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. 2016 Jun 29;5(1):920. doi: 10.1186/s40064-016-2647-x

Genome-wide identification of soybean WRKY transcription factors in response to salt stress

Yanchong Yu 1,2, Nan Wang 1, Ruibo Hu 3, Fengning Xiang 1,✉
PMCID: PMC4927560  PMID: 27386364

Abstract

Members of the large family of WRKY transcription factors are involved in a wide range of developmental and physiological processes, most particularly in the plant response to biotic and abiotic stress. Here, an analysis of the soybean genome sequence allowed the identification of the full complement of 188 soybean WRKY genes. Phylogenetic analysis revealed that soybean WRKY genes were classified into three major groups (I, II, III), with the second group further categorized into five subgroups (IIa–IIe). The soybean WRKYs from each group shared similar gene structures and motif compositions. The location of the GmWRKYs was dispersed over all 20 soybean chromosomes. The whole genome duplication appeared to have contributed significantly to the expansion of the family. Expression analysis by RNA-seq indicated that in soybean root, 66 of the genes responded rapidly and transiently to the imposition of salt stress, all but one being up-regulated. While in aerial part, 49 GmWRKYs responded, all but two being down-regulated. RT-qPCR analysis showed that in the whole soybean plant, 66 GmWRKYs exhibited distinct expression patterns in response to salt stress, of which 12 showed no significant change, 35 were decreased, while 19 were induced. The data present here provide critical clues for further functional studies of WRKY gene in soybean salt tolerance.

Electronic supplementary material

The online version of this article (doi:10.1186/s40064-016-2647-x) contains supplementary material, which is available to authorized users.

Keywords: WRKY, Soybean, Expression patterns, Salt stress

Background

Soybean (Glycine max) is a global cash crop. Apart from its major contribution to human and animal nutrition, the seed provides a feedstock for biodiesel production and represents a significant raw material for a number of pharmaceutical and industrial processes (Phang et al. 2008; Wang et al. 2010). In recent years, the demand for soybean is increasing rapidly, so it attracted more and more attention to improve soybean agronomic traits, such as stress tolerance. Soybean productivity is greatly compromised by soil salt. However, during the long period of evolution, soybean has evolved complex strategies to survive salt stress. These strategies are originated from the changes of various aspects, such as the genome, gene expression, metabolism and physiology (Phang et al. 2008). To present, functionally reported salt tolerance related genes in soybean are mainly categorized into several classes, including ion transporter coding genes (e.g. GmHKT1, GmSALT3, GmNHX1, GmCAX1 and GmCHX1) (Chen et al. 2011; Guan et al. 2014; Li et al. 2006; Luo et al. 2005a; Qi et al. 2014), transcription factors (TFs) (e.g. GmNAC11/-20/-29, GmDREB1, GmMYB76/-92/-174/-177, GmbZIP44/-62/-78, GmWRKY27 and GmERF7) (Hao et al. 2011; Jin et al. 2010; Liao et al. 2008a, b; Wang et al. 2015; Zhai et al. 2013) and others genes (e.g. glutathione S-transferase gene GsGST, late embryogenesis abundant gene GmLEA, calcineurin B-like protein coding gene GmCBL1 and flavone synthase gene GmFNSII) (Ji et al. 2010; Lan et al. 2005; Li et al. 2012; Phang et al. 2008; Wang et al. 2011; Yan et al. 2014; Zhou et al. 2010).

The product of a TF gene binds to a specific cis-regulatory sequence(s) in the promoter of its target gene. The WRKYs are among the largest class of plant TFs, and their promoter target (the W-box) has the sequence (T)(T)TGAC(C/T) (Rushton et al. 2010). WRKY TFs are recognized by the presence of a conserved DNA-binding region composed of about 60 residues (the “WRKY” domain), which harbors the WRKYGQK heptapeptide followed by a C2H2 or C2HC zinc finger motif (Rushton et al. 2010). In some cases, the heptapeptide can take the form WRKYGKK or WRKYGEK (Rushton et al. 2010). WRKY TFs have been classified into three main groups: those possessing two heptapeptides are clustered into group I; both group I and II members harbor one C2H2 type zinc finger motif, while the group III members feature a C2HC one. The large size of group II has been addressed by its division into five subgroups (IIa, IIb, IIc, IId and IIe), based on peptide sequence (Eulgem et al. 2000; Rushton et al. 2010).

Since the first reports of WRKY TFs (dePater et al. 1996; Ishiguro and Nakamura 1994; Rushton et al. 1995, 1996), considerable progress has been made in revealing the function of WRKY TFs. They are elucidated to be involved in various developmental and physiological processes (Rushton et al. 2010), such as seed development (Lagace and Matton 2004; Luo et al. 2005b), seed dormancy and germination (Xie et al. 2005, 2007; Zentella et al. 2007), senescence (Miao et al. 2004; Robatzek and Somssich 2002; Ulker et al. 2007) trichome morphogenesis (Johnson et al. 2002), metabolic pathways (Kato et al. 2007; Sun et al. 2003) and plant development (Cai et al. 2014; Devaiah et al. 2007; Guo et al. 2015; Yu et al. 2013, 2016). The particularly prominent roles of WRKY in plant appear to be the modulation of response to biotic and abiotic stresses (Chen et al. 2012; Eulgem and Somssich 2007; Pandey and Somssich 2009). In the root of the model plant Arabidopsis, 18 WRKY genes have been shown to be induced by exposure to salt stress (Jiang and Deyholos 2006); In rice, ten WRKY genes (of 13 analyzed) respond differentially to a range of abiotic stress treatment (Qiu et al. 2004), while in Brachypodium distachyon, over 60 % of a set of 86 WRKY genes assayed were up-regulated by heat and cold stress and over 50 % were down-regulated by salt, drought and/or oxidation stress (Wen et al. 2014). Among the soybean WRKY TFs, 25 out of 64 have been shown to be differentially expressed in response to at least one abiotic stress treatment (Zhou et al. 2008).

Base on the availability of the complete soybean genome sequence and several databases (PlantTFDB, SoyDB, SoyTFKB, NCBI and Phytozome), a previous study reported a genome-wide characterization of the WRKY family in soybean and a functional analysis of some genes involved in response to Phakopsora pachyrhizi (Bencke-Malato et al. 2014). However, in Phytozome (release v10), the new assembly (v2.0) replaces the Glyma1 assembly. The new database corrects several issues in pseudomolecule reconstruction in the Glyma1 assembly. According to the new database and a recent RNA-Seq result (Belamkar et al. 2014), Song et al. identified 176 GmWRKYs and analyzed their expression files in different tissues and in response to drought and salt stress (Song et al. 2016). However, beside Phytozome, there are many other soybean genome sequence databases. Furthermore, several other transcriptome experiment data sets of soybean under abiotic stress are provided by NCBI website. Here, integrating more databases and RNA-Seq results (Belamkar et al. 2014; Wei et al. 2015), we made a new genome-wide identification of soybean WRKYs and compared their response to salt stress in different tissues. In addition, we analyzed expression profiles of 66 GmWRKYs by quantitative RT-PCR (RT-qPCR). Our findings provide new clues for further investigation of WRKY gene in soybean salt tolerance.

Methods

Retrieval of GmWRKY sequences

A set of 185 GmWRKY sequences was recovered from Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html, release 10.2) using the keyword PF03106 as a search term, along with three further GmWRKY sequences from the NCBI database (http://www.ncbi.nlm.nih.gov). The presence of a WRKY domain(s) in all 188 GmWRKYs was confirmed by running the SMART program (http://smart.embl-heidelberg.de) (Letunic et al. 2015). These GmWRKY genes were further checked in PlantTFDB (http://planttfdb.cbi.pku.edu.cn/, release 3.0) (Jin et al. 2014) and SoyTFKB (http://www.igece.org/Soybean_TF/).

