Genome-wide identification and expression profile analysis of CCH gene family in Populus

Identification of CCH family genes in P. trichocarpa

To identify CCH proteins in P. trichocarpa, the protein database of Populus was downloaded from https://phytozome.jgi.doe.gov/pz/portal.html (Goodstein et al., 2012), and Hidden Markov Model (HMM) profile file (HMA.hmm) of the Heavy-metal-associated domain (HMA) (PF00403) was downloaded from the Pfam database (http://pfam.xfam.org/). The HMA.hmm file was exploited as a query to identify PtCCHs in the poplar protein database using the hmmer search command of the HMMER (v 3.0) software. On the other hand, A. thaliana AtCCH (AT3G56240) was used as the query sequence in a BlastP search against the P. trichocarpa genome database online. The relative parameters were as follows: target type = Proteome, Program = BLASTP-protein query to protein db, Expect (E) threshold = −5; the other parameters were the default. The sequences obtained by the above two methods were used for the subsequent selection, and the screening criteria was whether the protein had one conserved MXCXXC (M is methionine, X is any amino acid, C is cysteine) site (Arnold et al., 2006; Kiefer et al., 2009; Guex, Peitsch & Schwede, 2009; Biasini et al., 2014) and formed one βαββαβ structure in the N-terminal (SWISS-MODEL (http://swissmodel.expasy.org/) was used to construct the secondary structure), as well as the extended segment in the C-terminal. As AtCCH is a small cytoplasmic protein and has no identified functional domains in its C-terminal, we deleted the proteins with long C-terminal sequences which might influence the function of the metal binding domain in the N-terminal when formed the tertiary structures, as well as the proteins had the domains in their C-terminal if the functions of the domains have been identified. After eliminating those did not meet these criteria, the 21 remainders were identified as PtCCHs and the amino acid sequences were analyzed for the characteristics using online ExPASy programs (http://www.expasy.org/), such as molecular weight, isoelectric point, number of amino acids, aliphatic index, and grand average of hydropathicity (GRAVY) score (Brunner, Busov & Strauss, 2004; Gasteiger et al., 2005).

Exon/intron structure and conserved motifs analysis

The distribution patterns of exons and introns in the PtCCH genes were predicted using the Gene Structure Display Server (GSDS2.0, http://gsds.cbi.pku.edu.cn) (Hu et al., 2015), and the online MEME tool (Multiple Em for Motif Elicitation, version 4.11.3, http://meme-suite.org/tools/meme) (Bailey et al., 2009) was used to identify conserved motifs in the PtCCHs. The setting parameters were as follows: maximum number of different motifs to find = 10, minimum width = 7, maximum width = 50.

Multiple sequence alignment and phylogenetic analysis

The PtCCH amino acid sequences were aligned using the Clustal X program (Thompson et al., 1997) and the conserved sites of MXCXXC were checked (Himelblau et al., 1998; Puig et al., 2007a). MEGA5.0 software (Tamura et al., 2011) was used to construct phylogenetic trees by the Neighbor-Joining (NJ) method (No of bootstrap replications were 1,000) with the full predicted CCH amino acid sequences of P. trichocarpa, A. thaliana, and O. sativa. AtCCHs and OsCCHs were identified using the same method and the same criteria as in P. trichocarpa, and the sequences of AtCCHs and OsCCHs were retrieved from their genome databases (Goodstein et al., 2012).

Mapping PtCCH genes on Populus chromosomes and subcellular locations of PtCCHs

The location information of PtCCH genes were retrieved from Phytozome and PopGenIE (http://popgenie.org/chromosome-diagram), and then the chromosomal location maps were constructed (Sjödin et al., 2009). The positions of whole-genome duplication blocks of different chromosomes were defined according to the study of Tuskan et al. (2006). The subcellular locations of PtCCH proteins were predicted using the online WoLF PSORT tool (http://www.genscript.com/psort/wolf_psort.html) (Horton et al., 2007), and the option of organism type was plant.

Identification of tissue-specific expression patterns of PtCCH genes

The tissue-specific expression data of PtCCH genes in mature leaves, young leaves, roots, nodes, and internodes were derived from PopGenIE (http://popgenie.org) (Sundell et al., 2015), and used to generate visual images.

