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Article

Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear

by
Yunpeng Cao
1,†,
Yahui Han
2,†,
Dahui Li
1,
Yi Lin
1 and
Yongping Cai
1,*
1
School of Life Sciences, Anhui Agricultural University, Hefei 230036, China
2
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2016, 7(10), 89; https://0-doi-org.brum.beds.ac.uk/10.3390/genes7100089
Submission received: 9 July 2016 / Revised: 24 September 2016 / Accepted: 7 October 2016 / Published: 19 October 2016
Browse Figures
Versions Notes

Abstract

:
In plants, 4-coumarate:coenzyme A ligases (4CLs), comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei) were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS) genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR). We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

1. Introduction

The 4-coumarate:coenzyme A ligases (4CLs) play key roles in generating Coenzyme A (CoA) esters during the hydroxycinnamic acid production step of phenylpropanoid metabolism. The general phenylpropanoid pathway channels carbon flow into diverse branching pathways of secondary phenolic metabolism and generate various classes of phenolic secondary natural products, including flavonoids, lignin, suberins, and coumarins, which play important roles in plant development and environmental interactions [1]. According to the previous report by Hamberger et al. [2], 4CL genes could encode multiple isoenzymes with distinct substrate affinities that appeared to be related to specific metabolic functions. Numerous 4CL genes have been cloned from a large number of plant species, and it has been found that some of these genes were included into small gene families among the tested species. For example, there are four or five 4CL genes in poplar (Populus), Arabidopsis thaliana, maize (Zea mays), and rice (Oryza sativa) [2,3,4,5]. Moreover, the 4CL proteins in potato (Solanum tuberosum) and loblolly pine (Pinus taeda L.) are almost identical [6,7], whereas those in white poplar (Populus tremuloides), soybean (Glycine max), california poplar (Populus trichocarpa), and tobacco, are structurally divergent [8,9,10,11].
The differential expression profiles of 4CL genes suggest that these genes, which function in the formation of different classes of benzene propane compounds, have undergone subfunctionalization [3,12]. As members of an adenylate-forming enzyme superfamily, 4CL enzymes could participate in the formation of an adenylate intermediate. According to a wealth of genome sequence data from Malus domestica, Oryza sativa, Prunus persica, Pyrus bretschneideri, Prunus mume, and Arabidopsis, some genes encoding adenylate-forming enzymes are closely related to true 4CLs, but have no known specific biochemical function. These enzymes may function in the biosynthetic pathways of various metabolic and natural products in plants. For example, based on the phylogenetic relationship of 4CLs in Arabidopsis, four genes with distant similarity to true 4CL genes were identified [13]. Subsequently, due to their close phylogenetic relationship to true 4CLs (bona fide 4CLs in Figure 1), eight members of a larger class of Arabidopsis 4CL-type adenylate-forming enzymes were classified as 4CL-like genes [14]. These 4CL-like proteins contained similar structural components, such as conserved Box I and Box II domains and a conserved substrate binding domain [15,16]. Ehlting et al. [17] identified nine genes closely related to genes encoding 4CLs, and they were annotated as 4CL or 4CL-like genes based on clade [14,18,19].
In contrast to true 4CLs, functions of 4CL-like genes are not related to flavonoid or lignin biosynthesis [14,17], suggesting that they may encode enzymes without activity towards some 4CL hydroxycinnamate substrates, such as p-coumaric, 5-hydroxyferulic, ferulic, caffeic, and sinapic acids. Indeed, seven 4CL-like recombinant proteins (GenBank no. AAO64847, AAQ56837, AEE78480, AEE29981, AED94270, AEE34025, and AEE82486) did not show catalytic activities [20]. In a subsequent analysis, several 4CL-like genes were found to encode a type of acyl-coenzyme A synthetase, which accepted both medium- and long-chain fatty acids with the cyclopentenone 12-oxo-phytodienoic acid and/or 12-oxo-phytodienoic acid derivatives as substrates under some conditions [21,22]. 12-oxo-phytodienoic acid is an intermediate for jasmonic acid biosynthesis within the octadecanoid pathway. Furthermore, β-oxidation of 12-oxo-phytodienoic acid–CoA (resulting in acyl chain shortening) led to the formation of 12-oxo-phytodienoic acidCoA thioesters via the latter steps within the pathway [23,24]. One Arabidopsis 4CL-like gene (GenBank no. AEE29981) was found to encode a peroxisomal 12-oxo-phytodienoic acid, a CoA ligase related to jasmonic acid biosynthesis, which has since been designated OPCL1 [22,24]. However, to date, the biological functions of pear 4CL-like genes have not been elucidated. Based on the lack of activity of several 4CL-like enzymes against hydroxycinnamate substrates and their function as acyl-coenzyme A synthetases (ACS) to accept fatty acids, we used the name ACS to describe the products of 4CL-like genes in the pear (Pyrus bretschneideri Rehd.), yangmei (Prunus mume), peach (Prunus persica), and apple (Malus x domestica) in this report. Pears are among the most economically important fruit crops worldwide. Previous studies revealed that the stone cell content is an important factor impacting the taste of the fruit flesh in pears [25]. Pear stone cells are mainly composed of lignin [25,26]. Accordingly, to improve the quality of pear fruit, reducing the lignin content of the pear stone may be an effective method. In addition, although several 4CL-related genes that play roles in secondary metabolism have been characterized in some model species, such as rice and Arabidopsis [27], no study of this gene family has been conducted in pears. In the current study, we performed a genome-wide analysis of 4CL-related gene family members in the Chinese white pear [28], yangmei [29], apple [30], and peach [31] based on draft genome sequences, including gene models, phylogenetic relationships, genomic structures, chromosome locations, and other structural features. In addition, we performed quantitative real-time PCR (qRT-PCR) analysis to verify our results in pears. To the best of our knowledge, this work presented the first comprehensive study on the 4CL-related gene family in the Chinese pear. Furthermore, identification and analysis of the Pb4CL1 gene related to lignin synthesis in the Chinese pear will help to improve the quality of pear fruit in the future.

