Null mutation of the MdACS3 gene, coding for a ripening-specific 1-aminocyclopropane-1-carboxylate synthase, leads to long shelf life in apple fruit.

Expression of MdACS1, coding for 1-aminocyclopropane-1-carboxylate synthase (ACS), parallels the level of ethylene production in ripening apple (Malus domestica) fruit. Here we show that expression of another ripening-specific ACS gene (MdACS3) precedes the initiation of MdACS1 expression by approximately 3 weeks; MdACS3 expression then gradually decreases as MdACS1 expression increases. Because MdACS3 expression continues in ripening fruit treated with 1-methylcyclopropene, its transcription appears to be regulated by a negative feedback mechanism. Three genes in the MdACS3 family (a, b, and c) were isolated from a genomic library, but two of them (MdACS3b and MdACS3c) possess a 333-bp transposon-like insertion in their 5′ flanking region that may prevent transcription of these genes during ripening. A single nucleotide polymorphism in the coding region of MdACS3a results in an amino acid substitution (glycine-289 → valine) in the active site that inactivates the enzyme. Furthermore, another null allele of MdACS3a, Mdacs3a, showing no ability to be transcribed, was found by DNA sequencing. Apple cultivars homozygous or heterozygous for both null allelotypes showed no or very low expression of ripening-related genes and maintained fruit firmness. These results suggest that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and is a possible determinant of ethylene production and shelf life in apple fruit.

The plant hormone ethylene plays an important role in the regulation of fruit ripening (Capitani et al., 1999). Many ripening processes are driven by ethyleneregulated changes in gene expression, especially in climacteric fruit exhibiting a high respiration rate during ripening, such as apple (Malus domestica) and tomato (Solanum lycopersicum; Giovannoni, 2001). The level of ethylene in plants is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). In ripening apple fruit, ethylene production is strictly related to the level of MdACS1 expression (Sunako et al., 1999;Harada et al., 2000). Moreover, differential expression of MdACS1 allelic forms (MdACS1-1 and -2) among different cul-tivars causes differences in the extent of preharvest fruit drop, which is a physiological effect of the ethylene level in the fruit (Harada et al., 2000;Sato et al., 2004). However, the polygalacturonase gene (MdPG1), which is involved in softening of fruit flesh (shelf life), was transcribed differently in cultivars with the same MdACS1 allelic forms (Oraguzie et al., 2004), suggesting that other factor(s) are correlated with the trait of shelf life. On the basis of the level of ethylene production during fruit development, McMurchie et al. (1972) introduced the concept of system-1 and system-2 ethylene. System 1, ethylene autoinhibitory, is considered to function during normal vegetative growth and to be responsible for the basal level of ethylene production; system 2 has been proposed to operate in the upsurge of ethylene production during the ripening of climacteric fruit when ethylene is autostimulatory (Lelièvre et al., 1997;Barry et al., 2000). A large increase in ethylene production is triggered by exposing fruit to exogenous ethylene, a process that involves ACC synthase (Sitrit and Bennett, 1998). These observations suggested that system 2 is regulated by a positive feedback mechanism. The increase in the abundance of MdACS1 mRNA in ripening fruit is prevented to a large extent by treatment with 1 This work was supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) in Japan.
2 These authors contributed equally to the article. * Corresponding author; e-mail tharada@cc.hirosaki-u.ac.jp. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Takeo Harada (tharada@cc.hirosaki-u.ac.jp). [W] The online version of this article contains Web-only data. [OA] Open access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.135822 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, again indicating the involvement of the system-2 ethylene mechanism. Therefore, it seems that activation of system 2 is a critical step for entering ripening. We have observed a very low level of ethylene generation in fruit before the initiation of the burst of ethylene production.
In pear (Pyrus communis) and Japanese pear (Pyrus pyrifolia), other ACS genes in addition to the gene homologous to MdACS1 have been reported to determine the ripening behaviors of various cultivars (Itai et al., 1999;El-Sharkawy et al., 2004). Other apple ACS genes (including MdACS3) were reported by Rosenfield et al. (1996), but their correlation with ripening behavior remains unclear. In this study, we report the genomic structure and expression profile of MdACS3. Expression of MdACS3 in apple fruit was consistent with the small amount of ethylene production prior to the initiation of MdACS1 expression and was regulated by a negative feedback mechanism. In addition, we demonstrate that the allelic forms of MdACS3a are involved in determining the shelf life of apple fruit. The critical function of MdACS3a in regulating the transition from system-1 to system-2 ethylene synthesis and apple fruit ripening is discussed.