Plant materials, growth conditions, and copper stress treatments

P. simonii × P. nigra were grown at Northeast Forestry University Forest Farm, Harbin, China. Cuttings of P. simonii × P. nigra were cultivated in pots containing sterile mixture of turfy soil, vermiculite, and perlite (3:2:1) in the greenhouse under long-day conditions (16 h light/8 h dark) at 25 °C, and supplied with water every 3 days. The seedlings were cut when they were 10 cm tall and then rooted in water that was aerated constantly by aquarium pumps. After rooting, the plantlets were transferred into sterile vermiculite and supplied every 2 days with half-strength modified Hoagland (Assunção et al., 2003) nutrient solution composition of: 3 mM KNO₃, 2 mM Ca(NO₃)₂ ⋅ 4H₂O, 1 mM NH₄ H₂PO₄, 0.5 mM MgSO₄ ⋅ 7H₂O, 25 µM KCl, 12.5 µM H₃BO₃, 1 µM MnSO₄ ⋅ H₂O, 1 µM ZnSO₄ ⋅ H₂O, 0.247 µM Na₂MoO₄, 10 µM NaFeDTPA (Fe), and 0.5 µM CuSO₄ ⋅ 5H₂O; the pH was fixed at 5 with KOH. After 2 weeks, seedlings were treated as follows: To test the expression profiles response to copper stress, seedlings were transferred into half-strength modified Hoagland nutrient solution that contained 0, 0.5 (control), or 10 µM Cu applied as CuSO₄ ⋅ 5H ₂O. Plants were treated at different times in the day, and were maintained in these conditions for 3, 12, or 72 h, then roots, stems, young leaves (LPI 0–2), and mature leaves (LPI 7–9) (Larson & Isebrands, 1971) were harvested at the same time, 10 o’clock. To determine the tissue-specific expression patterns, the plant materials were collected directly without treatment at 10 o’clock as described above. The samples were frozen in liquid nitrogen and stored at −80 °C for further analysis. To obtain reproducible results, three biological replicates of each stress treatment were conducted, and each experiment was repeated three times with independent samples.

RNA extraction and qRT-PCR analysis

Total RNA was extracted from leaves, stems, and roots using pBIOZOL Plant Total RNA Extraction Reagent (BioFlux, Hangzhou, China) according to the protocol. After removing genomic DNA, first-strand of cDNA was obtained using a PrimeScript™ RT reagent Kit (Takara Bio, Dalian, China). qRT-PCR was performed using UltraSYBR Mixture (Low ROX) (CWBIO, Beijing, China) with three replicates on an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Amplifications were conducted in 20 µL reaction mixtures containing 10.0 µL 2 × UltraSYBR Mixture (with ROX), 4.0 ng cDNA template, and 0.2 µM of forward and reverse primers. The primers used for qRT-PCR were designed with Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA) according to PtCCH gene sequences (Table S1), and the annealing temperature was between 46 °C and 60 °C. The PtUBQ7 (XM_002306689.2) and PtCDC2 (XM_002305968.2) genes of P. trichocarpa were used as reference genes (Pettengill, Parmentier-Line & Coleman, 2012; Pettengill, Pettengill & Coleman, 2013), and the expression levels of them were stable, so we selected PtUBQ7 (XM_002306689.2) gene as reference gene to analyze the expression relative changes of PtCCH genes. The amplification conditions were as follows: denaturation at 95 °C for 10 min, 45 cycles of denaturation at 95 °C for 15 s, annealing between 60 °C and 68 °C for 60 s.

The 2^−ΔΔCT method was used to analyze the expression levels of PnCCHs in different tissues under control copper condition (0.5 µM CuSO₄) or copper stress conditions (0 µM CuSO₄ and 10 µM CuSO₄) (Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). The statistical significant differences of the tissue-specific expression profiles of PnCCHs were analyzed using duncan’s test (P < 0.05). The values of log₂ (sample/control) copper stress conditions and different treated times were calculated as the relative expression levels of every PnCCH genes, and the different color of the cells in the heatmaps indicated the expression level of the treated samples went up or down compared with their controls. The statistical significant differences of the expression levels were analyzed using t test (P < 0.05). All the values under copper stress conditions were compared with the corresponding values under control copper condition. Results are the mean ± SD of three replicates.

