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Abstract: 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACS or ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and both of them were strongly up-regulated by abscisic acid (ABA) and ethephon.

桑树MaACS和MaACO基因的鉴定和表达模式研究

研究目的：分离和鉴定桑树中参与乙烯生物合成的酶的编码基因MaACS和MaACO，研究其表达模式。
创新要点：基于最新公布的桑树基因组数据库数据，获得5个MaACS基因和2个MaACO基因，对其进行了生物信息分析，同时鉴定了其在不同桑树组织中、不同发育时期桑椹中和不同激素作用下的表达模式。
研究方法：通过生物信息学方法筛选和鉴定基因，利用荧光定量逆转录聚合酶链式反应（qRT-PCR）分析基因的表达量。
重要结论：MaACS和MaACO基因在根、茎、叶等不同组织中呈现出不同的表达模式，在桑椹发育过程中呈现出两种表达模式，其表达量被脱落酸和乙烯利上调。

关键词：桑椹；MaACS；MaACO；脱落酸；乙烯利

1.  Introduction

 The fruit is the reproductive organ for higher plants with a complex process of development and senescence, which are regulated by plant hormones (Gillaspy et al., 1993; Ozga and Reinecke, 2003). Among these hormones, ethylene, the simplest olefin, is involved in many aspects of the plant life cycle, including seed germination, seedling development, leaf expansion, blossoming, senescence, fruit ripening, and responses to biotic and abiotic stresses (Wang et al., 2002; Lin et al., 2009). Ethylene is a necessary factor for the initiation of fruit ripening and senescence and promotes the transcription and translation of responsive genes involved in cell-wall metabolism, membrane metabolism, and fruit softening by activating ethylene signaling transduction (Wang et al., 2002; Alonso and Stepanova, 2004).

 The biosynthesis of ethylene begins with the production of S-adenosylmethionine (SAM), which is catalyzed by SAM synthetase (EC 2.5.1.6) from methionine. SAM is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) and 5-methylthioadenosine (MTA) by ACC synthase (ACS; EC 4.4.1.14). ACC is subsequently oxidized by ACC oxidase (ACO; EC 1.14.17.4) to yield ethylene and CO₂ (Yang and Hoffmann, 1984; Kende, 1993). ACS and ACO are the key enzymes producing ethylene, are encoded by a multigene family, and have been isolated from numerous plant species. ACS and ACO genes show different expression profiles in plant tissues, and respond to different stimuli, including low oxygen, mechanical damage, high temperature, and hormones (Arteca and Arteca, 1999; Rieu et al., 2005; Choudhury et al., 2008).

 Ethylene is one of the most important factors regulating softening, senescence, and abscission of fruit, and the concentration of ethylene increases gradually during the development of climacteric fruit. ACS and ACO act as accelerators in fruit ripening by catalyzing the production of ethylene at the late fruit ripening stage, leading to irreversible senescence (Chen et al., 2003; Tatsuki et al., 2006; Cara and Giovannoni, 2008; Varanasi et al., 2011). It has been shown that once the expression levels of ACS and ACO genes were suppressed or mutated, the production of ethylene was down-regulated and the shelf life of fruit was also prolonged. Oeller et al. (1991) transformed the antisense messenger RNA (mRNA) of the ACS gene in tomato and inhibited fruit ripening. Ayub et al. (1996) generated transgenic melons by introducing an antisense ACO gene, and ethylene production of transgenic fruit was down-regulated compared with untransformed fruit. The silencing of LeACS2 by vacuum-infiltration and the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) method also leads to a significant delay in the post-harvest ripening and senescence of tomato fruit (Xie et al., 2006). Moreover, null mutations of the MdACS1 and MdACS3 genes have shown no or very low expression levels of ripening-related genes and maintained firmness in apple fruit (Malus domestica) (Sunako et al., 1999; Wang et al., 2009).

 Mulberry (Morus alba L.) is a deciduous tree and an economically important food crop for the domesticated silkworm (Bombyx mori L.). Mulberry has multiple uses in ecology, pharmaceuticals, and traditional Chinese medicine (He et al., 2013). Its bark is also used in paper production. In addition, mulberry fruit is one of the most popular fruits worldwide and is especially appreciated for its unique flavor in China. However, the commercialization of mulberry fruit is limited by its short maturity stages and shelf-life. The effective strategy to make use of mulberry fruit is to strengthen deep processing or to utilize ethylene inhibitors, such as silver thiosulfate, 1-methylcyclopropene (1-MCP), aminoethoxyvinylglycine, and 2,5-norbornadiene, which delay ripening and senescence (Blankenship and Dole, 2003). Furthermore, a potential tool to delay the ripening of mulberry fruit and to prolong its shelf-life is the use of transgenic approaches to down-regulate the expression of ethylene biosynthesis-related genes.