2. Materials and Methods

2.1. Sequence Identification and Collection

The conserved protein sequences of the AMP-binding domain PF00501were obtained using the Pfam database [32]. The Hidden Markov Model (HMM) algorithm implemented in DNATOOLS software [33] was used as a query to identify all AMP-binding- domain-containing sequences in pear, peach, apple, and yangmei. A total of 156 candidate 4CL-related genes were identified. Subsequently, the SMART database [34] and Pfam database [32] were used to verify each candidate 4CL-related protein sequence as a member of the 4CL-related family, which was vital for identifying an accurate number of candidate 4CL-related proteins. Finally, a total of 149 4CL-related genes were identified in this study. According to the naming method of Clarice de Azevedo Souza et al. [35], these genes were designated AEE, ACS, and 4CL, with species-specific identifiers used to distinguish among genes (Table 1).
A map of the chromosome locations of 4CL-related genes in pear was produced using MapInspect software (Version 1.0, Ralph van Berloo) based on their starting positions. A diagram of the exon/intron structures of these pear 4CL-related genes was generated using the online tool GSDS [36] based on genome annotation information. The conserved motifs encoded by each Pb4CL and PbACS gene were also investigated. These protein sequences were submitted to the online tool MEME (Multiple Expectation Maximization for Motif Elicitation) [37]. Parameters were set as follows: (1) optimum motif width was set to 6 and 200; and (2) maximum number of motifs was set to 20. For collinearity analysis, collinearity blocks between Pyrus bretschneideri Rehd. and the other species, including Arabidopsis, Prunus mume, Prunus persica, Malus x domestica, and Oryza sativa were downloaded from the Plant Genome Duplication Database [38] and were displayed in the collinearity map.
To analyze the promoter regions of these genes, the upstream sequences of the 4CL-related genes were analyzed based on the positions of the genes provided in the pear GigaDB database [28]. The PLACE database [39] was used to investigate putative cis-acting regulatory DNA elements in the promoter regions of the 4CL-related genes in the pear.

2.2. RNA Extraction and qRT-PCR Analysis

To investigate the expression patterns of Pb4CL/PbACS genes, pear fruits were sampled at 15, 39, 47, 55, 63, 79, 102, and 145 days after flowering (DAF). At least three fruits were harvested at each stage from 40-year-old pear trees grown in a horticultural field in Dangshan, Anhui, China. Total RNAs were extracted from the samples using Trizol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. DNase-treated RNA was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen). Gene-specific primers for quantitative reverse-transcription PCR (qRT-PCR) were designed to generate 80–200 bp products using Beacon Designer 7 software (Applied Biosystems, Foster, CA, USA), and the tubulin gene [40] was used as an internal reference with primers synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The ABI7500 (Applied Biosystems) instrument was used for quantitative reverse-transcription PCR analysis of cDNA samples from different developmental stages collected from cross-pollinated varieties. The experiments were repeated three times. The relative expression levels of these genes were calculated using the 2−ΔΔCT method [41]. The reactions contained the following: 10 μL of SYBR Premix Ex Taq II (2x) (Takara, Dalian, China), 1 μL of template cDNA, 0.5 μL of forward and reverse primers and free water in a final volume of 20 μL. PCR amplification was performed as follows: 50 °C for 2 min, 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s.
The experiments did not involve endangered or protected species. No specific permits were required for these locations/activities because the pears used in this study were obtained from a horticultural field in Dangshan, which were demonstration orchards at Auhui Agricultural University.

2.3. Expression Analysis of At4CL/ACS and Os4CL/ACS Genes

Affymetrix Arabidopsis and Oryza sativa microarray data were downloaded from the ArrayExpress [42], PLEXdb [43] and Rice Oligonucleotide databases [44]. Hierarchical clustering was performed with HemI 1.0 software [45] to analyze the expression patterns of 4CL/ACS family genes in Arabidopsis and Oryza sativa.