Structure and Organization of MdACS3
Three positive phage clones were isolated from the genomic library of cv Golden Delicious by screening with a probe DNA from the last exon of MdACS3. The sequence of one of the three clones corresponded to that of MdACS3 (accession no. U73816; Rosenfield et al., 1996). The nucleotide sequences of the other two clones were very similar (.90%) to that of MdACS3 at their predicted exons, but differed markedly at their predicted introns in both sequence (76%-43%) and size, as shown in Figure 1. We termed these three clones MdACS3a (accession no. AB243060, corresponding to the cDNA reported by Rosenfield et al., 1996), MdACS3b (accession no. AB243061), and MdACS3c (accession no. AB243062), respectively. The nucleotide sequences of the 5# flanking regions showed high homology with PcACS2a, PcACS2b, and PcACS3 in pear (El-Sharkawy et al., 2004). However, all MdACS3 sequences possessed a GA repeat at positions 2424, 2426, and 2456 of the respective MdACS3 clones, which is absent from the Pyrus genes (El-Sharkawy et al., 2004). Furthermore, MdACS3b and MdACS3c possessed a 333-bp insertion, not found in the MdACS3a and Pyrus genes, at around position 2570.

Three MdACS3 Genes Located at Distinct Loci
To investigate the relationship among these MdACS3 genes, we performed cleaved-amplified polymorphic sequence (CAPS) analysis using HindIII and EcoRI sites (Fig. 1) that produce diverse fragments from the respective MdACS3 genes (Fig. 2). The results indicated that the genomes of the six cultivars and 56 strains tested had all three MdACS3 genes. Then we performed linkage analysis of each MdACS3 gene using two F 1 progenies (Igarashi et al., 2008). MdACS3a was located on the 15th linkage group of cv Ralls Janet, whereas MdACS3b was located on the second group of cv Delicious (data not shown). As both of these two linkage groups appear to possess unique genes, this suggests that MdACS3a and MdACS3b are unlikely to be different alleles of the same gene. Furthermore, direct sequencing of the coding and flanking regions of these MdACS3s revealed the occurrence of several single nucleotide polymorphisms (SNPs), indicating that MdACS3c must also be an independent gene.  . CAPS analysis of the MdACS3 family. PCR products amplified from part of the last exon were digested by HindIII/EcoRI and then electrophoresed on 2% agarose gels. Lanes a, b, and c are the products from subcloned fragments containing MdACS3a, MdACS3b, and MdACS3c, respectively. Lanes 1 to 6 are the products from the genomic DNA of the following cultivars as the template. Lane 1, Golden Delicious; 2, Ralls Janet; 3, Fuji; 4, Delicious; 5, Kitaro; 6, Kotaro. The signal at 154 bp is a little faint compared with the other bands because the fragment is produced only from MdACS3b.

High Amount of Inserted Sequence in Malus, But Not in Pyrus
As shown in Figure 1, MdACS3b and MdACS3c possess extra sequence (333 bp) that is not present in MdACS3a. This sequence is flanked by the 8-bp terminal inverted repeat (5#-AATTTTTA-3#) located at 2570 of MdACS3a (accession no. AB243060). Querying this insertion in GenBank revealed that it is a miniature inverted-repeat transposable element (MITE;Bureau et al., 1996) in the Malus genome and we designated it as Mahk (Malus element identified by one of us [Hisayuki Kudo]). Furthermore, the entire sequence is characterized by A-T richness (220/330; Supplemental Fig. S1A) and potential to form a hairpin structure (Supplemental Fig. S1B), suggesting that the sequence is an inserted transposon (Wessler et al., 1995). To determine the genomic distribution of the MITE element in Malus, we performed Southern-blot hybridization ( Fig. 3) using HindIII-digested genomic DNA from eight Malus species, two Pyrus species, two Spiraea species, and one species each of Chaenomeles and Sorbus. Hybridization in all Malus species genomes showed a smeared signal, indicating that Mahk elements are present in high copy number. A similar result was obtained with DNA from quince (Chaenomeles lagenaria), whereas the other plants showed no or only very weak (pear, Japanese pear, and rowan [Sorbus commixta]) smear hybridization signals.

