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The Plant Journal (2008) 55, 212–223

doi: 10.1111/j.1365-313X.2008.03491.x

DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN Yasuhiro Ito1,*, Mamiko Kitagawa2, Nao Ihashi1, Kimiko Yabe1, Junji Kimbara2, Junichi Yasuda1, Hirotaka Ito2, Takahiro Inakuma2, Seiji Hiroi2 and Takafumi Kasumi3 1 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan, 2 Research Institute, Kagome Co. Ltd, 17 Nishitomiyama, Nasushiobara, Tochigi, 329-2762, Japan, and 3 Department of Agricultural and Biological Chemistry, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 11 December 2007; revised 26 February 2008; accepted 5 March 2008; published online 20 May 2008. *For correspondence (fax +81 29 838 8122; e-mail [email protected]).

Summary The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, and its ripening inhibitor (rin) mutation yields non-ripening fruit. In this study, the molecular properties of RIN and the rin mutant protein were clarified. The results revealed that the RIN protein accumulates in ripening fruit specifically and is localized in the nucleus of the cell. In vitro studies revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites. Analysis of binding site selection experiments revealed that the consensus binding sites of RIN highly resemble those of the SEPALLATA (SEP) proteins, which are Arabidopsis MADS box proteins that control the identity of floral organs. RIN exhibited a transcriptionactivating function similar to that exhibited by the SEP proteins. These results indicate that RIN exhibits similar molecular functions to SEP proteins although they play distinctly different biological roles. In vivo assays revealed that RIN binds to the cis-element of LeACS2. Our results also revealed that the rin mutant protein accumulates in the mutant fruit and exhibits a DNA-binding activity similar to that exhibited by the wild-type protein, but has lost its transcription-activating function, which in turn would inhibit ripening in mutant fruit. Keywords: MADS box, rin mutant, DNA binding, fruit ripening, SEPALLATA, tomato.

Introduction Members of the MADS box family of transcription factors are found in all eukaryotes, including yeasts, plants, insects, amphibians and mammals (Shore and Sharrocks, 1995). The MADS box motif is a distinctive domain for this transcription factor family that binds to a highly conserved DNA motif known as a CArG box. Within this family, the plant type II MADS box proteins are composed of an N-terminal MADS domain, followed by an I region and a K box, both of which are involved in protein–protein interactions, and a C-terminal domain that is necessary for ternary complex formation and transcription-activating function (Cho et al., 1999; EgeaCortines et al., 1999; de Folter and Angenent, 2006). Certain proteins belonging to the MADS box family have been extensively studied, and their important roles in regulating diverse biological functions have been clarified (Messenguy and Dubois, 2003; Shore and Sharrocks, 1995). In plants, the best-studied MADS box genes are those that determine 212

floral organ identity in Arabidopsis and Antirrhinum, for which the functions of the genes have been identified based on a simple hypothesis known as the ABC model (Coen and Meyerowitz, 1991). Additional extensive studies have revealed that the SEPALLATA (SEP) genes that also encode MADS box proteins are necessary for floral organ specification, and are defined as class E (Ditta et al., 2004; Honma and Goto, 2001; Pelaz et al., 2000). The molecular function of the SEP proteins is to facilitate the formation of specific transcription complexes and confer transcription activation potential upon these complexes (Ditta et al., 2004; Honma and Goto, 2001; Pelaz et al., 2000). In addition to floral organ identification, certain MADS box genes regulate important agricultural traits. For example, tomato jointless, which inhibits formation of the pedicel abscission zone and aids in the mechanical harvesting of tomatoes (Solanum lycopersicum), is a mutation in a MADS box gene (Mao et al., 2000). ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd

Characterization of ripening-regulator RIN 213 Parthenocarpic (seedless) apple fruit are achieved by mutations in MdPI, which is a gene homologous to PISTILLATA (PI) (Yao et al., 2001). The ripening inhibitor (rin) mutation found in a MADS box gene, RIN, completely inhibits tomato fruit ripening (Vrebalov et al., 2002). RIN is a possible key factor in manipulating fruit ripening (Causier et al., 2002; Tigchelaar et al., 1978), and, in fact, an F1 hybrid between a normal cultivar and the rin mutant line significantly extends the fruit shelf-life (Kitagawa et al., 2005). In addition, a breeding method for low-allergenicity tomatoes as a heterologous effect of the RIN/rin genotype in the F1 line has been proposed (Kitagawa et al., 2006). The rin mutation shows negative and pleiotropic effects on the tomato fruit-ripening process, including carotenoid biosynthesis, fruit softening, respiration increase and ethylene production, and its fruit fails to ripen (Tigchelaar et al., 1978). Expression of a number of genes required for ripening is strongly suppressed in the rin mutant, suggesting that these genes are transcriptionally regulated by the product of the rin locus (Barry et al., 2000; Knapp et al., 1989; Picton et al., 1993; Rose et al., 1997). The rin locus has been isolated and found to encode two putative MADS box transcription factors, RIN and MC (Vrebalov et al., 2002). RIN is apparently responsible for regulation of fruit ripening, and MC for regulation of calyx size (Vrebalov et al., 2002). In the rin mutant locus, the 1.7 kb deletion that partially removes the last intron and completely removes the last exon from RIN has been identified, and the transcriptional product of the mutant locus consists of an in-frame fusion gene between RIN with the last exon deleted and MC with the first exon deleted (Giovannoni, 2004). Although the rin mutation effect for fruit ripening has been extensively investigated (Barry et al., 2000; Knapp et al., 1989; Picton et al., 1993; Rose et al., 1997; Vrebalov et al., 2002), little is known about the molecular function of the RIN protein. In this study, we investigated the molecular function of RIN as a transcription factor. Results showed that the RIN protein shares similar molecular properties with the SEP proteins, such as homodimerization ability, DNA-binding site specificity, and transcriptional activation potential, although RIN and the SEP proteins play distinctly different biological roles. In addition, we have also characterized the protein encoded by the rin mutant gene and explain how fruit ripening is inhibited by the rin mutation. Results RIN protein accumulates in the nucleus of ripening-fruit cells The mRNA of RIN accumulates in the fruit during ripening (Kitagawa et al., 2005; Vrebalov et al., 2002). To analyze the expression specificities of the RIN protein, samples extracted from leaf, stem, flower and fruit were examined by immunoblotting analysis with anti-RIN antibody. Because