 The ethylene biosynthesis pathway has been detected in some plant species, like Arabidopsis thaliana, tomato (Lycopersicon esculentum), and apple. However, there is little knowledge on ethylene biosynthesis in mulberry. In this study, we report the isolation and characterization of five ACS genes and two ACO genes from mulberry, and demonstrate their expression levels in different organs and fruit with different development stages following treatment with abscisic acid (ABA) and ethephon. We hope this work will provide insights into the developmental functions of these genes and lay a foundation for further understanding the mechanism of mulberry fruit development and ripening.

2.  Materials and methods

2.1.  Plant materials and treatments

 Different tissues (e.g., root, stem, stem epidermis, petiole, leaf, and fruit) were collected from the mulberry cultivar “Changjiang 1”, which is grown in the mulberry garden of Southwest University, Chongqing, China. The mulberry fruit were sampled after full-bloom stages of 10, 20, 29, 36, and 40 d. All plant materials were frozen in liquid nitrogen and stored at −80 °C.

 The immature fruit on 20 d after full-bloom (DAF) with a partial red color were collected from trees treated with 264 mg/L ABA (containing 0.1% Tween-20) and 100 mg/L ethephon (containing 0.1% Tween-20) for 5 min at 25 °C; the control fruit were dipped in double-distilled water (ddH₂O) (Ren and Leng, 2010). After dipping, the fruit were kept at 25 °C for 5 d and were immediately frozen in liquid nitrogen and stored at −80 °C.

2.2.  Isolation of RNA and synthesis of the first strands of complementary DNA (cDNA)

 Total RNA was extracted from root, stem, stem epidermis, petiole, and leaf using the RNA Extraction Kit (TaKaRa, Japan) as described in the manufacturer’s instructions. Total RNA of fruit was extracted using the RNA Extraction Kit Transzol Plant (TransGen Biotech, China). To remove genomic DNA, the RNA samples were digested by DNase I (TaKaRa, Japan). The cDNA was synthesized from 3 μg of DNA-free RNA with the reverse transcriptional Moloney murine leukaemia virus (M-MLV; Promega) following the manufacturer’s protocol. Ten-fold diluted cDNA was used in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

2.3.  Identification and isolation of ACS and ACO

  ACS and ACO genes from other plant species were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), and were used as queries in BLAST searches against Morus Genome Database (http://morus.swu.edu.cn/morusdb/). This yielded non-redundant full mRNA sequences of ACS and ACO genes. The candidate genes were identified using BLASTN and SMART (http://smart.embl-heidelberg.de/). Five ACS genes and two ACO genes were finally selected for further research.

2.4.  Sequence alignment and phylogenetic analysis

 The similarities of ACS and ACO genes were analyzed by BLASTN. About 64 ACS and 46 ACO amino acid sequences were downloaded from the National Center of Biotechnology Information (NCBI) and used for alignment with putative protein sequences of mulberry ACS and ACO genes given by MUSCLE 3.6, and a phylogenetic tree was constructed by MEGA 5.0 software.

2.5.  qRT-PCR expression analysis of mulberry ACS and ACO genes

 The primers used for qRT-PCR were designed by the online tool of the GeneScript Company of China (http://www.genscript.com.cn/index.html) (Table 1). For the analyses of ACS and ACO gene expression, 20 μl total reaction volume composed of 10 μl of 2× SYBR® Premix Ex Taq II (TaKaRa, Japan), 0.8 μl of each primer (10 μmol/L), 0.4 μl of 50× ROX Reference Dye II (TaKaRa, Japan), 2 μl of cDNA, and 6 μl of ddH₂O. The thermal parameters for qRT-PCR were: 95 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s, with a final extension at 72 °C for 10 min. The MaACTIN3 (HQ163775.1) gene was used as an internal control. Relative expression levels were calculated using the fruit after full-bloom stage of 10 d set at 1.

3.  Results

3.1.  Identification and isolation of MaACS and MaACO genes

  ACS and ACO gene sequences from other plants were used to search the mulberry genome sequences and the obtained mRNA sequences were screened by the BLASTN program; their encoded amino acids were analyzed by the SMART program. Five ACS genes and two ACO genes were finally screened, which were named as MaACS1, MaACS2, MaACS3, MaACS4, MaACS5, MaACO1, and MaACO2. MaACO1 had previously been submitted to the NCBI (GenBank Accession No. KC709482). MaACO1 may be associated with tissue aging or senescence and respond to stress in mulberry (Pan and Lou, 2008).

