Genome-wide identification and characterization of WRKY gene family in Salix suchowensis

Datasets and sequence retrieval

The sequence of a shrub willow Salix suchowensis (S. suchowensis), which flowers within two years, was conducted with a combined approach using Roche/454 and Illumina/HiSeq-2000 sequencing technologies (Dai et al., 2014). The latest v5.2 S. suchowensis genome annotation information (version5_2.gff3) and protein sequences (Willow.gene.pep) were downloaded from our laboratory website (http://bio.njfu.edu.cn/ss_wrky/). Sequences of 72 Arabidopsis WRKY proteins were obtained from TAIR (release 10; http://www.arabidopsis.org/), and 104 poplar WRKY proteins were obtained from the Supplemental Information 3 file of poplar in He et al. (2012) (Eulgem, 2000).

Identification and distribution of WRKY genes in willow

The procedure performed to identify putative WRKY proteins in willow was similar to the method described in other species (Guo et al., 2014; He et al., 2012; Wu, 2005). The Hidden Markov Model (HMM) profile for the WRKY transcription factor was downloaded from the Pfam database (http://pfam.xfam.org/) with the keyword ‘PF03106’ (Punta et al., 2012). The HMM profile was applied as a query to search against the all willow protein sequences (Willow.gene.pep) using BLASTP program (E-value cutoff = 1e-3) (Camacho et al., 2009). Another procedure was performed to validate the putative accuracy. An alignment of WRKY seed sequences in Stockholm format from Pfam database was used by HMMER program (hmmbuild) to build a HMM model, and then the model was used to search the willow protein sequences by another HMMER program (hmmsearch) with default parameters (Eddy, 1998). Finally, we employed the SMART program (http://smart.embl-heidelberg.de/) to confirm the candidates from the two procedures correlated with the WRKY structure features (Letunic, Doerks & Bork, 2015).

Additionally, we calculated the length, molecular weight (MW), isoelectric point (PI) of these putative WRKY proteins by ExPasy site (http://au.expasy.org/tools/pi_tool.html). Every WRKY genes were mapped onto chromosomes (http://bio.njfu.edu.cn/ss_wrky/version5_2.fa) with an in-house Perl script (http://bio.njfu.edu.cn/willow_chromosome/BuildGff3_Chr.pl), and then renamed based on their orderly given chromosomal distribution. The distribution graph of every WRKY gene was drawn by MapInspect software (http://mapinspect.software.informer.com/).

Sequence alignments, phylogenetic analysis and classification of willow WRKY genes

Using the online tool SMART, we obtained the conserved WRKY core domains of predicted SsWRKY genes, and then multiple sequence alignment based on these domains was performed using ClustalX (version 2.1) (Larkin et al., 2007). After alignment, we used Boxshade (http://www.ch.embnet.org/software/BOX_form.html) to color the alignment result online. To gain a better classification of these SsWRKY genes, a further multiple sequence alignment including 103 SsWRKY domains and 82 WRKY domains from Arabidopsis (AtWRKY) was performed using ClustalW (Larkin et al., 2007), and a phylogenetic tree based on this alignment was built by MEGA 6.0 with the Neighbor-joining (NJ) method (Tamura et al., 2013). Bootstrap values have been calculated from 1,000 iterations in the pairwise gap deletion mode, which is conducive to the topology of the NJ tree by divergent sequences. Based on the phylogenetic tree constructed by SsWRKY and AtWRKY domains, these SsWRKY genes were classified into different groups and subgroups. In order to get a better comparison of WRKY family in Salicaceae, a phylogenetic tree including all SsWRKY domains and 126 WRKY domains from poplar (PtWRKY) was constructed with the similar method to Arabidopsis. Additionally, a phylogenetic tree based on full-length SsWRKY genes was also constructed to get a better classification. The ortholog of each SsWRKY gene in Arabidopsis and poplar was based on the phylogenetic trees of their respective WRKY domains, and the members of group I WRKY genes were considered as orthologs unless the same phylogenetic relationship can be detected between N-termini and C-termini in the tree. Another method described by Zou et al. (2016), BLAST-based method (Bi-direction best hit), was used to verify the putative orthologous genes (E-value cutoff = 1e-20) (Chen et al., 2007).