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Figure 1. Expression of RIN and the protein encoded by the rin mutant gene. (a) Immunoblotting analysis of RIN in various plant tissues. Nuclei extracted from leaf (L), stem (S), flower (F) and fruit harvested at the stages immature green (I), mature green (G), breaker (B), pink coloring (P) and red ripening (R) were subjected to SDS–PAGE, and the resulting gel was analyzed by immunoblotting using anti-RIN antibody. As a control, in vitro synthesized RIN protein (C) was also analyzed. (b) Accumulation of the protein encoded by the rin mutant gene in rin mutant fruit. Mutant fruit harvested at the mature green stage (G) and the stage corresponding to pink coloring (P) of the wild-type was subjected to immunoblotting analysis. The solid arrowhead indicates RIN and the open arrowhead indicates the rin mutant protein.

we could not detect RIN-specific signals from whole-cell extracts of ripening fruit, we analyzed nucleus extracts from cells of each type of tissue. As shown in Figure 1(a), in nucleus extracts of ripening fruits, the anti-RIN antibody specifically recognized a polypeptide in the 35 kDa region (Figure 1a), although the molecular mass estimated from the deduced amino acid sequence of RIN is aprroximately 28 kDa. Because the detected polypeptide showed the same mobility as in vitro-synthesized RIN protein (Figure 1a), the band at the 35 kDa region was identified as the RIN protein. RIN accumulated specifically in fruit during ripening, but not in non-ripening fruit or in other tissues, such as the leaf, stem and flower (Figure 1a). The mRNA of the rin mutant gene accumulates in the rin mutant fruit at stages corresponding to those for wild-type fruit ripening (Kitagawa et al., 2005; Vrebalov et al., 2002). As shown in Figure 1(b), immunoblotting analysis of rin mutant fruit using anti-RIN antibody revealed specific accumulation of a protein whose estimated molecular mass (47 kDa) was consistent with that of the deduced amino acid sequence encoded by the rin mutant gene (46.8 kDa). This result indicates that the rin mutant gene is indeed translated and that the product accumulates in the mutant fruit. To investigate the subcellular localization of RIN, we then performed an in vivo transient localization assay. The

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Figure 2. Subcellular localization of RIN. The RINMIK–EGFP fusion protein was transiently expressed in onion epidermal cells, and the fluorescence images were visualized using fluorescence microscopy. (a) Green fluorescence image merged with visual light image, (b) DAPI-stained nuclear image merged with visual light image, (c) merged images of green fluorescence, DAPI staining and visual light.

nuclear localization signal of MADS box proteins maps to the MADS domain (Immink et al., 2002). The plasmid expressing a fusion protein of the truncated RIN (amino acids 1-159) with enhanced green fluorescence protein (EGFP) was introduced into onion epidermal cells, and fluorescence of the EGFP was detected using fluorescence microscopy. The subcellular localization of intense EGFP fluorescence coincided with DAPI staining of the nucleus (Figure 2), indicating the nuclear localization of RIN. RIN binds to specific DNA sequences A gel retardation assay was performed to determine whether RIN could bind to specific DNA sequences or not. Because no information about the target sequences for RIN was available, we used three different types of MADS box protein binding sites: c-fos SRE, N10 and PAL (West et al., 1998). As shown in Figure 3(a), these three sequences were clearly shifted by the truncated RIN protein (amino acids 1-157; RINMIK), indicating that RINMIK binds to the typical MADS domain binding sites. The band shifted by the full-length RIN was also detected when an N10-type oligonucleotide was used in the binding reaction (Figure 3a). Because background fluorescence from an in vitro protein synthesis system migrated at the same position as a signal from the RIN–DNA complex, we could not accurately determine the presence of weak signals for a shifted band of c-fos SRE- or PAL-type oligonucleotides. When the rin mutant protein was used in the assay, signal for the N10-type oligonucleotide was clearly detected, and faint signals for c-fos SRE- and PAL-type oligonucleotides were detected (Figure 3a). The RIN–DNA complex migrated more slowly than the rin mutant protein–DNA complex under these electrophoresis conditions, although the estimated molecular mass of the mutant protein (46.8 kDa) is much larger than that of RIN

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Figure 3. DNA binding by RIN. (a) Gel retardation assay for RIN (R), C domain-deleted RIN (RINMIK; M) and the rin mutant protein (r) with the c-fos SRE, N10 or PAL binding sites. The protein synthesis reaction with empty vector (pEU-3a) was also analyzed as a negative control (E). The solid arrowhead indicates the shifted DNA by RIN, the open circle indicates that shifted by RINMIK, and open arrowheads indicate that shifted by the rin mutant protein. (b) Sequence-specific binding of RIN. Gel retardation assay of RINMIK with the sequences of N10 and probe B (a negative control whose central sequence is GGATGCATCC). A binding competition assay was performed using labeled N10 probe and 20- or 100-fold concentrations of non-labeled N10 or probe B.

(28 kDa). The unexpected reduced mobility of the RIN protein was also observed in denatured conditions (Figure 1a), indicating that RIN might be a strongly charged protein. Competition assays were performed to confirm the specific DNA-binding property of RIN. Probe B, which is a negative control oligonucleotide that does not bind to the MADS domain of AGAMOUS (AG) (Huang et al., 1993), was not shifted by the RINMIK protein (Figure 3b). The intensity of the shifted signal for the RINMIK–N10 oligonucleotide complex decreased when unlabeled N10 oligonucleotide was added as a competitor to the assay, but not when unlabeled probe B was added (Figure 3b). These results indicate that RIN is a sequence-specific DNA-binding protein. RIN forms a homodimer To determine whether RIN forms a homodimer, a gel retardation assay was performed using mixtures of full-length RIN and truncated RIN (RINMIK). As shown in Figure 4(a), when the co-translated product of the full-length RIN and

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Characterization of ripening-regulator RIN 215

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Figure 4. Homodimer formation for RIN. (a) Gel retardation analysis of RIN (RIN), RINMIK (MIK) and their co-translational products using the N10 probe. (b) Co-immunoprecipitation assay of RIN and tagged RIN. RIN (RIN) and cMyctagged RIN (t-RIN) were co-translated (C) or mixed after separate translation (M), and then precipitated using anti-cMyc antibody. The precipitate (Co-IP) and supernatant of the reaction were analyzed by immunoblotting using antiRIN antibody.