MdACS3 Expression Precedes MdACS1 Expression
Northern hybridization using different apple tissues (Fig. 4) revealed no transcription of MdACS3 in nonfruit organs, suggesting that its expression may be fruit specific. The cDNAs synthesized from total RNA of Golden Delicious on-tree fruit were analyzed by CAPS, in which the last exon of MdACS3a was amplified and double digested by HindIII/EcoRI and then electrophoresed on 2% agarose gel. The band pattern of the digested cDNA was the same as that from MdACS3a (Fig. 5), suggesting that only the transcript of MdACS3a is present in apple fruit.
To test the expression pattern of the MdACS3a gene during fruit ontogeny, we carried out northern hybridization of RNA fractions extracted from fruits that were sampled weekly from the full blossom to ripening stages (Supplemental Fig. S2). Expression of MdACS3a began before the commercial harvest day, but MdACS1 and MdACO1 were expressed only after commercial harvest. These results indicate that MdACS3a functions as a ripening-specific gene, like MdACS1 and MdACO1, at least 2 weeks before the burst of ethylene production caused by expression of MdACO1 and MdACS1. Figure 6 shows changes in ethylene production and expression of ripening-related genes, including MdACS3a, in Golden Delicious fruit. A strong signal of MdACS3a was observed on October 9, when MdACS1, MdACO1, and MdPG1 had not yet begun to be expressed, and persisted to October 27. Expression of MdACS3a decreased gradually from October 21 after the massive increase in MdACO1 and MdACS1 expression, suggesting that expression of MdACS3a is suppressed when fruit enter ripening with a burst of ethylene production. To investigate whether the expression of MdACS3a is affected by ethylene, we analyzed its transcript in 1-MCP-treated fruit (Golden Delicious). The expression of MdACS1, MdACO1, and MdPG1 was completely inhibited by 1-MCP treatment, but the signal of MdACS3a expression was observed until day 12 (Fig. 7).

A Null Mutation Exists in the Apple Genome
El-Sharkawy et al. (2004) and Itai et al. (2000) have reported that the allelic pattern of the MdACS3a ortholog in pear is involved in fruit ripening, causing us to investigate the relationship between MdACS3a and fruit ripening in apple. To analyze the allelic MdACS3a genes, we performed direct sequencing of MdACS3a genomic DNA of Golden Delicious. The presence of an allele can be identified where a sequenced nucleotide was judged as N due to the  appearance of two clear peaks at the same position (Supplemental Fig. S3C). Some SNPs were found, one of which was a G / T change in the protein-coding region, resulting in replacement of Gly-289 by a Val residue. We compared the predicted protein sequence with that of ACS genes in other species, revealing that G289 is located in the active site of ACC synthase (Yip et al., 1990;Liang et al., 1992;Lincoln et al., 1993). Furthermore, this amino acid substitution (G / V) has not been found in any ACS genes of other plant species reported so far (Yip et al., 1990;Lincoln et al., 1993;Itai et al., 1999;El-Sharkawy et al., 2004. These results indicate that G289V is a unique mutation in Malus causing an allele of ACS3a, ACS3a-G289V.