Evolutionary analysis of WRKY III genes in willow

The group of WRKY III genes, only found in flowering plants, is considered as the evolutionary youngest groups, and plays crucial roles in the process of plant growth (He et al., 2012; Wu, 2005). As described by Wang et al. (2015), the WRKY III genes also have a prominent impact on disease and drought resistance. Previous study of Zhang & Wang (2005) held the opinion that duplications and diversifications were plentiful in WRKY III genes, and they appeared to have confronted different selection challenges. Phylogenetic analysis of WRKY III genes was performed using MEGA6.0 with 65 WRKY III genes from Arabidopsis (AtWRKY), Populus (PtWRKY), grape (VvWRKY), willow (SsWRKY) and rice (OsWRKY). A NJ tree was constructed with the same method described before. Additionally, we estimated the non-synonymous (Ka) and synonymous (Ks) substitution ratio of SsWRKY III genes to verify whether selection pressure participated in the expansion of SsWRKY III genes. Each pair of these WRKY III protein sequences was first aligned using ClustalW. The alignments generated by ClustalW and the corresponding cDNA sequences were submitted to the online program PAL2NAL (http://www.bork.embl.de/pal2nal/) (Suyama, Torrents & Bork, 2006), which automatically calculates Ks and Ka by the codeml program in PAML (Yang, 2007).

Analysis of exon–intron structure, gene clusters, gene duplication events and conserved motif distribution of willow WRKY genes

The exon–intron structures of the willow WRKY genes were obtained based on the protein annotation files assembled ourselves (http://bio.njfu.edu.cn/ss_wrky/version5_2.gff3), and the diagrams were obtained from the online website Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn/) (Hu et al., 2015).

Gene clusters are very important for predicting co-expression genes or potential function of clustered genes in angiosperms (Overbeek et al., 1999). They can be defined as a single chromosome containing two or more genes within 200 kb (He et al., 2012; Holub, 2001).

Gene duplication events were always considered as the vital sources of biological evolution. Two or more adjacent homologous genes located on a single chromosome were considered as tandem duplication events (TDs), while homologous gene pairs between different chromosomes were defined as SDs (Liu & Ekramoddoullah, 2009). BLASTP (E-value cutoff = 1e-20) was performed to identify the gene duplication events in SsWRKY genes with the following definition (Gu et al., 2002; He et al., 2012): (1) the coverage of the aligned sequence ≥ 80% of the longer gene; and (2) the similarity of the aligned regions ≥ 70%. In this study, we set the cutoff of the similarity of the aligned regions as 65%, because the similarity of the unaligned regions may reduce the value in different species.

To better exhibit the structural features of SsWRKY proteins, the online tool MEME (Multiple Expectation Maximization for Motif Elicitation) was used to identify the conserved motifs in the encoded SsWRKY proteins (Bailey et al., 2006). The optimized parameters were employed as the following: any number of repetitions, maximum number of motifs = 20, and the optimum width of each motif was constrained to between 6–50 residues. The online program 2ZIP (http://2zip.molgen.mpg.de/) was used to verify the existence of the conserved Leu zipper motif (Bornberg-Bauer, Rivals & Vingron, 1998), whereas some other important conserved motifs, HARF, LXXLL (X, any amino acid) and LXLXLX, were identified manually.

Expression analyses of willow WRKY genes

The sequenced S. suchowensis RNA-HiSeq reads from five tissues including tender roots, young leaves, vegetative buds, non-lignified stems and barks generated in our previous study were separately mapped back onto the SsWRKY gene sequences using BWA (mismatch ≤ 2 bp, other parameters as default) (Li & Durbin, 2009), and the number of mapped reads for each WRKY gene was counted. Normalization of the mapped reads was done using RPKM (reads per kilo base per million reads) method (Wagner, Kin & Lynch, 2012). The heat map for tissue-specific expression profiling was generated based on the log₂ RPKM values for each gene in all the tissue samples using R package (Gentleman et al., 2004).