RINMIK was used for the assay with N10 oligonucleotide, a complex that showed intermediate mobility between that of each of the two protein complexes was detected (‘RIN/MIK’ shown in Figure 4a). This intermediate band represents a dimer between RIN and truncated RIN protein. Self-interaction of RIN was also demonstrated by immunoprecipitation experiments using cMyc epitope-tagged RIN. The RIN protein was not precipitated by cMyc monoclonal antibody, but the tagged RIN was precipitated efficiently (Figure 4b, lanes 1 and 2). When co-translated with the tagged RIN, RIN was efficiently precipitated by cMyc monoclonal antibody (Figure 4b, lane 3). In contrast, a very weak signal for precipitated RIN was detected when RIN and the tagged RIN were translated separately and then mixed after translation (Figure 4b, lane 4). These results indicate that once RIN has formed a homodimer, subunit exchange seldom occurs. In summary, RIN apparently forms a stable homodimer immediately after translation. Determination of a RIN binding consensus sequence in vitro To identify the spectrum of the DNA sites to which RIN can bind, we carried out a DNA-binding site selection experiment using RINMIK from a random double-stranded oligonucleotide pool. Figure 5(a) shows the selected RINMIK

binding DNA sequences that were individually confirmed to bind RINMIK (using a gel retardation assay; data not shown). These sequences can be categorized into four major groups: ‘MEF2-like’ [5¢-CTA(A/T)4TAG-3¢], ‘SRF-like’ [5¢-CC(A/T)6GG3¢], ‘intermediate’ [5¢-CC(A/T)6AG-3¢], and other sequences that contain a single G/C base pair within the central A/T-rich region. Sequences randomly selected from these four groups were also confirmed to bind full-length RIN and the rin mutant protein (Figure 5c). The sequence information is summarized in Figure 5(b), and the consensus for RINMIK binding sequence is (T/a)(T/a)(-C)CCA(A/T)(A/t)(A/T)ATAG(G)AA, where upper-case letters represent the most common nucleotide(s), lower-case letters represent a relatively frequent nucleotide, and bold letters represent the nucleotides that correspond to the CArG box. Figure 6(a) shows comparison of consensus CArG box sequences for several plant and non-plant MADS domain proteins determined by binding studies using a pool of random oligonucleotides. The consensus sequence of RIN highly resembles those of SEP4 and SEP1, whose CArG box core sequences contain ‘CCA’ as the first three nucleotides and ‘TAG’ as the last three, and only A or T in the central four nucleotides (Figure 6a). The correlation between the DNA binding sites of the MADS proteins and the amino acid sequences of the MADS domains was investigated by aligning the amino acid sequences of the MADS domains of the proteins shown in Figure 6(a). The amino acid sequence of the MADS domain of RIN (Figure 6b) shows higher identity with that of SEP4 and SEP1 (53/56 and 52/56 residues are identical, respectively) than with the other MADS box proteins compared here. These similarities in amino acid sequences agree with the similarities in binding sites between RIN and the SEP proteins. In vivo binding study of RIN to the genes required for ethylene biosynthesis and sensing in fruit ripening A chromatin immunoprecipitation (ChIP) assay was used to detect in vivo binding of RIN to the promoters of genes required for ripening. Chromatin was prepared from fruits harvested at either the pink coloring stage or the immature green stage, and was then immunoprecipitated with the anti-RIN antibody. Among the genes that are up-regulated during ripening of fruit, ACC synthase 2 (LeACS2), ACC oxidase 1 (LeACO1) and LeETR3 (Nr), which encode ethylene biosynthetic enzymes and an ethylene receptor, were examined in this study. Possible CArG motif sites [C(C/T)(A/ T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G)G] were found in the promoter regions of these genes (Figure 7a,b), but no typical CArG motif site was found in the 2283 bp 5¢-untranslated region of the LeACS4 gene (accession number M88487). For these possible CArG motif sites that we found, four primer pairs specific to sequences flanking the selected sites were designed (Figure 7a). When PCR was performed using

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Figure 5. Selected DNA-binding sites for RIN. (a) Sequences of 36 cloned binding sites selected by the truncated RIN protein (RINMIK). Numbers indicate the clone number, and ‘R’ indicates that the sequence is shown in the reverse direction for better comparison with the consensus. The gray-shaded box indicates the CArG box. Uppercase letters represent bases derived from the random part of the oligonucleotide subjected (see Experimental procedures), and lower-case letters represent constant ends. (b) Consensus sequence of the RIN binding sites. The sequences indicated in (a) were summarized and visualized using WebLogo (Crooks et al., 2004). The overall height of each stack indicates the sequence conservation at that position, and the height of symbols within the stack reflects the relative frequency of the corresponding nucleic acid at that position. (c) Gel retardation assay for RIN (R), RINMIK (M) and rin mutant protein (r) binding to the selected sites. Numbers indicate the clone number shown in (a).

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Characterization of ripening-regulator RIN 217

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Figure 6. Consensus DNA-binding sites of MADS box proteins and alignment of the MADS domains. (a) Consensus DNA-binding sites derived by selection from random oligonucleotide pools. N indicates any base, D indicates A, T or G, H indicates A, C or T, and V indicates A, C or G. (b) Alignment of MADS domains of MADS box proteins compared in (a). Accession numbers are: RIN, AAM15775; SEP4, NP_178466; SEP1, P29382; SQUA, CAA45228; AG, P17839; SHATTERPROOF1 (SHP1), NP_001078311; AGAMOUS-LIKE15 (AGL15), AAA65653; Myocyte-specific enhancer factor 2A (MEF2A), Q02078; Minichromosome maintenance 1 (MCM1), CAA88409; SRF, NP_003122.

primers specific to the target site of LeACS2, the amplified signal in the ChIP DNA from ripening fruit was significantly stronger than that observed in the input DNA or in pre-immune serum ChIP DNA, indicating enrichment of the LeACS2 fragment by ChIP (Figure 7c). In contrast, no significant enrichment was detected in the same assays for ChIP DNA from immature green fruit (Figure 7c). Reproducible results were obtained for all of the ChIP DNAs independently prepared from six ripening fruits and three immature green fruits. No significant enrichment was detected in the sites of ACO1 and Nr (data not shown). To determine whether in vitro-synthesized RIN could bind to the identified site, a oligonucleotide that included 26 bp of the LeACS2 target site was labeled and assayed by gel retardation assay. As shown in Figure 7(d), the truncated RIN protein clearly bound to the target site of LeACS2. RIN functions as a transcriptional activator and the rin mutation causes loss of function The transcription-activating machinery appears to function universally in all eukaruotic systems (Ma et al., 1988), and

Figure 7. In vivo binding assay for RIN at the CArG box site within the cis-element of genes required for ethylene biosynthesis and sensing. (a) Localization of CArG box sites within the cis-elements of Nr, LeACS2 and LeACO1. Boxes indicate the CArG box sites. Arrowheads indicate primers used for ChIP PCR. (b) DNA sequence of the CArG boxes within the cis-elements of Nr, LeACS2 and LeACO1. (c) ChIP PCR assay for the cis-element of LeACS2. Chromatin was prepared from either a pink coloring fruit or an immature green fruit, and was immunoprecipitated using RIN-specific antiserum (C) or pre-immune serum (P). Total input chromatin DNA treated with sonication (see Experimental procedures) was also used as a negative control (I). The PCR reaction was performed using primers amplifying the CArG motif region of LeACS2 or the control (actin). (d) Gel retardation assay of RINMIK binding to the selected site in LeACS2 (lane 2) and to the N10 probe (lane 1). The sequence shows the CArG box (underlined) and flanking region of LeACS2 used in the gel retardation assay.