3. Results and Discussion

3.1. The Phylogenetic Analysis of Pear 4CL-Related Genes

Using the sequence of the AMP-binding domain obtained from the Pfam database [32] as a query, we searched for 4CL-related genes from the genomic sequences of the four Rosaceae species, including the peach (Prunus persica) [31], apple (Malus x domestica) [30], yangmei (Prunus mume) [29], and pear (Pyrus bretschneideri) [28]. As a result, 149 sequences were identified (Table 1 and Table S1). A phylogenetic tree of the 149 4CL-related proteins from the four Rosaceae plants was constructed, along with those of the true 4CL proteins from Oryza sativa and Arabidopsis (Figure 1). The unrooted phylogenetic tree categorized the adenylate-forming proteins into two general clades with high bootstrap values. One large cluster contained representatives from all six plant species analyzed (the large arc in Figure 1). These adenylate-forming enzymes with various metabolic functions are conserved among these species and have a wide range of potential applications. For example, AtACN1 acts as an entry point to the glyoxylate cycle during seed germination [46].
The large clade includes both Arabidopsis ACN1 gene (AtCN1) [46] and many Arabidopsis genes for acyl-activating enzymes (AAEs) [46]. These AAEs are related to fatty acid metabolism [19]. In this study, proteins from the peach, apple, yangmei, and pear within this clade were designated as acyl-activating enzyme-like (AAEL) (Table S1) due to their sequence similarity to Arabidopsis AAE gene (AtAAE) [46]. The other clades of adenylate-forming proteins include one group of 4CL proteins and six groups of 4CL-like ACS proteins (Figure 1; clades A–F). These ACS proteins are closely related to bona fide 4CLs, forming a sister clade to clades A–F (Figure 1; clades A–F). As shown below, each of the clade A–F contains at least one representative from our examined plant species, indicating that they might have undergone gene duplication. These genes, which are probably orthologous or paralogous gene pairs, may contribute to the expansion of ACS gene family members during evolution. Both Arabidopsis and Oryza sativa proteins represented in the bona fide 4CL clade have been described and annotated [2,3,4,48]. Together with our annotated 4CL and ACS genes from the peach, apple, yangmei, and pear, they are listed in Table 1. ACS genes of the peach and yangmei are found in all clusters of A–F, whereas ACS genes from the remaining four plants are absent in some clusters. For example, PbACS genes are not present in cluster A. We propose that the gain or loss of some genes over the course of evolution may have helped the plant adapt to the environment. To further clarify the function of the 4CL genes in the pear, a phylogenetic tree was constructed for 4CL genes from the six species using the neighbor-joining method. Based on previous clustering approaches [2,48], these 4CL genes are clearly clustered into two categories with strong bootstrap values, as shown in Figure S1. In addition, previous studies showed that genes clustered in class I are mainly involved in lignin biosynthesis in plants, and genes clustered in class II may participate in flavonoid biosynthesis [2,48,50,51]. Then, we presented an analysis on the 4CL orthologous and paralogous gene pairs that clustered together at the terminal branch of the phylogenetic tree, and some functional clues about these genes were obtained. Therefore, we hypothesize that Pb4CL1 and Pb4CL3 are primarily involved in lignin biosynthesis in the pear, whereas Pb4CL2 and Pb4CL3 are primarily involved in flavonoid biosynthesis.

3.2. Structural Analysis of Pear 4CL-Related Genes

An unrooted phylogenetic tree containing all 4CL-related genes in the pear was constructed (Figure 2) using the neighbor-joining method with bootstrap values (1000 replicates). To facilitate the research and analysis, six groups (class I–VI) were classified according to bootstrap values (>50) (Figure 2). By contrast, some 4CL-related members were beyond the six groups because of low bootstrap values (<50) of the neighbor-joining tree. As shown in Figure 2, class V contains the most 4CL-related gene members (8), followed by class I (6), while class III and VI only contains three members. Notably, the 4CL-related genes from the pear formed 10 gene pairs, which had strong bootstrap support (>99%), with the exception of PbAEE5/PbAEE16.
The gene structures of the pear 4CL-related genes were drawn by GSDS [36] by comparing the DNA sequences with their corresponding coding sequences. Observation of the gene structure in pear 4CL-related genes (Figure 2) suggested the number of introns ranged from 1 to 17, with the exception of PbAEE9 and PbACS2 having no introns, and PbAEE1 and PbAEE17 contains most introns (17) among the pear 4CL-related gene family. In addition, our analysis of exon/intron structure supports the phylogeny reconstruction. The 4CL-related genes within the same groups displayed similar exon/intron structures, particularly in terms of the number of introns, although there were some exceptions. For example, in group V, PbACS2 lacks introns, whereas PbACS7 and PbACS8 contain two introns. In addition, the intron length ranges from several bases to approximately 35,500 bases, which demonstrates the high sequence diversity among members of the 4CL-related gene family.

3.3. Collinearity Analyses

Genomic comparison is a rapid method for transferring genomic knowledge from a well-studied group to a less-studied group. Therefore, based on their collinearity with Arabidopsis and Oryza sativa genes, we can infer the functions of 4CL-related genes in the pear to some extent. To further investigate the origin and evolutionary process of pear 4CL-related genes in Rosaceae species, we performed a comparative analysis of the genomes of the pear, peach, and yangmei. Based on the plant genome duplication event database [53], we analyzed the collinearity of 4CL-related genes of Prunus persica, Pyrus bretschneideri, Prunus mume, Arabidopsis, and Oryza sativa (Figure 3A,B), revealing no collinearity between the pear 4CL-related genes of rice. Remarkably, 33.3% of PbAEE genes share collinearity with fragments in Arabidopsis, Prunus persica and Prunus mume, whereas no PbAEEs share collinearity with fragments in rice. However, among PbACS and Pb4CL genes, we found collinear fragments only in Arabidopsis, for PbACS3/AtACS9 and Pb4CL2/At4CL3. We did not detect collinear fragments in genes related to PbACSs and Pb4CLs in Rosaceae species such as peach and yangmei. These results suggest that the genomes of these species have undergone multiple rounds of significant chromosomal rearrangements, fusions and selective gene deletions. Due to this selective gene deletion, chromosomal colinearity blocks that may have arisen during the speciation of Prunus persica, Prunus mume, Pyrus bretschneideri, Oryza sativa, and Arabidopsis would be severely obscured [54].
Genes in the pear showed a closer homologous relationship with those in flowering peaches and plums belonging to the Rosaceae, as well as Arabidopsis, whereas they have a distant relationship with genes in rice. This is consistent with the evolution of the plant species. Several AEE, 4CL, or ACS genes existing in Pyrus bretschneideri, Prunus persica, Prunus mume, and Arabidopsis could form orthologous gene pairs. Notably, no homologous genes of the pear 4CL-related gene family were found in the rice genome. Therefore, we postulated that pear 4CL-related genes should occur at a certain degree of specific evolution to adapt to the environment during the process of differentiation from monocotyledonous plants. In the study of the MYB gene family, Cao et al. found that the AtMYB genes in subfamily S12 may be a specific gene type to adapt to phytophagous conditions [55,56].