Enzyme Activity of ACS3a and ACS3a-G289V
To test whether this amino acid substitution affects the enzyme activity of ACS3a, full-length cDNAs of ACS3a and ACS3a-G289V were constructed into the expression vector pET11d. As a positive control, cDNA of MdACS1 (Sunako et al., 1999) was also constructed into the vector. DNA sequencing was performed to confirm that no spurious mutations were introduced. Proteins expressed in Escherichia coli cells were fractioned and assayed for enzyme activity. Twodimensional electrophoresis was performed to confirm the existence of the ACS3a and ACS3a-G289V proteins. The protein spots corresponded to a molecular mass of 50 kD, identical with the predicted molecular mass of ACS3a (Supplemental Fig. S4). Enzyme activity assay showed that the activity of ACS3a was 1/40 of that of ACS1, whereas ACS3a-G289V was inactive (Fig. 8), suggesting that G289 is essential for the activity of ACS3a. The ACS3a allelic genotype of each cultivar was determined from the relative abundance of SNPs in genomic and cDNA sequences, which were analyzed by direct sequencing (Supplemental Fig. S3). Cultivars with either G alone or T alone in both their genomic and cDNA sequences were identified as ACS3a/ ACS3a or ACS3a-G289V/ACS3a-G 289V, respectively (Supplemental Fig. S3, A or B). Cultivars having both G and T in their genomic and cDNA sequences were identified as ACS3a/ACS3a-G289V (Supplemental Fig.  S3C).
During the SNP analyses, some cultivars were assessed as ACS3a/ACS3a-G289V in their genomic sequence, but only ACS3a-G289V was found in the cDNA sequences. For example, in apple cv Kitaro, two peaks (G and T) appeared in the sequencing trace for genomic DNA but only one peak (T) appeared in the cDNA sequence (Supplemental Fig. S3D). This result suggests that a null gene, with no ability to be transcribed, exists in some apple varieties, and it was designated as Mdacs3a. Thus the allelic genotype of Kitaro is acs3a/ACS3a-G289V. Consequently, we were able to infer the genotype of apple cv Fuji from that of Kitaro, the progeny derived from a cross between cultivars Fuji and Hatsuaki (ACS3a-G289V/ACS3a-G289V), indicating that Fuji is ACS3a/acs3a. Supplemental Table S1 lists the ACS3a allelic genotypes of the apple cultivars we have investigated. Thus, we conclude that ACS3a is inactive in ACS3a-G289V/ACS3a-G289V and acs3a/ACS3a-G289V cultivars.
A Simple Sequence Repeat Linked to ACS3a-G289V To develop a DNA marker to identify MdACS3a alleles, we analyzed the promoter region of each allele from several apple cultivars. The promoter region (290 to 2530) of each cultivar was cloned and sequenced. A dinucleotide GA repeat sequence (simple sequence repeat [SSR]) was found at 2420 of the promoter, and contained 20 repeats in mutated ACS3a-G289V but only nine repeats in wild-type ACS3a (Fig. 9A). Sequencing  the counterpart region of ACS3a amplified from the wild species Malus coronaria (ACS3a-G289V/ACS3a-G289V) showed 20 repeats in the SSR, suggesting that the repeat polymorphism is linked to the respective SNPs of ACS3a and ACS3a-G289V.
The SSR region of each cultivar was amplified and run on 6% polyacrylamide gels (Fig. 9B). It is possible to identify from the band pattern whether apple cultivars contain the ACS3a-G289V allele. Supplemental Fig. S5 shows the presence of ACS3a-G289V allele in the apple cultivars and wild species we have investigated. ACS3a-G289V was found in the wild species M. coronaria, indicating that this mutation occurred before apple domestication. In addition, ACS3a-G289V exists not only in cultivars bred in Japan but also in those bred in other countries such as New Zealand, America, and Australia (Supplemental Fig. S5).
Expression Profile of Ripening-Related Genes in Kitaro, acs3a/ACS3a-G289V Figure 10 shows changes in ethylene production and expression of ripening-related genes in apple cultivars Kitaro and Kotaro that are derived from the same cross parents. Ethylene production was much higher in Kotaro than in Kitaro. Expression of MdACS1 was not detected in Kitaro by northern blotting, but distinct bands were observed in Kotaro after storage at 24°C for 3 to 9 d. Trace expression of MdACS3a was detected in Kitaro, whereas the expression was quite strong in Kotaro. Although MdACO1 was expressed in both cultivars, it was much stronger in Kotaro. Expressions of MdPG1, which is related to the softening of apple fruit (Wakasa et al., 2006), was not detected in Kitaro but expressed clearly in Kotaro. A similar pattern was obtained from the comparison of cv Gala (ACS3a/ACS3a-G289V) and cv Koukou (acs3a/ACS3a-G289V) (Supplemental Fig. S6). These results show that acs3a/ ACS3a-G289V cultivars have a longer shelf life than ACS3a/ACS3a-G289V cultivars, suggesting that the shelf life of apple fruit is under the control of ACS3a allelotypes.