several plant MADS proteins have been demonstrated to function as transcriptional activators in yeast cells as well as in plant cells (Honma and Goto, 2001). To determine whether RIN functions as a transcriptional activator, we performed a transcriptional activity assay using a yeast system. In this system, a target protein is expressed as a fusion protein with the GAL4 DNA-binding domain (GAL4DNA BD) in a yeast cell, and if the target protein has a transcriptional activation potential, then the yeast cell expresses marker genes (ADE2, HIS3 and MEL1), can survive on histidine- and adeninedeficient medium, and exhibits a-galactosidase activity. The assay results showed that, when the GAL4DNA BD–RIN fusion protein was expressed in a yeast cell, the marker genes were clearly induced (‘RIN’ in Figure 8b), indicating that RIN has a transcriptional activator domain. To identify the transcriptional activator domain of RIN, we also assayed constructs that encode various truncated RIN proteins (Figure 8a). Although all the constructs expressing

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Figure 8. Transcriptional activity of RIN in yeast cells. (a) Schematic of the proteins produced from yeast vectors. (b) Viability test for yeast cells containing plasmids expressing the proteins shown in (a) on SD/)Trp or SD/)Trp/)Ade/)His/X-a-Gal medium. When the plasmids contain a gene that encodes a transcriptional activation protein, the transformed yeast cells can grow on SD/)Trp/)Ade/-His/X-a-Gal medium, and the colony turns blue as a result of the a-galactosidase activity of MEL1.

protein containing the whole C domain of RIN (‘IKC’ and ‘KC’ shown in Figure 8b) induced the marker genes, those expressing proteins whose C domain was partially deleted (KC178 and MIKC215 in Figure 8b) or entirely deleted (MIK and K in Figure 8b) did not. These results indicate that the C domain of RIN has transcriptional activation potential, and that the C-terminal 27-amino-acid region is prerequisite for this function. In addition, the transcriptional activation potential of the rin mutant protein and of MC, which is another component of the rin mutant protein (Figure 8a), was also assayed. As shown in Figure 8(b), neither the rin mutant protein nor MC had transcriptional activator potential. Discussion Extensive molecular characterization of the MADS box transcription factor family has revealed that a dimer is the fundamental function unit of MADS box transcription factors. The crystal structure of MADS box domains indicates that the dimerized MADS box domain binds to CArG box DNA (Pellegrini et al., 1995; Santelli and Richmond, 2000). Among MADS box proteins in plants, APETALA1 (AP1), AG, SQUAMOSA (SQUA), PLENA (PLE) and SEP1 bind to DNA as homodimers (Huang et al., 1996; Riechmann et al., 1996; West et al., 1998), whereas APETALA3 (AP3), PI, DEFICENS (DEF), and GLOBOSA (GLO) bind to DNA when they form

heterodimers with their counterparts but not when used alone (Riechmann et al., 1996; Schwarz-Sommer et al., 1992). Furthermore, dimerization is necessary for nuclear localization of MADS box proteins (Immink et al., 2002; McGonigle et al., 1996). In this study, we showed that RIN synthesized in vitro formed stable homodimers that bind to CArG box DNA, and that the truncated RIN–EGFP fusion protein expressed transiently in the plant cell was localized in the nucleus. Our results suggest that the RIN homodimer can act as a fundamental functional unit in cells of ripening fruit. In a ripening tomato cell, however, at least seven MADS box genes are expressed in early ripening or fully ripe tomato fruit (Giovannoni, 2004). We cannot exclude the possibility that MADS box proteins encoded by these genes might have a higher affinity with RIN and form a heterodimer with RIN to function in vivo. We also determined the consensus sequence to which RIN binds and demonstrated that RIN has transcriptional activation potential. As shown in Figure 6(a), the consensus binding sequence of RIN highly resembles that of SEP4 and SEP1 (Huang et al., 1995, 1996). SEP proteins also apparently have a transcriptional activator domain (Honma and Goto, 2001). These findings indicate that RIN and the SEP proteins have similar molecular functions although they play distinctly different biological roles. Phylogenetic analysis of the MADS box family in tomato and Arabidopsis revealed that RIN is classified within the SEP clade (Hileman et al., 2006), suggesting that the RIN gene was generated from duplication of the SEP genes after the diversification of Arabidopsis and tomato, and might have taken on a new function in tomato development (Hileman et al., 2006). Despite RIN and SEP proteins recognizing similar DNA-binding sites in vitro, these transcription factors are expected to activate transcription of distinctly different genes in the cell. Based on the floral quartet model, a tetrameric MADS box protein complex binds to two target DNA motifs, which results in more specific recognition of target promoters than recognition by a single dimer (Egea-Cortines et al., 1999; Theissen and Saedler, 2001). Although a vast number (>17 000) of the 10 bp CArG box consensus-like sequences are found around genes in the Arabidopsis genome, the number of genes with at least two CArG boxes is only approximately 1700 (de Folter and Angenent, 2006). The SEP3 protein interacts with AP1, the PI–AP3 complex and AG, and formation of these protein complexes possibly activates the specific regulation of floral organ identification (Honma and Goto, 2001). These findings suggest that, similar to the SEP proteins, RIN forms multimers with other transcription factor(s), presumably with MADS box protein(s), and that recognition of several binding sites by the multimer determines the genes that should be transcribed at the fruit-ripening stage. Our ChIP assay revealed that the CArG box site in the cis-elements of the LeACS2 gene did indeed bind to RIN in a ripening fruit cell, but the typical CArG box sites found in LeACO1 and Nr

