Genome-wide identification and analysis of the Populus trichocarpa TIFY gene family

Plant Physiology and Biochemistry 115 (2017) 360e371

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Genome-wide identification and analysis of the Populus trichocarpa TIFY gene family Yue Wang a, 1, Feng Pan a, 1, Danmei Chen a, Wenyuan Chu a, Huanlong Liu a, Yan Xiang a, b, * a b

Laboratory of Modern Biotechnology, School of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei, 230036, China Key Laboratory of Biomass Improvement and Conversion, Anhui Agriculture University, Hefei, 230036, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 February 2017 Received in revised form 11 April 2017 Accepted 12 April 2017 Available online 13 April 2017

The plant-specific TIFY proteins are widely present in land plants and play the important roles in the regulation of plant stress-responses. In this study, we carried out a bioinformatics analysis of TIFY genes in Populus trichocarpa by determining the phylogenetic relationship, chromosomal location and gene structure and expression profiles analysis under stresses. The 24 TIFY genes were identified and classified into four subfamilies (ZML, JAZ, PPD and TIFY). The 24 TIFY genes were irregularly located on 13 of the 19 chromosomes; ten gene pairs were involved in large-scale interchromosomal segmental duplication events; we identified 17 collinear TIFY gene pairs in the Populus trichocarpa genome. Numerous abiotic stress cis-elements were widely found in the promoter regions. Analysis of the Ka/Ks ratios indicated that the paralogs of the PtTIFY family principally underwent purifying selection. Microarray data and qRT-PCR analysis revealed that 24 PtTIFY genes were differentially expressed in various tissues. Quantitative realtime RT-PCR analysis of TIFY genes expression in response to salt, JA hormones and low-temperature stress revealed their stress-responses profiles. The results of this study provided valuable information for further exploration of the TIFY gene family in Populus trichocarpa. © 2017 Published by Elsevier Masson SAS.

Keywords: Populus trichocarpa TIFY genes Phylogenetic analysis Stress-responses profiles

1. Introduction A previous report revealed that the tify domain was first identified in the Arabidopsis gene AT4G24470 and was annotated as a Zinc-finger (ZIM) domain. Vanholme et al. (2007) proposed to use ‘tify’ instead of ZIM to represent the domain, pointing to its most conserved amino acid pattern (TIF[F/Y]XG). The TIFY transcription factors are characterized by a highly conserved tify domain, Jas and CCT motifs. The tify domain contains ~28 amino acids and a core motif, TIF[F/Y]XG, in which 13 out of 28 amino acid sites are invariant. All genes encoding proteins containing a tify domain should group together in the TIFY family. According to secondary structure prediction analysis, the TIFY domain was predicted to form an alpha-alpha-beta fold (Bai et al., 2011). The TIFY family was

* Corresponding author. Laboratory of Modern Biotechnology, School of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei, 230036, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (F. Pan), [email protected] (D. Chen), [email protected] (W. Chu), 1641999267@qq. com (H. Liu), [email protected], [email protected] (Y. Xiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2017.04.015 0981-9428/© 2017 Published by Elsevier Masson SAS.

classified into four subfamilies (TIFY, JAZ, PPD and ZML) based on its contained domains. The JAZ proteins are characterized by the presence of a C-terminal Jas domain that interacts with the MYC2 proteins to repress the JA signaling pathway (Chini et al., 2009; Chung et al., 2009). ZML proteins contain a CCT motif, a GATA zinc-finger domain and a tify domain. The PPD family contains three domains: (1) a PPD domain at the N-terminus, (2) a modified Jas domain that the PY motif is missing, (3) a tify domain. Plants can experience various adversity stresses during their life-cycles. Several studies have demonstrated that the TIFY genes play the critical roles in plant stress-responses, and it has been shown that JAZ proteins function as inhibitors in the jasmonate signaling pathway. Multiple factors can rapidly activate the expression of JAZ proteins in the plant developmental stages, such as leaf and root growth (White, 2006), flower development and senescence. In rice, most OsTIFY genes were responsive to at least one type of abiotic stress, such as drought, salt stress and cold stress (Ye et al., 2009). The OsJAZ1 was not induced under N deficiency, however; most of the other OsJAZ genes were up-regulated at both seven days and 15 days (Singh et al., 2015). The VvJAZ genes in grapevine were induced by osmotic stress, low temperature,

