Molecular characterization and transcription analysis of DNA methyltransferase genes in tomato (Solanum lycopersicum)

Abstract DNA methylation plays an important role in plant growth and development, gene expression regulation, and maintenance of genome stability. However, only little information regarding stress-related DNA methyltransferases (MTases) genes is available in tomato. Here, we report the analysis of nine tomato MTases, which were categorized into four known subfamilies. Structural analysis suggested their DNA methylase domains are highly conserved, whereas the N-terminals are divergent. Tissue-specific analysis of these MTase genes revealed that SlCMT2, SlCMT3, and SlDRM5 were expressed higher in young leaves, while SlMET1, SlCMT4, SlDRM7, and SlDRM8 were highly expressed in immature green fruit, and their expression declined continuously with further fruit development. In contrast, SlMETL was highly expressed in ripening fruit and displayed an up-regulated tendency during fruit development. In addition, the expression of SlMET1 in the ripening of mutant rin and Nr tomatoes is significantly higher compared to wild-type tomato, suggesting that SlMET1 was negatively regulated by the ethylene signal and ripening regulator MADS-RIN. Furthermore, expression analysis under abiotic stresses revealed that these MTase genes were stress-responsive and may function diversely in different stress conditions. Overall, our results provide valuable information for exploring the regulation of tomato fruit ripening and response to abiotic stress through DNA methylation.


Introduction
DNA methylation plays a crucial role in gene expression regulation, maintenance of genome stability, and it controls the transcription of invading and mobile DNA elements (Law and Jacobsen, 2010;Feng and Jacobsen, 2011). Plants possess four types of DNA methyltransferases (MTases), namely methyltransferase (MET), chromomethylase (CMT), domains rearranged methyltransferase (DRM), and DNA methyltransferase homologue 2 (DNMT2) (Law and Jacobsen, 2010). MET maintains CG methylation of heterochromatic regions enriched with transposable elements (TEs) and repeats, and genic regions (Cokus et al., 2008;Lister et al., 2008). CMT and DRM mediate CHG and CHH (H=A/C/T) methylation (Law and Jacobsen, 2010;Kohler et al., 2012). DNMT2 has a novel transfer RNA (tRNA) methyltransferase activity (Goll et al., 2006;Jeltsch et al., 2006), but its role in C5 DNA methylation remains largely unknown (Pavlopoulou and Kossida, 2007). DNA methyltransferases genes have been found in many plant species, such as tobacco, rice, Arabidopsis, wheat, maize, Physcomitrella, and legumes (Dai et al., 2005;Wada, 2005;Pavlopoulou and Kossida, 2007;Fulnecek et al., 2009;Sharma et al., 2009;Malik et al., 2012;Rohini et al., 2014). DNA methylation is primarily catalyzed by the DNA methyltransferase family. DNA methyltransferase plays an important role in plant development, transcriptional regulation, and metabolic pathway control. For example, the triple mutation of drm1drm2cmt3 leads to delayed growth, small plant size, and partial barrenness in Arabidopsis (Cao and Jacobsen, 2002). DNA methylation is also involved in tomato fruit ripening. The Colorless non-ripening (Cnr) mutation inhibits normal tomato ripening due to methylation of the SBP-CNR gene promoter (Manning et al., 2006;Giovannoni, 2007). Chen et al. (2015) recently reported on the role of a chromomethylase (SlCMT3) for the stable methylation of the promoter region of the Cnr gene. these environmental perturbations (Zhou et al., 2007). Epigenetic mechanisms, including DNA methylation and histone modification, play important roles in regulating gene expression in plant responses to environmental stress (Razin and Cedar, 1992;Cullis, 2005;Boyko et al., 2007;Boyko and Kovalchuk, 2008). For instance, salinity and water stress can trigger demethylation at coding regions of certain genes and subsequently initiate their expression (Choi and Sano, 2007). To the contrary, satellite sequences can be hypermethylated, especially in CHG sequences after salt stress (Dyachenko et al., 2006). Low-temperature stress reduces the amount of methyltransferase in corn (Zea mays L.) (Steward et al., 2000).
In this study, based on the complete sequence of tomato genomes, as well as expression profiles at different tissues/stages and abiotic stresses (low temperature and salt), the nine tomato MTases were analyzed and characterized through an approach combining bioinformatics and expression experiments. Our study provides valuable information for functional research of DNA methyltransferase genes in tomato.