DISCUSSION
Levels of ethylene production in ripening fruit are broadly in agreement with the presence of particular MdACS1 allelic forms (Sunako et al., 1999;Harada et al., 2000). However, there are differences even within the same allelic forms, suggesting the effect of other ripening-related MdACS gene(s) (Rosenfield et al., 1996), as shown in pear fruit (Itai et al., 1999;El-Sharkawy et al., 2004). By screening a genome DNA library, we identified another ACC synthase gene subfamily in apple, MdACS3. Two of the genes in the subfamily, MdACS3b and c, are not transcribed to mRNA because of a transposon insertion, and thus only MdACS3a is functional. Furthermore, MdACS3a in some cultivars is present as a mutant allele, MdACS3a-G289V, in which a single nucleotide polymorphism leading to a single amino acid substitution results in an inactive protein. In addition, another allele, Mdacs3a, is found as a null allele because of failure of transcription. The existence of these three alleles made it possible to relate differences in ethylene production to shelf life among apple cultivars.
In apple, expression of MdACS3 preceded that of MdACS1 and MdACO1, and decreased after the robust expression of MdACS1 and MdACO1 and the burst of ethylene production ( Fig. 6; Wang et al., 2009). Moreover, 1-MCP treatment did not affect the expression of MdACS3a (Fig. 7), similar result was also reported by Tatsuki et al. (2007). These results clearly demonstrate that the expression of MdACS3a is regulated by a negative feedback mechanism in apple fruit. This negative regulation has also been reported in tomato (Nakatsuka et al., 1998;Barry et al., 2000). Taking into account that system-1 ethylene is negatively autoregulated (Lelièvre et al., 1997;Barry et al., 2000), it is probable that in apple MdACS3 functions in regulating system-1 ethylene biosynthesis and the transition to system 2, and that the burst of system-2 ethylene  negatively feeds back to system 1, resulting in the decrease in MdACS3 expression.
According to the phylogenetic relationships of the Maloideae reported by Campbell et al. (1995), the position of Chaemomeles is closer to that of Malus than to that of Pyrus. Strong smear hybridization signals for the MITE found in this study were observed in digested genomic DNA of both Malus species and quince, but not in that of Pyrus (Fig. 3). These data support the result of Campbell et al. (1995). Therefore, it appears that the amplification of Mahk occurred in Malus at least after the divergence from Pyrus.
ACC synthase belongs to the group of pyridoxal-5#-phosphate-dependent enzymes, and residue K283 is necessary for pyridoxal-5#-phosphate binding (Capitani et al., 1999). Moreover, the enzyme functions as a dimer whose active site is formed from the interaction of residues from the monomeric subunits (Tarun and Theologis, 1998). Therefore, if the G289V mutation in MdACS3a affects the formation of the active site as well as the case of K278A in AtACSs (Tsuchisaka and Theologis, 2004), it would influence heterodimeric interactions with other MdACS, which expresses after MdACS3a in apple fruit. Furthermore, Tsuchisaka and Theologis (2004) reported that AtACS7, a unique ACS missing the hypervariable carboxyl terminus, can form functional heterodimers with other ACS only when it provides the K278 residue. Interestingly, the MdACS3a is the homolog of AtACS7 missing the C terminus. In our study, apple cultivars could be classified into several genotypes according to their homozygosity for ACS3a alleles (Supplemental Table S2). For ACS3a/ACS3a, the enzymatic activity is normal; however, for the ACS3a-G289V/ACS3a-G289V and acs3a/ACS3a-G289V genotypes, ACS3a must be inactive. For ACS3a/ACS3a-G289V genotype, the ACS3a couple dimer can function, but the ACS3a/ACS3a-G289V dimer would be inactive. The same situation may occur in the case of heterodimerization between MdACS3a-G289V and MdACS1.
Ethylene production was much lower in the Kitaro than in Kotaro, and the expression of ripening-related genes was also greatly suppressed in Kitaro (Fig. 10). Figure 10. Changes in ethylene production and expression levels of ripening-related genes in cultivars Kitaro and Kotaro. Numbers indicate days after commercial harvest (day 0). "-14" means fruit at 14 d before harvest; "3" and "12" mean days of storage at 24°C. Ribosomal RNA was used as loading control. A similar result was found in the comparison of Gala and Koukou (Supplemental Fig. S6). These differences can be explained by the respective MdACS3a allelotypes of these cultivars, because Kitaro and Koukou are acs3a/ACS3a-G289V with inactive ACS3a, whereas Kotaro and Gala are ACS3a/ACS3a-G289V with active ACS3a. In Kitaro and Koukou (Supplemental Fig. S7), system-2 ethylene synthesis may not occur because acs3a/ACS3a-G289V cannot produce sufficient ethylene to initiate the transition to system-2 ethylene synthesis, and thus downstream genes, especially MdPG1, were not expressed in Kitaro and Koukou. This supports the hypothesis that ACS3a acts as a switch to initiate the transition from system-1 to system-2 ethylene synthesis, subsequently leading to a burst of ethylene production. Thus, ACS3a is likely to be the main enzyme that controls ethylene production and shelf life of apple fruit. This conclusion was also supported from the data of other cultivars (Supplemental Table S2).
Alignment of MdACS3a orthologs of more than 100 plant species revealed that the G289V mutation is found only in Malus, including wild species and domesticated cultivars from America, Australia, Europe, New Zealand, and Japan (Supplemental Fig. S5). It was not found in pear, although this species is very closely related to apple. These results indicate that the G289V mutation arose after separation of Malus from the Maloideae and was then maintained during domestication and inherited stably among the domesticated cultivars.
The difference between MdACS3a and the null gene Mdacs3a is still unclear, and understanding the difference will lead to development of a molecular marker for Mdacs3a. No transposon or other insertion was found in the promoter region of the null gene, suggesting that the difference might occur much further upstream of the promoter than we have investigated. Matarasso et al. (2005) reported that a Cys protease (LeCp) regulates the expression of tomato LeACS2 by binding to a cis-element (TAAAAT motif), and Cp was found to be induced by fruit ripening (Alonso and Granell, 1995). Sequence analysis revealed that alleles of MdACS3a also possess this motif. Further study is needed to investigate whether mutation of this motif causes the null allele Mdacs3a. In addition, other factors that regulate gene expression, such as DNA methylation, should be taken into consideration.