Identification and characterization of 85 WRKY genes in willow (Salix suchowensis)

In this study, we obtained 92 putative WRKY genes by using HMMER to search the HMM profile of WRKY DNA-binding domain against willow protein sequences, and validated the accuracy of the consequence by BLASTP. After submitting the 92 putative WRKY genes to the online program SMART, seven genes without a complete WRKY domain were removed, while the other 85 WRKY genes were selected as possible members of the WRKY superfamily.

WRKY genes contain one or two WRKY domains, comprising a conserved WRKYGQK heptapeptide at the N-termini and a novel zinc finger motif (C-X_4–7-C-X_22–23H-X-H/C) at the C-termini (Eulgem, 2000). The variations of WRKY core domain or zinc finger motif may lead to the binding specificities of WRKY genes, but this remains to be largely demonstrated (Brand et al., 2013; Rinerson et al., 2015; Yamasaki et al., 2005). In order to identify the variations in WRKY core domains, a multiple sequence alignment of 85 SsWRKY core domains was conducted, and the result was shown in Fig. 1. Among the selected 85 WRKY genes, 81 (95.3%) were identified to have highly conserved sequence WRKYGQK, whereas the other four WRKY genes (SsWRKY14, SsWRKY23, SsWRKY38 and SsWRKY78) had a single mismatched amino acid in their core WRKY domains (Fig. 1). In SsWRKY14 and SsWRKY38, the WRKY domain has the sequence WRKYGKK, while SsWRKY23 contains a WKKYGQK sequence, and SsWRKY78 contains WRKYGRK sequence. Eulgem (2000) previously described that the zinc finger motif (C-X_4–5-X_22–23-H-X₁-H or C-X₇-C-X₂₃-H-X₁-C) is another vital feature of the WRKY family. As illustrated in Fig. 1, four WRKY domains (SsWRKY76C, SsWRKY64, SsWRKY12 and SsWRKY28) do not contain any distinct zinc finger motif, but they were still reserved in the succeeding analyses, as performed in barley and poplar (He et al., 2012; Mangelsen et al., 2008). Additionally, some zinc-finger-like motifs, including C-X₄-C-X₂₁-H-X₁-H in SsWRKY23 and C-X₅-C-X₁₉-H-X₁-H in SsWRKY73 and SsWRKY17, were identified in willow WRKY genes. Both the two zinc-finger-like motifs were also found in poplar (PtWRKY39, 57, 42 and 53).

Detailed characteristics of SsWRKY genes are listed in Table 1, including the WRKY gene specific group numbers, chromosomal distribution, Arabidopsis and poplar orthologs. The MW, PI and the length of each WRKY protein sequence are also shown in Table 1. According to the particularization (Table 1), the average length of these protein sequences is 407 residues, and the lengths ranged from 109 residues (SsWRKY23) to 1,593 residues (SsWRKY78). Additionally, the PI ranged from 5.03 (SsWRKY38, SsWRKY60) to 10.27 (SsWRKY28), and the MW ranged from 12.9 (SsWRKY23) to 179.0 kDa (SsWRKY78).

Locations and gene clusters of willow WRKY genes

Nearly 84 of the 85 putative SsWRKY genes could be mapped onto 19 willow chromosomes and then renamed from SsWRKY1 to SsWRKY84 based on their specific distributions on chromosomes. Only one SsWRKY gene (willow_GLEAN_10002834), renamed as SsWRKY85, could not be conclusively mapped onto any chromosome. As shown in Fig. 2, Chromosome (Chr) 2 possessed the largest number of SsWRKY genes (11 genes), followed by Chr14 (10 genes). Eight SsWRKY genes were found on Chr6, six on Chr1 and Chr16, and five on Chr5. Additionally, four chromosomes (Chr4, Chr11, Chr17, Chr18) had four SsWRKY genes, as well as three SsWRKY genes were found on Chr8, Chr13 and Chr19. Chr10 and Chr15 had two SsWRKY genes, and only one SsWRKY gene was identified on Chr7, Chr9 and Chr12. The distribution of each SsWRKY genes was extremely irregular, indicating the reduction of the TDs in willow WRKY genes.