Y. Wang et al. / Plant Physiology and Biochemistry 115 (2017) 360e371

drought, ABA treatment and salt stress (Zhang et al., 2012). The expression of ZmJAZ genes in maize was found to be abundantly responsive to certain types of abiotic stress (e.g. drought) (Zhang et al., 2015). Overexpression of the Glycine soja gene GsTIFY10 in Arabidopsis enhanced plant tolerance to bicarbonate stress during most developmental stages (Zhu et al., 2011). In apple, MdJAZ3 was up-regulated under high salinity, but not drought, while MdJAZ7 was down-regulated under drought stress, but was unaffected by high salinity (Li et al., 2015). In chickpea, CaJAZs showed upregulation of CaJAZ10 and CaJAZ1a and down-regulation for CaJAZ6 and CaJAZ8 in response to early N deficiency (Singh et al., 2015). The PPD1 protein in Arabidopsis has been demonstrated to coordinate tissue growth, modulate lamina size and limit the curvature of the leaf blade (Li et al., 2015). The CCT domain was first discovered in the transcription factor TOC1 and CONSTANS (CO) proteins as the mediating protein-protein interactions in the plant photoperiodic signaling. Populus trichocarpa, black cottonwood, was the first tree species to have its genome sequenced. Populus trichocarpa is not only an ecologically and economically important tree species, but also as a model plant for xyloid production, it is being intensively studied using transgenes, but the bioinformatics analysis of TIFY genes in the woody plant species Populus trichocarpa is lacking (Wang et al., 2015). Therefore, a study of Populus trichocarpa TIFY genes would be valuable to understand the important information of these genes. Here, we performed a bioinformatics analysis of the TIFY gene family in Populus trichocarpa. We constructed a phylogenetic tree of the members of this gene family, determined chromosomal location and gene duplication, identified conserved domains and motifs, assessed the influences of positive purifying selection and determined the transcriptional expression profiles of the Populus trichocarpa TIFY genes in response to adversity stresses (Wang et al., 2015). Our analysis provides valuable information that will enable further characterization of TIFY genes in Populus trichocarpa. 2. Materials and methods 2.1. Identification of TIFY family genes in Populus trichocarpa Previously identified JAZ, PPD and ZML genes of the Arabidopsis TIFY gene family were submitted to the NCBI (http://www.ncbi. nlm.nih.gov) and Pfam database (http://pfam.sanger.ac.uk) to acquire information on conserved protein domains in this family (Finn et al., 2010). To identify all TIFY proteins in Populus trichocarpa, BLASTP searches (E-value<1e
6) were carried out in the Phytozome database (http://www.phytozome.net) with the Arabidopsis, rice, grape and apple TIFY proteins as queries. Data for all the other plant genomes was also obtained from the Phytozome database based on previous reports. Information on Populus trichocarpa TIFY genes, including chromosomal location, sequence ID, genomic sequence, protein sequence, coding sequence (CDS), protein molecular mass(MW), protein isoelectric point (pI) and ORF length were acquired from the Phytozome database and ExPASy programs (http://web.expasy.org/protparam/). All obtained protein sequences were examined for the presence of a tify domain (PF06200) using the Pfam and NCBI. 2.2. Phylogenetic analysis of the TIFY gene family Phylogenetic reconstructions are central to the fields of evolution and comparative genomics. Methods for evaluating genetic distance use multiple sequence alignments as distance-matrix methods including neighbor-joining and UPGMA that are the most straightforward for implementation and do not invoke an evolutionary model (Atteson, 2006). A multiple sequence

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alignment of predicted Populus trichocarpa, Arabidopsis (Vanholme et al., 2007), grape (Zhang et al., 2012), rice (Ye et al., 2009) and apple (Li et al., 2015) TIFY protein sequences were imported into MEGA6.0 to generate phylogenetic trees with the Neighbor-Joining (NJ) method (Tamura et al., 2013). 2.3. Chromosomal location Based on chromosomal position information provided by the Phytozome database, a chromosomal location image for the PtTIFY genes was obtained using the MapInspect software (http://www. plantbreeding.wur.nl/uk/software_mapinspect.html). 2.4. Microsynteny analysis and gene duplication A syntenic block was defined as a region where three or more conserved homologs were located within 15 genes upstream and downstream between genomes (Wang et al., 2015). The syntenic blocks within the Populus trichocarpa genome were obtained from the Plant Genome Duplication Database (http://chibba.agtec.uga. edu/duplication), a web service providing synteny information by collinearity between chromosomes (Zhang et al., 2012). Microsynteny analysis was performed based on comparisons of the specific regions containing TIFY genes using the MicroSyn software (Wang et al., 2015). Paralogs can be obtained from segmental duplication. Segmental duplications (SDs) are long DNA sequences that have approximately identical sequences (90e100%) and are present in multiple locations as the results of duplication events (Behura and Severson, 2013). We used the online VISTA (http:// pipeline.lbl.gov/cgi-bin/gateway2) program to analyze the paralogous pairs (Wu et al., 2015b). 2.5. Exon-intron structure and conserved domain analysis An intron is any part of a gene that removed by RNA splicing during the RNA maturation, and an exon will be present in the mature mRNA (Raff et al., 2002). Exon-intron structures of Populus trichocarpa TIFY genes were predicted using the GSDS (http://gsds. cbi.pku.edu.cn/). In genetics, a sequence motif is a special, identifiable nucleotide or amino-acid sequence pattern. The motif structures were predicted using the online MEME (http://meme. nbcr.net/meme/cgi-bin/meme.cgi) (Bailey et al., 2009). 2.6. Promoter analysis The 2000-bp putative promoter regions of the Populus trichocarpa TIFY genes were downloaded from the Phytozome database. The PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/) (Postel et al., 2002) were adopted to identify putative ciseelements. 2.7. Calculation of Ka/Ks values The Ka/Ks ratio is a more powerful test of selection pressure than others available in population genetics acting on the proteincoding genes. Paralogous pairs with a Ka/Ks ratio >1 means activating evolution under advantageous selection, suggesting that at least some of the mutations must be profitable. If a Ka/Ks ratio ¼ 1 means that the mutations are neutral. A Ka/Ks < 1 means the mutations restriction with the disadvantageous or purifying selection. Methods for estimating the Ka and Ks substitution rates use an alignment of multiple nucleotide sequences of homologous genes that code for proteins. In our study, paralogous pairs were aligned using MEGA6.0. Synonymous substitution (Ks) and nonsynonymous substitution (Ka) rates were evaluated by Dnasp

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(Librado and Rozas, 2009). A sliding window analysis of Ka per nonsynonymous locus Ka/Ks ratios were generated with a window size of 150 bp and a step size of 9 bp (Wang et al., 2015; Lin et al., 2014).

