interaction between OsMADS6 and Other MADS Box Proteins

To confirm the lack of interaction, the region containing the K domain and 14 amino acids of the C-terminal region of the OsMADS3 and OsMAS4 proteins were fused to the binding domain vector pBDGAL4. Most of the C-terminal regions of OsMADS3 and OsMADS4 were not included in the construction to avoid a potential activator domain. Introduction of these plasmids into the YRG-2 strain containing pAD/OSMADS6-KC did not activate the LacZ gene (data not shown). These results showed that OsMADS6 interacts with OsMADS1, 5, 7, 8, 14, 15, 17, and 18, members of the AP1/AGL9 family, but not to the B and C class of MADS box proteins, OsMADS4 and OsMADS3; it also failed to interact with OsMADS6 itself.

Identification of the Protein-to-Protein Interaction Motif

To narrow down the motif responsible for the protein-to-protein interaction, the KC14 region of OsMADS6 was divided into three regions; KI (86th to 110th amino acid), KII (109th to 137th amino acid), and KIII (138th to 170th amino acid) (Fig. 4). The three regions were connected into the binding domain of GAL4 and introduced into the yeast strain YRG-2 containing pAD/OsMADS14-KC. The results in Table III show that only the KII region activated the LacZ gene, suggesting that this region plays an important role in the protein-to-protein interaction. The experiment also indicated that the KIII region alone did not bind to OsMADS14, but enhanced the interaction between these proteins. When pAD/OsMADS14-KC14 was used as the activation domain plasmid instead of pAD/OsMADS14-KC, similar results were observed, although the -galactosidase activities generally decreased (data not shown). Also, when those regions of OsMADS6 were fused into the activation domain and OsMADS14 was connected to the binding domain, similar results were obtained, except that pAD/OsMADS6-KII did not activate the LacZ gene in the presence of pBD/OsMADS14-KC14 (Table III). None of the activation domains or binding domain plasmids used in these experiments activated the LacZ gene by itself (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 4. Amino acid sequence alignment of the region containing the K domain of OsMADS6 with those of OsMADS1, OsMADS5, OsMADS7, OsMADS8, OsMADS14, OsMADS15, OsMADS17, and OsMADS18. The region of OsMADS6 was divided into three regions: the KI region (amino acids 86-110), the KII region (amino acids 109-137), and the KIII region (amino acids 138-170). The entire K domain of OsMADS6 is underlined. The replaced amino acids are indicated below each Leu with arrows. The conserved hydrophobic residues, such as Leu, Ile, Val, and Met, are shaded. The numbers indicate the positions of mutagenized Leu residues and the first and last amino acids of the K region elucidated in this study.

Identification of the Amino Acid Residues Responsible for the Protein-to-Protein Interaction

These mutant fragments were connected to the binding domain of GAL4 and transferred into the YRG-2 strain containing the activation domain plasmid pAD/OsMADS14-KC. The results in Table IV show that mutations in Leu-110 and Leu-118 diminished the interacting ability of the KII fragment. Similarly, mutations of Leu-126 and Leu-134 also significantly affected the activity. These results suggest that the four periodical Leus in the KII region are necessary for interaction between the K box regions. When the mutations at Leu-110 and Leu-118 were introduced into the KII+KIII fragment, the enzyme activity was reduced, but still retained a significant level of activity. However, when mutations were introduced into both KII and KIII regions by replacing Leu-110, Leu-118, Leu-159, and Leu-166, the activity was almost completely diminished. These results, together with the results shown in Table III, indicate that the KIII region alone is not sufficient for protein interaction, but is able to enhance the interaction between MADS box proteins.

When the mutant fragments were used in the construction of activation domain plasmids and their interaction ability in the yeast strain carrying the binding domain plasmid pBD/OsMADS14-KC14 was tested, similar results were obtained except that pAD/OsMADS6-KII and pBD/OsMADS14-KC14 did not activate the LacZ gene (Table IV). Taken together, these results suggest that the Leu residues in the KII region are important for the protein interaction and the Leu residues in the KIII region are involved in enhancing the interaction.