CONCLUSION
This study describes molecular evidence that accounts for the difference of apple fruit shelf life among cultivars. Out of three genes of ripening-specific MdACS3 family, only one (MdACS3a) expresses at the transition from system 1 to system 2 ethylene biosynthesis. We demonstrated here that the existence of three alleles of MdACS3a made it possible to relate differences in ethylene production to shelf life among apple cultivars. These results have allowed us to propose that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and may be the main determinant of ethylene production and shelf life in apple fruit.

Plant Materials
Young expanding leaves of apple (Malus domestica) cultivars and wild species sampled from the experimental farms of Aomori Apple Experimental Station (Japan) and Hirosaki University were used as a source of genomic DNA, which was extracted as described by Sunako et al. (1999). Fruits on tree (Golden Delicious) were collected weekly from full bloom to ripening stage and were treated as described by Wakasa et al. (2003). Fruit materials used for RNA gel-blot analysis were obtained as described by Wakasa et al. (2006). Briefly, mature fruits, collected at commercial harvest day from an orchard of the Aomori Apple Experimental Station, were kept at 24°C and sampled every 3 d. The commercial harvest day of each cultivar is September 29 for Gala, October 9 for Kitaro, October 21 for Golden Delicious, October 23 for Kotaro, and October 28 for Koukou, respectively. Young expanding leaves, in vitrocultured shoots, roots emerged from seeds, and receptacles and pistils of fullbloom flowers of Golden Delicious were also sampled for analysis of gene expression. Young leaves of the following plants collected at the experimental farms of Hirosaki University and Aomori Apple Experimental Station were also used for Southern-blot analysis: cultivated apple (Malus domestica 'Fuji'), wild apple species (Malus sieboldii, Malus hupehensis, Malus transitoria, Malus arnoldiana, Malus platycarpa, Malus robusta, Malus floribunda), Japanese pear (Pyrus pyrifolia 'Kosui'), pear (Pyrus communis 'La France'), quince (Chaenomeles lagenaria), rowan (Sorbus commixta), and spiraea (Spiraea thunbergii; Spiraea cantoniensis).