DNA and protein sequence analysis
The protein sequences of Arabidopsis and rice MTases (Table S1) were used to search for the amino acid sequences of tomato MTases in the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Sol Genomics Network (SGN) (http://solgenomics.net/) databases using the Blastp tool with the filter-off option and a cut-off value of 1 e -10 . The genomic DNA sequences of these nine genes were obtained from the SGN. In order to analyze the exons and introns of genomic DNA, sequence alignment between CDS (coding sequence) and genomic DNA was done by MultAlin. The gene structures of the DNA MTases in tomato were generated using the GSDS. Molecular weight (Mw), isoelectric points, and grand average of hydropathicity (GRAVY) were estimated with the ExPASy compute Mw tool. Conserved structure domains were annotated based on ScanProsite and the Pfam protein family database. Motif detection was dependent on MEME (Timothy et al., 1994). The phylogenetic tree was constructed using MEGA 5.02 software and the neighbor-joining method with the following parameters: bootstrap analysis of 1,000 replicates, Poisson model, and pairwise deletion. The numbers at the nodes indicate the bootstrap values. Promoter element analysis was performed using plant CARE and PLACE, which is a database of motifs found in plant cis-acting regulatory DNA elements.

Plant material
Tomato (Solanum lycopersicum Mill. cv. Ailsa Craig) seedlings were grown under greenhouse conditions (16 h days at 27°C and 8 h nights at 19°C). For or-gan-specific expression profiling of genes, tomato roots, stems, leaves, sepals, flowers and fruit pericarp tissues of different periods were harvested. Roots and stems were collected from 45-day-old tomato seedlings based on their uniformity. The leaves were taken from three different parts of 65-day-old tomato plants, namely young leaves (3 leaves of new growth), mature leaves (5 to 7 leaves from top to bottom) and senescent leaves (8 to 10 leaves from top to bottom). Sepals and petals were collected at the same time. Flowers were marked at anthesis and fruit development was recorded as days post-anthesis (DPA). Fruits ripening was divided into five stages, namely IMG (immature green, 28 DPA), MG (mature green, 35 DPA, full fruit expansion but no obvious color change), B (breaker, fruit showing the first signs of ripening-associated color change from green to yellow), B4 (4 days after breaker) and B7 (7 days after breaker).

Expression analysis of DNA MTase genes by gene microarray
Microarray expression data were obtained from the tomato Gene Chip platform of Genevestigator (https://www.genevestigator.com/gv/). The nucleotide sequences of DNA MTase genes were used as query sequences to blast against all of the gene probe sequences from the Affymetrix Gene Chip (http://www.affymetrix.com/), and the best homologous probes were selected and used to carry out search in the Affymetrix Tomato Genome Array platform.

Stress treatments
Potted 35-day-old tomato seedlings chosen based on their uniformity were used for all stress treatments. For salt stress treatment, the roots of tomato seedlings were submerged in a solution containing 250 mM NaCl for 0, 1, 2, 4, 8, 12, and 24 hours, and the young leaves of the treated seedlings and controls were collected. For low temperature stress treatment, the whole potted tomato seedlings were incubated at 4°C for 0, 1, 2, 4, 8, 12, and 24 hours, after which the leaves were collected (Zhu et al., 2014). All stress treatments were performed with three biological replicates.

RNA isolation and quantitative RT-PCR analysis
Total RNA was extracted from tomato tissues with the Trizol reagent (Invitrogen, Shanghai, China). Genomic DNA pollution was eliminated with DNase I (Promega, Beijing, China) in the presence of RNase inhibitor (Takara Biotechnology, Japan). Poly (A)+RNA was used as a template for synthesis of first-strand cDNA. Complementary DNA was synthesized by M-MLV reverse transcriptase (Promega, Beijing, China) at 37°C for 1 h. The quantitative RT-PCR reaction system and conditions were performed as in our previous report (Guo et al., 2016). The tomato CAC and EF1a genes were used as internal controls under normal growth conditions (Expósito-Rodríguez et al., 2008) and abiotic stress (Nicot et al., 2005), respectively. The analysis of gene relative expression levels was conducted using the 2 -DDCT method (Livak and Schmittgen, 2001). All primers used for quantitative RT-PCR are listed in Supplementary Table S2. The mean values of three independent experiments were calculated, and the standard deviations (± SD) were indicated.

Statistical analysis
All experiments were conducted with three biological replicates. Statistical data were analyzed by Origin 8.0 software, and performed using the Student's t-test (SPSS 22.0). Values of p < 0.05 were considered significant. Data are presented as mean ± SD.