Multiple sequence alignment and phylogenetic analysis

A multiple alignment of the WRKY sequences was performed using the ClustalW program implemented in MEGA v6.06 software package (http://www.megasoftware.net/) (Tamura et al. 2013). The sequences were also subjected to a phylogenetic analysis using the neighbor-joining method; the resulting tree was based on 1000 bootstrap replicates, the p-distance model and pairwise deletion.

Gene structure and conserved motifs analysis

The exon–intron structure of each gene was derived by comparing its coding sequence with the corresponding genomic DNA sequence, using the GSDS program (http://gsds.cbi.pku.edu.cn/) (Hu et al. 2015). The online program MEME v4.10.1 (http://meme-suite.org/tools/meme) was used to identify the conserved motifs present; the relevant parameters were: number of repetitions = any; maximum number of motifs = 16; optimum width of each motif = 6–70 residues.

Genomic location and gene duplication

Each WRKY gene was positioned in the genome by reference to the full genome sequence. The gene duplications in GmWRKY genes were identified based on the investigations described in previous study (Schmutz et al. 2010), and Circos software was used to provide a graphical representation of the position of homeologous chromosome segments (Krzywinski et al. 2009).

RNA-seq analysis

A transcriptomic analysis was based on archival RNA-seq data collected from a set of salt stress experiments, mounted on the NCBI GEO database (Belamkar et al. 2014). Experiments GSM1377923, -24 and -25 represented three independent replicates of plants sampled before any exposure to salt; GSM1377935, -36 and -37 related to plants sampled after a 1 h exposure to the stress; GSM1377938, -39 and -40 after a 6 h exposure; and GSM1377941, -42 and -43 after a 12 h exposure. The other transcriptomic analysis was also based on archival RNA-seq data derived from the NCBI GEO database (Wei et al. 2015), experiment GSE57960 was related to plants sampled after a 12 h exposure to salt stress, the aerial part of plants was used for sequencing. The reads per kilobase of exon model per million mapped reads (RPKM) algorithm was used for normalization and mean normalized values were used for the analysis. The transcription response was given in the form of fold changes relative to the 0 h control. Cluster v3.0 software (University of Tokyo, Human Genome Center) was used to perform hierarchical clustering, which was visualized using Java TreeView software (Saldanha 2004). The relevant parameters were: similarity measurement: correlation (uncentered); linkage method: average linkage method.

Plant materials

Seed of cv. Williams 82 was germinated on a sheet of moist filter paper, and the seedlings were grown under a regime of 28/20 °C, 14 h photoperiod, light intensity 800 μmol m−2 s−1 and relative humidity 55 %. Two weeks old seedlings were exposed to 200 mM NaCl for either 0, 2, 6 or 24 h, after which the whole seedling was harvested, snap-frozen in liquid nitrogen and stored at −80 °C.

RT-qPCR analysis

Total RNA was extracted from the frozen plant material using the TRIzol reagent (Invitrogen, USA) following to the manufacturer’s instructions. The resulting RNA was treated with RNase-free DNaseI (Promega, USA) to remove genomic DNA contamination, and the cDNA First strand was synthesized with 3 μg total RNA by TransScript One-step gDNA Removal and cDNA synthesis SuperMix (TransGen, China) following the manufacturer’s protocol. The subsequent RT-qPCRs and data analysis were performed using a Bio-Rad Real-Time PCR detection system (Bio-Rad) based on the SYBR Green I master mix, as reported previously (Bustin et al. 2009; Seo et al. 2009). According to a previous study, GmELF1b was most stably expressed under salt stress, so it was used as a reference gene (Le et al. 2012). All reactions were carried out in triplicate, using samples harvested from independent plants. The relevant primer sequences are given in Additional file 1: Table S1.

Results and discussion

Identification of WRKY genes in soybean

The WRKYs represent one of the largest families of plant TFs. The acquisition of full genome sequences has simplified the enumeration of WRKY copy number, so that it is now clear that there are 81 WRKY copies in tomato (Huang et al. 2012), 55 in cucumber (Ling et al. 2011), 104 in poplar (He et al. 2012), 59 in grapevine (Wang et al. 2014), 116 in cotton (Dou et al. 2014) and 119 in maize (Wei et al. 2012). In a previous study, 182 putative WRKY gene models were identified (Bencke-Malato et al. 2014). In recent, phytozome updated the soybean assembly; the new assembly (v2.0) replaced the Glyma1 assembly. Therefore, we performed a comprehensive analysis of soybean WRKY sequences obtained from Phytozome, PlantTFDB and NCBI, and finally identified a total of 185 non-redundant putative WRKY genes. Compared with previous 182 WRKY genes, six genes GmWRKY49 (Glyma.05G203900), GmWRKY53 (Glyma.06G061900), GmWRKY72 (Glyma.07G161100), GmWRKY108 (Glyma.10G171000), GmWRKY130 (Glyma.14G085500) and GmWRKY131 (Glyma.14G100100) are novel ones, while three previous genes GmWRKY17 (Glyma06g06530), GmWRKY38 (Glyma18g48460) and GmWRKY132 (Glyma14g11440) are considered obsolete according to the current version of the annotated genome. However, the three obsolete genes have been retained for further study. Thus, a total of 188 annotations of GmWRKYs were presented in this study (Table 1). All the 188 retrieved sequences were proved to contain WRKY domains using SMART analysis.

Table 1.