PtPPD2 and PtJAZ1 to PtJAZ12, based on the domains and motifs that they contained. The length of PtTIFY proteins ranged from 149 amino acids (aa) to 454 aa. The 24 PtTIFY genes were distributed on 13 chromosomes linked to segmental duplication events. Details of the PtTIFY gene family are provided in Table 1.

2.8. Microarray analysis of expression profiles To obtain further insight into the expression levels of PtTIFY genes during natural selection, we performed a comprehensive analysis of the TIFY genes using publicly available microarray data (Wu et al., 2015b).The microarray data of Populus trichocarpa was obtained from the GEO database at the NCBI. The corresponding probes of the tify genes were identified using an online ProbeMatch tool available at the NetAffx Analysis Center (http://www. affymetrix.com/). The data for the TIFY genes identified by the TIFY-probe in GSE13990 were imported into the Cluster to construct heat-map. 2.9. Plant growth and treatments Clonally propagated six-week-old poplar seedlings that grown in Tissue Culture Lab to be used to analyze gene expression in all experiments. For the salt-stress, plants were watered with a NaCl solution at a concentration of 200 mM; the plants were subjected to cold treatments by placing them at 4  C. For the jasmonic acid (JA) treatments, the leaves were sprayed with 100 mM jasmonic acid (JA) solution. In all cases, untreated seedlings were used as the controls. All treatments had five-time points (1, 3, 6, 12 and 24 h) (Wang et al., 2015; Zhang et al., 2015), after which the samples were collected, immediately frozen in liquid nitrogen, then stored at
80  C prior to RNAs extraction. Three biological and technical replicates were performed for each sample. 2.10. RNAs extraction and qRT-PCR analysis All RNAs extraction and First-strand cDNAs synthesis were carried out as directed by the manufacturer. Gene-specific primers for TIFY genes in Populus trichocarpa were designed and examined for specificity using Primer Premier5.0 and the NCBI primerBlast tool. The Populus trichocarpa housekeeping ubiquitin gene (UBQ, gene ID: Potri.001G418500) was used as the reference standard because of its stable expression patterns (Hui et al., 2014). The relative expression levels evaluated by 2
DDCT.

3.2. Phylogenetic analysis of ZML, PPD and JAZ proteins from five plant species According to similarities and differences in their physical or genetic characteristics among various species, a phylogenetic tree could show the inferred evolutionary relationships using branching diagram. Unrooted trees illustrate the correlation of the leaf nodes without making assumptions about ancestry. We constructed a phylogenetic tree with the TIFY protein sequences. As shown in Fig. 1, all of the tify proteins were grouped into eight clades in the phylogenetic tree; the ZIM and ZML proteins were grouped into one clade; the PPD proteins also composed a unique clade. The proteins that contained a Jas domain were divided into six clades (I to VI); in particular, the JAZ proteins from Populus trichocarpa were distributed among all of the clades except for JAZ V. This result is well consistent with the reports of two previous studies of JAZ proteins from grape and apple, which indicated a more widespread phylogenetic relationship within these proteins, and showed that these species diverged mutually early during the processes of terrestrial plants evolution and had undergone significant mutations (Li et al., 2015). Although the phylogenetic relationships could not be explicated unambiguously due to the limited number of species represented, the analysis did generate some interesting results. For instance, the JAZ V clade consists only of JAZ proteins from Oryza sativa. The JAZ II clade contains proteins from rice, poplar, Arabidopsis and grape, but not apple. The remaining four JAZ clades (I, III, IV and VI) contain different numbers of proteins from five different plant species. The JAZ proteins from apple and grape make up a large proportion of the proteins in the JAZ VI clade, but JAZ VI contains only a single protein from Populus trichocarpa, PtJAZ9. All of these results indicated that genetic material divergence has occurred as the separation of the eudicots (Populus trichocarpa, apple, grape and Arabidopsis) and the monocots (rice) (Zhang et al., 2012). 3.3. Chromosomal location, microsynteny analysis and gene duplication

3. Results 3.1. Identification of TIFY family genes in Populus trichocarpa Twenty-four TIFY genes were identified in the Populus trichocarpa genome. Two protein sequences contained only a tify domain and were predicted to belong to the TIFY subfamily. Fourteen predicted protein sequences contained both a tify domain and a Jas motif; two Jas motif of these lacked the crucial PY-NLS sequence, which is characteristic of partial Jas domain in PPD proteins, they were grouped as members of the PPD subfamily, while the other 12 proteins were classified into the JAZ subfamily. Eight protein sequences that contained both a GATA zinc-finger, a tify domain and a CCT motif to belong to the ZML subfamily. Since the research principally concentrated on the investigation of TIFY family members that contain either CCT or Jas motifs, proteins predicted to belong to the TIFY subfamilies (PtTIFY1, 2) that contain only a tify domain were not analyzed further. Taken together, we identified two TIFY, eight ZML, two PPD and 12 JAZ genes in Populus trichocarpa. The TIFY genes were named sequentially from PtTIFY1 to PtTIFY2, PtZML1 to PtZML8, PtPPD1 to