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Characterization of ripening-regulator RIN 219 (Figure 7a) did not. These results support the hypothesis that RIN forms a complex with other transcription factor(s) to recognize specific DNA sites among possible CArG box sites in a genome. The presence of LeACS2 transcripts in ripening tomato fruit has been well documented (Barry et al., 2000; Lincoln et al., 1993; Olson et al., 1991; Yip et al., 1992). In addition, the expression of LeACS2 in fruit can be induced by wounding stress or ethylene treatment (Lincoln et al., 1993; Olson et al., 1991). Moreover, LeACS2 can be induced by ethylene treatment even in rin mutant fruit (Barry et al., 2000). Based on their analysis of expression of ACS genes, Barry et al. (2000) suggested that RIN induces up-regulation of LeACS1A and LeACS4 during transition to the ripening stage, and that the consequent increase in ethylene production induces LeACS2 expression. Our ChIP assay PCR, however, revealed that the RIN protein bound with high affinity to the promoter region of the LeACS2 gene, suggesting that RIN directly induces transcription of LeACS2. One possible explanation for this discrepancy in the results between the two studies is that transcription of LeACS2 might be induced by both RIN-dependent and RIN-independent mechanisms. The promoter of LeACS2 contains a potential cis-acting regulatory element that is responsible for inducement by either ethylene treatment or wounding stress (Lincoln et al., 1993). This potential regulatory element is located about 500 bp upstream of the RIN-binding site identified in this study. These two distinct regulatory sites might be independently responsible for the initiation of the transcription that is induced by different signal transduction mechanisms. If so, LeACS2 expression in ripening fruit should be initiated and maintained by both ethylene and the RIN-mediating regulation. Because only a limited number of MADS box proteins have transcriptional activation potential (Busi et al., 2003; Cho et al., 1999; Honma and Goto, 2001), this function is an important feature for RIN. In addition, accumulation of mRNA and the product of the RIN gene coincides with the beginning of fruit ripening (Figure 1a) (Kitagawa et al., 2005; Vrebalov et al., 2002), suggesting that expression of RIN regulates onset of transcription of the genes required for fruit ripening. We also detected in vivo binding of RIN to the cis-elements of the LeACS2 gene, suggesting that RIN directly controls expression of this gene that encodes a key enzyme in ethylene biosynthesis during fruit ripening. In summary, RIN apparently controls fruit ripening by determining the timing of transcriptional activatation, contributing partially to the selection of the gene to transcribe, and supplying a transcription complex with a transcriptional activating domain. Coordination of RIN and interacting protein(s) enables highly synchronized and specific expression of the genes required for fruit ripening. The rin mutant fruit completely fails to ripen and does not show up-regulation of the gene expression required for

ripening (Barry et al., 2000; Kitagawa et al., 2005; Knapp et al., 1989; Rose et al., 1997). The product of the rin mutant gene is translated and then accumulates in the mutant fruit cell (Figure 1b), suggesting that the rin mutation causes loss of a particular function of the RIN protein. We then demonstrated that the rin mutant protein exhibited in vitro DNAbinding activity with the same specificity as the wild-type protein, but had lost its transcription-activating function. These results suggest that loss of transcription-activating function by the rin mutation suppresses up-regulation of the transcription of genes required for fruit ripening, and consequently totally inhibits fruit ripening. This interpretation suggests that the rin mutant protein might be a possible negative regulator because it binds to target sites but does not have a transcription-activating function. We observed that the fruit of the RIN/rin heterozygote ripened at an intermediate level between the normal (RIN/RIN) and the rin mutant (rin/rin), and that transcription of the genes required for ripening was partially suppressed (Kitagawa et al., 2005). This partial suppression can be explained by competition between the transcription factor complex with the normal RIN protein and that with the mutant protein. In this competition model, the transcription factor complex with the mutant protein has comparable DNA-binding activity to that with the normal protein, and the normal- and mutanttype complexes compete for binding to the target sites. Consequently, the reduced frequency of access of the normal-type complex to the target sites would decrease initiation of transcription. Another explanation is that the partial suppression in the heterozygote is simply due to low production of the normal RIN protein. In this low-production model, the amount of transcription factor complex with the normal protein that is produced in the heterozygote is too low to induce sufficient amounts of transcripts, and the mutant protein has no function. Further experiments are necessary to confirm the effect of the rin mutant protein. The rin mutant gene encodes a fusion protein of RIN lacking the C-terminal 27 amino acids and MC that lacks the MADS box domain (Giovannoni, 2004). The C domain of the MADS box proteins is highly variable region and seems to play a crucial role in the functions of these proteins. Ectopic expression results for AG cDNA that lacks the C region revealed that this region is required for AG function (Mizukami et al., 1996). Ternary complex formation between DEF, GLO and SQUA requires the C domain of the proteins (EgeaCortines et al., 1999). The C-terminal region of AP1 and its homologs are responsible for the transcriptional activation function (Cho et al., 1999). Our results indicate that the C domain of RIN exhibits transcription-activating function, and that the C-terminal 27 residues, which are deleted in the rin mutant protein, are crucial for this function (Figure 8). Partial deletion of this important functional domain appears to cause the rin mutant protein to lose its ability to induce fruit ripening.

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220 Yasuhiro Ito et al. Experimental procedures Plasmid construction The plasmid that expresses a fusion protein of part of RIN (amino acids 1-159) and EGFP in a plant cell was constructed as follows. A CaMV 35S promoter–GUS–nos terminator fragment from pBI121 (Clontech, http://www.clontech.com) that had been digested using two enzymes (HindIII and EcoRI) was inserted into pUC19 digested with the same enzymes. The resulting plasmid was digested with SacI, blunt-ended with T4 DNA polymerase, digested with BamHI, and then ligated with an EGFP fragment from pEGFP (Clontech) that had been first digested with EcoRI, then blunt-ended with T4 DNA polymerase, and finally digested with BamHI. The resulting plasmid was inserted along with a fragment encoding the MIK region of RIN (amino acids 1-159) that had been amplified using the primer pair 5¢-CGGATCCAATATGGGTAGAGGGAAAGTAG-3¢ and 5¢-ATGC CATGGATTGTTCCTTTTGTTGAAG-3¢ and digested with BamHI and NcoI. The resulting plasmid was designated pUC-MIK-EGFP. The plasmid to produce the RIN protein via an in vitro translation system was constructed as follows. The RIN cDNA was amplified using the primer pair 5¢-CCATGGGTAGAGGGAAAGTAG-3¢ and 5¢-TCAAAGCATCCATCCAGGTAC-3¢, and was then inserted into the EcoRV site of pBluescriptSK(+) (Stratagene, http://www.stratagene.com/). The resulting plasmid, named pBT-Rin5, was digested with NcoI and SacI, and then the fragment of the RIN coding region was inserted into the same restriction sites of the pEU-3a in vitro translation vector (Ito et al., 2002), and the resulting plasmid was designated pEU-RIN. The plasmid pEU-cMyc-RIN, expressing cMyc epitopetagged RIN, was constructed as follows. The DNA fragment encoding the cMyc epitope-tagged RIN was amplified from pGBKRIN (described below) using the primer pair 5¢-ATACCATGGAGGAGCAGAAG-3¢ and 5¢-TATAGGCAATGTATTATTGC-3¢. The resulting fragment was digested with NcoI and then inserted into the same restriction sites of pEU-RIN. To construct pEU-rin, the cDNA of the rin mutant gene was amplified using the primer pair rinF2 (5¢-AGACCATGGGTAGAGGGAAAGTAG-3¢) and rinR3 (5¢-CTTATACTCATTTGTCTGTC-3¢), digested with NcoI and SacI, and inserted into the same restriction sites of pEU-3a. To create pEURINMIK, pGBK-RINMIK (described below) was digested with NcoI and BamHI, and the resulting DNA fragment containing the MIK region was inserted into the same restriction sites of pEU-3a. To create pEU-MC, the MC coding region was amplified using primers MC-F1 (5¢-GACCATGGGAAGAGGAAAAGTTG-3¢) and rinR3, then digested with NcoI and SacI, and finally inserted into pEU-3a. The plasmids pGEM-SRE, pGEM-N10, pGEM-PAL, pGEM-B and pGEMACS2 were constructed as follows. The oligonucleotide pairs 5¢-AGCTTTACCATATTAGGTAAAGCTTG-3¢/5¢-GATCCAAGCTTTACCTAATATGGTAA-3¢ (pGEM-SRE), 5¢-AGCTTTACTATTTATAGTAAAGCTTG-3¢/5¢-GATCCAAGCTTTACTATAAATAGTAA-3¢ (pGEMN10), 5¢-AGCTTTACCTAATTAGGTAAAGCTTG-3¢/5¢-GATCCAAGCTTTACCTAATTAGGTAA-3¢ (pGEM-PAL), 5¢-AGCTTTAGGATGCATCCTAAAGCTTG-3¢/5¢-GATCCAAGCTTTAGGATGCATCTAA-3¢ (pGEM-B), and 5¢-AGCTATTCTAAAAAAAGTATCACATA-3¢/5¢-GATCTATGTGATACTTTTTTTAGAAT-3¢ (pGEM-ACS2) were annealed, and each resulting DNA fragment was inserted into HindIII/BamHIcleaved pGEM-7zf(+) (Promega, http://www.promega.com/). To test the transcriptional activation ability of RIN in a yeast system, the coding region of RIN from pBT-Rin5 digested with NcoI and BamHI was inserted into pGBKT7 (BD Matchmaker Library Construction & Screening Kit, Clontech), and the resulting plasmid was designated pGBK-RIN. pEU-rin was digested with NcoI and EcoRI and resulting rin mutant gene fragment was inserted into pGBKT7, resulting in the plasmid pGBK-rin. DNA fragments of various portions of RIN