The WRKY gene family in Soybean

Gene name Gene IDa Conserved heptapeptideb Group Chromosome Amino acid
GmWRKY1 Glyma.01G043300 WRKYGQK IIb Chr1 509
GmWRKY2 Glyma.01G053800 WRKYGQK/WRKYGQK I Chr1 455
GmWRKY3 Glyma.01G056800 WRKYGQK IIc Chr1 297
GmWRKY4 Glyma.01G128100 WRKYGEK/WRKYGQK I Chr1 507
GmWRKY5 Glyma.01G189100 WRKYGQK IId Chr1 321
GmWRKY6 Glyma.01G222300 WRKYGQK IIe Chr1 245
GmWRKY7 Glyma.01G224800 WRKYGQK III Chr1 322
GmWRKY8 Glyma.02G007500 WRKYGQK IIb Chr2 484
GmWRKY9 Glyma.02G010900 WRKYGQK IIc Chr2 320
GmWRKY10 Glyma.02G020300 WRKYGQK IIb Chr2 480
GmWRKY11 Glyma.02G112100 WRKYGQK/WRKYGQK I Chr2 455
GmWRKY12 Glyma.02G115200 WRKYGQK IIc Chr2 293
GmWRKY13 Glyma.02G141000 WRKYGQK IId Chr2 355
GmWRKY14 Glyma.02G203800 WRKYGQK/WRKYGQK I Chr2 505
GmWRKY15 Glyma.02G232600 WRKYGQK/WRKYGQK I Chr2 580
GmWRKY16 Glyma.02G285900 WRKYGQK IIc Chr2 337
GmWRKY17 Glyma.02G293400 WRKYGQK IIb Chr2 401
GmWRKY18 Glyma.02G297400 WRKYGQK/WRKYGQK I Chr2 588
GmWRKY19 Glyma.02G306300 WRKYGQK/WRKYGQK I Chr2 507
GmWRKY20 Glyma.03G002300 Lost　 III Chr3 271
GmWRKY21 Glyma.03G042700 WRKYGEK/WRKYGQK I Chr3 507
GmWRKY22 Glyma.03G109100 WRKYGQK IIc Chr3 238
GmWRKY23 Glyma.03G159700 WRKYGQK IId Chr3 341
GmWRKY24 Glyma.03G176600 WRKYGQK/WRKYGQK I Chr3 448
GmWRKY25 Glyma.03G220100 WRKYGQK IId Chr3 253
GmWRKY26 Glyma.03G220800 WRKYGQK IIc Chr3 287
GmWRKY27 Glyma.03G224700 WRKYGQK IIb Chr3 541
GmWRKY28 Glyma.03G256700 WRKYGQK III Chr3 362
GmWRKY29 Glyma.04G054200 WRKYGKK IIc Chr4 161
GmWRKY30 Glyma.04G061300 WKKYGQK IIa Chr4 222
GmWRKY31 Glyma.04G061400 WRKYGQK IIa Chr4 220
GmWRKY32 Glyma.04G076200 WRKYGQK IId Chr4 279
GmWRKY33 Glyma.04G115500 WRKYGQK/WRKYGQK I Chr4 761
GmWRKY34 Glyma.04G173500 WRKYGQK IIb Chr4 531
GmWRKY35 Glyma.04G218400 WRKYGQK IIc Chr4 234
GmWRKY36 Glyma.04G218700 WRKYGKK IIc Chr4 196
GmWRKY37 Glyma.04G223200 WRKYGQK III Chr4 337
GmWRKY38 Glyma.04G223300 WRKYGQK III Chr4 317
GmWRKY39 Glyma.04G238300 WRKYGQK III Chr4 364
GmWRKY40 Glyma.05G029000 WRKYGQK IIb Chr5 594
GmWRKY41 Glyma.05G096500 WRKYGQK IId Chr5 334
GmWRKY42 Glyma.05G123000 WRKYGQK IIb Chr5 361
GmWRKY43 Glyma.05G123600 WRKYGQK IIe Chr5 430
GmWRKY44 Glyma.05G127600 WRKYGQK IIc Chr5 358
GmWRKY45 Glyma.05G160800 WRKYGQK IIe Chr5 255
GmWRKY46 Glyma.05G165800 WRKYGKR III Chr5 1355
GmWRKY47 Glyma.05G184500 WRKYGKK IIc Chr5 188
GmWRKY48 Glyma.05G185400 WRKYGQK IIc Chr5 216
GmWRKY49 Glyma.05G203900 Lost　 IIc Chr5 99
GmWRKY50 Glyma.05G211900 WRKYGQK IIe Chr5 288
GmWRKY51 Glyma.05G215900 WRKYGQK III Chr5 363
GmWRKY52 Glyma.06G054500 WRKYGKK IIc Chr6 175
GmWRKY53 Glyma.06G061900 WRKYGQK IIa Chr6 309
GmWRKY54 Glyma06g06530* WRKYGQK IIa Chr6 294
GmWRKY55 Glyma.06G077400 WRKYGQK IId Chr6 300
GmWRKY56 Glyma.06G125600 WRKYGQK III Chr6 364
GmWRKY57 Glyma.06G142000 WRKYGQK III Chr6 319
GmWRKY58 Glyma.06G142100 WRKYGQK III Chr6 331
GmWRKY59 Glyma.06G147100 WRKYGKK IIc Chr6 196
GmWRKY60 Glyma.06G147500 WRKYGQK IIc Chr6 236
GmWRKY61 Glyma.06G168400 WRKYGKK IIc Chr6 160
GmWRKY62 Glyma.06G190800 WRKYGQK IIb Chr6 615
GmWRKY63 Glyma.06G212900 WKKYGQK IIa Chr6 242
GmWRKY64 Glyma.06G219800 WRKYGQK/WRKYGQK I Chr6 470
GmWRKY65 Glyma.06G242200 Lost/WRKYGQK I Chr6 176
GmWRKY66 Glyma.06G307700 WRKYGQK IIb Chr6 628
GmWRKY67 Glyma.06G320700 WRKYGQK/WRKYGQK I Chr6 776
GmWRKY68 Glyma.07G023300 WRKYGQK IIa Chr7 311
GmWRKY69 Glyma.07G057400 WRKYGQK III Chr7 369
GmWRKY70 Glyma.07G116300 WRKYGQK IIc Chr7 237
GmWRKY71 Glyma.07G133700 WRKYGQK IId Chr7 317
GmWRKY72 Glyma.07G161100 Lost/WRKYGQK I Chr7 252
GmWRKY73 Glyma.07G227200 WRKYGQK/WRKYGQK I Chr7 533
GmWRKY74 Glyma.07G238000 WRKYGQK IIc Chr7 391
GmWRKY75 Glyma.07G262700 WRKYGQK IIb Chr7 576
GmWRKY76 Glyma.08G011300 WRKYGEK IIc Chr8 147
GmWRKY77 Glyma.08G018300 WRKYGQK IIe Chr8 292
GmWRKY78 Glyma.08G021900 WRKYGQK III Chr8 359
GmWRKY79 Glyma.08G078100 WRKYGQK IIb Chr8 181
GmWRKY80 Glyma.08G078700 WRKYGQK IIe Chr8 429
GmWRKY81 Glyma.08G082400 WRKYGQK IIc Chr8 371
GmWRKY82 Glyma.08G118200 WRKYGQK IIe Chr8 261
GmWRKY83 Glyma.08G142400 WRKYGKK IIc Chr8 184
GmWRKY84 Glyma.08G143400 WRKYGQK IIc Chr8 235
GmWRKY85 Glyma.08G218600 WRKYGQK IIa Chr8 313
GmWRKY86 Glyma.08G240800 WRKYGQK/WRKYGQK I Chr8 523
GmWRKY87 Glyma.08G320200 WRKYGQK IIb Chr8 486
GmWRKY88 Glyma.08G325800 WRKYGQK/WRKYGQK I Chr8 577
GmWRKY89 Glyma.09G005700 WRKYGQK IIb Chr9 541
GmWRKY90 Glyma.09G029800 WRKYGQK IIe Chr9 506
GmWRKY91 Glyma.09G034300 WRKYGQK IIc Chr9 331
GmWRKY92 Glyma.09G061900 WRKYGQK IId Chr9 296
GmWRKY93 Glyma.09G080000 WRKYGQK IIb Chr9 458
GmWRKY94 Glyma.09G127100 WRKYGQK IIb Chr9 242
GmWRKY95 Glyma.09G129100 WRKYGQK IIe Chr9 372
GmWRKY96 Glyma.09G240000 WRKYGQK IIb Chr9 541