As described by Holub (2001), a single chromosome region containing two or more genes within 200 kb was defined as gene clusters (He et al., 2012). According to this description, a total of 23 SsWRKY genes were clustered into 11 clusters in willow (Fig. 2). The chromosomal distribution of gene cluster was irregular, and only seven chromosomes were identified to have gene clusters. Three clusters, including seven SsWRKY genes, were found on Chr2, and two clusters were found on both Chr6 and Chr14. Only one cluster was distributed on each of Chr3, Chr8, Chr10 and Chr18, whereas none was identified on other 11 chromosomes. Further analysis of SsWRKY chromosomal distribution showed that a high WRKY gene density region in only 2.23 Mb regions on Chr2, which had also been observed in rice and poplar (He et al., 2012; Wu, 2005).

Phylogenetic analysis and classification of WRKY genes in willow

In order to get a better separation of different groups and subgroups in SsWRKY genes, a total of 185 WRKY domains, including 82 AtWRKY domains and 103 SsWRKY domains, were used to construct the NJ phylogenetic tree. On the basis of the phylogenetic tree and structural features of WRKY domains, all 85 SsWRKY genes were clustered into three main groups (Fig. 3). Nineteen members containing two WRKY domains and C₂H₂-type zinc finger motifs were categorized into group I, except SsWRKY78, which contains only one WRKY domain and two zinc finger motifs. Domain acquisition and loss events appear to have shaped the WRKY family (Ross, Liu & Shen, 2007; Rossberg et al., 2001). Thus, SsWRKY78 may have evolved from a two-domain WRKY gene but lost one WRKY domain during evolution. Additionally, as shown in Fig. 3, SsWRKY78 shows high similarities to SsWRKY40N, implying a common origin of their domains. The similar phenomenon was also found in PtWRKY90 of poplar (He et al., 2012).

The largest number of SsWRKY genes, comprising a single WRKY domain and C₂H₂ zinc finger motif, were categorized into group II. SsWRKY genes of group II could be further divided into five subgroups: IIa, IIb, IIc, IId and IIe. As shown in Fig. 3, subgroup IIa (four members) and IIb (eight members) were clustered into one clade, as well as subgroup IId (13 members) and IIe (11 members). Strikingly, SsWRKY genes in subgroup IIc (21 members) and group IC are classified into one clade, suggesting that group II genes are not monophyletic and the group IIc WRKY genes may evolve from the group I genes by the loss of the WRKY domain in N-terminal. As shown in Figs. 3 and S1, SsWRKY23, SsWRKY34 and their orthologous genes (AtWRKY49, PtWRKY39, PtWRKY57, PtWRKY34 and PtWRKY32) seem to form a new subgroup closer to the group III. However, SsWRKY23 and SsWRKY34 exhibit the zinc finger motif C-X₄-C-X₂₁-H-X-H and C-X₄-C-X₂₃-H-X-H as observed in the subgroup IIc and group IC. Therefore, they were classified into subgroup IIc in this study.

Different from the C₂H₂ zinc finger pattern in group I and II, group III WRKY genes (seven members), broadly considered as playing vital roles in plant evolution process and adaptability, contained one WRKY domain and a C-X₇-C-X₂₃-H-X-C zinc finger motif. However, in rice and barley, a new CX₇CX_nHX₁C (n ≥ 24) zinc finger motif was identified in group III (Mangelsen et al., 2008; Wu, 2005), which was never found in poplar, grape, Arabidopsis and willow, suggesting that this feature perhaps only belong to monocotyledonous species.