were amplified using primers RIN-MADS-F (5¢-ATGCCATGGAGA GATACCACAGATACA-3¢) and RIN-MADS-R (5¢-CGGAATTCAAAG -CATCCATCCAGG-3¢) for the IKC region (amino acids 68-242), RINK-F (5¢-AGACCATGGACCAAGAGTATTTGAAG-3¢) and RIN-MADS-R for the KC region (amino acids 92-242), RIN-K-F and RIN-K-R1 (5¢GCCGAATTCTTATTCCTTTTGTTGAAGTTC-3¢) for the K region (amino acids 92-159), RIN-K-F and RIN-K-R2 (5¢-GCCGAATTCTTAAAAGGTAACACCAAGTTC-3¢) for the K region and truncated C domain (amino acids 92-178), rinF2 and RIN-K-R1 for the MIK region (amino acids 1-157), and rinF2 and RIN-K-R3 (5¢-GCCGAATTCTTAACTTATAGGCAATGTATT-3¢) for the MIK region and truncated C domain (amino acids 1-215). Each amplified DNA fragment was digested using NcoI and EcoRI, and then inserted into pGBKT7. The resulting plasmids were designated pGBK-IKC, pGBK-KC, pGBK-K, pGBK-KC178, pGBK-MIK and pGBK-MIKC215, respectively. The LeMADS MC fragment from pEU-MC that had been digested with NcoI and EcoRI was inserted into pGBKT7, resulting in the plasmid pGBK-MC.

Immunoblotting analysis Tomato fruits were harvested at five maturation stages: immature green, mature green, breaker, pink coloring (4 days after breaker stage) and red ripe (7 days after breaker stage). Nuclear extracts from fresh fruits and frozen leaves, flowers and stems were prepared as described previously (Manzara et al., 1991). Proteins were separated on 8% w/v or 10% w/v SDS–PAGE and transferred to Immobilon-P PVDF membranes (Millipore, http://www.millipore. com). After blocking overnight in PBST (137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4, 1.5 mM KH2PO4 and 0.1% v/v Tween-20) containing 2% w/v skim milk, the blot was incubated for 1 h with anti-RIN antiserum at a dilution of 1:10 000 with Can Get Signal solution 1 (Toyobo, http://www.toyobo.co.jp). Unbound antiserum was removed by washing in PBST (3 · 10 min). The blots were then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare Bio Science, http://www.gehealth care.com) previously diluted to 1:10 000 using Can Get Signal solution 2 (Toyobo). After washing the blots again in PBST (3 · 10 min), ECL Western blotting detection reagents (GE Healthcare Bio Science) were used to detect the signals. For each immunoblot, a duplicate gel was stained using a Rapid Stain CBB kit (Nacalai tesque, http://www.nacalai.com) to confirm equal loading of proteins.

Transient expression assay of the EGFP fusion protein In the transient expression assay, the pUC-MIK-EGFP plasmid was first delivered into onion (Allium cepa) epidermal cells using a Rehbock Particle Gun 190 (Rehbock Co., Tokyo, Japan). The transformed tissues were then incubated on Murashige and Skoog agar medium in darkness for 16 h at 25C. Nuclei were stained with 10 lg ml)1 4¢,6-diamino-phenylindole (DAPI) for 10 min. EGFP fluorescence and DAPI-stained nuclear images were acquired using a BZ-8000 fluorescence microscope (Keyence, http://www.keyence.com). Images were then analyzed by using BZ analyzer software (Keyence).

Gel retardation assay Fluorescein isothiocyanate (FITC)-labeled probes were constructed as follows. First, oligonucleotides 5¢-ACTCGAGGAATTCGGTAC-3¢ and 5¢-ACGCGTTGGGAGCTCTC-3¢, which are matching sequences flanking the insertion site of pGEM7zf(+), were end-labeled with

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Characterization of ripening-regulator RIN 221 FITC and used for labeling DNA fragments cloned in pGEM7zf(+) via PCR reaction. Proteins were prepared by in vitro translation using TNT coupled reticulocyte lysate systems (Promega, http:// www.promega.com). Protein–DNA binding reactions were performed according to the method described previously (Riechmann et al., 1996), and then free and bound DNAs were separated on a 5% polyacrylamide/bisacrylamide (60:1) gel in 1· TBE. Fluorescence images of the FITC-labeled probe were detected using a Typhoon 8600 (GE Healthcare Bio Science) and subsequently adjusted for contrast/brightness using Adobe Photoshop (http://www.adobe. com).