GmWRKY97 Glyma.09G244000 WRKYGQK IIc Chr9 238
GmWRKY98 Glyma.09G250500 WRKYGQK/WRKYGQK I Chr9 734
GmWRKY99 Glyma.09G254400 WRKYGQK IIc Chr9 192
GmWRKY100 Glyma.09G254800 WRKYGQK IIe Chr9 348
GmWRKY101 Glyma.09G274000 WRKYGQK III Chr9 300
GmWRKY102 Glyma.09G280200 WIKYGQK/WRKYGQK I Chr9 543
GmWRKY103 Glyma.10G011300 WRKYGQK IIc Chr10 323
GmWRKY104 Glyma.10G032900 WRKYGQK IId Chr10 392
GmWRKY105 Glyma.10G111400 Lost　 IIb Chr10 305
GmWRKY106 Glyma.10G113800 WRKYGKK IIa Chr10 120
GmWRKY107 Glyma.10G138300 WRKYGQK IIb Chr10 482
GmWRKY108 Glyma.10G171000 WHQYGLK IIc Chr10 367
GmWRKY109 Glyma.10G171100 WRKYGQK IIc Chr10 192
GmWRKY110 Glyma.10G171200 WRKYGQK IIc Chr10 336
GmWRKY111 Glyma.10G230200 WRKYGQK IIe Chr10 297
GmWRKY112 Glyma.11G021200 WRKYGQK IIe Chr11 214
GmWRKY113 Glyma.11G053100 WRKYGQK IId Chr11 321
GmWRKY114 Glyma.11G163300 WRKYGQK/WRKYGQK I Chr11 548
GmWRKY115 Glyma.12G097100 WRKYGQK IIb Chr12 614
GmWRKY116 Glyma.12G152600 WRKYGQK/WRKYGQK I Chr12 467
GmWRKY117 Glyma.12G212300 WRKYGQK IIe Chr12 263
GmWRKY118 Glyma.13G102000 WRKYGQK IId Chr13 324
GmWRKY119 Glyma.13G117600 WRKYGQK IIb Chr13 383
GmWRKY120 Glyma.13G267400 WRKYGQK III Chr13 294
GmWRKY121 Glyma.13G267500 WRKYGQK III Chr13 296
GmWRKY122 Glyma.13G267600 WRKYGQK III Chr13 300
GmWRKY123 Glyma.13G267700 WRKYGQK III Chr13 270
GmWRKY124 Glyma.13G289400 WRKYGQK IIe Chr13 265
GmWRKY125 Glyma.13G310100 WRKYGQK IIb Chr13 614
GmWRKY126 Glyma.13G370100 WRKYGQK IIa Chr13 309
GmWRKY127 Glyma.14G006800 WRKYGQK/WRKYGQK I Chr14 508
GmWRKY128 Glyma.14G016200 WRKYGQK/WRKYGQK I Chr14 585
GmWRKY129 Glyma.14G028900 WRKYGQK IIc Chr14 335
GmWRKY130 Glyma.14G085500 Lost　 IId Chr14 276
GmWRKY131 Glyma.14G100100 WRKYGKK IIc Chr14 68
GmWRKY132 Glyma14g11440* WRKYGKK IIc Chr14 137
GmWRKY133 Glyma.14G102900 WRKYGQK IIa Chr14 278
GmWRKY134 Glyma.14G103100 WRKYGQK IIa Chr14 282
GmWRKY135 Glyma.14G135400 WRKYGQK IId Chr14 316
GmWRKY136 Glyma.14G185800 WRKYGQK III Chr14 329
GmWRKY137 Glyma.14G186000 WRKYGQK III Chr14 303
GmWRKY138 Glyma.14G186100 WRKYGQK III Chr14 240
GmWRKY139 Glyma.14G199800 WRKYEDK III Chr14 332
GmWRKY140 Glyma.14G200200 WRKYGQK/WRKYGQK I Chr14 575
GmWRKY141 Glyma.15G003300 WRKYGQK IIa Chr15 330
GmWRKY142 Glyma.15G110300 WRKYGQK IIb Chr15 599
GmWRKY143 Glyma.15G135600 WRKYGQK IIe Chr15 523
GmWRKY144 Glyma.15G139000 WRKYGQK IIc Chr15 356
GmWRKY145 Glyma.15G168200 WRKYGQK IId Chr15 293
GmWRKY146 Glyma.15G186300 WRKYGQK IIb Chr15 451
GmWRKY147 Glyma.16G026400 WRKYGQK III Chr16 373
GmWRKY148 Glyma.16G031400 WRKYGQK IIc Chr16 195
GmWRKY149 Glyma.16G031900 WRKYGQK IIe Chr16 335
GmWRKY150 Glyma.16G054400 WRKYGQK IIc Chr16 195
GmWRKY151 Glyma.16G176700 WRKYGQK IIe Chr16 274
GmWRKY152 Glyma.16G177000 WRKYGQK IIe Chr16 408
GmWRKY153 Glyma.16G219800 WRKYGQK III Chr16 265
GmWRKY154 Glyma.17G011400 WRKYGQK IIb Chr17 489
GmWRKY155 Glyma.17G035400 WRKYGQK IIc Chr17 398
GmWRKY156 Glyma.17G042300 WRKYGQK IIb Chr17 391
GmWRKY157 Glyma.17G057100 WRKYGQK IId Chr17 320
GmWRKY158 Glyma.17G074000 WRKYGQK/WRKYGQK I Chr17 505
GmWRKY159 Glyma.17G097900 WRKYGQK IIb Chr17 600
GmWRKY160 Glyma.17G168900 WRKYGQK IId Chr17 332
GmWRKY161 Glyma.17G197500 WRKYGQK IId Chr17 316
GmWRKY162 Glyma.17G222300 WRKYGQK IIa Chr17 312
GmWRKY163 Glyma.17G222500 WRKYGQK IIa Chr17 278
GmWRKY164 Glyma.17G224800 WRKYGKK IIc Chr17 164
GmWRKY165 Glyma.17G239200 WRKYGQK IId Chr17 278
GmWRKY166 Glyma.18G056600 WRKYGQK/WRKYGQK I Chr18 542
GmWRKY167 Glyma.18G081200 WRKYGQK/WRKYGQK I Chr18 577
GmWRKY168 Glyma.18G092200 WRKYGQK IIb Chr18 478
GmWRKY169 Glyma.18G124700 WRKYGQK IIb Chr18 529
GmWRKY170 Glyma.18G183100 WRKYGQK IId Chr18 308
GmWRKY171 Glyma.18G208800 WRKYGQK/WRKYGQK I Chr18 541
GmWRKY172 Glyma.18G213200 WRKYGQK III Chr18 299
GmWRKY173 Glyma.18G238200 WRKYGQK IIe Chr18 351
GmWRKY174 Glyma.18G238600 WRKYGQK IIc Chr18 192
GmWRKY175 Glyma.18G242000 WRKYGQK/WRKYGQK I Chr18 744
GmWRKY176 Glyma.18G256500 WRKYGQK IIb Chr18 541
GmWRKY177 Glyma.18G263400 WRKYGQK/WRKYGQK I Chr18 520
GmWRKY178 Glyma18g48460* WRKYGQK IIc Chr18 225
GmWRKY179 Glyma.19G020600 WRKYGQK IIb Chr19 495
GmWRKY180 Glyma.19G094100 WRKYGQK IIc Chr19 188
GmWRKY181 Glyma.19G177400 WRKYGQK/WRKYGQK I Chr19 471
GmWRKY182 Glyma.19G217000 WRKYGQK IId Chr19 264
GmWRKY183 Glyma.19G217800 WRKYGQK IIc Chr19 290
GmWRKY184 Glyma.19G221700 WRKYGQK IIb Chr19 516
GmWRKY185 Glyma.19G254800 WRKYGQK III Chr19 362
GmWRKY186 Glyma.20G028000 WRKYGQK/WRKYGQK I Chr20 439
GmWRKY187 Glyma.20G030500 WRKYGQK IIb Chr20 163
GmWRKY188 Glyma.20G163200 WRKYGQK IIe Chr20 321
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aGenes that are not annotated in the new assembly (v2.0) are marked with the star symbol