Identification and sequence conservation of CCH genes in P. trichocarpa

A total of 21 PtCCH genes (named PtCCH1–21) were identified in the P. trichocarpa genome according to their encoded proteins that contained the conserved domains and typical secondary structures like the AtCCH of A. thaliana. The 21 PtCCH genes were analyzed and the parameters, including locus name, chromosome location, protein length, molecular weight, isoelectric point, aliphatic index, and grand average of hydropathicity (GRAVY) score, are listed in Table 1. The putative PtCCH protein sequences varied from 115 to 179 amino acids (aa) in length, the isoelectric points ranged from 4.96 to 9.64, and all of them were hydrophilic because of the GRAVY values were negative. The 21 PtCCH amino acid sequences shared 23–100% identities (Table S2), and the proteins were predicted to be localized in cytosol, chloroplast, or mitochondria.

The multiple sequence alignment was conducted to examine the conserved domains of the PtCCHs, and the result showed that all the PtCCH proteins shared the consensus metal-binding motif MXCXXC in the N-terminal, and some PtCCHs had a lysine-rich (Lys, K) region (Fig. 1) (Lin & Culotta, 1995; Agrawal et al., 2002; Lee et al., 2005). These two structure characteristics are involved in the function of copper chaperones (Rosenzweig et al., 1999; Mira et al., 2001b). The secondary structure prediction indicated that all the PtCCHs possessed, at their N-terminal, the conserved βαββαβ-fold structure that is present in the ATX1-like family of metallochaperones (Fig. S1).

Chromosomal location and duplication of PtCCHs in P. trichocarpa

To study the gene distribution and duplication in the P. trichocarpa genome, we mapped all of the identified PtCCH genes to their corresponding chromosomes. The 21 PtCCH genes were found to be physically located on 10 of the 19 Populus chromosomes (none on chromosomes III, VIII, IX, XII, XIII, XIV, XV, XVI, and XVIII) (Fig. 2). The distribution of the PtCCH genes on each chromosome was obviously heterogeneous: ChrV harbored the highest number (5) of PtCCH genes; ChrI harbored two PtCCH genes (PtCCH1 and PtCCH2); ChrVI, ChrX, and ChrXI harbored three PtCCH genes each; while only a single PtCCH gene was found on ChrII, ChrIV, ChrVII, ChrXVII, and ChrXIX.

According to the study of Tuskan et al. (2006), a whole-genome duplication (WGD) event occurred 60–65 million years ago in the Salicaceae family (salicoid duplication), and this created the identified paralogous segments in the Populus genome. Except for six genes (PtCCH1, 2, 14, 19, 20, 21), fifteen PtCCH genes were located in duplicate blocks (Fig. 2). Five pairs of genes (PtCCH1/2, PtCCH8/9, PtCH10/11, PtCCH15/16, and PtCCH17/18) were arranged in tandem repeats on ChrI, ChrV, ChrVI, ChrX, and ChrXI, respectively.

Phylogenetic analysis of the PtCCH gene family

We constructed a phylogenetic tree to analyze the evolutionary relationships of the PtCCH genes and to classify the PtCCHs in P. trichocarpa. Twenty-one full-length PtCCH sequences were used to generate an unrooted phylogenetic tree (Fig. 3A). The 21 sequences clustered into three distinct subgroups: SI, SII, and SIII. The SI subgroup contained PtCCH3, 5, 6, 7, 8, 9, 13, 15, 16, and 20; the SII subgroup contained PtCCH1, 4, 12, 17, 18, 19, and 21; and the SIII subgroup contained the remaining four PtCCHs.

To analyze the relationships of the PtCCHs to CCHs in other species, we constructed a phylogenetic tree using full-length amino acid sequences of the 21 P. trichocarpa CCHs, 13 A. thaliana CCHs (AtCCH6 was the AtCCH which has been functionally characterized and used as the query sequence to screen the CCH genes), and 12 Oryza sativa CCHs (Table S3). All these CCH sequences clustered into three groups: SI, SII, and SIII. The SI and SII groups contained 22 and 16 members, respectively, and the SIII group had eight members (Fig. 4). The SI group had four subgroups, SI–1, SI–2, SI–3, and SI–4; the SII group had two subgroups, SII–1 and SII–2; and the SIII group had three subgroups, SIII–1, SIII–2, and SIII–3. Among the three species, CCHs that had the same and similar number of motifs clustered into the same clade. The phylogenetic analysis showed, noticeably, subgroup SI–3 contained only PtCCHs, and subgroup SIII–1 contained AtCCH and OsCCH but no PtCCH. Otherwise, the CCH proteins encoded by paralogous pairs of PtCCH genes clustered together in the same subgroups; for instance, PtCCH3 and PtCCH8/9 were assigned to SI-3, and PtCCH1 and PtCCH19 were assigned to SII–1. Also, the products of the tandem duplication pairs, PtCCH8/9 and PtCCH17/18, were assigned to SI–3 and SII–2, respectively.