 The full-length genomic sequences of MaACS genes ranged from 1660 bp (MaACS5) to 2732 bp (MaACS2) and the open reading frame (ORF) length ranged from 1341 bp (MaACS5) to 1491 bp (MaACS2), encoding polypeptides ranging from 446 to 496 amino acids with predicted molecular masses ranging from 50.16 to 55.57 kDa. The full-length genomic sequences of MaACO genes were 1666 bp (MaACO1) and 1526 bp (MaACO2) and the ORF lengths were 960 and 969 bp, encoding polypeptides of 319 and 322 amino acids with predicted molecular masses of 36.38 and 36.48 kDa, respectively (Table 2).

3.2.  Structure of MaACS and MaACO genes

 To reveal the intron and exon organizations of MaACS and MaACO genes, we compared all obtained ORF sequences with the corresponding genomic DNA sequences (Fig. 1). The MaACS sequences could be divided into two groups. The first group included MaACS1, MaACS2, MaACS3, and MaACS4, which have a 4-exon/3-intron structure, and MaACS5 has a 3-exon/2-intron structure. Both MaACO1 and MaACO2 have a 4-exon/3-intron structure.

3.3.  Multiple-sequence alignments and phylogenetic analysis of MaACS genes

 Sequence similarity matrix analysis of MaACS amino acids has revealed that the sequence identity ranged from 50% to 69% (Table 3). The sequence identities were higher between MaACS1 and MaACS2 (69%) and between MaACS3 and MaACS4 (68%).




 The amino acids encoded by MaACS genes were aligned by the Clustal W program and have the pyridoxal phosphate (PLP)-dependent enzyme domain, which is a co-factor associated with ACS catalyzing the conversion of S-adenosyl-L-methionine (AdoMet) to ACC, predicted by the SMART program. It was shown that ACS proteins could be divided into three main types differing in the C-terminal sequences. Type I proteins have an extended C-terminus containing three conserved serine residues that are targets for phosphorylation by mitogen-activated protein kinase 6 (MPK6) and a conserved serine residue that is a phosphorylation site for calcium-dependent protein kinase (CDPK) (Tatsuki and Mori, 2001; Chae et al., 2003; Liu and Zhang, 2004; Sebastià et al., 2004). Type II proteins have a shorter C-terminus that has only the CDPK phosphorylation site. Type III proteins have a very short C-terminal extension that lacks both phosphorylation sites. The result of multiple-sequence alignment against MaACS proteins showed that MaACS1 and MaACS2 were type I, MaACS3 and MaACS4 were type II, and MaACS5 was type III (Fig. 2).




 The amino acids of MaACS were aligned by the Muscle 3.6 program and a phylogenetic tree was constructed by MEGA 5.0. The resulting tree showed that MaACS1 and MaACS2, MaACS3 and MaACS4, and MaACS5 were three branches, respectively (Fig. 3a). A phylogenetic tree was also constructed for ACS from mulberry with other plants via the neighbor-joining method (Fig. 3b). The tree was divided into three groups: I, II, and III; MaACS1 and MaACS2 were group I, MaACS3 and MaACS4 were group II, and MaACS5 was group III. This result is consistent with the classification based on the difference in the C-terminal sequences of MaACS.




 ACS proteins can act as homodynamic or heterodimeric proteins to form active heterodimers enhancing the isozyme diversity of the ACS gene family and providing physiological multifunctionality by operating in different SAM concentrations in various cells and tissues during plant growth and development (Tsuchisaka and Theologis, 2004). In this study, Y92 and K278 residues of MaACS proteins are predicted as the active sites associated with heterodimeric interactions (Fig. 2).