bThe variants of conserved WRKYGQK peptide are shown in red color and some conserved WRKYGQK sequences are lost in several members

In previous genome-wide studies, the commonly accepted nomenclature for WRKY members was based on their location order on chromosomes (Dou et al. 2014; Ling et al. 2011). Identically, in the present study, GmWRKYs was designated from GmWRKY1 to GmWRKY188 based on their exact physical position from the top to the bottom on the soybean chromosomes 1–20 (Table 1). For genes producing more than one transcript, only the primary sequence was named. This nomenclature system was different from previous study (Bencke-Malato et al. 2014). A full comparison of currently known WRKY genes is given in Additional file 2: Table S2.

In silico mapping revealed that the WRKY genes were distributed over all 20 soybean chromosomes. Chromosome 7 harbored the highest number of GmWRKY genes (16, 8.51 %), while chromosome 11, 12 and 20 harbored only three (1.60 %). The largest WRKY product was encoded by GmWRKY46 (1355 residues), and the shortest was GmWRKY131 (68 residues) (Fig. 1; Table 1).

Fig. 1.

Fig. 1

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The distribution of WRKY genes on the 20 chromosomes of soybean

Classification of WRKY genes in soybean

As described previously, WRKY family is typically categorized into three main groups defined by the number of WRKY domains present and the configuration of their zinc finger (Rushton et al. 2010). The 188 soybean WRKY genes were also categorized into the three main groups (Additional file 3: Fig. S1). The group I members numbered 32 (GmWRKY65 and -72 harbored a single N-terminal WRKY domain); there were 130 sequences assigned to group II, sub-divided into subgroup IIa (14 members), IIb (33 members), IIc (42 members), IId (21 members) and IIe (20 members); the remaining 26 sequences belonged to group III (Table 1; Fig. 1). In Arabidopsis and poplar, group I houses the largest number of WRKYs, while in rice, group III is the largest (He et al. 2012). However, the largest group in soybean is group II, implying that this group had experienced more gene duplications during the evolutionary course.