GAAGCAAGTTGTTGAAG-3¢) and CPACO1R1 (5¢-TGAGAGGTTCACAAATTCTC-3¢), or CPACO1F2 (5¢-GCTAGCTTCCGCGTTTACAC-3¢) and CPACO1R2 (5¢-GTATGAATTTCCTAGAGATG-3); for LeACS2, CPACS2F1 (5¢-AATTCTTTACGCAATCTTAC-3¢) and CPACS2R1 (5¢TATTAAGCAGGTGTCTGGAC-3¢); for Nr, CPNRF1 (5¢-CCTTTTGGTAAGTTCAGGAG-3¢) and CPNRR1 (5¢-TACTTGAATTCACTGTCTAC-3¢); for the coding region of actin, Tom52F3 (5¢-GTTGTCTTACATTGCTCTTG-3¢) and Tom52R (TAGATCCTCCGATCCAGACA -3¢). PCRs were resolved on 2% agarose gels.

Transcriptional activity assay in yeast Selection of RIN binding sequences from random oligonucleotides A pool of random double-stranded oligonucleotides of sequence 5¢-GTAAAACGACGGCCAGTGAATTCGGTACCCCGGGT-N26-TGGA TCCGGAGAGCTCCCAACGCGT-3¢ was labeled by a PCR reaction using primers 5¢-GTAAAACGACGGCCAGT-3¢ and FITC-labeled 5¢ACGCGTTGGGAGCTCTC-3¢. DNA-binding sites were selected according to the method described previously (Huang et al., 1993). After six rounds of selection, bound DNA pools were amplified again with the same primer set, then digested with EcoRI and BamHI, and cloned into the same site of pGEM7zf(+). DNA sequences were determined using an ABI310 Gene Analyzer (Applied Biosystems, http://www.appliedbiosystems.com) with a BigDye Terminator Cycle sequencing kit (Applied Biosystems). WebLogo software (Crooks et al., 2004) was used to represent the consensus sequence graphically.

Immunoprecipitation assay For immunoprecipitation experiments, RIN and cMyc-tagged RIN were synthesized using TNT coupled reticulocyte lysate systems. Immunoprecipitation reactions with anti-cMyc monoclonal antibody were carried out using a BD Matchmaker Co-IP kit (Clontech). Precipitated proteins were separated by 10% w/v SDS–PAGE, and then detected by immunoblotting analysis with anti-RIN antibody.

Chromatin immunoprecipitation To cross-link genomic DNA and protein in fruit tissue, sliced fruit was submerged in extraction buffer [0.4 M sucrose, 10 mM Tris–HCl, pH 8.0, 5mM b-mercaptoethanol, 0.1 mM PMSF and proteinase inhibitor cocktail (Nacalai)] with 1% formaldehyde and vacuumed for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M and application of vacuum for an additional 5 min. After rinsing with ice-cold water, fruit tissue was frozen in liquid nitrogen. Chromatin isolation was performed by the method described previously (Bowler et al., 2004). The chromatin solution was sonicated for 10 sec four times on 10% power (setting 3) using a Sonifier 450 (Branson, http://www.sonifier.com). The sonicated chromatin suspension was immunoprecipitated with anti-RIN serum or pre-immune serum, and DNA was recovered using the method described previously (Bowler et al., 2004). To identify the potential in vivo target sites of RIN, 5¢ non-coding regions of the genes for LeACO1 (accession number X58273), LeACS2 (accession number X59139) and Nr (LeETR3; accession number AY600437) were analyzed using Genetyx software (Genetyx, http:// www.sdc.co.jp/genetyx/) to determine possible CArG box sequences. PCR reaction was performed for the recovered DNA with the following oligonucleotides: for LeACO1, CPACO1F1 (5¢-GGA-

Transcriptional activity of RIN in the yeast cell was assayed as described previously (Cho et al., 1999). The yeast strain used was AH109 (Clontech), which contains three reporters (ADE2, HIS3 and MEL1) under the control of three distinct GAL4 upstream activating sequences and TATA boxes. To assess the transcription-activating function, plasmid vectors that express the GAL4 DNA-binding domain fused either with whole or partial sections of RIN, with the rin mutant protein, or with MC as described above were transformed into AH109 by the lithium acetate-mediated method (Gietz et al., 1992), and transformants were selected on the SD/-Trp media (Clontech). To assess marker gene expression, transformants were cultured on SD/)Trp/)Ade/)His/X-a-Gal medium.

Acknowledgements We thank Dr Y. Ishiguro for valuable discussions during this study. This research was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References Barry, C.S., Llop-Tous, M.I. and Grierson, D. (2000) The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiol. 123, 979–986. Bowler, C., Benvenuto, G., Laflamme, P., Molino, D., Probst, A.V., Tariq, M. and Paszkowski, J. (2004) Chromatin techniques for plant cells. Plant J. 39, 776–789. Busi, M.V., Bustamante, C., D’Angelo, C., Hidalgo-Cuevas, M., Boggio, S.B., Valle, E.M. and Zabaleta, E. (2003) MADS-box genes expressed during tomato seed and fruit development. Plant Mol. Biol. 52, 801–815. Causier, B., Kieffer, M. and Davies, B. (2002) Plant biology. MADSbox genes reach maturity. Science, 296, 275–276. Cho, S., Jang, S., Chae, S., Chung, K.M., Moon, Y.H., An, G. and Jang, S.K. (1999) Analysis of the C-terminal region of Arabidopsis thaliana APETALA1 as a transcription activation domain. Plant Mol. Biol. 40, 419–429. Coen, E.S. and Meyerowitz, E.M. (1991) The war of the whorls: genetic interactions controlling flower development. Nature, 353, 31–37. Crooks, G.E., Hon, G., Chandonia, J.M. and Brenner, S.E. (2004) WebLogo: a sequence logo generator. Genome Res. 14, 1188– 1190. Ditta, G., Pinyopich, A., Robles, P., Pelaz, S. and Yanofsky, M.F. (2004) The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Curr. Biol. 14, 1935–1940. Egea-Cortines, M., Saedler, H. and Sommer, H. (1999) Ternary complex formation between the MADS-box proteins SQUAMO-