Identification of tomato DNA MTases and sequence analysis
Firstly, the data for 11 and 10 MTases in Arabidopsis and rice (Table S1) was collected from NCBI, respectively. Based on these data, nine MTases were identified in tomato through Blastp (Table 1). The open reading frame (ORF) length of these genes varies from 1.1 to 4.6 kb, and their protein length ranged from 381 to 1559 amino acids. All the deduced polypeptides are hydrophilic. In addition, Figure 1 shows the intron-exon organization (number of introns and exons) of nine MTases in tomato. The coding regions of CMT subfamily genes are interrupted by 14-21 introns ( Figure 1). MET gene (SlMET1) length is approximately 4.6 kb in tomato, harboring 12 exons. The length of the DRM subfamily genes in tomato varies from 1.8-2.1 kb with nine exons. DNMT2 gene (SlMETL) is smallest in length (1.1 kb) harboring nine exons. Genomic distribution of these tomato MTase genes was also analyzed. Nine tomato MTases genes are dispersedly located on chromo-somes, with one MTase variant mostly located on a single chromosome (Table 1), suggesting at least partial influence of WGD in the diversification of the MTases family in tomato, rather than gene duplication.

Conserved domains and phylogenetic analysis
Alignment of the amino acid sequences of these nine tomato DNA MTases revealed that tomato MTases genes possess a regulatory region and a catalytic region with conserved motifs that are arranged in a specific order. Six highly conserved motifs I, IV, VI, VIII, IX, and X were identified in the methyltransferase domain via MEME analysis in the nine MTases ( Figure 2). We found that each subfamily of tomato MTase has a characteristic arrangement of these motifs in the catalytic region. MET members showed the order of motifs as I, IV, VI, VIII, IX, and X. In CMT members, chromodomain was present between the conserved motifs I and IV with the rest of the arrangement similar to the MET members. It is interesting to note that SlCMT4 appeared to lack the IX and X domains. DRM DNA MTase genes in tomato 3 Length of amino acids, molecular weight (kDa), isoelectric point (pI), and grand average of hydropathicity (GRAVY) of the deduced polypeptide. c GenBank, SGN or TIGR accession number of tomato MTases genes. members showed the order of motifs as VI, VIII, IX, X, I, and IV except in SlDRM7, which only possesses the IV motif. Only one ubiquitin-associated domain (UBA) was present in the DRM family members. Similar to MET, DNMT2 member showed the order of motifs as I, IV, VI, VIII, IX, and X, but no regulatory region (Figure 2).
MTases, including replication foci domain (RFD), bromo adjacent homology (BAH), and methyltransferase domains were classified as MET subfamily members, whereas members with the Chr domain, along with BAH, and methyltransferase domain were placed in the CMT subfamily ( Figure 2). Members harboring both UBA and methyltransferase domains were grouped into a DRM subfamily ( Figure 2). DNMT2 subfamily members seem to lack any amino-terminal regulatory domain and include only a methyltransferase domain (Figure 2). In tomato, a total of three MTase genes were identified as CMT, one as MET, four as DRM, and one as DNMT2 members ( Figure  3); in Arabidopsis, three members belonged to CMT (AtCMT1, 2, and 3), four to MET (AtMET1, AtMET2a, AtMET2b, and AtMET3), three to DRM (AtDRM1, 2, and 3) and one to DNMT2 (AtDNMT2) families. Similarly, there were three CMTs (OsMET2a, OsMET2b, and OsMET2c), two METs (OsMET1-1 and OsMET1-2), four DRMs (OsDRM1aa, OsDRM1ba, OsDRM3, and OsZmet3) and one DNMT2 (OsDNMT2) in rice (Sharma et al., 2009). As shown in Figure 3, four clades (CMT, MET, DNMT2, and DRM) were clearly distinguished with support values close to 100. The CMT subfamily contained nine proteins, among which were three tomato proteins (SlCMT2, SlCMT3, and SlCMT4). The clades MET and DNMT2 included only SlMET and SlMETL, respectively. The DRM clade contained four tomato proteins (SlDRM5, SlDRM6, SlDRM7, and SlDRM8). Thus, our evolutionary analysis results showed good consistency with the classification results.