Genomic Library Screening and Sequence Analysis
A genomic DNA library obtained from Golden Delicious (Sunako et al., 1999) was screened with a 32 P-radiolabeled ACS3 probe made by PCR using primers ACS3-7 and ACS3-14 (Supplemental Tables S1 and S3). Genomic DNA from Golden Delicious was cloned as described by Sunako et al. (1999). The positive phage clones were used to make a restriction map, and fragments containing the ACS3 gene were subcloned into the pBluescript II KS vector (Stratagene) and sequenced.

Measurements of Ethylene Production Rates
Intact fruits were enclosed in a gas-tight container (0.8 L) equipped with septa and kept at 24°C for 1 h, then 1 mL of gas was sampled through the headspace of the container by a syringe. The ethylene concentration in the sample was measured with a gas chromatograph (Shimadzu) equipped with a flame ionization detector. Five fruits per sample were measured.

Measurements of Flesh Firmness
Flesh firmness was measured with a hand-held penetrometer (FT-327; Facchini) fitted with an 11-mm-diameter probe. Four skin discs (approximately 2.5 cm in diameter) were removed from opposite sides of each fruit. The probe was pressed into the tissue of the cut surface to a depth of 8 to 9 mm in a single smooth motion. Five fruits per sample were measured.

1-MCP Treatment of Fruit
Golden Delicious fruits were treated with 1 mL L 21 of 1-MCP (EthylBloc; Rohm and Haas) for 15 h at 24°C. After treatment, fruits were held at 24°C and sampled every 3 d.

DNA Extraction and Southern-Blot Analysis
Genomic DNA extraction and Southern-blot analysis were performed as described by Sunako et al. (1999). Genomic DNA of apple cultivars and wild species and other species was digested with HindIII, separated in a 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with a Mahk probe that had been prepared by PCR with the primer ACS3bTE-1 and ACS3bTE-4 (Supplemental Table S3).

RNA Extraction and Northern-Blot Analysis
RNA extraction and northern gel-blot analysis were performed as described by Sunako et al. (1999). The probe for ACS3 expression was prepared by PCR using the ACS3a clone and the oligonucleotide primers ACS3-1 and ACS3-2 (Supplemental Table S3). Probes for other genes were prepared as described in Wakasa et al. (2006).

CAPS Analysis of PCR and Reverse Transcription-PCR Products
MdACS3 genes in the genome of apple cultivars were amplified by PCR, which was performed with primers ACS3-17 and ACS3-8 for the first PCR, and ACS3-7 and ACS3-14 (Supplemental Table S3) for the second PCR. The product digested with EcoRI and HindIII was electrophoresed on a 2% agarose gel. Two micrograms of total RNA extracted from the fruit was used for firststrand cDNA synthesis (Superscript II RT, Invitrogen) with primer ACS3-8, and the subsequent PCR was carried out with the same primers as for amplification of genomic DNA.
Overexpression of MdACS1 and MdACS3a in Escherichia coli cDNAs for MdACS3a and MdACS3a-G289V were amplified by PCR with ACS3a cDNA as template and the primers ACS3a infu-1 and ACS3a infu-2 (Supplemental Table S3). cDNA for MdACS1 (accession no.U89156) was amplified with the primers ACS1 infu-1 and ACS1 infu-2 (Supplemental Table  S3). The PCR products were then ligated to the NcoI/BamHI double-digested 5.7 kb pET1ld (Stratagene) vector by using In-Fusion Dry-Down PCR cloning kit (CLONTECH) according to the manufacturer's instructions. Those recombinants containing the correct sequence were identified and retransformed into an Escherichia coli host, BL21 (DE3; Novagen). BL21 (DE3) cells harboring a pET11d-ACS3 and pET11d-ACS1 recombinant plasmid were grown on an Luria-Bertani (LB) plate in the presence of 100 mg/mL ampicillin. LB medium (5 mL) containing 100 mg/mL ampicillin was inoculated with a single colony and incubated at 37°C overnight with constant shaking. The overnight culture (5 mL) was transferred into 500 mL of LB medium. Cells were grown at 30°C with constant shaking. When the cell density reached OD600 of 0.5, isopropylb-D-thiogalactopyronoside was added to the cell culture to a concentration of 0.1 mM. The culture was then transferred to room temperature (20°C-25°C) for 5 more hours with constant shaking. The cells were harvested by centrifugation at 4,000g for 10 min at 4°C. Cell pellets were stored at 270°C for later use.