Segmental and tandem duplications have been demonstrated to be two of the main causes of gene family expansion in plants (Cannon et al., 2004). According to the chromosomal location map (Fig. S1), 24 TIFY genes of Populus trichocarpa were distributed irregularly across 13 chromosomes, with no TIFY protein genes present on the other six chromosomes. We identified 17 collinear TIFY gene pairs in the Populus trichocarpa genome (Fig. S2), which might have resulted from ancient processes during the course of evolution. We did not identify any tandem duplication clusters, suggesting that tandem duplication may not be a major duplication pattern to the expansion of the TIFY gene family in Populus trichocarpa. However, ten paralogous gene pairs (Table 2) were identified that were related to large-scale interchromosomal segmental duplication events. As described in previous reports, whole genome duplication events within the species can account for most of the expansion of the TIFY family (Bai et al., 2011), and our results showed that the paralogous pairs arose from segmental duplication events. Given that PtJAZ-7/-8 and PtJAZ-10/-11 are highly similar to one another, we can conclude that these gene pairs had a common ancestor that underwent duplication and divergence.

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Table 1 Gene coordinates and characteristics of the 24 predicted TIFY proteins in the Populus trichocarpa genome. Name

PtTIFY1 PtTIFY2 PtPPD1 PtPPD2 PtZML1 PtZML2 PtZML3 PtZML4 PtZML5 PtZML6 PtZML7 PtZML8 PtJAZ1 PtJAZ2 PtJAZ3 PtJAZ4 PtJAZ5 PtJAZ6 PtJAZ7 PtJAZ8 PtJAZ9 PtJAZ10 PtJAZ11 PtJAZ12

Gene Identifier

Potri.006G247500 Potri.018G033700 Potri.002G048500 Potri.005G214300 Potri.002G110800 Potri.002G110900 Potri.005G152500 Potri.005G152800 Potri.007G116500 Potri.007G116700 Potri.010G251600 Potri.017G042200 Potri.001G062500 Potri.001G166200 Potri.003G068900 Potri.003G165000 Potri.006G139400 Potri.006G217200 Potri.008G133400 Potri.010G108200 Potri.011G083900 Potri.012G044900 Potri.015G035800 Potri.018G047100

Chr.

6 18 2 5 2 2 5 5 7 7 10 17 1 1 3 3 6 6 8 10 11 12 15 18

Location coordinates (50 - 30 )

Chr06:25449398..25454621 Chr18:2692940..2697905 Chr02:3161909..3166359 Chr05:22734458..22738357 Chr02:8198186..8203728 Chr02:8206492..8211812 Chr05:14348124..14353280 Chr05:14386485..14398559 Chr07:13730413..13732003 Chr07:13736385..13742259 Chr10:22342132..22345674 Chr17:3570422..3576737 Chr01:4842780..4845551 Chr01:13947989..13949828 Chr03:9718300..9720218 Chr03:17563038..17565340 Chr06:11681034..11683548 Chr06:22993284..22997384 Chr08:8848283..8851667 Chr10:12780105..12783705 Chr11:8532739..8533794 Chr12:4116344..4120741 Chr15:3132632..3136562 Chr18:4408305..4414489

CDS length (bp)

1293 1323 1128 1365 1083 873 1098 867 636 1155 924 1221 591 810 804 606 831 867 753 1134 450 1086 1191 654

Protein

Exons

Length (a.a.)

PI

MW (Da)

430 440 375 454 360 290 365 288 211 384 307 406 196 269 267 201 276 288 250 377 149 361 396 217

9.56 9.37 6.91 9.17 4.87 5.93 5.13 5.67 4.89 4.9 7.8 5.36 9.91 8.74 7.75 9.89 8.87 8.11 9.61 9.16 9.66 9.59 9.47 9.11

45044.5 46113.6 41390.6 50224.7 39278.4 31465.7 39708.2 31463.7 22662.4 43220.8 33369.7 46027.4 21735.9 29319.8 29023.5 22388.6 29923 31027.8 26291.8 39902.2 17279.7 37978.7 41730.3 23620

6 6 9 9 10 7 10 7 3 11 7 10 6 5 5 6 5 6 5 7 2 6 7 5

CDS, coding sequence; bp, base pair; aa, amino acids; pI, isoelectric point; MW, molecular weight; Da, Dalton.

Table 2 Ks, Ka, and Ka/Ks values calculated for paralogous TIFY gene-pairs (Pt-Pt) in the Populus trichocarpa genome.

Fig. 1. Phylogenetic tree for tify domain-containing proteins from Populus trichocarpa, Arabidopsis, grape, rice and apple. The phylogenetic tree was constructed using the neighbor-joining method as implemented in MEGA6.0 from a TIFY protein sequence alignment. Bootstrap values from 1000 replicates are displayed at each node. The proteins on the tree can be divided into eight clades and the different clades are indicated by different colors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Exon-intron structure and conserved amino acid domain analysis The divergence in the exon-intron structure can play the key roles in the evolution of gene families to support phylogenetic

Paralogous pairs

Ka

Ks

Ka/Ks ratio

Date (MY)

PtPPD1-PtPPD2 PtTIFY1-PtTIFY2 PtJAZ2-PtJAZ3 PtJAZ6-PtJAZ12 PtJAZ1-PtJAZ4 PtJAZ10-PtJAZ11 PtJAZ7-PtJAZ8 PtZML1-PtZML3 PtZML6-PtZML8 PtZML2-PtZML4

