Introduction

Transcription factors (TFs) contain distinct types of DNA-binding domains and transcriptional regulation regions, and are capable of activating or repressing the transcription rates of multiple target genes (Riechmann et al. 2000; Wray et al. 2003). TFs are important regulators of diverse cellular processes and the complexity of living organisms necessitates a large number of TFs. Teosinte branched1/cycloidea/ proliferating cell factor1 (TCP) proteins constitute one of the largest families of plant-specific TFs (Martin-Trillo and Cubas 2010). The TCP gene family was named after the TCP domain from the first identified members: TB1 (teosinte branched1 from maize), CYC (cycloidea from Antirrhinum) and PCFs (PCF proteins from rice) (Doebley et al. 1997; Kosugi and Ohashi 1997; Cubas et al. 1999; Luo et al. 1999). Sequence comparison of TCP proteins identified two conserved regions, including a TCP domain with a noncanonical basic helix-loop-helix (bHLH) structure and an R domain (Doebley et al. 1997; Kosugi and Ohashi 1997; Cubas et al. 1999; Luo et al. 1999). Although the bHLH domain is present in all TCP proteins, only some of the TCP genes had the R domain (Brameier 2010).

Recently, many TCPs were found to have important roles in regulating various developmental processes, such as branching, floral organ morphogenesis and leaf growth (Aguilar-Martinez et al. 2007; Nag et al. 2009; Martin-Trillo and Cubas 2010; Danisman et al. 2012). OsTB1, the rice homologue of the maize TB1 gene, was considered as a negative regulator of lateral branching (Takeda et al. 2003). Functional analysis of the TCP genes BRC1 and BRC2 in Arabidopsis demonstrated that these genes were involved in suppressing axillary bud outgrowth (Aguilar-Martinez et al. 2007). TCP2, TCP3, TCP4, TCP10 and TCP24 in Arabidopsis were all targeted by miR319, and have been implicated in regulating leaf morphogenesis (Palatnik et al. 2003). A group of AS2-binding TCP TFs (TCP3, TCP4, TCP10 and TCP24) were identified as regulators in leaf development by binding directly to the promoters of BP and KNAT2 to repress their expression (Li et al. 2012a). Reporter gene analysis and the use of SRDX fusions suggested that AtTCP14 and AtTCP15 modulate cell proliferation in the developing leaf blade and specific floral tissues (Hiratsu et al. 2003; Kieffer et al. 2011). In addition, AtTCP15 played an important role in regulating endoreduplication during Arabidopsis development (Li et al. 2012b). In Arabidopsis, TCP16 functioned in pollen development, including variations in size, shape and staining patterns, suggesting that TCP16 was important for early pollen development (Takeda et al. 2006). AtTCP20, acting upstream of AtTCP9, served as a pivotal link between the regulation of growth and cell division pathways, and controlled leaf development via the jasmonate signalling pathway (Li et al. 2005; Aguilar-Martinez et al. 2007; Nag et al. 2009; Martin-Trillo and Cubas 2010; Danisman et al. 2012). LjCYC2, a CYC homologue, was shown to function in establishing dorsal identity together with Keeled wings in Lotus 1 (Kew1), which regulated the control of lateral petal identity, suggesting a common molecular origin for the mechanisms controlling floral zygomorphy in Lotus japonicus (Feng et al. 2006). Two CYC-like TCP proteins were characterized in the genetic control of floral zygomorphy in Pisum sativum L. (Wang et al. 2008).

Evolution of the TCP gene family in the Asteridae suggested that gene duplication, followed by functional divergence, might be the mechanism responsible for regulatory gene family diversification and its impact on morphological evolution in the Lamiales (Reeves and Olmstead 2003). To date, various members of the TCP family from Arabidopsis (23 members) and rice (22 members) have been identified, which can be divided into three classes by phylogenetic analysis. Expression pattern analyses of the AtTCPs and OsTCPs in stems, leaves and flowers showed that approximately half of the genes (22 TCP genes) were expressed in all the three tissues tested, suggesting that TCPs may play regulatory roles at multiple developmental stages in Arabidopsis and rice (Brameier 2010) In contrast to the intensive research on TCP in model and crop plants such as Arabidopsis and rice, there are very limited reports on apple. Recently, the draft genome sequence of apple has been decoded, which provided an excellent opportunity for genomewide analyses of all the genes belonging to specific gene families (Velasco et al. 2010). The genomewide analysis of the RING finger, DREB, dehydrin and Hsf gene families have been reported in apple (Li et al. 2011; Giorno et al. 2012; Liang et al. 2012; Zhao et al. 2012). However, no genomewide information on the apple TCP gene family is currently available.