Transcription pattern of DNA MTase genes in wild-type tomato and mutants
To elucidate the tissue/organ expression patterns of MTase genes in tomato, quantitative RT-PCR was carried out using cDNAs from different tissues and development stages. Figure 4 shows that SlCMT2 was highly expressed in young leaves, mature green fruits, and stems, while its expression was down-regulated continuously during leaf development. SlCMT3 was also predominantly expressed in young leaves and its transcription level declined continuously with further fruit ripening. SlCMT4 was highly expressed in flowers and immature green fruits relative to other tissues, while its expression was down-regulated continuously during fruit development. The expression pattern of SlMET1 was very similar to that of SlDRM7. Their transcripts both reached a maximum level in immature green fruits. SlDRM5 was highly expressed in young leaves. During fruit development, SlDRM5 transcripts reached a maximum in immature green fruit and then decreased. Interestingly, the expression of SlDRM6 in the reproductive stage was higher than in the vegetative growth stage. SlDRM8 expression was slightly higher in flowers, sepals, and immature green fruits than in other tissues. SlMETL expression 4 Guo et al.   Table S1. Os -Oryza sativa, At -Arabidopsis.
was higher in ripening fruits and displayed an up-regulated tendency during fruit development. Spatial and temporal expression of SlMET1, SlCMT2, SlDRM5, SlDRM7, SlDRM8, and SlMETL were basically consistent with microarray expression data ( Figure S1). Besides, it is worthy of note that the expression level of SlMET1 in the tomato ripening mutants rin and Nr was significantly higher compared to wild-type tomato ( Figure 5).

Tomato DNA MTases are involved in abiotic stress response
To further study the potential functions of these tomato DNA MTases genes, we carried out expression analyses under low temperature and salt stress conditions by quantitative RT-PCR. For low-temperature treatment (Figure 6), we noted that the expression of SlMET1 and SlDRM5 was inhibited by low temperature and decreased gradually. The transcript levels of SlCMT3, SlCMT4, SlDRM7, SlDRM8, and SlMETL were also decreased under low temperature stress, especially SlCMT3 and SlDRM7, which were sharply down-regulated at 1 h. Additionally, SlCMT2 and SlDRM6 were up-regulated slightly during the first 12 hours of treatment, but a significant decrease in SlCMT2 mRNA was detected at 24 h.
For salt treatment (Figure 7), the induction of SlCMT2 gene expression was observed; it peaked at 4 h and returned to basal level at 24 h. The expression of SlCMT3 in leaves was significantly up-regulated at 12 h by about 13-fold. SlCMT4 was slightly down-regulated at 1 h and up-regulated subsequently in leaves. SlDRM5 and SlMETL were induced, and their transcripts peaked at 4 h in leaves. The expression of SlDRM6 was increased gradually and peaked at 4 h in leaves, with an expression pattern similar to that of SlDRM7. Comparatively, the transcript levels of SlMET1 and SlDRM8 were less affected in leaves. The above results suggest that these MTases genes may be involved in the response to salt stress.