0.15 0.07 0.14 0.09 0.11 0.10 0.16 0.07 0.08 0.06

0.30 0.26 0.33 0.32 0.32 0.19 0.17 0.22 0.28 0.22

0.52 0.28 0.41 0.28 0.35 0.51 0.95 0.32 0.28 0.26

16.44 14.34 18.31 17.57 17.48 10.47 9.32 11.92 15.63 12.15

groupings (Shiu and Bleecker, 2003). Our results indicated that a positive correlation exists between gene phylogeny and structure. Based on phylogenetic results (Fig. 2), we identified 10 paralogous pairs of PtTIFY proteins: PtJAZ-1/-4, PtJAZ-2/-3, PtJAZ-6/-12, PtJAZ-7/8, PtJAZ-10/-11; PtTIFY-1/-2; PtPPD-1/-2; PtZML-1/-3, PtZML-2/-4 and PtZML-6/-8; the proteins that clustered together at the tips of branches of the phylogenetic tree generally possessed a similar gene structure. Most PtJAZ genes had 5 to 7 exons, except for PtJAZ9 which had only 2 exons. The PtZML genes had 7 to 11 exons, except for PtZML5 which had only three. This finding suggests that numerous TIFY genes were conserved, and we speculate that the PtJAZ9 and PtZML5 lost exons through duplication events over evolutionary time. The total 20 potential conserved sequence motifs were identified by MEME. Each of these putative motifs was examined by searching NCBI. Motifs 1, 2 and 4 were identified to encode the tify DNA-binding domain, but the others have no functional annotations. As shown in Fig. 3, Motif1 is the most common motif found in all 24 TIFY proteins, and it is the tify domain. Motif2 is present in seven ZML subfamily members, except for PtZML5, while motif4 was identified only in the ZML subfamily; motifs 2 and 4 might be GATA zinc-finger and CCT motifs, respectively. Motif 3 was found in

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Fig. 2. Phylogenetic relationships, exon-intron structures and conserved domains of the predicted PtTIFY proteins. A: a phylogenetic tree constructed with the N-J method in MEGA 6.0. The proteins on the tree can be divided into four distinct subfamilies, which are indicated with different colored backgrounds. B: The exons, introns and untranslated regions (UTRs) are represented by yellow rectangles, gray lines and blue rectangles, respectively. C: Distribution of domains in the Populus trichocarpa TIFY proteins. The relative positions of each domain within each protein are shown by colored bars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

22 TIFY proteins except for PtTIFY-1/-2, and motif4 was always present with motif3 in the ZML subfamily, and the two motifs could possibly be merged into a single domain in the ZML subfamily. These results showed that the members of a given subfamily contain very similar conserved sequence motifs and that numerous paralogous pairs within subfamilies had the same motifs that are also present in the same relative orientation, such as PtJAZ-10/-11 and PtZML-6/-8. In proteins, a motif can be formed by the threedimensional arrangement of amino acids which may not be adjacent, so a structural motif describes the connectivity between secondary structures; protein motifs often include loops of variable number and length or indefinite structure, and some characteristic motifs may comprise the functional divergence of TIFY genes. Moreover, the domain analysis was also consistent with the exon/ intron structure analysis and agreed well with the phylogenetic analysis, although we found that PtZML5 has a deletion of the GATA-zinc finger domain. 3.5. Analysis of cis-elements in the promoter regions of PtTIFY genes Cis-elements in the promoter regions play a key role in the regulation of the tissue-specific genes expression profiles (Le et al., 2012), and we identified putative stress-responsive cis-elements in the putative promoter regions. Numerous abiotic stress cis-

elements were found widely in the promoter regions of TIFY genes in Populus trichocarpa, such as S000408, S000415 and S000414 for drought-responsive, S000453 for salt-responsive and S000407 for cold-responsive (Wu et al., 2015a). For instance, JAZ12 possessed up to 12 drought-responsive elements (S000413), JAZ7 had up to 10 cold-responsive elements (S000407), and JAZ3 had up to nine salt-responsive elements (S000453). The details about the putative promoter regions of TIFY genes were shown in Table 3. 3.6. Calculation of Ka/Ks ratios The Ka/Ks ratio is an indicator of selective pressure acting on a protein-coding gene. Some systematic bias in certain species may occur more easily in the process of nucleotides substitutions(e.g. G to T transversions occurred more easily than do G to A transitions) due to species diversity, a high mutations rate will raise the proportion of amino acid changes. The analysis demonstrated that the Ka/Ks ratios of the ten paralogous pairs (Table 2) were <1, which indicates that the PtTIFY gene family has been influenced principally by the high purifying selection and that the PtTIFY genes are evolving slowly and have conserved characteristics at the protein level. According to the divergence rate of 9.1  10
9 synonymous mutations per synonymous locus per year, the divergence time (T) was evaluated as T ¼ Ks/(2  9.1  10
9) Mya (Hurst, 2002; Yang

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Fig. 3. Distribution of conserved sequence motifs in the PtTIFY proteins. Schematic representation of the 20 conserved motifs in PtTIFY proteins. Motifs in the PtTIFY proteins were identified by the online MEME program. The different motifs were represented by colored boxes containing numbers. The colored boxes were ordered manually based on the results of the MEME analysis. The amino acid sequences of the 20 motifs are given in Table S1.

and Bielawski, 2000); the 10 paralogous pairs were evaluated to have diverged between 9.32 and 18.31 million years ago (Mya). We performed a sliding window (Fig. S3) analysis of Ka/Ks ratios between each paralogous pair of proteins. As predicted from the basic Ka/Ks analysis, the sliding window analysis directly demonstrated that numerous loci/regions were under neutral to disadvantages mutations, but a minority of loci/regions were evolving under positive selection. The Ka/Ks ratios of some loci/regions were approaching two, and we removed Ks values > 2.0 because of the risk of saturation (Maher et al., 2006). The Ka/Ks ratios conform to the conserved characteristics of the tify domain.