Given the importance of TCPs in diverse biological and physiological processes and their potential application for the development of stress-tolerant transgenic plants, a systematic analysis of the apple TCP gene family was carried out for the first time. The chromosomal location and gene structure of the putative TCP genes were analysed carefully. In addition, the TCPs were subjected to phylogenetic analyses with their Arabidopsis counterparts. These comparisons enabled the identification of gene orthologues and clusters of orthologous groups that can be subjected to further functional characterization. Further, we analysed the expression patterns using microarray and expressed sequence tag (EST) data. To our knowledge, this is the first genomewide analysis of the apple TCP family, which provides valuable information for understanding the classification and putative functions of TCPs. Ultimately, these findings will lead to potential applications for the improvement of apples via genetic engineering.

Materials and methods

Identification of TCPs in apple

To identify members of the TCP gene family, multiple database searches were performed. The Arabidopsis rice and Populus TCP sequences were used as queries to perform repetitive blast searches against the GDR database (Jung et al. 2014). Additionally, all proteins sequences were then used as queries to perform multiple database searches against proteome and genome files downloaded from GDR database. Stand-alone versions of BLASTP and TBLASTN (basic local alignment search tool, http://blast.ncbi.nlm.nih.gov) available from NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) were used with the e-value cutoff set to 1e-003 (Mount 2007). Moreover, the predicted TCP gene family sequences were downloaded from the Apple GFDB database (Apple gene function and gene family database, http://www.applegene.org/) (Yao et al. 2011). All protein sequences derived from the candidate TCP genes were examined using the domain analysis programmes, Pfam (protein family, http://pfam.sanger.ac.uk/) and SMART (simple modular architecture research tool, http://smart.embl-heidelberg.de/) with the default cutoff parameters (Letunic et al. 2012; Finn et al. 2014) The pIs (isoelectric points) and molecular weights of the TCP TFs were obtained with the help of proteomics and sequence analysis tools on the ExPASy proteomics server (http://expasy.org/).

Sequence alignment and phylogenetic analysis of TCP TFs in apple

TCP sequences were aligned using the program ClustalX with BLOSUM30 as the protein weight matrix. The MUSCLE (multiple sequence comparison by log-expectation) program (ver. 3.52) was also used to perform multiple sequence alignments to confirm the ClustalX data output (http://www.clustal.org/) (Edgar 2004). Phylogenetic trees based on the protein sequences of the MdTCPs were constructed using the NJ (neighbour-joining) method of the program MEGA5.0 (molecular evolutionary genetics analysis) with p distance and using the complete deletion option parameters engaged (Tamura et al. 2011). The reliability of the trees obtained was tested using bootstrapping with 1000 replicates. Images of the phylogenetic trees were also drawn using MEGA5.0.

Chromosomal location and gene structure of MdTCPs

The chromosomal locations and gene structures were retrieved from the apple genome data that were downloaded from the GDR database. The remaining genes were selected using a Perl-based program and mapped to the chromosomes with MapDraw (Liu and Meng 2003), further the gene structures of the MdTCPs were generated with the GSDS (gene structure display server, http://gsds.cbi.pku.edu.cn/).

Expression analysis of TCP TFs in microarray

The microarray data of gene expression in apple fruits during fruit ripening process was downloaded from the gene expression omnibus database using the GSE series accession number GSE24523. The sequences of the identified MdTCP-containing genes were used as queries to blast against probe sequence (GPL11164) to find corresponding unigene IDs used in microarray data. The microarray data during rootstock–scion interactions process (GSE4762) was also downloaded from the gene expression omnibus database. And MdTCP-containing genes were used as queries to blast against probe platform (GPL3715) to find corresponding unigene IDs used in microarray data. Phylogenetic analysis was performed to determine the corresponding unigene IDs when sequences of high similarity were acquired. The microarray data were made into a database by Perl-based programs and then clustered using Cluster3.0 with Euclidean distances and the hierarchical cluster method of complete linkage clustering. The clustering tree was constructed and viewed in Java Treeview.