Two-Dimensional Electrophoresis
Protein concentration was quantified using the Bio-Rad protein assay with bovine serum albumin as a standard. Two-dimensional electrophoresis was performed with the ReadyPrep 2-D starter kit (Bio-Rad) according to the instruction manual. Briefly, immobilized pH gradient strips (7 cm, pH 3-10; Bio-Rad ReadyStrip, Bio-Rad) were rehydrated overnight with 125 mL of sample buffer containing 87.5 mg of total protein. Proteins were focused in a PROTEAN IEF cell (Bio-Rad) at 20°C for 12 h, applying 250 V (15 min), 4,000 V (120 min), and 4,000 V for a total of 20 KVh. After isoelectric focusing, strips were equilibrated in equilibration buffers I and II for 10 min, respectively. Two-dimensional SDS-PAGE was run in acrylamide gels (Ready Gel precast gel, Bio-Rad) at 200 V for 45 min. Gels were stained with colloidal CBB G-250.

ACC Synthase Activity Assay
ACC synthase extract was prepared according to the method of Li et al. (1996). The ACC synthase extract was subsequently concentrated with a Nanosep centrifugal device (PALL) according to the manufacturer's instructions. The amount of ACC was determined by the method of Lizada and Yang (1979). ACC synthase activity was assayed by incubating the ACC synthase extract with AdoMet in a reaction buffer for 1 h at 30°C (Li et al., 1996). One unit of ACC synthase activity is defined as 1 nmol of ACC formed per hour at 30°C. Protein concentrations were determined with Bio-Rad protein assay reagent (http://www.bio-rad.com).

Nucleotide Sequence and SSR Analysis
For sequence analysis of genomic DNA of MdACS3a to determine the genotype, a first PCR was performed with primers MdACS3a-1F and MdACS3a-2R (Supplemental Table S3) and genomic DNA as template. For SNP sequencing, a second PCR was conducted with the primers MdACS3a-2F and MdACS3a-2R with the first PCR product as the template. All cycle sequencing reactions for DNA sequence analysis were performed using Big Dye terminator chemistry (Applied Biosystems) according to the manufacturer's protocols, and sequences were determined using an ABI 310 automated DNA sequencer.
For SSR analysis, primers MdACS3a-1F and MdACS3a-1R were used to amplify the SSR region from the first PCR product. PCR product was run on 6% polyacrylamide gel and stained with silver according to Bassam et al. (1991).
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB243060, AB243061, and AB243062.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Sequence and secondary structure of Mahk.
Supplemental Figure S2. Changes in MdACS3a expression during fruit ontogeny.
Supplemental Figure S3. Sequencing traces of genomic DNA and cDNA of each MdACS3a allelic genotype.
Supplemental Figure S4. Two-dimensional electrophoresis analysis of ACS3a proteins expressed in E. coli.
Supplemental Figure S5. Presence of ACS3a-G289V in apple cultivars and wild species.
Supplemental Figure S6. Changes in ethylene production, flesh firmness, and ripening-related genes.
Supplemental Figure S7. Fruit of cultivars Kitaro and Koukou.
Supplemental Table S2. Apple cultivars and these ripening characters.
Supplemental Table S3. Primers used for the PCR amplification in this study.