3.7. Microarray and qRT-PCR analysis of expression profiles Twenty-three genes were identified in GSE13990 by TIFY-probe, while one gene (PtJAZ7) was not found. As shown in Fig. 4, although expression levels vary, there are some special cases. Specifically, the PtJAZ subfamily genes had high expression in tissues/organs. In contrast, the ZML, PPD and TIFY subfamily genes had lower expression levels in most of the tissues/organs. Moreover, the PtJAZ8 showed strong expression in all the tissues/organs, and PtJAZ6 had its highest expression levels in the xylem. The PtJAZ2, 3, 5 and 12 genes were highly expressed in roots and flowers; the PtJAZ6, 10 and 12 were highly expressed in roots and leaves. Among

the 24 PtTIFY genes, seven showed the highest levels of accumulation in the roots (PtJAZ2, 3, 5, 6, 8, 10 and 12) and five in the leaves (PtJAZ3, 6, 8, 10 and 12). Six PtJAZ genes (1, 2, 3, 5, 8 and 12) were expressed at higher levels than other genes in catkins, and only PtZML4 in the ZML subfamily showed a higher expression in young leaves. These results suggested that the PtTIFY genes have varying profiles of expression. To obtain further insight into the expression profiles of PtTIFY genes, we extracted the total RNAs from phloem, xylem, mature leaves, young leaves and roots of poplar to perform the qRT-PCR analysis, respectively. There were some different results between the qRT-PCR analysis and Microarray analysis, which might be caused by the differences of their experimental materials, such as conditions, poplar ages, sample collection times, etc. As shown in Fig. 5, the PtJAZ4, PtJAZ9, PtZML7 and PtPPD1 had low expression levels in all the tissues, while the PtJAZ8 showed the strong expression levels in all the tissues. The PtJAZ6, PtJAZ7 and PtJAZ12 had abundant expression levels in the phloem, xylem and young leaves, but PtJAZ1 had low expression levels in these tissues. The PtJAZ7, PtJAZ8 and PtJAZ12 were highly expressed in the roots; the PtJAZ8 and PtJAZ10 had high expression levels in the mature leaves; six genes (PtJAZ6, -7, -8, -10, -11 and -12) presented high expression levels in the young leaves; four genes (PtJAZ6, -7, -8 and -12) had high expression levels in the phloem and xylem.

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Table 3 Summary of stress inducible cis-elements is in the promoter regions of JAZ genes in Populus trichocarpa. Abiotic stress

cis-element

Gene JAZ1

Drought-stress

Cold-stress

Salt-stress

Heat-stress

Wound-stress

S000133 S000153 S000174 S000175 S000176 S000177 S000402 S000408 S000413 S000414 S000415 S000418 total S000153 S000402 S000407 S000418 total S000402 S000418 S000453 total S000030 S000418 total S000244 S000457 total

CCACGTGG CCGAC CACATG CTAACCA CNGTTR TAACTG ACCGAC WAACCA CATGTG ACGTG ACGT RCCGAC CCGAC ACCGAC CANNTG RCCGAC ACCGAC RCCGAC GAAAAA CCAAT RCCGAC AACGTGT TGACY

JAZ2

JAZ3

JAZ4

JAZ5

JAZ6

JAZ7

JAZ8

1

1

JAZ9

JAZ10

JAZ11

JAZ12

1 1

1 2

3

1 1

3 2 2

5 1 10 1

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Fig. 4. Microarray analysis of expression profiles. A heat-map shows the hierarchical clustering of the relative expression of 23 PtTIFY genes across the six different tissues analyzed. The vertical color scale shown to the right of the image represents log2 expression values: red indicates a high level and blue represents a low level of transcript abundance. Microarray data under the series accession number GSE13990 was obtained from the NCBI GEO database. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. The qRT-PCR analysis of expression profiles. A heat-map shows the hierarchical clustering of the relative expression of 24 PtTIFY genes across the five different tissues analyzed. The vertical color scale shown to the right of the image represents log2 expression values: red indicates a high level and blue represents a low level of transcript abundance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.8. Expression profiles of PtTIFY genes in response to different stress treatments To explore the relative expression levels and better understand the functions of PtTIFY family genes, the six-week-old poplar seedlings that were grown in the Tissue Culture Lab were treated by salinity, JA and cold stresses. All treatments had five time points (1, 3, 6, 12 and 24 h), and after that RNAs were extracted from poplar leaves at these different time points. We used the qRT-PCR to analyze the expression levels in response to several different treatments. As shown in Figs. 6e8, the results revealed a widespread gene expression profiles. Analysis of the JAZ subfamily demonstrated that most genes were up-regulated by jasmonic acid (JA) and sodium chloride (NaCl) stress treatments, but were both up-regulated and down-regulated in response to cold treatment. In general, we found that PtJAZ genes were highly up-regulated by JA, NaCl and cold treatments, while the expression levels of PtPPD and PtZML genes were only slightly induced by the same stresses. There was a general tendency for expression levels to either rise early in the experiment or to decline rapidly from an initial peak, especially for the JA and NaCl treatments. Some paralogous pairs shared similar expression profiles, although some differences between them were also observed. Interestingly, PtJAZ3 always had the highest expression levels of all the genes in response to the three treatments, and this may deserve further study. Cold treatment. Analysis of expression data showed that two genes (PtJAZ3, 5) were up-regulated in response to cold treatment. The PtJAZ3 and PtJAZ5 were both up-regulated at five-time points