RNA extraction and cDNA synthesis

The young leaves and other tissues of the M. hupehensis (an excellent apple rootstock widely used for grafting in China) were used to determine the expression patterns of the apple TCP genes. The M. hupehensis trees were 12 years of age and planted in the experimental orchard of Shandong Institute of Fruit Tree Science (Taian, China).

The total RNA was extracted using the PureLinkTM RNA mini kit (Invitrogen, Carlsbad, USA) and treated with RNase-free DNase I. Two micrograms of the total RNA was used to synthesize the first-strand cDNA using the PrimeScript First Strand cDNA synthesis kit (Takara, Dalian, China).

Quantitative real-time PCR analysis

The qRT-PCR reaction was performed in 25 μL volumes containing 10 μM of each primer (table 1), 50 ng of cDNA and 12.5 μL of SYBR Premix Ex Taq II. The PCR amplification conditions included an initial heat denaturing step at 95 ∘C for 3 min and then 40 cycles of 95 ∘C for 20 s, 56 ∘C for 20 s and 72 ∘C for 20 s. The fluorescence was measured at the end of each cycle. A melting-curve analysis was performed by heating the PCR product from 55 to 95 ∘C. The expression data for the apple TCP genes were presented as relative units after their normalization to the apple actin gene using the 2−ΔΔCT method. The qRT-PCR experiment was carried out at least three times under identical conditions using actin as an internal control. Details of primers are listed in table 1.

Table 1 Primers used for the qRT-PCR analysis.
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Results

Identification of TCP TFs in apple

To identify TCP genes from the apple genome, BLASTP searches of the entire apple genome database (GDR, Genome database for Rosaceae, http://www.rosaceae.org) using well-studied plant (Arabidopsis, rice and Populus) TCPs as queries were first performed. The hidden Markov model (HMM) of the simple modular architechture research tool (SMART) and Pfam tool were then exploited as query to confirm the putative TCP genes. Finally, 52 typical TCP genes containing full open reading frame (ORF) were identified. Later, these genes were analysed manually using InterProScan (Jones et al. 2014) and ClustalX program to confirm the presence of a TCP domain. We provisionally named them MdTCP1 through MdTCP52, based on their chromosomal locations (table 2). Table 2 also shows the gene identifier, genomic position, pI, number of amino acids (aa), protein size, exon numbers and homologous genes. The ORF lengths ranged from 348 (MdTCP50) to 1839 bp (MdTCP27), with an average of 1049 bp. The corresponding proteins contained aa from 115 to 612 (average 348 aa), with a predicted molecular mass range of 12,816–69,007 Da; the pIs ranged from 5.41 (MdTCP32) to 10.65 (MdTCP3).

Table 2 TCP transcription factors in apple.
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Phylogenetic relationships and gene structure analysis of TCP gene family in apple

To evaluate the evolutionary relationship among the MdTCP proteins, the full-length aa sequences of 52 MdTCPs and 24 TCPs from Arabidopsis were subjected to a multiple sequence alignment using the MEGA5.0 program. The multiple sequence alignment file was then used to construct an unrooted phylogenetic tree. As shown in figure 1, the MdTCPs were divided into three classes (class 1, 2 and 3) as monophyletic clades with at least 50% bootstrap support, containing 22, 4 and 26 members, respectively. The small number of proteins in class 2 was consistent with the classifications of Arabidopsis and rice (table 3). In addition, 12 sister pairs of paralogous MdTCP genes were identified (figure 2), with strong bootstrap support (> 90%). The gene structures of the MdTCPs were analysed using the gene structure display server 2.0 (GSDS). As shown in figure 2, most MdTCP genes (32 members, 61.5%) have no introns. Nine genes have one intron (17.3%), seven have two introns (13.5%), two have three introns (3.8%) and two have five introns (3.8%). The sequence alignment analysis of these TCP proteins showed that the specific TCP domain with bHLH was highly conserved and the R domain was not present in apple TCP proteins (figure 3). We also compared the TCP gene structures between Arabidopsis and apple (figure 4). In Arabidopsis, the exon numbers ranged from one to four, 82% genes contained only one exon and the average number is 1.30. In MdTCPs, the exon numbers ranged from one to six, 63% genes contained only one exon and the average number is 1.73. These results suggested that the TCP genes did not exhibit gene structure diversification independently in different organisms.