3.4. Genomic Distribution and Gene Duplication

In almost all Rosaceae plants, one or multiple genome duplication events have occurred over the course of evolution [28,30,57]. To further investigate the effects of gene differentiation and gene duplication on 4CL-related gene families, we used MapInspect software to localize 4CL-related gene family members on the 13 pear chromosomes. As shown in Figure 4, the 4CL-related genes were found to be unevenly distributed on 13 pear chromosomes. Five gene models (PbAEE7, PbAEE13, PbAEE17, PbACS8, and Pb4CL3) could not be conclusively mapped to these chromosomes because they were localized to scaffolds, which may have resulted from sequencing anomalies. The number of 4CL-related genes per chromosome varies widely. Chromosome 9 contains the largest number of pear 4CL-related genes (five), followed by chromosome 17 (four). Each of chromosomes 1, 2, 3, 6, 10 and 16 has only one 4CL-related gene. Relatively high densities of 4CL-related genes were found on the tops of chromosomal regions, such as chromosome 9. According to Holub’s definition of a gene cluster [58], we found only one gene cluster (with three members) in the 4CL-related family. We propose that the members of this gene cluster underwent differentiation after chromosomal duplication.
We also investigated gene duplication events to reveal the expansion mechanism of the 4CL-related gene family during evolution. According to Gu et al.’s definition of gene duplication events [59], we found that PbAEE16/PbAEE5 on chromosome 9 has undergone tandem duplication, whereas nine gene pairs (PbAEE1/PbAEE11, PbAEE2/PbAEE3, PbAEE4/PbAEE13, PbAEE8/PbAEE9, PbAEE5/PbAEE16, PbAEE7/PbAEE17, Pb4CL1/Pb4CL3, Pb4CL2/Pb4CL4, and PbACS7/PbACS8) have undergone segmental duplication (Figure 4). Notably, among the 10 sister pairs, one gene pair (PbACS3/PbACS4) is located just outside the segmental duplication regions. However, their phylogenetic relationships and gene structures suggest that these genes have a similar evolutionary history, indicating that this gene pair has indeed undergone segmental duplication. We therefore assert that this gene pair has undergone gene fragment duplication (blue box logo in Figure 4) [60]. These results suggest that gene duplication events played an important role in the expansion of pear 4CL-related family members.

3.5. Analysis of Conserved Motifs in Pb4CL and PbACS and Their Promoter Regions

We used the MEME online search tool to identify conserved motifs presented in 4CL/ACS proteins in the pear (Figure 5). Each motif from MEME was annotated and identified by searching the Pfam and SMART databases. Twelve sequences were categorized into two classes, which is consistent with the classification derived by the phylogenetic analysis. AMP-binding domains were represented by motifs 1, 2, 3, 4, 7, 8, 9, and 10. As shown in Figure 3, all sequences contain several motif 1-, 3-, and 5-encoding genes. Motif 7 was found in all eight PbACS genes, whereas Motif 2, encoding the amino acid synthesis domain, was found in all four Pb4CL genes. Bakolitsa et al. showed that the amino acid synthesis domain functions in amino acid synthesis [61]. We further postulate that the 4CL gene is a key gene in the phenylpropanoid pathway. Therefore, identifying an amino acid synthesis domain in the 4CL gene provides a reference for the subsequent identification of 4CL genes in other species. Furthermore, some subfamily-specific motifs with unknown functions were also identified, suggesting that these motifs are likely required for subfamily-specific functions. For example, motif 2 is specific to the 4CL subfamily. Detailed information about the conserved amino acid sequences and lengths of the 20 motifs are shown in Table S2.
We also analyzed the promoter sequences in a 1500 bp-region upstream of the transcription start site (ATG) of the predicted Pb4CL and PbACS genes. Five regulatory elements, ABRE [62,63], LTRE [63], DRE [62], BOXP [64], and BOXL [65], were detected by searching these promoter sequences against the PLACE database [39]. Surprisingly, we found that all sequences contained putative ABRE elements in their promoter regions (Figure S2), indicating that ABA can affect the expression levels of PbACSs and Pb4CLs. Concurrently, we also found that the Pb4CLs and PbACS3 genes contained BOXP and BOXL components, suggesting that these genes could be involved in the regulation of lignin biosynthesis. By comparing the distribution of the five regulatory elements (ABRE, LTRE, DRE, BOXP, and BOXL) in the promoter sequences, the four sister pairs (Pb4CL1/Pb4CL3, Pb4CL2/Pb4CL4, PbACS3/PbACS4, and PbACS7/PbACS8) were found to exhibit significant differences in their promoter regions, suggesting that the duplicated genes may not have some regulatory features in common, but may instead function in similar regulatory pathways. For example, each gene in these duplicated gene pairs contains at least one ABRE element in its promoter sequence.