	DISCUSSION

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We have isolated the OsMADS6 gene by screening a young rice flower library using OsMADS1 as a probe. On the basis of deduced amino acid sequences, the OsMADS6 gene can be classified into the AGL6 subfamily (Theissen et al., 1996), which belongs to the AP1/AGL9 family (Purugganan et al., 1995) (Fig. 5). In addition, we have isolated various MADS box genes that encode for protein-to-protein interaction partners of OsMADS6 by the yeast two-hybrid screening method.

View larger version (21K): [in this window] [in a new window]   Figure 5. Phylogenetic tree showing the relationship among AP1/AGL9 family proteins. Rice MADS box proteins are indicated in bold type. The horizontal branches are proportional to the number of base substitutions. 1, 7, 8, 18, 23, 24, and 25, Arabidopsis; 9, 19, 20, and 26, snapdragon; 10, 14, and 15, maize; 11, 12, 13, 16, 17, 27, 28, 29, and 30, rice; 21, petunia; 3, potato; 4 and 22, tomato; 5, Silene latifolia; 2 and 6, Sinapis alba.

It was observed that the region containing the K domain and the C-terminal region of OsMADS6 had transcription activation ability in yeast. The region responsible for the activation appears to be located in the C-terminal region, since deletion of the region eliminated the activation ability. Recently, we reported that OsMADS16, a rice AP3 homolog, contains a motif responsible for the transcription activation ability in the C-terminal region (Moon et al., 1999). We also observed the transcription activation ability of other rice MADS box proteins such as OsMADS1, OsMADS3, OsMADS5, OsMADS7, OsMADS8, and OsMADS14 (unpublished data), and found that one of the roles of the C-terminal region of MADS box proteins is transcription activation.

Almost all of the genes isolated by two-hybrid screening using OsMADS6 corresponded to MADS box proteins. This suggests that MADS box proteins efficiently interact with each other in yeast. The isolated clones belonged to seven MADS genes that can be classified into three groups in the AP1/AGL9 family (Table I; Fig. 5). The OsMADS1, OsMADS5, and OsMADS7 genes belong to the AGL2 subfamily, the OsMADS17 gene belongs to the AGL6 subfamily, and the OsMADS14, OsMADS15, and OsMADS18 genes belong to the SQUA subfamily (Theissen et al., 1996). In addition to these seven MADS box proteins, OsMADS6 also interacted with OsMADS8, which is also a member of the AGL2 subfamily (Kang et al., 1997). However, OsMADS6 did not interact with OsMADS3 and OsMADS4, rice homologs of AG and PI, respectively (Table II). In addition, OsMADS6 failed to interact with itself.

In studies using the yeast two-hybrid system, it was previously reported that AG, a C-class protein in Arabidopsis, interacted with AP1/AGL9 family proteins such as AGL2, AGL4, AGL6, and AGL9, most of which are expressed at the early flowering stage (Fan et al., 1997). Moreover, MADS box proteins expressed at the early (SQUA), intermediate (DEFH200 and DEFH72), and late (DEFH49) stages of flower development were also identified as interaction partners of PLE, a C-class MADS box protein in snapdragon, by the two-hybrid screening method (Davies et al., 1996). Sequence similarity and expression patterns showed that DEFH200 and DEFH72 were very similar to FBP2 and TM5, and DEFH49 was very similar to AGL2. These results indicate that C-class proteins and AP1/AGL9 family proteins interact with each other in Arabidopsis and snapdragon. However, we demonstrated that in rice the AP1/AGL9 family proteins interact with each other within the family, but do not interact with OsMADS3, a C-class protein.