Figure 1
figure 1

Phylogenetic relationships of Arabidopsis and apple TCP genes. The phylogenetic tree was constructed based on a complete protein sequence alignment of TCPs in Arabidopsis and apple by the (NJ) method with bootstrapping analysis (1000 replicates). The classes are marked by blue fragments. Scale bar represents 0.1 aa substitution per site.

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Figure 2
figure 2

Phylogenetic relationships and exon/intron structure of TCP genes in apple. The phylogenetic tree was constructed based on a complete protein sequence alignment of TCPs by the (NJ) method with bootstrapping analysis (1000 replicates). Sister paralogous gene pairs are indicated by red line. The exons and introns are represented by the green boxes and black lines, respectively, and the number of the exons and introns are marked behind the gene structure. The sizes of exons, introns and untranslated regions are drawn to scale as indicated at the bottom of the figure.

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Figure 3
figure 3

Sequence alignment of the conserved motif of TCP proteins in apple. Conserved aa are shown in colour. Dots denote gaps.

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Figure 4
figure 4

TCP gene structures in Arabidopsis.

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Table 3 Number of TCPs in Arabidopsis, rice and apple.
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Chromosomal location of TCPs on apple genomes

Chromosomal location analyses showed that 49 MdTCP genes were located on 16 chromosomes, dispersed throughout the genome (figure 5). No TCP gene was found on chromosome 3, while chromosome 5 had the most TCP members (seven genes). Interestingly, among the 12 sister pairs of paralogous genes, two pairs MdTCP19/20 and MdTCP3/4 were tightly colocated in the apple genome and seven sister pairs (MdTCP24/48, MdTCP15/39, MdTCP25/49, MdTCP16/40, MdTCP33/45, MdTCP22/47 and MdTCP34/46) were linked to at least one of the 15 potential chromosomal/segmental duplications combined with genomewide duplication on different chromosomes. Therefore, it was suggested that segmental duplication and transposition events both played roles in the evolution of TCP gene family in apple.

Figure 5
figure 5

Chromosomal mapping analysis of the TCP gene family in apple. The chromosome number is indicated at the top of each chromosome representation. Scale measures a 10 Mb chromosomal distance. The gene names on the right side of each chromosome correspond to the approximate locations of each TCP gene. Segmented duplicate homologous blocks are indicated with a blue shadow.

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Microarray analysis of the expression patterns of TCPs

Gene expression pattern analysis can provide important information for understanding the roles of genes. The microarray data of GEO (GSE24523) showed the transcriptional variation of apple genes from four weeks before ripening (WBR) to ripening. By BLAST searching against the apple unigene database from GDR, 28 MdTCPs were identified (figure 6). Based on hierarchical clustering, the expression patterns of these 28 MdTCP genes were divided into two groups, namely, groups I and II (figure 6). The expression of group II genes, such as MdTCP11, MdTCP13, MdTCP14, MdTCP25 and MdTCP49, increased slightly from four WBR to ripening, whereas MdTCP16, MdTCP22, MdTCP32 and MdTCP47, showed slightly decreased expression among the apple cultivar, Honey Crisp (HC). In addition, the expression of MdTCP16 (group II) decreased slightly from four WBR to ripening, whereas genes in group I, such as MdTCP1, MdTCP8 and MdTCP40, increased in the apple cultivar, Cripps Pink (CP) (figure 6). However, the expression of other genes did not show significant changes during the ripening process.

Figure 6
figure 6

Expression patterns of the MdTCPs from microarray during fruit development in apple. The colour scale representing the relative signal values is shown above (green, low expression; black, medium expression; red, high expression). HC, Honey Crisp; CP, Cripps Pink; week-4, four weeks before ripening; week-2, two weeks before ripening; week-0, ripening. Twenty-four samples are shown as GSM618107–GSM618130 (GEO accession numbers).