3.6. Expression of 4CLs and ACSs during Ripening in Pear Fruit

For the 4CL/ACS genes, we were interested in which ones play important roles during the development of pear fruit. In the present study, qRT-PCR analysis of pear 4CL/ACS genes revealed that these genes exhibited diverse expression patterns at 15, 39, 47, 55, 63, 79, 102, and 145 DAF (Figure 6). This result indicated that these expressed genes are functionally active, with them being expressed in several or all eight stages during the fruit development of the pear (Figure 6). The expressions of PbACS2 and PbACS6 in clade B, and Pb4CL3 in bona fide 4CLs were significantly increased at 15 DAF (Figure 6), implying that these genes might play important roles in the early stage of pear fruit development. Expression of Pb4CL1, Pb4CL2, Pb4CL4, and PbACS1 was obviously increased at 55 DAF, with consistent change trends of lignin content in pear fruit [25,26], indicating that these genes might be involved in the regulation of the lignin synthesis of pear fruit.
The 4CLs were shown to be involved in regulating the biosynthesis of metabolites, such as lignin and flavonoids. To help identify the Pb4CLs and PbACSs that participate in the biosynthesis of lignin or flavonoids in pear fruit during development, a composite phylogenetic tree of 4CL genes in the six species was constructed (Figure S1). The 4CLs in class I were previously shown to participate in lignin biosynthesis, whereas the 4CLs in class II function in flavonoid biosynthesis according to previous studies [2,48]. Therefore, the qRT-PCR results suggest that in the pear, only Pb4CL1 is involved in lignin biosynthesis, whereas Pb4CL2 and Pb4CL4 may participate in flavonoid biosynthesis during fruit development. We also compared the expression profiles of the four duplicated gene pairs, finding that the duplicated genes within a sister pair exhibited similar expression patterns at 15, 39, 47, 55, 63, 79, 102, and 145 DAF. Differential expression patterns between the two duplicated genes were also observed. For example, the highest level of Pb4CL1 expression was observed at 55 DAF, whereas that of Pb4CL3 was observed at 15 DAF.

3.7. Expression Profiles of Rice and Arabidopsis 4CL and ACS Genes

Gene expression patterns can provide important clues about gene function. In addition, previous studies showed that orthologous genes were more likely to share correlated expression patterns compared with non-orthologous genes [66,67]. To further understand the function of 4CL/ACS genes in pear, we used publicly available genome-wide transcript profiling data from ArrayExpress [42], PLEXdb [43] and Rice Oligonucleotide databases [44] to investigate the expression patterns of 4CL/ACS genes in Arabidopsis and Oryza sativa.

3.7.1. Arabidopsis 4CL and ACS Genes

To compare the expression patterns of 4CLs and ACSs of the Pyrus bretschneideri with those of Arabidopsis, we obtained gene expression information for Arabidopsis in various tissues and during various stages of development from the Array Express and PLEXdb chip databases and constructed a heat map (Figure 7). During Arabidopsis development, the expression of At4CL1, At4CL2, and At4CL4 increased, followed by a decrease, whereas AtACS1, AtACS2, AtACS5, AtACS7, and AtACS8 exhibited almost no expression. Interestingly, the expression patterns of 4CLs and ACSs in Arabidopsis are highly similar to those of pear genes in the same cluster. For example, the expression patterns of Pb4CL1, Pb4CL2, Pb4CL3 and At4CL1, At4CL2, and At4CL4 were essentially the same, and the expression patterns of ACSs, such as AtACS2 and PbACS6 (in cluster B), were similar. However, the expression patterns of a few genes from the pear were different from those of Arabidopsis in the same cluster. For example, the expression of AtACS6 (in cluster C) was relatively high during each period, whereas the expression of PbACS2 was low beginning at 15 DAF (Figure 7).

3.7.2. Rice 4CL and ACS Genes

We also compared the expression profiles of Pb4CL and PbACS genes in different tissues of the pear with those in rice, based on rice tissue expression microarray data from the Rice Oligonucleotide Array Databases and PLEXdb chip database and using the methods described above. The results showed that the expression level of OsACS8 and OsACS9 was specifically high in seedlings at the four-leaf stage (Le), instead of in other tissues (Figure 8). These different expression patterns were present in other OsACSs, with relatively higher levels in most tissues. For example, the expression levels of Os4CL2, Os4CL3, Os4CL4, and Os4CL5 were high in various tissues over different periods (Figure 8). These results suggested that pear genes in the same cluster with rice genes could have similar expression patterns.