In order to obtain a better study in woody plant species, a phylogenetic tree based on the WRKY domains between willow and poplar was constructed (Fig. S1). The tree showed that most of the WRKY domains from willow and poplar were clustered into sister pairs, suggesting that gene duplication events played prominent roles in the evolution and expansion of WRKY gene family. Furthermore, a total of 20 SsWRKY domains show extremely the same domains (similarity: 100%) to poplar, i.e., SsWRKY39 and PtWRKY9, SsWRKY39 and PtWRKY9, SsWRKY39 and PtWRKY9, SsWRKY39 and PtWRKY9, and so on. Further functional analyses of these genes in willow or poplar will provide a useful reference for another one.

The ortholog of SsWRKY genes in Arabidopsis and poplar

The clustering of orthologous genes emphasizes the conservation and divergence of gene families, and they may contain the same functions (Ling et al., 2011). In this study, a phylogeny-based method was used to identify the putative orthologous SsWRKY genes in Arabidopsis and poplar (Figs. 3 and S1), and BLAST-based method (Bi-direction best hit) was used to confirm the true orthologs. The WRKY genes of group I contained two WRKY domains, and both of them were used to construct the phylogenetic trees. To avoid the mistakes of orthologous genes in group I, the members of group I WRKY genes were considered as orthologous genes unless the same phylogenetic relationship can be detected between N-termini and C-termini in the phylogenetic tree. For example, SsWRKY37 and AtWRKY44 were considered as an orthologous gene pair because they clustered into a clade of their N-termini and C-termini (Fig. 3), while SsWRKY80 and PtWRKY30 were excluded from orthologous gene pairs due to their different clusters of N-termini and C-termini (Fig. S1). Totally, 75 orthologous gene pairs were found between willow and Arabidopsis, less than 82 orthologous gene pairs between willow and poplar (Table 1), which was congruent with the evolutionary relationship among the three plant species.

Evolutionary analysis of WRKY III genes in willow

The WRKY III genes were considered as the evolutionary youngest groups, and played crucial roles in the process of plant growth and resistance. In order to further probe the duplication and diversification of WRKY III genes after the divergence of the monocots and dicots, a phylogenetic tree was constructed using 65 WRKY III genes from Arabidopsis (13), rice (29), poplar (10), willow (7) and grape (6). As shown in Fig. S2, willow SsWRKY III genes were closer to the eurosids I group (poplar and grape) than eurosids II group (Arabidopsis) and monocots (rice). Meanwhile, most Arabidopsis and rice WRKY III genes formed the relatively independent clades, suggesting that two gene duplication events, including tandem and segmental duplication, perhaps were the main factors in the expansion of WRKY III genes in Arabidopsis and rice. The results also indicated that WRKY III genes might arise after the divergence of the Arabidopsis (eurosids I) and eurosids II (poplar, willow and grape). The study by Ling et al. (2011) in cucumber showed the similar results and hence proved the validity. Additionally, we found that seven rice WRKY III genes (OsWRKY55, 84, 18, 52, 46, 114 and 97) contained the variant domain WRKYGEK, which was not found in other four dicots (Arabidopsis, poplar, grape and willow), implying that this may be a feature of WRKY III genes in monocots and these OsWRKY genes may respond to different environmental signals.

According to the comparison of the number of WRKY III genes in the five observed plants, the number is smaller in eurosids I (poplar, grape and willow) than Arabidopsis (eurosids II) and rice (monocots), which may be caused by different patterns of duplication events. Genes generated by duplication events are not stable, and can be retained or lost due to different selection pressure and evolution (Zhang, 2003). In order to determine which selection pressure played prominent roles in the expansion of willow WRKY III genes, we estimated the Ka/Ks ratios for all pairs (21 pairs) of willow WRKY III genes. As shown in Table S1, all the Ka/Ks ratios were less than 0.5, suggesting willow WRKY III genes had mainly been subjected to strong purifying selection and they were slowing evolving at the protein level.