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Additionally, using BLAST searches against the probe sequences (GPL3715), the expression patterns of 51 MdTCPs (except MdTCP29) during the rootstock–scion interaction process were identified. Based on hierarchical clustering, the expression patterns of the MdTCP genes were divided into three groups, namely, groups I, group II and group III, with 22, 15 and 14 TCP genes, respectively (figure 7). The expression of group I decreased slightly in Ambrosia/ B9, Gala/B9 and Melrose/B9, but increased in the other seven rootstock–scion types. The transcripts of group II increased slightly in Ambrosia/B9 and Gala/B9, while most decreased in other rootstock–scion types. Intriguingly, the expression of group III showed significant differences during the rootstock–scion interaction process. Taken together, the different expression patterns of MdTCP genes indicated that they might be directly or indirectly involved in the fruit development and rootstock–scion interaction processes.

Figure 7
figure 7

Expression profile of the MdTCPs during rootstock–scion interactions. The colour scale represents the relative signal value from weak (green) to strong (red). Ten types of rootstock–scion interactions in altogether 27 samples were analysed.

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Expression profiles of MdTCP genes

Based on the sequence similarity comparisons and phylogenetic analyses, we randomly selected 12 apple TCP genes for further study. Among them, three, four and five genes were selected from classes 1, 2 and 3, respectively. RT-PCR was used to detect their expression patterns in different tissues and in response to abiotic stresses. As shown in figure 8, 10 genes were differentially expressed in all of the examined tissues. However, tissue-specific expression was also observed. For example, the expression of MdTCP8 was too low to be detected in fruits, while MdTCP45 could be only detected in the stem, leaf and fruit (figure 8). When treated with stressed conditions, the expression levels of most genes (except MdTCP3, MdTCP25 and MdTCP49) were apparently modulated (figure 9). Among them, seven genes (MdTCP4, MdTCP8, MdTCP9, MdTCP10, MdTCP16, MdTCP31 and MdTCP40) were suppressed by heat (37 ∘C), cold (4 ∘C), high salinity (150 mM NaCl) and osmotic stress (10% PEG). Intriguingly, MdTCP33 and MdTCP45 were significantly induced by heat, salt and osmotic stress, respectively (figure 9). Overall, the expression profile analysis indicated that MdTCP genes were widely distributed and might play multiple roles in apple development and abiotic stress tolerance.

Figure 8
figure 8

Expression profiles of the selected MdTCP genes in different apple tissues The data were normalized to the expression level of apple Actin. The mean expression value was calculated from three independent replicates. The vertical bars indicate the standard deviation (SD).

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Figure 9
figure 9

Expression analysis of MdTCP genes to different abiotic stresses. One-month-old seedlings were treated with 37 ∘C, 4 ∘C, 150 mM NaCl or 10% PEG6000 for 6 h, respectively. After treatment, the aerial part was collected for RNA extraction and real time PCR analysis. The data were normalized to the expression level of apple Actin. The mean expression value was calculated from three independent replicates. The vertical bars indicate the standard deviation (SD).

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Discussion

TCP TFs play important roles in diverse processes, especially in developmental programmes (Cubas et al. 1999; Li et al. 2005; Takeda et al. 2006; Aguilar-Martinez et al. 2007; Herve et al. 2009; Nag et al. 2009; Giraud et al. 2010; Martin-Trillo and Cubas 2010; Danisman et al. 2012). To date, only a limited number of TCP TFs have been identified and functionally characterized even in model plants such as Arabidopsis and rice, and no dataset of apple TCP TFs are available. In this study, we identified 52 TCPs containing full ORFs in apple using genomewide analysis. However, the missing ones may reside in the apple genome due to the screening methods that we utilized. Compared with the TCPs from Arabidopsis and rice, the number in apple is much larger, indicating that the TCP gene family in apple has expanded (Brameier 2010). We speculate that the presence of more TCP genes in apple genome may reflect the greater need for the involvement of these genes in the complicated transcriptional regulations in the woody perennial species.