Analyses of gene structure and conserved motif distribution

To gain further insight into the possible relationships among different gene family members, the exon/intron organization of the PtCCH genes was compared in the context of the phylogenetic tree (Fig. 3B). The relative lengths of the introns and exons were provided detailed, and revealed a highly-conserved distribution of intron regions (from one to two in numbers) among the PtCCH genes, which is similar to the distribution of intron regions among Arabidopsis CCH genes (one to two introns) and O. sativa CCH genes (null to three introns). Fifteen of the 21 PtCCH genes had only one intron, and the other six had two introns. Notably, closely related genes generally had similar exon/intron structures, although the lengths of the introns and exons varied. There were some exceptions when compared with the other members of the same subgroup; for example, PtCCH15, PtCCH16, and PtCCH20 in SI, PtCCH1 and PtCCH19 in SII, and PtCCH14 in SIII. These results imply a tight relationship between the phylogeny and gene structure of the PtCCHs, and the regularity of the gene structure might be related to the evolutionary trends and the conservation of the gene family members.

Ten motifs (named motif 1–10) were predicted in the PtCCH sequences using MEME software (Table S4). As shown in Fig. 3C, the PtCCH proteins in the same subgroup generally contained similar motifs. Motif 3, which was present in all the PtCCHs, contains the MXCXXC site which is essential for copper binding. Most members of the PtCCH family shared three motifs, motif 3, motif 1, and motif 2, which were linked in the same order. A few proteins, such as PtCCH2 and PtCCH11, showed quite different protein structures compared with the others. In SI, except PtCCH7 and PtCCH16 without motif 6, all the other PtCCHs showed the same motif distribution and arrangement. Motif 5 appeared only in the SI subgroup. In SII, except PtCCH12 without motif 7, the other motifs in PtCCH4, 12, 17, 18, and 21 were the same. Interestingly, motif 10 was selectively distributed among a specific pair, PtCCH1 and PtCCH19, which had the same motif types and arrangements. The PtCCHs in subgroup SIII had few motifs (one to three) compared with the PtCCHs in the SI and SII subgroups. Overall, the PtCCHs in specific subgroups appear to have evolved conserved and diverged motifs during evolution.

Tissue-specific expression profiles of CCH genes in the Populus genome

We used the tissue-specific expression data of PtCCH genes in mature leaves, young leaves, roots, nodes, and internodes from the Plant Genome Integrative Explorer (http://PlantGenIE.org) (Sundell et al., 2015) to generate visual images (Figs. 5A–5R). The expression data showed that PtCCH2, PtCCH10, PtCCH11, PtCCH12, PtCCH14, PtCCH15, and PtCCH16 were highly expressed in mature leaves; PtCCH3, PtCCH6, PtCCH8, PtCCH17, PtCCH18, and PtCCH20 were highly expressed in roots; and PtCCH1 and PtCCH19, which were clustered into the same clade, were highly expressed in roots/internodes/nodes. The expression levels of PtCCH4, PtCCH5, PtCCH7, PtCCH13 were higher in mature leaves/roots, young/mature leaves, young leaves, internodes, respectively, and PtCCH21 was highly expressed in roots, and young and mature leaves. Expression data for PtCCH9, which has the same nucleotide sequences as PtCCH8, was not available in http://PlantGenIE.org.