4. Conclusions

4CL enzymes participate in the regulation of lignin and flavonoid biosynthesis in plants. Proteins encoded by ACS genes may catalyze reactions involving different fatty acids or other acyl substrates. These genes are present in gene families in plants. Currently, although 4CL-related gene families were identified and characterized in several model plants (Oryza sativa and Arabidopsis), no systematic analysis of these families has previously been reported in the Rosaceae. Using DNATOOLS software, we identified 149 4CL-related genes in four Rosaceae plants, the peach, yangmei, apple, and pear. We classified these genes and analyzed their evolutionary relationships (and those of rice and Arabidopsis), as well as their physical locations, promoter regions, collinearity, gene structures and expression patterns using qRT-PCR analysis. The results of this study provide a characterization of the 4CL-related gene family in pear and the phylogenetic relationships of 4CL-related genes among Prunus persica, Malus x domestica, Prunus mume, Oryza sativa, Pyrus bretschneideri, and Arabidopsis. To further explore the role of these genes in the pear, we also analyzed the expression profiles of several 4CL and ACS family genes in the pear, as well as those in Arabidopsis and Oryza sativa, in various tissues and during different developmental stages. The results suggest that only Pb4CL1 plays a key role in lignin biosynthesis and metabolic pathways in pear fruit. Our results clarify the biological function of 4CL/ACS genes in pear development and have a significant influence on our knowledge of woody plant 4CL-related genes.

Supplementary Materials

The following are available online at www.mdpi.com/2073-4425/7/10/89/s1, Table S1: Gene names and sequences used in Figure 1, Figure S1: Phylogenetic relationships of rice, Arabidopsis, apple, yang mei, peach and pear 4CL proteins. The tree was generated with MEGA 6.0 using the NJ method, Table S2: Major MEME motif sequences in pear 4CL/ACS proteins, Figure S2: Distribution of major DNA elements in the promoter sequences of the 12 4CL and ACS genes in pear. Putative ABRE, LTRE, DRE BOXL and BOXP core sequences are represented by different symbols, as indicated.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant 30771483, 31171944 and 31640068). We extend our thanks to the reviewers and editors for their careful reading and helpful comments on this manuscript.