3.4.  Multiple-sequence alignments and phylogenetic analysis of MaACO genes

 ACO is a member of a superfamily of non-haem iron-dependent oxygenases, most of which utilize Fe(II) as a co-factor and 2-oxoglutarate (2OG) as a co-substrate (John, 1991; Bidonde et al., 1998). The domain of MaACO encoding protein sequences was analyzed by the SMART program and NCBI. The results revealed that the sequences of amino acids ranging from 156 to 256 have the 2OG-Fe(II) oxygenase domain (Fig. 4), which indicates that ACO is a member of the ketoglutaric acid dioxygenase family. Crystal structure studies of the Petunia hybrida ACO showed that it forms a complex with Fe(II) and Cu(II) and contains a single Fe(II) atom binding site coordinated by three residues (His177, Asp179, and His234), and Arg175 and Arg244 are proposed to be involved in binding bicarbonate; this leads to the activation of the ACO enzyme (Zhang et al., 2004). Here, as reported previously, H177, D179, and H234 in MaACO1 and MaACO2 proteins were predicted and identified as Fe(II) binding sites, and R175 and R244 were predicted as 2OG binding sites (Fig. 4).




 Multiple-sequence alignment results showed that ACO amino acids are relatively conserved with high similarities in higher plants. The results of phylogenetic analysis of MaACO suggested that VvACO (XP_002278331.1), AtACO (AAK68810.1), and ZmACO (NP_001151658.1) could be clustered into the same clades, and MaACO1 and MaACO2 could be clustered into a branch with another 43 ACO proteins from NCBI (Fig. 5).

3.5.  Tissue specificity in qRT-PCR expression analyses of MaACS and MaACO genes

 Tissue-specific expression analysis revealed that MaACS and MaACO genes were differentially expressed in various mulberry tissues (Fig. 6). Within the MaACS gene family, MaACS1 and MaACS4 showed similar expression patterns, with transcripts strongly expressed in fruit and more weakly in roots, stem, stem epidermis, petioles, and leaves. MaACS2 and MaACS5 showed somewhat similar expression patterns, and had higher expression in roots, old leaves, and fruit, and lesser levels in others, although MaACS5 transcripts were also low in roots. MaACS3 transcripts were more abundant in petiole and fruit tissues, and the expression level was higher in petioles than in fruit.




 The expression of MaACO1 and MaACO2 showed different patterns. The transcript of MaACO1 had higher levels in roots and old leaves, and lower levels in other tissues. MaACO2 had higher levels in stem epidermis, petioles, and immature and old leaves, and lower levels in other tissues.

3.6.  qRT-PCR expression analysis of MaACS and MaACO genes in fruit

 The expression of the different MaACS and MaACO genes showed two patterns during the development of mulberry fruit (Fig. 7). MaACS1, MaACS2, MaACS4, MaACS5, and MaACO1 had strong peaks of expression in early stages of fruit, and decreased throughout development. However, transcript abundance of MaACS3 and MaACO2, unlike other genes, increased rapidly throughout fruit development.




 The mulberry fruit on 20 DAF were treated with ABA and ethephon, and fruit turned the full red color (data not shown). Transcript abundance of MaACS and MaACO genes were up-regulated by ABA and ethephon (Fig. 8). This indicated that ABA and ethephon could regulate the transcription of ethylene biosynthesis-related genes.

4.  Discussion

 ACS and ACO are the key enzymes producing ethylene, and are typically encoded by multiple ACS and ACO genes, and some members of these gene families are differentially regulated to respond to different cues in various tissues throughout plant development. In this study, we have isolated and analyzed five ACS genes and two ACO genes from mulberry genome database. Sequence alignments and phylogenetic analyses of MaACO1 and MaACO2 showed that sequence identity is 82% and their amino acids are conserved compared with ACO proteins from other species. Multiple-sequence alignments and phylogenetic analyses of MaACS genes show that ACS genes have the PLP-dependent enzyme domain and could be divided into three types (I, II, and III) in higher plants based on the difference in the C-terminal sequences. Like Arabidopsis thaliana, tomato, apple, and grape (Vitis vinifera), ACS is classified into three types in mulberry: MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III.

 Fruit can be classified into two groups, climacteric and non-climacteric fruit, based on pattern of respiration and ethylene release during maturation and ripening. Further, the ethylene biosynthesis in fruit is controlled by two systems (McMurchie et al., 1972): system I is responsible for ethylene production during normal vegetative growth in all tissues including climacteric and non-climacteric fruit; system II only exists in climacteric fruit, and is essential for the burst of ethylene production during the ripening of fruit (Seymour et al., 1993). The transition from system I to system II is considered to be an important step in fruit ripening (Lelièvre et al., 1997; Cara and Giovannoni, 2008). In climacteric fruit, system I-like ACS and ACO genes showed lower expression levels in the later stages of fruit, but transcript abundance of system II-like genes increased rapidly throughout fruit development. The expression of the different MaACS and MaACO genes showed two patterns during the development of mulberry fruit. MaACS1, MaACS2, MaACS4, MaACS5, and MaACO1 had strong peaks of expression in early stages of fruit, and decreased throughout development. However, transcript abundance of MaACS3 and MaACO2, unlike other MaACS and MaACO genes, increased rapidly throughout fruit development. However, these data could not support the view that MaACS3 and MaACO2 are responsible for system II ethylene biosynthesis in ripening fruits and that other genes are responsible for system I, because all MaACS and MaACO genes were stimulated by ethephon as shown in Fig. 8; more studies are needed to support MaACS and MaACO gene classification.