Tissue-specific expression patterns of PnCCHs in P. simonii × P. nigra roots, stems, and mature and young leaves were measured and analyzed by qRT-PCR. The primers were designed based on the nucleic acid sequences of the PtCCHs (Table S1). PtCCH8 and PtCCH9 have identical sequences and may be tandem repeats (Fig. 2). PtCCH15 and PtCCH16 are the different transcripts that were described in the gene information of P. trichocarpa genome database and their putative proteins have highly similar amino acid sequences, as well as PtCCH17 and PtCCH18; therefore, we did not design primers for PtCCH16 and PtCCH18. We could not design primers for PtCCH21 because its nucleotide sequences shared high identity with other genes in the Populus database (Tuskan et al., 2006). Beside this, we did not obtain the amplification results of PnCCH13 and PnCCH14. So, the primers for PtCCH9, 13, 14, 16, 18, and 21 genes are not included in Table S1. The results showed that PnCCH3, PnCCH4, PnCCH6, PnCCH7, PnCCH8, PnCCH17, and PnCCH20 were most highly expressed in roots, and PnCCH1, PnCCH5, and PnCCH15 were most highly expressed in mature leaves. PnCCH10 and PnCCH11, PnCCH12 and PnCCH19 had the highest expression levels in young leaves, roots/stems, respectively, while PnCCH2 was most highly expressed in young and mature leaves (Figs. 5A–5R). The expression patterns of the remaining PnCCHs could not be measured by qRT-PCR.

Expression patterns of PnCCH genes under copper stress

Previous studies showed that the function of AtCCH was copper-dependent, and that AtCCH mRNA was induced in the absence of copper and reduced with excess copper (Himelblau et al., 1998; Shin, Lo & Yeh, 2012). To investigate the functions of PnCCHs, the expression patterns of the PnCCH genes under copper stresses (deficiency and excessive) were analyzed by qRT-PCR. All the PnCCH genes exhibited expression variations in response to deficiency (0 µM CuSO₄) and/or excessive copper (10 µM CuSO₄). In different tissues, the expression levels of PnCCH genes relative to controls (0.5 µM CuSO₄) differed (Fig. 6, Table S5). In young leaves, the expression levels of PnCCH3 were higher than the controls under either deficiency or excessive copper conditions for 72 h, and the differences were significant. PnCCH7 and PnCCH8 had increased expression levels with significant differences under excessive copper for 72 h and 12 h, respectively. The expression levels of PnCCH2 and PnCCH12 under excessive copper, and PnCCH4, PnCCH6, PnCCH10, PnCCH15, and PnCCH17 under copper deficiency could not be induced (Figs. 6A–6B). In mature leaves, the expression levels of PnCCH3 and PnCCH7 increased significantly under copper deficiency for 12 h, and the expression levels of PnCCH6, PnCCH8, and PnCCH15 were higher than the controls significantly under the deficiency or excessive copper conditions for 72 h. The expression levels of PnCCH12 under copper deficiency and PnCCH4, PnCCH5, PnCCH11, PnCCH12, PnCCH17, and PnCCH19 under the copper excessive condition were not induced (Figs. 6C–6D).

In stems, the expression levels of the PnCCH genes increased under copper deficiency, except for PnCCH6, PnCCH11, and PnCCH20; notably, the expression level of PnCCH4 was distinctly higher than the control after treatment for 12 h. When treated with excess copper, PnCCH3, PnCCH7, and PnCCH8 had increased transcript levels significantly in stems compared with those of the controls after treatment for 72 h, while the expression levels of PnCCH2, PnCCH12, PnCCH19, and PnCCH20 were not be induced by excessive copper treatment. Unlike its lack of response under copper deficiency, the expression of PnCCH6 was higher than the controls significantly after excessive copper treated for 72 h (Figs. 6E–6F). In roots, the expression of most PnCCH genes was induced under the copper deficiency condition after treatment for different times, except for PnCCH19, which had lower expression levels relative to the controls significantly. The expression levels of PnCCH12, PnCCH15, PnCCH17, and PnCCH19 were significantly decreased compared with controls after copper deficiency treatment for 12 h, and the expression levels of PnCCH1, PnCCH4, PnCCH17 and PnCCH19 were decreased significantly after copper deficiency treatment for 72 h (Fig. 6G). Under excessive copper treatment, the expression levels of PnCCH1, PnCCH4, PnCCH6, PnCCH15, PnCCH17, PnCCH19, and PnCCH20 were lower than those of the controls significantly after treated for 72 h; and the expression levels of PnCCH4 and PnCCH17 increased distinctly after treatment for 3 h and 12 h (Fig. 6H).