Although most of the sequences harbored the well conserved WRKYGQK motif, variants were present in 24 of the sequences: WRKYGKK in 11, WRKYGEK in three, WKKYGQK in two, and WRKYGKR, WRKYEDK, WIKYGQK and WHQYGLK each in one. Strikingly, A WRKYGQK-like stretch was lacking in GmWRKY20, -49, -105 and -130, while the group I members WRKY65 and -72 had both lost their N terminal WRKYGQK-like stretch (Table 1; Additional file 3: Fig. S1). The largest number of variants belonged to group IIc, 11 out of 24. The WRKYGQK sequence was highly conserved in subgroups IIb, IId and IIe, as well as in the C terminal WRKY domain of group I members (Table 1; Additional file 3: Fig. S1). This is consistent with the implication that this group experienced more gene duplications. There was also some variation in the zinc finger motif (including its complete absence) in 11 of the sequences (WRKY6, -42, -52, -65, -72, -79, -94, -106, -112, -139 and -165) (Additional file 3: Fig. S1).

Gene duplication of soybean WRKY genes

Duplication events contribute not only to functional redundancy, but also generate functional novelty (Moore and Purugganan 2005). The modern soybean genome has undergone two whole genome duplication (WGD) events, the first, associated with the evolution of the legume clade occurred ~59 million years ago (Lavin et al. 2005), while the second, which was responsible for the creation of the Glycine genus, occurred ~13 million years ago (Schmutz et al. 2010). To investigate whether the expansion of GmWRKY genes had primarily happened during both WGD events, we mapped the GmWRKYs to the duplicated blocks (Fig. 2). Consistent with previous study (Schmutz et al. 2010), the blocks between chromosomes involved more than just two chromosomes. Of 188 GmWRKYs, 180 (95.7 %) genes were located in the blocks (the exceptions were GmWRKY46, -63, -65, -72, -94, -105, -106, and -139) (Fig. 2), indicating that WGD was the primary reason for the expansion of GmWRKYs (Fig. 2).

Fig. 2.

Fig. 2

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Chromosomal location of the soybean WRKY genes. The illustrated genome-wide chromosome organization caused by whole genome duplication events is accomplished using the Circos software based on the duplication coordinates defined in the current genome assembly v2.0. Segmental duplicated blocks are color coded. Paralogous pairs are connected with lines

Besides WGD, tandem duplication event is the other approach for gene expansion. Precise mapping analysis showed the presence of 14 adjacent genes possibly due to tandem duplication (Fig. 2; Additional file 4: Fig. S2a). These 14 WRKY genes were localized in 6 distinct tandem duplicate gene clusters, with four clusters containing two tandem genes (GmWRKY120/123, GmWRKY121/122, GmWRKY131/132 and GmWRKY151/152) and two clusters possessing three ones (GmWRKY108/109/110 and GmWRKY136/137/138). All the 14 tandem duplicated WRKY genes were mapped onto the duplicated blocks, implying that local duplications occurred earlier than the WGD.

Gene structure and conserved motifs of GmWRKYs

Gene structural diversity may reflect the evolution of multigene families (Hu et al. 2010). In order to look into the structural diversity of GmWRKY genes, we first constructed a phylogenetic tree based on the full-length GmWRKY polypeptide sequences, and they were also categorized into seven subfamiles as above (Additional file 4: Fig. S2a). From the tree, we could find that each clade consists of two to four genes, which well matched the two WGD events and confirmed that the expansion of GmWRKY happened during both WGD events. We then analyzed the exon–intron organization in the coding sequences of each soybean WRKY genes HD-ZIP genes (Additional file 4: Fig. S2b). Previous study showed that most Populus WRKY genes contain two to four introns (He et al. 2012). Similarly, the majority of soybean WRKY members harbored two to four introns. For instance, over 60 % members of subgroups IIc (26/42), IId (17/21), IIe (14/20) and III (23/26) harbored two introns; over 60 % group I members (17/32) harbored four; most members in group IIa harbored three (7/14) or four (5/14) (Additional file 4: Fig. S2b). In contrast, the gene structure appeared to be more variable in groups IIb, the number of introns in this group varied from one to six (Additional file 4: Fig. S2b). In Populus, although there were only eight members in group IIb, the numbers of introns varied from three to six (He et al. 2012). These results indicated that WRKY genes in different species were relatively conserved during the evolution. Furthermore, genes shared similar exon–intron organization within the same subgroup, while they were strikingly distinct in the gene structure among different groups, suggesting that they were not only conserved, but diverged during the evolution.

To better understand the conservation and diversification of WRKY genes in soybean, putative motifs of GmWRKYs were predicted using MEME software and finally 16 distinct motifs were identified (Additional file 4: Fig. S2c). As expected, most of the closely related members in the phylogenetic tree shared common motif compositions, suggesting that the WRKY proteins within the same subfamily might be of similar functions. However, like putative motifs predicted in ZmWRKYs (Gao et al. 2014), the biological significance of most of the putative motifs in GmWRKYs was also unclear because they did not have homologs when searching against Pfam (http://pfam.sanger.ac.uk/search) and SMART (Simple Modular Architecture Research Tool) databases. The same phenomenon also existed in Populus NAC and HD-ZIP proteins (Hu et al. 2010, 2012). According to previous study, WRKY proteins harbor typical WRKY domains and zinc-finger motifs (Eulgem et al. 2000; Rushton et al. 2010). Here, motif 1, 2 and 9 comprised the WRKY domain, motif 3 and 10 were the partial zinc-finger motifs followed motif 2 and 9 (Additional file 5: Table S3). The product size of the group I and subgroup IIb genes was larger than that of members of the other groups (Table 1), consistent with their harboring a greater number of motifs (Additional file 4: Fig. S2c). In contrast, although subgroup IIc possessed the largest number of members, they harbored the least number of motifs (one to three). Even though the C-terminal regions of GmWRKYs were highly divergent, we could also identify several conserved motifs which were present in GmWRKYs from specific subgroups, for example, motifs 3, 5 and 6 in group I, motif 14 in subgroup IIb, and motif 13 in subgroups IIa and IIb (Additional file 4: Fig. S2c). Whether these motifs play functional roles remained to be further elucidated.

Expression profiles of GmWRKYs in response to salt stress

The WRKY gene family is heavily implicated in the plant response to abiotic stress (Chen et al. 2012), as indicated by a number of microarray-based transcriptomic data sets. Several studies have reported the influence of abiotic stress on WRKY genes based on these data sets (Dou et al. 2014; Satapathy et al. 2014; Wei et al. 2012). In soybean (cv. Kefeng No. 1), the response of a set of 64 WRKY genes following the plant’s exposure to salt stress has been described (Zhou et al. 2008), but this number represents only about one-third of the total WRKY genes. In order to give insight to the function of GmWRKYs in plant response to salt tolerance, we analyzed the soybean (cv. Williams 82) gene expression profiles under salt stress (Belamkar et al. 2014). Finally, 66 of the 188 GmWRKY genes were transcriptionally regulated under salt stress (Fig. 3a; Additional file 6: Table S4). 65 genes were up-regulated, with only WRKY71 being down-regulated (Fig. 3a). The response of WRKY was typically quite rapid (Eulgem et al. 2000), most notably in the case of GmWRKY20, -47, -76, -126, -134, -153, -164, which responded by an at least five fold rise in transcript abundance after a 1 h exposure to the stress. In some cases, the response was transient: some examples were the genes WRKY44, -51, -54, -78, -81, -85, -102 and -107, for which transcript abundance peaked after a 6 h exposure and then fell away (Fig. 3a; Additional file 6: Table S4). The most responsive gene (WRKY134) belonged to subgroup IIa, which was increased to ~226 fold after a 6 h exposure (Fig. 3a; Additional file 6: Table S4). By contrast, the expression of GmWRKY71, a member of group IId was down-regulated in response to salt stress (Fig. 3a).