and reached its highest levels at 24 h. However, expression differences were observed among the remaining genes. For instance, expression of PtZML5 peaked at 1 h, and expression of PtJAZ9 peaked at 3 h, but then decreased significantly at 24 h. The PtJAZ3 had the highest relative change in expression levels of all the JAZ genes. JA treatment. Analysis of expression data showed that ten genes (PtJAZ1, 2, 3, 4, 5, 6, 7, 9, 11 and 12) were up-regulated in response to JA treatment, but PtZML3, 4, 6 were down-regulated. Five genes (PtJAZ2, 3, 4, 5 and 9) showed the highest up-regulation. Six genes (PtZML1, 2, 8, PtTIFY1, 2, PtPPD2) always had the higher expression levels after 1 h of treatment and then declined. From our data, we can conclude that the JAZ subfamily genes play a more important JA-responses role than genes in the other subfamilies. NaCl treatment. Analysis of expression data showed that the majority of the JAZ subfamily genes were up-regulated by NaCl treatment, while genes in other subfamilies only showed lower levels in response to NaCl treatment. The expressions of seven genes (PtJAZ1, 2, 3, 4, 5, 7 and 9) were up-regulated in response to NaCl treatment; PtJAZ2, 3, 4 and 9 exhibited the most significant upregulation, indicating that these genes may play the different roles in response to NaCl treatment. 4. Discussion The TIFY family are plant-specific transcription factors that play significant roles in various aspects of plant growth and development, such as mechanical damage resistance and induction of defense-related gene expression (Demianski et al., 2011). The TIFY

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Fig. 6. Expression profiles of PtTIFY genes in response to cold treatment as determined by qRT-PCR. A heat-map shows the hierarchical clustering of the relative expression of 24 PtTIFY genes under cold treatment. Green indicates lower and blue represents higher transcript abundance compared to the relevant control. The plant materials were incubated 4  C and sampled after 1, 3, 6, 12 and 24 h of treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

family is present in most terrestrial plants, but does not exist in the unicellular algae C. reinhardtii or in the multicellular green algae V. carteri, which indicates that these genes might have been essential in the emergence of terrestrial plants. The TIFY genes were thought to have originated after the divergence of algae and the land plants (Bai et al., 2011). Additionally, the TIFY subfamily is absent in two monocotyledons, Brachpodium distachyon and Sorghum bicolor; the PPD subfamily was only found in the dicotyledons and spikemoss Selaginella moellendorffii. It is noteworthy that the PPD subfamily is not present in monocotyledons (Ye et al., 2009). A possible reason is that the profiles of plant growth and development in monocots are quite different from the eudicots and other vascular plants, and factors other than the PPD genes may be more necessary for regulation of plant growth and developmental processes in monocots (White, 2006). Previous research has shown that proteins in different transcription factor families share some common domains (Riechmann et al., 2001). The TIFY domain is a common and conserved domain that widely exist in all land plants. Members of the TIFY family have been reported in Arabidopsis, rice, grape and apple. Among these species, the JAZ subfamily always comprises a large proportion of tify domain-containing proteins. Twenty-four putative TIFY genes were identified in the Populus trichocarpa genome. These genes were found to be located on 13 chromosomes. Based on domain architecture, TIFY family proteins can be further classified into four subfamilies; TIFY, JAZ, PPD and ZML. Phylogenetic analysis showed that JAZ subfamily was classified into six clades (I to VI), while the ZML and PPD proteins cluster into a single clade each. The JAZ proteins from apple and grape make up a large proportion of the proteins in the JAZ VI clade, but the JAZ VI clade contains only on

JAZ protein from Populus trichocarpa, JAZ9; the PtJAZ9 gene only has two exons and is distinct from other JAZ VI clade protein genes with respect to exon/intron structure. This suggests that the JAZ protein genes in these species diverged from one another very early during land plant evolution. We can deduce that the JAZ subfamily showed more divergence than the PPD and ZML subfamilies. The divergence of the JAZ protein subfamily in these species showed that plant specificity is often the result of evolution. Approximately 32% of the predicted genes in poplar were retained in duplicated pairs that resulted from whole genome duplication events, according to previous reports (Hurst, 2002), although we did not identify tandem duplication clusters in our analysis. However, 10 paralogous pairs and 17 synteny blocks were identified, which may reflect the fact that the PtTIFY genes had undergone large-scale segmental duplication events. The phylogenetic relationships and sequence structural features showed that the TIFY proteins are also closely related to each other. Although it has been reported that duplicated genes rarely diverge with respect to their biochemical function; gene duplication is considered to provide the raw material for evolution; duplicated genes may undergo substantial changes in their structures and/or regulatory mechanisms allowing them to assume novel roles (Xu et al., 2012, Wapinski et al., 2007). The plant specificity is often the result selective gene loss or gain during evolution and can involve chromosomal rearrangements and fusions, such as gene duplication with the three principal types of exon/intron diversification: exon-intron gain/loss, exonization/ pseudo-exonization and insertion/deletion (Xu et al., 2012). We are unable to deduce from the work presented here that whether the paralogs have assumed same the roles. Most PtJAZ genes had 5 to 7 exons, except for PtJAZ9 which had only 2 exons. The PtZML