Exon–intron structures of SsWRKY genes

The exon–intron structures of multiple gene families play crucial roles during plant evolution. As shown in Fig. 4, the SsWRKY gene phylogenetic tree and the corresponding exon–intron structures are shown in A and B, respectively. Exon–intron structures of each group were shown in Fig. 4B, a large number of WRKY genes had two to five introns (94%, 80 of 85), including eight WRKY genes contained one intron; 39 contained two introns; 13 contained three introns; 15 contained four introns and 5 contained five introns. The number of exons in remaining WRKY genes was quite different: SsWRKY49, SsWRKY76 and SsWRKY78 had 6, 11 and 10 introns, respectively; SsWRKY17 had the largest number of introns (17 introns), while no intron was found in SsWRKY12. The intron acquisition or loss occurred during the evolution of WRKY gene family, while WRKY genes in the same group shared the similar number of introns (Guo et al., 2014). In our study, most of WRKY genes in group I had three to six introns, expect SsWRKY76 and SsWRKY78, which might acquire some introns during evolution. The number of introns of WRKY genes in group II was extremely different, ranging from one to five introns, except SsWRKY17 with 17 introns and SsWRKY12 with zero intron might obtain or loss some introns during evolution. Strikingly, WRKY genes in group III had the most stable number of introns with all of seven WRKY III genes had two introns, suggesting that WRKY III genes may be the most stable genes in the environmental stress. The stable number of introns in SsWRKY III genes was consistent with the results of Ka/Ks analysis, which reflected that purifying selection pressure played vital roles in willow WRKY III genes.

A great deal of studies in WRKY genes proved that nearly all of the WRKY genes contained an intron in their WRKY core domains (Eulgem, 2000; Guo et al., 2014; He et al., 2012; Huang et al., 2012; Ling et al., 2011; Zou et al., 2004). According to the further analysis of SsWRKY genes, two major types of splicing introns, R-type and V-type, introns were observed in numerous SsWRKY domains. The R-type intron was spliced exactly at the R residue, about five amino acids before the first Cys residue in the C₂H₂ zinc finger motif. The V-type intron was localized before the V residue, six amino acids after the second Cys residue in the C₂H₂ zinc finger motif. As shown in Fig. 4B, the R-type introns could be observed in more groups, including group IC, subgroup IIc, IId, IIe and group III, while V-type introns were only observed in subgroup IIa and IIb. However, there was no intron found in group IN. The similar results were also observed in Arabidopsis, poplar and rice, suggesting that the special distribution of introns in WRKY domains was a feature of WRKY family (Eulgem, 2000; He et al., 2012; Wu, 2005).

Identification of gene duplication events and conserved motifs in willow

Gene duplication events were always considered as the vital sources of biological evolution (Chothia et al., 2003; Ohno, Wolf & Atkin, 1968). TDs were defined as two or more adjacent homologous genes located on a single chromosome, while homologous gene pairs between different chromosomes were defined as SDs (Liu & Ekramoddoullah, 2009). In our study, a total of 33 homologous gene pairs, including 66 SsWRKY genes, were identified to participate in gene duplication events (Table S2). The composition of gene duplication events in each group in ascending order was group I: 73.7% (14 of 19), group II: 78% (46 of 59) and group III: 85.7% (6 of 7). Among the 33 homologous gene pairs, none of them appeared to have undergone TDs, on the contrary, all of the 66 genes (77.6% of all SsWRKY genes) participated in SDs, implying that SDs played major roles in the expansion of willow WRKY genes.

WRKY genes shared more functional and homologies in their conserved WRKY core domains (about 60 residues), while the rest sequences of WRKY genes shared a little (Eulgem, 2000). In order to get a more comprehensive understanding of the structural feature in WRKY domains, the conserved motifs of SsWRKY genes were predicted using the online program MEME (Fig. S3; Table S3). Among the 20 putative motifs, motifs 1, 2, 3 and 5, broadly distributed across SsWRKY genes, were characterized as the WRKY conserved domains. The motif 6 was characterized as nuclear localization signals (NLS), which mainly distributed in subgroup IId and IIe and group III. Some other motifs with poorly defined recently were also predicted by MEME: the motif 4 was only found in group IC and subgroup IIc; motifs 7 and 9 were limited to subgroup IIa and IIb; the motif 8 was found in group I and a few genes of subgroup IIc; motifs 10, 13, 15 and 17 were unique in subgroup IId; the motif 12 was only observed in subgroup IIb; the motif 16 was mainly found in group II; the motif 18 was found in subgroup IIc; motifs 19 and 20 were only observed in subgroup I. The distinct conserved motifs of different groups could be an important foundation for future structural and functional study in WRKY gene family.