Discussion
DNA methylation is an important epigenetic modification established by DNA methyltransferase. Although tomato is a model plant for studying fleshy fruit development and ripening, little is known regarding a comprehensive DNA MTase genes in tomato 5   analysis of MTases in tomato. In the present study, we analyzed tomato MTases and identified three members of CMT, one MET, four DRMs, and one DNMT2 in tomato. Each of the tomato MTases genes has a homologous gene in Arabidopsis, suggesting that MTases in tomato might have similar roles as in Arabidopsis. In addition, the systematic expression pattern of tomato MTases in different tissues/development stages and abiotic stress provides evidence for diverse functions in various aspects of plant development and abiotic stress responses. The structural analysis suggested that catalytic DNA methylase domains are highly conserved, whereas the Nterminus, which is regarded as a regulatory region, is divergent ( Figure 2). Thus, these nine tomato MTase genes may play different roles in regulating tomato growth and development. MET subfamily members are very similar to the mammalian DNMT1 class (Law and Jacobsen, 2010). Our structural analysis of tomato CMTs (SlCMT2, SlCMT3, and SlCMT4) suggested that the N-terminus of CMT harbors the BAH and Chr domains, which could possibly enhance the binding attraction of CMTs to methylated histones, similar to Zea mays CMT3 (Du et al., 2012). Four DRM members were identified in tomato. The N-terminus of DRM possesses the UBA domain, where sequence motifs occur that are usually involved in ubiquitin-mediated proteolysis and contributing to ubiquitin (Ub) binding or ubiquitin-like (UbL) domain binding. Recent findings have established DNMT2 as a tRNA methyltransferase that plays an important function under stress conditions (Schaefer and Lyko, 2010;Thiagarajan et al., 2011). We also investigated one member (SlMETL) of the DNMT2 family in tomato, lacking a conserved N-terminal regulatory domain, but possessing a catalytic C-terminal domain, which seems to be characteristic for all DNMT2s.
So far, the characteristics and functions of MTases in Arabidopsis have been studied clearly (Finnegan and Dennis 1993), but there is very little knowledge of their expression profiles in different tissues/developmental stages in tomato (Teyssier et al., 2008). In this study, we investigated the expression pattern of the nine DNA MTases genes in different tissues/stages (Figure 4), suggesting overlapping and specific roles during tomato development. The higher expression of SlMET1 in IMG fruits in tomato suggested its role in the maintenance of methylation in early stages of DNA MTase genes in tomato 7 Figure 7 -Quantitative RT-PCR analysis of the MTase genes in young leaves under NaCl stress. Tomato seedlings were grown with 250 mM NaCl. The relative expression levels were normalized to 1 in unstressed leaves (0 h). Data are reported as mean ± SD of three independent experiments. The asterisks indicate statistically significant differences between the treated and unstressed seedlings (p < 0.05). fruit development. This is different from the expression of MET members in Arabidopsis and rice, which was higher in the early stages of flower and seed development (Saze et al., 2003;Xiao et al., 2003;Kinoshita et al., 2004;Sharma et al., 2009;Schmidt et al., 2013). The ANAERO2CONSENSUS and CANBNNAPA elements (Ellerström et al., 1996) regulating fruit and embryo development respectively, were identified in the promoter of SlMET1 (Table S3), suggesting SlMET1 might be related with fruit development, which was confirmed by its high expression in fruit. SlCMT4 was highly expressed in flower, immature green fruit, and young leaf, which was coincident with a previous report (Teyssier et al., 2008). SlMETL showed the highest expression in B4 fruits, and SlDRM6 expression in reproductive stage was significantly higher than in vegetative growth stage, suggesting that these proteins may play an important role in tomato reproductive stage. Interestingly, SlCMT3 was specifically expressed in young leaves, suggesting that SlCMT3 may play critical roles in tomato leaf development. Consistent with its function in the DNA methylation maintenance, the tomato CMT was predominantly expressed in actively replicating cells in young leaves and roots. Additionally, it is noteworthy that SlMET1 and SlDRM7 were specifically expressed in immature green fruit, suggesting their useful application in fruit ripening and development.
Epigenetic modifications play an important role in response to environmental stimuli (Chinnusamy and Zhu, 2009;Gutzat and Mittelsten, 2012). For example, most of the MTases genes in pigeon pea are responsive to NaCl and extreme temperature (Rohini et al., 2014). To further study the potential functions of the nine tomato MTases genes, we examined their expression under various stress conditions by quantitative RT-PCR. We found that most of the DNA MTases genes in tomato are responsive to stress treatments, including NaCl and low temperature (Figures 6 and  7), and the differential expression profiles indicated that they may function diversely in different stress conditions. Although SlDRM5 and SlDRM6 appeared highly similar in protein structure ( Figure 2) and transcription in native leaves (Figure 4), the transcriptional responses to salt stress were remarkably different, being increased by about 2 times for SlDRM5 and 4.5 times for SlDRM6 after 4 h of treatment (Figure 7). This probably correlates with number of GAAAAA (GT1GMSCAM4) promoter cis-elements, known to be responsible in wound repair (Table S3).
DNA methylation is involved widely in the regulation of the temporal and spatial gene expression in plants. DNA methyltransferase inhibitor 5-azacytidine induces tomato fruit premature ripening (Zhong et al., 2013), and it is demonstrated that DNA methylation contributes to the regulation of fruit ripening. In this study, we observed that SlMET1 was highly expressed in immature green fruit and then declined during fruit ripening, which was consistent with a previous report by Teyssier et al. (2008). Interest-ingly, the expression levels of SlMET1 in the tomato ripening mutants rin and Nr are higher than in wild type tomato ( Figure 5), suggesting that SlMET1 is negatively regulated by the ethylene signal and ripening-related transcriptional factor MADS-RIN. We speculate that the abnormal fruit ripening in the mutants Nr and rin might be related to the concurrent hypermethylation of multiple ripening-related genes by DNA methyltransferase SlMET1.
In summary, based on bioinformatics and transcriptional pattern analysis, the nine MTase genes identified in tomato could be involved in tomato development and abiotic stress responses. This study also provided valuable information about tomato MTase genes associated with fruit ripening.
The following online material is available for this article: Table S1 -Basic information of DNA methyltransferases from Arabidopsis and rice. Table S2 -Primer pairs used in quantitative RT-PCR analysis. Table S3 -Putative cis-elements enriched in the promoters of tomato MTases genes. Figure S1 -Microarray expression data.

Associate Editor: Marcia Pinheiro Margis
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