The temporal expression patterns of the rice AP1/AGL9 family genes, such as OsMADS1, OsMADS5, OsMADS6, OsMADS7, OsMADS8, and OsMADS14, were similar (Chung et al., 1994; Kang and An, 1997; Kang et al., 1997). These six MADS box genes were expressed at the early stage of the flower development and their expressions were extended into later stages of flower development. However, the spatial expression patterns of these MADS box genes were different. In mature flowers the OsMADS1 transcript was present in paleas/lemmas and carpels (Chung et al., 1994), OsMADS5 in anthers and weakly in carpels (Kang and An, 1997), OsMADS7 and OsMADS8 in carpels and weakly in anthers (Kang et al., 1997), OsMADS14 in sterile lemmas, paleas/lemmas, stamens, and carpels, and OsMADS6 in lodicules, sterile lemmas, and carpels (Fig. 3B). It is likely that an AP1/AGL9 protein cooperates with another AP1/AGL9 protein coexisting in specific cell types at a particular developmental stage.

Ectopic expression of OsMADS6 and OsMADS14 in rice exhibited extreme early flowering and dwarfism, which were also observed in rice plants ectopically expressing OsMADS1, OsMADS5, OsMADS7, and OsMADS8 (unpublished results). However, the early flowering and dwarfism in transgenic plants of those four MADS genes was not as extreme as in those of OsMADS6 and OsMADS14, indicating that OsMADS6 and OsMADS14 might regulate a very early stage of flower development. Further in vivo studies are now being undertaken to identify whether a heterodimer between OsMADS6 and its interaction partners participates in these developmental processes.

It is known that MADS box proteins interact with each other to form dimers (Davies et al., 1996; Fan et al., 1997). In the present study, motifs responsible for the interaction between OsMADS14 and OsMADS6 proteins were investigated using the yeast two-hybrid screening method. Our results showed that the K domain plays a very important role in the protein-to-protein interaction. In Arabidopsis, it was previously reported that AG forms a dimer with various AGL proteins via K-domain-mediated interaction (Fan et al., 1997). It was also reported that the K domain was essential for protein-to-protein interaction between GLO and DEF (Davies et al., 1996).

It has also been suggested that the AG and AGL proteins contain two helices in the K domain, which may participate in the protein-to-protein interaction (Ma et al., 1991). The KI and KII regions of OsMADS6 contain one helix each. In the present study, we demonstrated that the KII region carrying the second helix plays a more important role in the interaction. The site-directed mutation analysis revealed that Leu-126 and Leu-134 of the KII region play an important role in the interaction. These amino acids are the hydrophobic residues suggested to cluster on one face of the putative helices (Theissen et al., 1995). Therefore, it can be proposed that the putative helix structure plays an important role in the K-domain-mediated interaction between OsMADS14 and OsMADS6.

In Arabidopsis, it was reported that the C region of the AG and AGL proteins enhanced the K-domain-mediated interaction between these proteins (Fan et al., 1997). In the present study, we showed that the 14 amino acid residues located immediately downstream of the K domain enhanced the interaction between OsMADS6 and OsMADS14. In Arabidopsis, ag-4 mutants that result from deletions of 12 or 14 amino acids downstream of the K domain retain a partial AG activity (Sieburth et al., 1995). Therefore, it appears that the region is not essential for function, but is necessary for full activity, probably by enhancing the interaction between AG and its partner. The deleted amino acids correspond to the residues between amino acids 142 and 153 or 142 and 155 of OsMADS6, which are located in the KIII region.

We demonstrated by site-directed mutagenesis experiments that Leu-110, Leu-118, Leu-126, and Leu-134 in the KII region and at Leu-159 and Leu-166 residues in the KIII region of OsMADS6 play a very important role in the protein interaction. These periodical hydrophobic residues are conserved in OsMADS6 and its interaction partners such as OsMADS1, OsMADS5, OsMADS7, OsMADS8, OsMADS14, OsMADS15, OsMADS17, and OsMADS18 (Fig. 4), supporting the idea that these amino acids are important for the function of MADS box proteins.

	FOOTNOTES

   Received January 29, 1999; accepted May 17, 1999.

	ACKNOWLEDGMENTS

We wish to thank Sung Key Jang for valuable suggestions during yeast two-hybrid experiments, Dong-Hoon Jeong, Jongmin Nam, and Jinwon Lee for DNA sequencing, and Chahm An for critical reading of the manuscript.

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