Author Contributions

Yunpeng Cao and Yahui Han conceived and designed the experiments; Yunpeng Cao and Yahui Han performed the experiments; Yunpeng Cao and Yahui Han analyzed the data; Yunpeng Cao, Yahui Han, Dahui Li, Yi Lin, and Yongping Cai contributed reagents/materials/analysis tools; Yunpeng Cao and Yahui Han wrote the paper.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Figure 1. Phylogenetic relationships of 4-coumarate:coenzyme A ligase (4CL)-related proteins in Malus x domestica, Prunus persica, Prunus mume, Oryza sativa, Arabidopsis, and Pyrus bretschneideri. Asterisks represent bootstrap values of at least 70%. The large dashed arc indicates a large clade of acyl-activating enzymes (AAEs) from four Rosaceous plants (Malus x domestica, Prunus persica, Prunus mume, and Pyrus bretschneideri) as well as Oryza sativa and Arabidopsis. The red dashed ellipse indicates the clade of 4CL and acyl-coenzyme A synthetases (ACS) genes. The gene name species prefixes: Pb, Pyrus bretschneideri; Pp, Prunus persica; Pm, Prunus mume; Md, Malus x domestica; At, Arabidopsis thaliana; and Os, Oryza sativa. Gene names and models are listed in Table 1 and Table S1. The scale represents 0.2 amino acid changes.
Figure 1. Phylogenetic relationships of 4-coumarate:coenzyme A ligase (4CL)-related proteins in Malus x domestica, Prunus persica, Prunus mume, Oryza sativa, Arabidopsis, and Pyrus bretschneideri. Asterisks represent bootstrap values of at least 70%. The large dashed arc indicates a large clade of acyl-activating enzymes (AAEs) from four Rosaceous plants (Malus x domestica, Prunus persica, Prunus mume, and Pyrus bretschneideri) as well as Oryza sativa and Arabidopsis. The red dashed ellipse indicates the clade of 4CL and acyl-coenzyme A synthetases (ACS) genes. The gene name species prefixes: Pb, Pyrus bretschneideri; Pp, Prunus persica; Pm, Prunus mume; Md, Malus x domestica; At, Arabidopsis thaliana; and Os, Oryza sativa. Gene names and models are listed in Table 1 and Table S1. The scale represents 0.2 amino acid changes.
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Figure 2. Phylogenetic and exon–intron structure analyses of the 30 predicted pear 4CL-related genes. The neighbor joining (NJ) tree is shown on the left. The phylogenetic tree was constructed with MEGA 6.0 [52] using the full-length amino acid sequences of the 30 pear 4CL-related proteins. Bootstrap values are represented by black numbers. The gene structures are shown on the right. Exons, introns and untranslated regions (UTRs) are indicated by green boxes, gray lines, and blue boxes, respectively. The length of each 4CL-related gene can be estimated based on the scale at the bottom.
Figure 2. Phylogenetic and exon–intron structure analyses of the 30 predicted pear 4CL-related genes. The neighbor joining (NJ) tree is shown on the left. The phylogenetic tree was constructed with MEGA 6.0 [52] using the full-length amino acid sequences of the 30 pear 4CL-related proteins. Bootstrap values are represented by black numbers. The gene structures are shown on the right. Exons, introns and untranslated regions (UTRs) are indicated by green boxes, gray lines, and blue boxes, respectively. The length of each 4CL-related gene can be estimated based on the scale at the bottom.
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Figure 3. Collinearity analysis of 4CL-related regions among pear, rice, Arabidopsis, apple, peach, and yangmei. To identify the species of origin for each chromosome, a species identifier is included before the chromosome name: At, Arabidopsis thaliana; Os, Oryza sativa; Pb, Pyrus bretschneideri; Pp, Prunus persica; and Pm, Prunus mume. Different colored bars connect collinear regions between Pyrus bretschneideri and the other plant species.
Figure 3. Collinearity analysis of 4CL-related regions among pear, rice, Arabidopsis, apple, peach, and yangmei. To identify the species of origin for each chromosome, a species identifier is included before the chromosome name: At, Arabidopsis thaliana; Os, Oryza sativa; Pb, Pyrus bretschneideri; Pp, Prunus persica; and Pm, Prunus mume. Different colored bars connect collinear regions between Pyrus bretschneideri and the other plant species.
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Figure 4. Chromosomal locations and gene duplication of 4CL-related genes in pear. Chromosome numbers are indicated at the top of each vertical black bar, the names on the left side of each chromosome correspond to the approximate locations of each 4CL-related gene. Dashed lines represent segmental duplicated genes, and the tandem duplicated gene pairs are joined by a red line. The scale is in megabases (Mb).
Figure 4. Chromosomal locations and gene duplication of 4CL-related genes in pear. Chromosome numbers are indicated at the top of each vertical black bar, the names on the left side of each chromosome correspond to the approximate locations of each 4CL-related gene. Dashed lines represent segmental duplicated genes, and the tandem duplicated gene pairs are joined by a red line. The scale is in megabases (Mb).
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Figure 5. Distribution of conserved motifs in pear 4CL and ACS numbers. Motifs of 4CL and ACS proteins were identified with the Multiple Expectation Maximization for Motif Elicitation (MEME) online tool. Note that the length of each box in the proteins does not represent the actual motif size, and different motifs are indicated by different colored boxes based on results of the MEME analysis. The conserved amino acid sequences and length of each motif are shown in Table S2. Clades are defined in Figure 1.
Figure 5. Distribution of conserved motifs in pear 4CL and ACS numbers. Motifs of 4CL and ACS proteins were identified with the Multiple Expectation Maximization for Motif Elicitation (MEME) online tool. Note that the length of each box in the proteins does not represent the actual motif size, and different motifs are indicated by different colored boxes based on results of the MEME analysis. The conserved amino acid sequences and length of each motif are shown in Table S2. Clades are defined in Figure 1.
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Figure 6. Expression levels of Pb4CL/PbACS genes during the eight stages of pear fruit development at 15 days after flowering (DAF), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF and 145 DAF. These expression patterns were obtained using qRT-PCR, and the relative expression was log2 transformed. B: clade B genes; C: clade C genes; D: clade D genes; E: clade E genes; F: clade F genes; and 4CLs: 4CL genes. Clades are defined in Figure 1.