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 212–223

222 Yasuhiro Ito et al. SA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J. 18, 5370–5379. de Folter, S. and Angenent, G.C. (2006) Trans meets cis in MADS science. Trends Plant Sci. 11, 224–231. Gietz, D., St Jean, A., Woods, R.A. and Schiestl, R.H. (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425. Giovannoni, J.J. (2004) Genetic regulation of fruit development and ripening. Plant Cell, 16(Suppl.), S170–S180. Hileman, L.C., Sundstrom, J.F., Litt, A., Chen, M., Shumba, T. and Irish, V.F. (2006) Molecular and phylogenetic analyses of the MADS-box gene family in tomato. Mol. Biol. Evol. 23, 2245–2258. Honma, T. and Goto, K. (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 409, 525–529. Huang, H., Mizukami, Y., Hu, Y. and Ma, H. (1993) Isolation and characterization of the binding sequences for the product of the Arabidopsis floral homeotic gene AGAMOUS. Nucleic Acids Res. 21, 4769–4776. Huang, H., Tudor, M., Weiss, C.A., Hu, Y. and Ma, H. (1995) The Arabidopsis MADS-box gene AGL3 is widely expressed and encodes a sequence-specific DNA-binding protein. Plant Mol. Biol. 28, 549–567. Huang, H., Tudor, M., Su, T., Zhang, Y., Hu, Y. and Ma, H. (1996) DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation. Plant Cell, 8, 81–94. Immink, R.G., Gadella, T.W., Jr, Ferrario, S., Busscher, M. and Angenent, G.C. (2002) Analysis of MADS box protein–protein interactions in living plant cells. Proc. Natl Acad. Sci. USA, 99, 2416–2421. Ito, Y., Ozawa, A., Sawasaki, T., Endo, Y., Ochi, K. and Tozawa, Y. (2002) OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase. Biosci. Biotechnol. Biochem. 66, 2727–2731. Kitagawa, M., Ito, H., Shiina, T., Nakamura, N., Inakuma, T., Kasumi, T., Ishiguro, Y., Yabe, K. and Ito, Y. (2005) Characterization of tomato fruit ripening and analysis of gene expression in F1 hybrids of the ripening inhibitor (rin) mutant. Physiol. Plant. 123, 331–338. Kitagawa, M., Moriyama, T., Ito, H. et al. (2006) Reduction of allergenic proteins by the effect of the ripening inhibitor (rin) mutant gene in an F1 hybrid of the rin mutant tomato. Biosci. Biotechnol. Biochem. 70, 1227–1233. Knapp, J., Moureau, P., Schuch, W. and Grierson, D. (1989) Organization and expression of polygalacturonase and other ripening related genes in Ailsa Craig ‘Neverripe’ and ‘Ripening inhibitor’ tomato mutants. Plant Mol. Biol. 12, 105–116. Lincoln, J.E., Campbell, A.D., Oetiker, J., Rottmann, W.H., Oeller, P.W., Shen, N.F. and Theologis, A. (1993) LE–ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis. J. Biol. Chem. 268, 19422– 19430. Ma, J., Przibilla, E., Hu, J., Bogorad, L. and Ptashne, M. (1988) Yeast activators stimulate plant gene expression. Nature, 334, 631–633. Manzara, T., Carrasco, P. and Gruissem, W. (1991) Developmental and organ-specific changes in promoter DNA–protein interactions in the tomato rbcS gene family. Plant Cell, 3, 1305–1316. Mao, L., Begum, D., Chuang, H.W., Budiman, M.A., Szymkowiak, E.J., Irish, E.E. and Wing, R.A. (2000) JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development. Nature, 406, 910–913.

McGonigle, B., Bouhidel, K. and Irish, V.F.(1996) Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression. Genes Dev. 10, 1812–1821. Messenguy, F. and Dubois, E. (2003) Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development. Gene, 316, 1–21. Mizukami, Y., Huang, H., Tudor, M., Hu, Y. and Ma, H. (1996) Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations. Plant Cell, 8, 831–845. Olson, D.C., White, J.A., Edelman, L., Harkins, R.N. and Kende, H. (1991) Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits. Proc. Natl Acad. Sci. USA, 88, 5340–5344. Pelaz, S., Ditta, G.S., Baumann, E., Wisman, E. and Yanofsky, M.F. (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature, 405, 200–203. Pellegrini, L., Tan, S. and Richmond, T.J. (1995) Structure of serum response factor core bound to DNA. Nature, 376, 490–498. Picton, S., Gray, J., Barton, S., AbuBakar, U., Lowe, A. and Grierson, D. (1993) cDNA cloning and characterisation of novel ripeningrelated mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant. Plant Mol. Biol. 23, 193–207. Pollock, R. and Treisman, R. (1990) A sensitive method for the determination of protein–DNA binding specificities. Nucleic Acids Res. 18, 6197–6204. Pollock, R. and Treisman, R. (1991) Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 5, 2327–2341. Riechmann, J.L., Krizek, B.A. and Meyerowitz, E.M. (1996) Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proc. Natl Acad. Sci. USA, 93, 4793–4798. Rose, J.K., Lee, H.H. and Bennett, A.B. (1997) Expression of a divergent expansin gene is fruit-specific and ripening-regulated. Proc. Natl Acad. Sci. USA, 94, 5955–5960. Santelli, E. and Richmond, T.J. (2000) Crystal structure of MEF2A core bound to DNA at 1.5 A˚ resolution. J. Mol. Biol. 297, 437– 449. Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P.J., Hansen, R., Tetens, F., Lonnig, W.E., Saedler, H. and Sommer, H. (1992) Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO J. 11, 251–263. Shiraishi, H., Okada, K. and Shimura, Y. (1993) Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro. Plant J. 4, 385–398. Shore, P. and Sharrocks, A.D. (1995) The MADS-box family of transcription factors. Eur. J. Biochem. 229, 1–13. Tang, W. and Perry, S.E. (2003) Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J. Biol. Chem. 278, 28154–28159. Theissen, G. and Saedler, H. (2001) Plant biology. Floral quartets. Nature, 409, 469–471. Tigchelaar, E.C., McGlasson, W.B. and Buescher, R.W. (1978) Genetic regulation of tomato fruit ripening. HortScience, 13, 508– 513. Vrebalov, J., Ruezinsky, D., Padmanabhan, V., White, R., Medrano, D., Drake, R., Schuch, W. and Giovannoni, J. (2002) A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science, 296, 343–346.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 212–223

Characterization of ripening-regulator RIN 223 West, A.G., Causier, B.E., Davies, B. and Sharrocks, A.D. (1998) DNA binding and dimerisation determinants of Antirrhinum majus MADS-box transcription factors. Nucleic Acids Res. 26, 5277–5287. Wynne, J. and Treisman, R. (1992) SRF and MCM1 have related but distinct DNA binding specificities. Nucleic Acids Res. 20, 3297– 3303. Yao, J., Dong, Y. and Morris, B.A. (2001) Parthenocarpic apple fruit production conferred by transposon insertion mutations in a

MADS-box transcription factor. Proc. Natl Acad. Sci. USA, 98, 1306–1311. Yip, W.K., Moore, T. and Yang, S.F. (1992) Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions. Proc. Natl Acad. Sci. USA, 89, 2475–2479.

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