Fig. 3.

Fig. 3

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The transcription response of the WRKY genes in response to salt stress. Transcript abundance levels have been normalized and hierarchical clustered. Blue colored blocks indicate a decreased and yellow ones an increased level of transcription relative to the control. a The set of 66 genes transcriptionally altered in soybean root by the stress. Genes with remarkable changed expressions are labeled in red. b–d The transcription profiles of genes belonging to subgroups b IIb and c IIc and to d group III. hr number of hours of exposure to the stress. e The set of 49 genes transcriptionally altered in the aerial part of soybean by the stress

In soybean, a notable number of responsive genes belonged to subgroup IIb (18 of 33), although their level of induction by salt treatment was only modest (Fig. 3b). In addition, most of subgroup IIc (14/42) and group III (14/26) members were significantly induced and these genes tended to be dramatically up-regulated after a 6 h exposure (Fig. 3c, d). In Arabidopsis root, the 18 salt induced members belong to group I (4/18), II (11/18) and III (3/18), respectively (Jiang and Deyholos 2006). These data indicated that either in soybean or Arabidopsis, group II members made major contribution in salt response, suggesting that WRKY functions in response to salt stress in different organisms appeared to be conserved during evolution.

The material used in the above RNA-seq was soybean root which was not able to represent other parts. We then analyzed the other RNA-seq data which were derived from the aerial part of soybean plants (cv. SuiNong 28) (Wei et al. 2015). A total of 49 GmWRKY genes were transcriptionally regulated under salt stress (Fig. 3e; Additional file 7: Table S5). 47 genes were down-regulated, with only two (WRKY155 and WRKY183) being up-regulated (Fig. 3e). These results were quite different with the above RNA-seq analysis, indicating that GmWRKY genes showed distinct response profiles in different tissues.

Investigation of GmWRKY gene expressions by RT-qPCR

The transcriptional profiles we analyzed above could provide clues for revealing the function of GmWRKYs in plant response to salt tolerance. However, the material used in RNA-seq was soybean root or aerial part which was not able to represent the entire plant. To shed light on the expression profiles of GmWRKY genes, 2 weeks old soybean seedlings (cv. Williams 82) were exposed to 200 mM NaCl for 0, 2, 6 or 24 h, respectively, and then the total RNA of the whole plant was isolated used for RT-qPCR analysis. 66 GmWRKY genes were tested and exhibited distinct expression patterns in response to salt stress, of which 12 showed no significant change (Fig. 4a), 35 were decreased (Fig. 4b), while 19 were induced (Fig. 4c). GmWRKY38, -120 and -185 were substantially decreased, especially GmWRKY120. In contrast, GmWRKY20, -89, -114 and -142 were remarkably induced. These expression patterns were different with the above RNA-seq analysis, indicating that GmWRKY genes showed distinct response profiles in the whole plants compared to different tissues. The response of WRKY to abiotic stresses was generally rapid and transient (Eulgem et al. 2000). Likely, most of the GmWRKY genes responded rapidly, their expressions were decreased (31/35) or induced (16/19) after only a 2 h exposure (Fig. 4b, c). In addition, the response of GmWRKYs was transient, such as GmWRKY36, -82, -83, -141, -153, -159 and -66 (Fig. 4b, c).

Fig. 4.

Fig. 4

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RT-qPCR-based transcription profiling of 66 WRKY genes. a Unaffected WRKY genes. b Salt-inhibited WRKY genes. c Salt-inducible WRKY genes. Error bars represent SD (n = 3). (t test, *P < 0.05; **P < 0.01)

In tomato and cucumber, most WRKYs are up-regulated by salt stress (Huang et al. 2012; Ling et al. 2011). In contrast, the majority of Brachypodium distachyonWRKYs are down-regulated by the stress (Wen et al. 2014). In soybean, most WRKYs were up-regulated in root, while down-regulated in the aerial part by the stress (Fig. 4). Species differences presumably reflected a major degree of functional divergence in the WRKY gene family.

Conclusion

The present study has taken a genome-wide view of the soybean WRKY gene family, and characterized their transcriptional response to salt stress. An analysis of their phylogeny, chromosomal location, gene structure and content of conserved motifs has allowed the genes to be classified into the standard set of groups. The expansion in copy number of the GmWRKYs has occurred largely as a result of the two well recognized ancient whole genome duplication events. To date, only three GmWRKY genes have been functionally investigated (Jiang and Deyholos 2009), leaving unknown the function of the remaining more than 180. The responsiveness to salt stress of about one-third of the GmWRKY complement confirms the potential of gene manipulation within this gene family as means of improving the salt tolerance of important crop species.

Authors’ contributions

YY were involved in designing the research. YY and RH collected and analyzed the data. YY and NW performed the experiments. YY and FX wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This research was financially supported by the National High Technology Research and Development Program “863” (Grant No. 2013AA102602-4), National Special Science Research Program of China (Grant No. 2013CB967300), National Natural Science Foundation (Grant Nos. 30970243, 31270328, 31471515), National Key Technology R&D Program of the Ministry of Science and Technology (Grant No. 2011BAI06B01), China Postdoctoral Science Foundation funded project (Grant No. 2014M551893), National Transgenic Project of China (Grant No. 2013ZX08010002-002), Science & Technology Plan of Shandong Province (Grant No. 2013GNC11010), and Research Program for International S&T Cooperation Projects of Shandong Province (Grant No. 2011176). We thank Dr. Robert Koebner (UK) for English editing of the manuscript and Chengming Fan (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for his skillful technical assistance in the present study.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

NCBI

National Center for Biotechnology Information

PlantTFDB

Plant Transcription Factor Database

SoyDB

Database of Soybean Transcription Factors

SoyTFKB

Soybean Transcription Factor Knowledge Base

SMART

Simple Modular Architecture Research Tool

GSDS

Gene Structure Display Server

MEME

Multiple Em for Motif Elicitation

Additional files

40064_2016_2647_MOESM1_ESM.doc (146.5KB, doc)

10.1186/s40064-016-2042-7 Primers used in this study.

40064_2016_2647_MOESM2_ESM.doc (264KB, doc)

10.1186/s40064-016-2042-7 A comparison between the WRKY genes identified in the current study and those described previously (Bencke-Malato et al. 2014).

40064_2016_2647_MOESM3_ESM.eps (1.2MB, eps)

10.1186/s40064-016-2042-7 Multiple alignment of the conserved WRKY domain sequences.

40064_2016_2647_MOESM4_ESM.eps (5.2MB, eps)

10.1186/s40064-016-2042-7 The phylogeny, gene structure and conserved motifs of the soybean WRKY gene family. (a) A multiple sequence alignment of 188 full length polypeptide sequences. The seven groups/subgroups (I, IIa-e and III) are depicted by different colors. (b) Exon–intron structures. (c) The 16 conserved motifs as identified by MEME software. Detailed sequence information given in Additional file 4: Table S3.

40064_2016_2647_MOESM5_ESM.doc (35.5KB, doc)

10.1186/s40064-016-2042-7 Conserved WRKY motif sequences as predicted by MEME software.

40064_2016_2647_MOESM6_ESM.doc (90.5KB, doc)

10.1186/s40064-016-2042-7 Normalized transcript levels of 66 GmWRKY genes in root under salinity stress conditions.

40064_2016_2647_MOESM7_ESM.doc (67.5KB, doc)

10.1186/s40064-016-2042-7 Normalized transcript levels of 49 GmWRKY genes in aerial part under salinity stress conditions.

Contributor Information

Yanchong Yu, Email: fuhuodeyihui@163.com.

Nan Wang, Email: wangnan1991gogo@163.com.