Some other important motifs, including Leu zipper motif, HARF, LXXLL and LXLXLX, could be also identified in WRKY genes. Using the online program 2ZIP, the conserved Leu zipper motif, described as a common hypothetical structure to DNA binding proteins (McInerney et al., 1998), was identified in only two SsWRKY genes (SsWRKY61 and SsWRKY39). With manual inspection, the conserved HARF (RTGHARFRR[A/G]P) motifs, whose putative functions were not distinguished clearly, were only observed in seven WRKY genes of subgroup IId, including SsWRKY82, 33, 45, 81, 9, 30 and 56. In the meantime, the conserved LXXLL and LXLXLX (L: Leucine; X: any amino acid) motifs, which respectively defined as the co-activator and active repressor motifs, were also found in SsWRKY genes. A total of seven SsWRKY genes (SsWRKY19, 45, 72, 61, 76, 30 and 59) contained the helical motif LXXLL, whereas eight genes (SsWRKY66, 26, 35, 81, 83, 75, 73 and 3) shared the LXLXLX motif. The plenty of conserved motifs in WRKY genes with different lengths and variant functions, suggesting that the WRKY genes might play more vital roles in gene regulatory network.

Distinct expression profiles of SsWRKY genes in various tissues

In order to gain more information about the roles of WRKY genes in willow, RNA-seq data from the sequenced genotype were used to quantify the expression level of WRKY genes in five tissues of Salix suchowensis. As illustrated in Fig. 5, the expression of all 85 SsWRKY genes were detected in at least one of the five examined tissues, such as 84 genes in roots, 80 in stems, 84 in barks, all in buds and 73 in leaves. Meanwhile, the cluster analysis of the expression pattern in five tissues showed that SsWRKY genes shared more similarities between stem and leaf, as well as bark and bud, and root was more similar to the clade formed by bark and bud. The results detected here were consistent with their biological characteristics. SsWRKY38, not detected in roots and leaves, was also lowly expressed in other tissues. Similarly, SsWRKY74, not detected in stems, barks and leaves, was only expressed in roots and buds with extremely low levels. Among the five genes not expressed in stems, SsWRKY66, 74 and 79 were also not detected in leaves. The largest number of expressed or unexpressed SsWRKY genes (12 genes) was found in buds or leaves, respectively, suggesting that WRKY genes might play more roles in buds than leaves.

According to the expression annotation of 85 SsWRKY genes by RPKM method in Fig. 5 and Table S4, the total transcript abundance of SsWRKY genes in tender root (RPKM = 1,181.21), bark (RPKM = 1,363.01) and vegetative bud (RPKM = 928.58) was relatively larger than that in other two tissues, including non-lignified stem (RPKM = 537.88) and young leaf (RPKM = 349.84). As shown in Table S4, SsWRKY81 (RPKM = 97.75), the most expressed SsWRKY genes in roots, was also expressed in other four tissues, though the expression levels were relatively low; SsWRKY56 (RPKM = 32.54), the most expressed SsWRKY genes in stem, was also highly expressed in other examined tissues. Similarly, SsWRKY67, the most expressed SsWRKY genes in barks (RPKM = 188.16), was also detected in vegetative buds (RPKM = 82.07) and young leaves (RPKM = 26.11) with high expression levels. Similarly, SsWRKY6 (RPKM = 26.31), the most expressed genes in leaves, was also highly detected in other tissues. A few genes, i.e., SsWRKY52, SsWRKY2 and SsWRKY35, were expressed highly in barks, but lowly in other four tissues. The results mentioned above may be an important foundation for the specific expression analysis of each WRKY gene in willow.