We constructed a phylogenetic tree based on the full length of the TCP proteins and three classes were clustered, which was consistent with the previous studies (Brameier 2010). MdTCP8–MdTCP11, MdTCP25 and MdTCP49 belonged to class 3 and shared the highest homology with the miR319-targetd TCP genes and AS2-binding genes, which implied that these MdTCP genes may be involved in leaf development through similar mechanisms (Palatnik et al. 2003; Li et al. 2012a). Likewise, MdTCP13, MdTCP14, MdTCP34, MdTCP38 and MdTCP46 in class 1, which resembled TCP14 and TCP15 in Arabidopsis, might be involved in regulating cell proliferation in the developing leaf blade and specific floral tissues (Hiratsu et al. 2003; Kieffer et al. 2011). MdTCP22, MdTCP35 and MdTCP47 in class 1 might act as key regulators with similar function to AtTCP20 in flower development (Li et al. 2005; Kieffer et al. 2011). As shown in figure 2, most TCP genes from the same subfamily shared relatively similar exon/intron structures in terms of intron number and exon length between Arabidopsis and apple, which provided an excellent reference to explore the functions of the MdTCPs. The structural analyses of apple TCP TFs will mirror the diverse functions of TCP genes and encourage future functional research. However, the detailed biological functions of most TCP genes require further investigation.

Gene duplications, including segmental and tandem duplications, play important roles not only in genomic rearrangement and expansion but also in the diversification of gene functions, implicating them as the primary driving forces throughout the evolutionary process of genomes (Moore and Purugganan 2003; Cannon et al. 2004). It has been reported that a recent genomewide duplication event in apple occurred 60–65 million years ago, which resulted in gene expansion (Velasco et al. 2010). In this study, chromosomal location analysis showed that the apple TCP genes were dispersed throughout 16 out of 17 chromosomes with different densities, and mutiple sister pairs were linked to chromosomal segmental duplications. Consistent with this there was a clear paralogous pattern of gene family divergence by gene duplication in apple. Evolutionary divergence analysis suggested that the whole genome duplication, chromosomal segment duplications and transposition events, might have contributed to the expansion of apple TCP TFs.

Microarray and EST data revealed the expression patterns of 28 MdTCP genes during fruit development, and 51 MdTCPs during rootstock–scion interaction process. For example, MdTCP11, MdTCP13, MdTCP14, MdTCP25, MdTCP49, MdTCP22 and MdTCP47, increased or decreased slightly from four WBR to ripening among the apple cultivars HC and CP, respectively (figure 6). This result implied that the MdTCP genes might be involved in the apple ripening process, especially in leaf and flower development, which correlated with the phylogenetic relationship analysis. Additionally, most MdTCPs showed significant differences during rootstock–scion interaction process. According to the microarray and EST analyses, MdTCP genes might be intimately involved in fruit development and the rootstock–scion interaction processes. Moreover, nine out of 12 randomly selected MdTCP genes exhibit obvious responsiveness to different kinds of abiotic stresses, implying that MdTCPs are also involved in abiotic stress signalling or tolerance, which is worth of further investigation.

This study presented a systematic analysis of TCP gene family in apple, with special emphasis on fruit development and rootstock–scion interaction process. Our results lay the foundation for functional characterization of MdTCP genes and will lead to further understanding of the structure-function relationship between these family members. Additionally, our study provided comprehensive information and novel insights into the evolution and divergence of the TCP genes in plants. Potentially, this study will aid in the understanding of the molecular basis of many agronomically important traits of apple, such as fruit development, stress tolerance and other physiological processes.

Conclusion

In this study, 52 TCP genes were identified in the apple genome. The MdTCPs were divided into three classes and 49 MdTCP genes were distributed across 16 chromosomes, except Chr03, with different densities. Expression analysis showed that the MdTCPs were altered during the ripening process and rootstock–scion interaction process. Expression profile analyses of MdTCPs were performed in different tissues and in response to different stress conditions. All of the selected genes were expressed in at least one of the tissues tested and most of them were responsive to abiotic stress treatments, indicating that the MdTCPs were involved in various developmental and physiological processes in apples. To the best of our knowledge, this is the first report of a genomewide analysis of the apple TCP gene family. This study provides valuable information for understanding the classification and functions of the TCP genes in apples.