 Within the MaACS genes, the expression pattern of MaACS3 was different from those of other MaACS genes, and its transcript abundance was increased gradually during fruit development, especially increased by 38.8-fold from 29 to 36 DAF of fruit. In addition, the expression level of MaACS3 was higher than those of the other four MaACS genes in the fruit on 40 DAF. Therefore, the high accumulation of MaACS3 is closely associated with the production of ethylene in later stages of mulberry fruit. It is possible that mulberry fruit is climacteric on the basis of the results from Luo (2003a; 2003b), and mulberry fruit certainly decays readily. It is probable that using MaACS3 as the target gene to suppress its expression in the later stages of mulberry fruit development will prolong shelf-life.

 Tissue-specific expression analysis revealed that the MaACS and MaACO genes were differentially expressed in various mulberry tissues. Within the MaACS gene family, the transcript abundance in the fruit on 10 DAF was much higher than that in other tissues. However, the expression of MaACO genes showed different patterns, the transcriptional levels of MaACO1 and MaACO2 in fruit being equal to or lower than those in other tissues. It is indicated that MaACS genes, unlike MaACO genes, tend to be expressed in fruit compared with other tissues. Promoters are the key factor driving time and space expression of the correspondent gene, and fruit-specific promoters can control gene expression in fruit but not in other tissues. There are some promoters, like tomato E8/E4 promoter, peach (Prunus persica) ACO1 promoter, and apple ACO1 promoter, being expressed specifically in fruit (Deikman et al., 1992; Blume and Grierson, 1997; Moon and Callahan, 2004), and the target genes fused to these promoters could overexpress specifically in fruit. There are no reports on fruit-specific promoters using the promoter of ACS genes. Based on the expression analysis of MaACS genes in different tissues and fruit at different stages, MaACS genes are potential targets to develop fruit-specific promoters. Considering that MaACS1, MaACS2, MaACS4, and MaACS5 have lower expression levels in the later stages of fruit (Fig. 7b), it is probable that we could use MaACS3 promoter as a candidate to research the fruit-specific promoter in mulberry.

 ABA and ethylene are considered as the key factors regulating fruit maturity and senescence. Some research has confirmed that ABA plays an important role in triggering ethylene biosynthesis and softening during the ripening of fruit (Zhang et al., 2009; Zaharah et al., 2013). ABA can stimulate the production of ethylene by promoting the activities of ethylene biosynthesis enzymes ACS and ACO, and regulating the expression of its encoded genes, thus starting the ripening process. ABA, like ethephon, can rapidly up-regulate the expression levels of MaACS and MaACO genes. This indicates that ABA can promote the biosynthesis of ethylene in mulberry fruit by enhancing the expression of ACS and ACO genes. Sun et al. (2010) thought that the productions of ABA and ethylene at the onset of berry ripening and after harvest are affected by each other. Thus, the relationship between these two hormones and how they affect fruit ripening need further studies.

5.  Conclusions

 We are the first to report ACS and ACO genes from mulberry and demonstrate their expression profiles. Tissue-specific expression analysis revealed that MaACS and MaACO genes showed different patterns in various mulberry tissues and two patterns throughout the development of mulberry fruit. MaACS genes also tended to be expressed in fruit rather than in other tissues. Further, the transcription levels of MaACS and MaACO genes could be up-regulated by ABA and ethephon. In future practice, we may be able to use MaACS3 as the target gene to develop a fruit-specific promoter and to prolong the shelf-life of mulberry fruit by down-regulating its expression.

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* Project supported by the National Natural Science Foundation of China (No. 31360190), the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201403064), and the China Agriculture Research System (No. CARS-22-ZJ0102) Chang-ying LIU, Rui-hua LÜ, Jun LI, Ai-chun ZHAO, Xi-ling WANG, Umuhoza DIANE, Xiao-hong WANG, Chuan-hong WANG, Ya-sheng YU, Shu-mei HAN, Cheng LU, and Mao-de YU declare that they have no conflict of interest.