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Fig. 7. Expression profiles of PtTIFY genes in response to JA treatment as determined by qRT-PCR. A heat-map shows the hierarchical clustering of the relative expression of 24 PtTIFY genes under JA treatment. Green indicates lower and blue represents higher transcript abundance compared to the relevant control. The leaves were sprayed with 100 mM jasmonic acid (JA) solution and sampled after 1, 3, 6, 12 and 24 h of treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

genes had 7 to 11 exons, except for PtZML5 which had only three. Moreover, we found that the PtZML5 protein had a deletion of the GATA-zinc finger domain. We can conclude that PtJAZ9 and PtZML5 evolved through a substantial change in structure. The exon/intron arrangement and the sequence motif structure is not complex. Motif15 was only found in PtJAZ2 and PtJAZ3 proteins. Most paralogous pairs have similar structures, but there can be certain differences. For example, PtJAZ10 has six exons, while the paralogous gene PtJAZ11 has seven. Evolutionary theory explains the existence of homologous structures adapted to different purposes as the result of descent from a common ancestor with subsequent modification; such divergence either in exon/intron length or exon/ intron number can potentially lead to the generation of functionally distinct paralogs (Xu et al., 2012). The exon/intron and motif structures showed that some genes might have undergone multiple rounds of significant chromosomal rearrangement and fusions, tendency to lose/gain exons since evolution selection, having substantial changes in their structures. The Ka/Ks ratios supported the conserved characteristics of the tify domain and the slow rate of TIFY gene evolution and conserved characteristics at the protein level. Abiotic stresses, such as drought, salinity and extreme temperatures are limiting factors in plant growth and development. Proteins in the TIFY family of putative transcription factors have been reported to play important roles in plant stress-responses, with the JAZ proteins being the best characterized to date. The JAZ proteins are stress proteins which play the key roles in mediating the JAresponses (Hua et al., 2015). For instance, overexpression of OsTIFY11a in rice resulted in significantly increased tolerance to both

salt and dehydration stress; the grape gene VvJAZ11 exhibited a high up-regulation in response to heat stress; the majority of apple JAZ genes were significantly up-regulated by both ABA and MeJA treatments. Transcription of maize genes ZmTIFY4, 5, 8, 26 and 28 was induced, while transcription of ZmTIFY16, 13, 24, 27, 18 and 30 was suppressed in response to drought stress. Proteins in the JAZ subfamily are involved in the jasmonate signaling pathway, representing one of the vital defense mechanisms. Within the JA signaling cascades, JAZ proteins that are induced by jasmonates play a central role. The JAZ proteins modulate JA-responsive gene expression by inhibiting DNA-binding transcription factors in the absence of jasmonates, but JAZ proteins interact with COI1 in the presence of jasmonates, releasing transcription factors from inhibition and activating JA-responsive gene transcription (Melotto et al., 2008; Yan et al., 2007). Thus, members of the JAZ subfamily of TIFY proteins play important roles in many aspects of growth and development in plants. The PtJAZ subfamily genes were abundantly expressed in most tissues/organs as shown by publicly available microarray data and qRT-PCR analysis. In contrast, the other TIFY subfamilies showed lower expression levels in most of the tissues/ organs. In addition, the qRT-PCR analysis also showed that JAZ subfamily genes play more important roles than other TIFY subfamily genes in response to different stress treatments. These results are in agreement with previous expression analysis of TIFY genes from the grape, apple and rice.

5. Conclusion In this study, we identified total 24 PtTIFY genes in the genome

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Fig. 8. Expression profiles of PtTIFY genes under NaCl treatment as determined by qRT-PCR. A heat-map shows the hierarchical clustering of the relative expression of 24 PtTIFY genes under NaCl treatment. Green indicates lower and blue represents higher transcript abundance compared to the relevant control. Salt-stress was carried out by watering the plants with a 200 mM solution of sodium chloride (NaCl) solution. Plants were sampled after 1, 3, 6, 12 and 24 h of treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of Populus trichocarpa and classified them into four protein subfamilies (two TIFY, eight ZML, two PPD and 12 JAZ) based on the conserved domains present. Ten gene pairs were identified arose from large-scale interchromosomal segmental duplication events. The phylogenetic analysis separated the PtJAZ proteins into six clades that diverged mutually early during the processes of land plant evolution, and this was supported by their exon/intron structures, motifs and the domains present. Our results showed that the PtTIFY gene family has been influenced principally by the purifying selection and that the TIFY genes evolved slowly and have conserved characteristics. Microarray data and qRT-PCR analysis of the PtTIFY genes showed that 24 genes have organ-specific roles. The qRT-PCR analysis suggested that PtJAZ proteins play vital roles in various aspects of plant growth and stress-responses. This study not only provides a basis for assigning functions to the individual PtTIFY genes but also provides valuable information for further exploration of TIFY genes in Populus trichocarpa.

Author contribution statement YW and FP designed and conceived the experiment, carried out the principal bioinformatics analysis, drafted the manuscript. Performed the experiments: YW, FP. Edited the data, figures and tables: DMC, WYC, HLL. Contributed reagents/materials/analysis tools: YX. All authors read and approved the final manuscript. Acknowledgments We thank the members of the Laboratory of Modern Biotechnology for their assistance in this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2017.04.015.

Funding

References

This work was supported by grants from the National Research Council of Science and Technology Support Plan Corpus (No. 2015BAD07B070104).

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Conflicts of interest The authors declare no conflict of interest.

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