Figure 6. Expression levels of Pb4CL/PbACS genes during the eight stages of pear fruit development at 15 days after flowering (DAF), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF and 145 DAF. These expression patterns were obtained using qRT-PCR, and the relative expression was log2 transformed. B: clade B genes; C: clade C genes; D: clade D genes; E: clade E genes; F: clade F genes; and 4CLs: 4CL genes. Clades are defined in Figure 1.
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Figure 7. Expression patterns of ACS/4CL genes in Arabidopsis in different organs. R, root; St, stem; L, leaf; S3, siliques with seeds, stage 3 (mid-globular to early heart embryos); S4, siliques with seeds, stage 4 (early to late heart embryos); S5, siliques with seeds, stage 5 (late heart to mid torpedo embryos); S6, seeds, stage 6, without siliques; mid to late torpedo embryos; S7, seeds, stage 7, without siliques (late torpedo to early walking-stick embryos); S8, seeds, stage 8, without siliques (walking-stick to early curled cotyledons embryos); S9 seeds, stage 9, without siliques (curled cotyledons to early green cotyledons embryos); and S10, seeds, stage 10, without siliques (green cotyledons embryos). Clades are defined in Figure 1.
Figure 7. Expression patterns of ACS/4CL genes in Arabidopsis in different organs. R, root; St, stem; L, leaf; S3, siliques with seeds, stage 3 (mid-globular to early heart embryos); S4, siliques with seeds, stage 4 (early to late heart embryos); S5, siliques with seeds, stage 5 (late heart to mid torpedo embryos); S6, seeds, stage 6, without siliques; mid to late torpedo embryos; S7, seeds, stage 7, without siliques (late torpedo to early walking-stick embryos); S8, seeds, stage 8, without siliques (walking-stick to early curled cotyledons embryos); S9 seeds, stage 9, without siliques (curled cotyledons to early green cotyledons embryos); and S10, seeds, stage 10, without siliques (green cotyledons embryos). Clades are defined in Figure 1.
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Figure 8. Expression patterns of ACS/4CL genes in rice in different organs. Se5, seeds at 5 DAP; Se10, seeds at 10 DAP; Sh, shoots; Le20, leaves at 20 days; Le, seedlings at the four-leaf stage; Em25, embryos at 25 DAP; En25, endosperm at 25 DAP; An, anthers; and Pi, pistils. Clades are defined in Figure 1.
Figure 8. Expression patterns of ACS/4CL genes in rice in different organs. Se5, seeds at 5 DAP; Se10, seeds at 10 DAP; Sh, shoots; Le20, leaves at 20 days; Le, seedlings at the four-leaf stage; Em25, embryos at 25 DAP; En25, endosperm at 25 DAP; An, anthers; and Pi, pistils. Clades are defined in Figure 1.
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Table
Table 1. Oryza sativa, Arabidopsis, Malus x domestica, Prunus mume, Prunus persica, and Pyrus bretschneideri 4-coumarate:coenzyme A ligase (4CL) and acyl-CoA synthetase (ACS) genes.
Table 1. Oryza sativa, Arabidopsis, Malus x domestica, Prunus mume, Prunus persica, and Pyrus bretschneideri 4-coumarate:coenzyme A ligase (4CL) and acyl-CoA synthetase (ACS) genes.
NameClade 1Gene ModelNameClade 1Gene Model
Pyrus bretschneideri Prunus mume
Pb4CL14CLsPbr024635.1 2PmACS5DPm006110
Pb4CL24CLsPbr039972.1PmACS6FPm024403
Pb4CL34CLsPbr001283.1PmACS7FPm024473
Pb4CL44CLsPbr013445.1PmACS8APm007022
PbACS1DPbr005761.1PmACS9FPm024474
PbACS2CPbr026213.1PmACS10FPm006911
PbACS3FPbr027219.1Prunus persica
PbACS4FPbr036926.1Pp4CL14CLsppa003747m 5
PbACS5FPbr036272.1Pp4CL24CLsppa003854m
PbACS6BPbr026415.1Pp4CL34CLsppa022401m
PbACS7EPbr040326.1PpACS1Bppa003893m
PbACS8EPbr041161.1PpACS2Cppa017093m
Malus × domestica PpACS3Cppa022562m
Md4CL14CLsMDP0000260512 3PpACS4Dppa003506m
Md4CL24CLsMDP0000295794PpACS5Bppa003871m
Md4CL34CLsMDP0000293578PpACS6Eppa022057m
Md4CL44CLsMDP0000691789PpACS7Fppa003658m
MdACS1AMDP0000257967PpACS8Fppa003674m
MdACS2DMDP0000716496PpACS9Appa003797m
MdACS3EMDP0000171033PpACS10Fppa003742m
MdACS4DMDP0000376256Oryza sativa
MdACS5EMDP0000425292Os4CL14CLsOs08g14760 6
MdACS6EMDP0000206055Os4CL24CLsOs02g46970
MdACS7BMDP0000277093Os4CL34CLsOs02g08100
MdACS8BMDP0000267064Os4CL44CLsOs06g44620
MdACS9FMDP0000179301Os4CL54CLsOs08g34790
MdACS10FMDP0000289629OsACS1BOs03g05780
MdACS11AMDP0000878279OsACS2COs10g42800
MdACS12FMDP0000129547OsACS3COs08g04770
MdACS13FMDP0000262301OsACS4DOs03g04000
MdACS14FMDP0000284973OsACS5FOs01g67530
MdACS15FMDP0000915947OsACS6FOs01g67540
MdACS16FMDP0000206737OsACS7FOs07g17970
MdACS17FMDP0000671749OsACS8DOs07g44560
MdACS18FMDP0000278716OsACS9AOs04g24530
MdACS19FMDP0000303675Arabidopsis
MdACS20BMDP0000300934At4CL14CLsAt1g51680 7
MdACS21FMDP0000282038At4CL24CLsAt3g21240
MdACS22FMDP0000275090At4CL34CLsAt1g65060
MdACS23DMDP0000249003At4CL44CLsAt3g21230
MdACS24FMDP0000723168AtACS1DAt1g20480
MdACS25AMDP0000249364AtACS2DAt1g20490
Prunus mume AtACS3DAt1g20500
Pm4CL14CLsPm013887 4AtACS4DAt1g20510
Pm4CL24CLsPm019600AtACS5AAt1g62940
Pm4CL34CLsPm008736AtACS6CAt4g05160
PmACS1CPm021042AtACS7BAt4g19010
PmACS2CPm026124AtACS8FAt5g38120
PmACS3BPm026358AtACS9DAt5g63380
PmACS4EPm017552
1 Clades are defined in Figure 1; 2 Pyrus bretschneideri gene models are found in the GigaDB Genome database [28]; 3 Apple gene models are found in the Phytozome database [30]; 4 Prunus mume and 5 Prunus persica gene models are found in the Rosaceae Genome Database [47]; 6 Rice gene models are found in the JGI Genome database [35]. The 4CL gene models are from Hamberger et al. [48]; 7 Arabidopsis gene models are found in the Institute for Genome Research, TAIR database [49]. The 4CL gene models are from Souza et al. [35].

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Cao, Y.; Han, Y.; Li, D.; Lin, Y.; Cai, Y. Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear. Genes 2016, 7, 89. https://0-doi-org.brum.beds.ac.uk/10.3390/genes7100089

AMA Style

Cao Y, Han Y, Li D, Lin Y, Cai Y. Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear. Genes. 2016; 7(10):89. https://0-doi-org.brum.beds.ac.uk/10.3390/genes7100089

Chicago/Turabian Style

Cao, Yunpeng, Yahui Han, Dahui Li, Yi Lin, and Yongping Cai. 2016. "Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear" Genes 7, no. 10: 89. https://0-doi-org.brum.beds.ac.uk/10.3390/genes7100089

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