Ruibo Hu, Email: hurb@qibebt.ac.cn.

Fengning Xiang, Phone: 86-531-88363629, Email: xfn0990@sdu.edu.cn.

References

  1. Belamkar V, Weeks NT, Bharti AK, Farmer AD, Graham MA, Cannon SB. Comprehensive characterization and RNA-Seq profiling of the HD-Zip transcription factor family in soybean (Glycine max) during dehydration and salt stress. BMC Genom. 2014;15:950. doi: 10.1186/1471-2164-15-950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bencke-Malato M, Cabreira C, Wiebke-Strohm B, Bucker-Neto L, Mancini E, Osorio MB, Homrich MS, Turchetto-Zolet AC, De Carvalho MCCG, Stolf R, Weber RLM, Westergaard G, Castagnaro AP, Abdelnoor RV, Marcelino-Guimaraes FC, Margis-Pinheiro M, Bodanese-Zanettini MH. Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection. BMC Plant Biol. 2014;14:236. doi: 10.1186/s12870-014-0236-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–622. doi: 10.1373/clinchem.2008.112797. [DOI] [PubMed] [Google Scholar]
  4. Cai YH, Chen XJ, Xie K, Xing QK, Wu YW, Li J, Du CH, Sun ZX, Guo ZJ. Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice. PLoS ONE. 2014;9:e102529. doi: 10.1371/journal.pone.0102529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen H, He H, Yu D. Overexpression of a novel soybean gene modulating Na+ and K+ transport enhances salt tolerance in transgenic tobacco plants. Physiol Plantarum. 2011;141:11–18. doi: 10.1111/j.1399-3054.2010.01412.x. [DOI] [PubMed] [Google Scholar]
  6. Chen LG, Song Y, Li SJ, Zhang LP, Zou CS, Yu DQ. The role of WRKY transcription factors in plant abiotic stresses. BBA-Gene Regul Mech. 2012;1819:120–128. doi: 10.1016/j.bbagrm.2011.09.002. [DOI] [PubMed] [Google Scholar]
  7. dePater S, Greco V, Pham K, Memelink J, Kijne J. Characterization of a zinc-dependent transcriptional activator from Arabidopsis. Nucleic Acids Res. 1996;24:4624–4631. doi: 10.1093/nar/24.23.4624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devaiah BN, Karthikeyan AS, Raghothama KG. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 2007;143:1789–1801. doi: 10.1104/pp.106.093971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dou LL, Zhang XH, Pang CY, Song MZ, Wei HL, Fan SL, Yu SX. Genome-wide analysis of the WRKY gene family in cotton. Mol Genet Genomics. 2014;289:1103–1121. doi: 10.1007/s00438-014-0872-y. [DOI] [PubMed] [Google Scholar]
  10. Eulgem T, Somssich IE. Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol. 2007;10:366–371. doi: 10.1016/j.pbi.2007.04.020. [DOI] [PubMed] [Google Scholar]
  11. Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000;5:199–206. doi: 10.1016/S1360-1385(00)01600-9. [DOI] [PubMed] [Google Scholar]
  12. Gao J, Peng H, He XJ, Luo M, Chen Z, Lin HJ, Ding HP, Pan GT, Zhang ZM. Molecular phylogenetic characterization and analysis of the WRKY transcription factor family responsive to Rhizoctonia solani in maize. Maydica. 2014;59:32–41. [Google Scholar]
  13. Guan RX, Qu Y, Guo Y, Yu LL, Liu Y, Jiang JH, Chen JG, Ren YL, Liu GY, Tian L, Jin LG, Liu ZX, Hong HL, Chang RZ, Gilliham M, Qiu LJ. Salinity tolerance in soybean is modulated by natural variation in GmSALT3. Plant J. 2014;80:937–950. doi: 10.1111/tpj.12695. [DOI] [PubMed] [Google Scholar]
  14. Guo DS, Zhang JZ, Wang XL, Han X, Wei BY, Wang JQ, Li BX, Yu H, Huang QP, Gu HY, Qu LJ, Qin GJ. The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis. Plant Cell. 2015;27:3112–3127. doi: 10.1105/tpc.15.00829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hao YJ, Wei W, Song QX, Chen HW, Zhang YQ, Wang F, Zou HF, Lei G, Tian AG, Zhang WK, Ma B, Zhang JS, Chen SY. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J. 2011;68:302–313. doi: 10.1111/j.1365-313X.2011.04687.x. [DOI] [PubMed] [Google Scholar]
  16. He HS, Dong Q, Shao YH, Jiang HY, Zhu SW, Cheng BJ, Xiang Y. Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa. Plant Cell Rep. 2012;31:1199–1217. doi: 10.1007/s00299-012-1241-0. [DOI] [PubMed] [Google Scholar]
  17. Hu RB, Qi G, Kong YZ, Kong DJ, Gao QA, Zhou GK. Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa. BMC Plant Biol. 2010;10:145. doi: 10.1186/1471-2229-10-145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hu RB, Chi X, Chai G, Kong Y, He G, Wang X, Shi D, Zhang D, Zhou G (2012) Genome-wide identification, evolutionary expansion, and expression profile of homeodomain-leucine zipper gene family in poplar (Populus trichocarpa). PLoS One 7(2):e31149 [DOI] [PMC free article] [PubMed]
  19. Hu B, Jin JP, Guo AY, Zhang H, Luo JC, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296–1297. doi: 10.1093/bioinformatics/btu817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huang SX, Gao YF, Liu JK, Peng XL, Niu XL, Fei ZJ, Cao SQ, Liu YS. Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum. Mol Genet Genomics. 2012;287:495–513. doi: 10.1007/s00438-012-0696-6. [DOI] [PubMed] [Google Scholar]
  21. Ishiguro S, Nakamura K. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato. Mol Gen Genet. 1994;244:563–571. doi: 10.1007/BF00282746. [DOI] [PubMed] [Google Scholar]
  22. Ji W, Zhu YM, Li Y, Yang LA, Zhao XW, Cai H, Bai X. Over-expression of a glutathione S-transferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco. Biotechnol Lett. 2010;32:1173–1179. doi: 10.1007/s10529-010-0269-x. [DOI] [PubMed] [Google Scholar]
  23. Jiang YQ, Deyholos MK. Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol. 2006;6:25. doi: 10.1186/1471-2229-6-25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jiang YQ, Deyholos MK. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol. 2009;69:91–105. doi: 10.1007/s11103-008-9408-3. [DOI] [PubMed] [Google Scholar]
  25. Jin TC, Chang Q, Li WF, Yin DX, Li ZJ, Wang DL, Liu B, Liu LX. Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell Tiss Org. 2010;100:219–227. doi: 10.1007/s11240-009-9628-5. [DOI] [Google Scholar]
  26. Jin JP, Zhang H, Kong L, Gao G, Luo JC. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res. 2014;42:D1182–D1187. doi: 10.1093/nar/gkt1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnson CS, Kolevski B, Smyth DR. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell. 2002;14:1359–1375. doi: 10.1105/tpc.001404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, Dubouzet JG, Yazaki K, Sato F. Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol. 2007;48:8–18. doi: 10.1093/pcp/pcl041. [DOI] [PubMed] [Google Scholar]
  29. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–1645. doi: 10.1101/gr.092759.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lagace M, Matton DP. Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta. 2004;219:185–189. doi: 10.1007/s00425-004-1253-2. [DOI] [PubMed] [Google Scholar]
  31. Lan Y, Cai D, Zheng YZ. Expression in Escherichia coli of three different soybean late embryogenesis abundant (LEA) genes to investigate enhanced stress tolerance. J Integr Plant Biol. 2005;47:613–621. doi: 10.1111/j.1744-7909.2005.00025.x. [DOI] [Google Scholar]
  32. Lavin M, Herendeen PS, Wojciechowski MF. Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary. Syst Biol. 2005;54:575–594. doi: 10.1080/10635150590947131. [DOI] [PubMed] [Google Scholar]
  33. Le DT, Aldrich DL, Valliyodan B, Watanabe Y, Van Ha C, Nishiyama R, Guttikonda SK, Quach TN, Gutierrez-Gonzalez JJ, Tran LSP, Nguyen HT (2012) Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS One 7(9):e46487 [DOI] [PMC free article] [PubMed]
  34. Letunic I, Doerks T, Bork P. SMART: recent updates, new developments and status in 2015. Nucleic Acids Res. 2015;43:D257–D260. doi: 10.1093/nar/gku949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li WYF, Wong FL, Tsai SN, Phang TH, Shao GH, Lam HM. Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. Plant, Cell Environ. 2006;29:1122–1137. doi: 10.1111/j.1365-3040.2005.01487.x. [DOI] [PubMed] [Google Scholar]
  36. Li ZY, Xu ZS, He GY, Yang GX, Chen M, Li LC, Ma YZ. Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis. Biochem Biophys Res Commun. 2012;427:731–736. doi: 10.1016/j.bbrc.2012.09.128. [DOI] [PubMed] [Google Scholar]
  37. Liao Y, Zou HF, Wang HW, Zhang WK, Ma B, Zhang JS, Chen SY. Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants. Cell Res. 2008;18:1047–1060. doi: 10.1038/cr.2008.280. [DOI] [PubMed] [Google Scholar]
  38. Liao Y, Zou HF, Wei W, Hao YJ, Tian AG, Huang J, Liu YF, Zhang JS, Chen SY. Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis. Planta. 2008;228:225–240. doi: 10.1007/s00425-008-0731-3. [DOI] [PubMed] [Google Scholar]
  39. Ling J, Jiang WJ, Zhang Y, Yu HJ, Mao ZC, Gu XF, Huang SW, Xie BY. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genom. 2011;12:471. doi: 10.1186/1471-2164-12-471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Luo GZ, Wang HW, Huang J, Tian AG, Wang YJ, Zhang JS, Chen SY. A putative plasma membrane cation/proton antiporter from soybean confers salt tolerance in Arabidopsis. Plant Mol Biol. 2005;59:809–820. doi: 10.1007/s11103-005-1386-0. [DOI] [PubMed] [Google Scholar]
  41. Luo M, Dennis ES, Berger F, Peacock WJ, Chaudhury A. MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci USA. 2005;102:17531–17536. doi: 10.1073/pnas.0508418102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Miao Y, Laun T, Zimmermann P, Zentgraf U. Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol. 2004;55:853–867. doi: 10.1007/s11103-005-2142-1. [DOI] [PubMed] [Google Scholar]
  43. Moore RC, Purugganan MD. The evolutionary dynamics of plant duplicate genes. Curr Opin Plant Biol. 2005;8:122–128. doi: 10.1016/j.pbi.2004.12.001. [DOI] [PubMed] [Google Scholar]
  44. Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity. Plant Physiol. 2009;150:1648–1655. doi: 10.1104/pp.109.138990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Phang TH, Shao G, Lam HM. Salt tolerance in soybean. J Integr Plant Biol. 2008;50:1196–1212. doi: 10.1111/j.1744-7909.2008.00760.x. [DOI] [PubMed] [Google Scholar]
  46. Qi XP, Li MW, Xie M, Liu X, Ni M, Shao GH, Song C, Yim AKY, Tao Y, Wong FL, Isobe S, Wong CF, Wong KS, Xu CY, Li CQ, Wang Y, Guan R, Sun FM, Fan GY, Xiao ZX, Zhou F, Phang TH, Liu X, Tong SW, Chan TF, Yiu SM, Tabata S, Wang J, Xu X, Lam HM. Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing. Nat Commun. 2014;5:4340. doi: 10.1038/ncomms5340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Qiu YP, Jing SJ, Fu J, Li L, Yu DQ. Cloning and analysis of expression profile of 13 WRKY genes in rice. Chin Sci Bull. 2004;49:2159–2168. [Google Scholar]
  48. Robatzek S, Somssich IE. Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev. 2002;16:1139–1149. doi: 10.1101/gad.222702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rushton PJ, Macdonald H, Huttly AK, Lazarus CM, Hooley R. Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of α-Amy2 genes. Plant Mol Biol. 1995;29:691–702. doi: 10.1007/BF00041160. [DOI] [PubMed] [Google Scholar]
  50. Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 1996;15:5690–5700. [PMC free article] [PubMed] [Google Scholar]
  51. Rushton PJ, Somssich IE, Ringler P, Shen QJ. WRKY transcription factors. Trends Plant Sci. 2010;15:247–258. doi: 10.1016/j.tplants.2010.02.006. [DOI] [PubMed] [Google Scholar]
  52. Saldanha AJ. Java Treeview-extensible visualization of microarray data. Bioinformatics. 2004;20:3246–3248. doi: 10.1093/bioinformatics/bth349. [DOI] [PubMed] [Google Scholar]
  53. Satapathy L, Singh D, Ranjan P, Kumar D, Kumar M, Prabhu KV, Mukhopadhyay K. Transcriptome-wide analysis of WRKY transcription factors in wheat and their leaf rust responsive expression profiling. Mol Genet Genomics. 2014;289:1289–1306. doi: 10.1007/s00438-014-0890-9. [DOI] [PubMed] [Google Scholar]
  54. Schmutz J, Cannon SB, Schlueter J, Ma JX, Mitros T, Nelson W, Hyten DL, Song QJ, Thelen JJ, Cheng JL, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu SQ, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du JC, Tian ZX, Zhu LC et al (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183 [DOI] [PubMed]
  55. Seo PJ, Xiang FN, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park WJ, Park CM. The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol. 2009;151:275–289. doi: 10.1104/pp.109.144220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Song H, Wang P, Hou L, Zhao S, Zhao C, Xia H, Li P, Zhang Y, Bian X, Wang X. Global analysis of WRKY genes and their response to dehydration and salt stress in soybean. Front Plant Sci. 2016 doi: 10.3389/fpls.2016.00009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sun CX, Palmqvist S, Olsson H, Boren M, Ahlandsberg S, Jansson C. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. Plant Cell. 2003;15:2076–2092. doi: 10.1105/tpc.014597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ulker B, Mukhtar MS, Somssich IE. The WRKY70 transcription factor of Arabidopsis influences both the plant senescence and defense signaling pathways. Planta. 2007;226:125–137. doi: 10.1007/s00425-006-0474-y. [DOI] [PubMed] [Google Scholar]
  60. Wang Z, Libault M, Joshi T, Valliyodan B, Nguyen HT, Xu D, Stacey G, Cheng JL. SoyDB: a knowledge database of soybean transcription factors. BMC Plant Biol. 2010;10:14. doi: 10.1186/1471-2229-10-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang YX, Suo HC, Zhuang CX, Ma H, Yan XL. Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis. J Plant Physiol. 2011;168:2260–2267. doi: 10.1016/j.jplph.2011.07.014. [DOI] [PubMed] [Google Scholar]
  62. Wang LN, Zhu W, Fang LC, Sun XM, Su LY, Liang ZC, Wang N, Londo JP, Li SH, Xin HP (2014) Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera. BMC Plant Biol 14:103 [DOI] [PMC free article] [PubMed]
  63. Wang FF, Chen HW, Li QT, Wei W, Li W, Zhang WK, Ma B, Bi YD, Lai YC, Liu XL, Man WQ, Zhang JS, Chen SY (2015) GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants. Plant J 83:224–236 [DOI] [PubMed]
  64. Wei KF, Chen J, Chen YF, Wu LJ, Xie DX. Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in Maize. DNA Res. 2012;19:153–164. doi: 10.1093/dnares/dsr048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wei W, Li QT, Chu YN, Reiter RJ, Yu XM, Zhu DH, Zhang WK, Ma B, Lin Q, Zhang JS, Chen SY (2015) Melatonin enhances plant growth and abiotic stress tolerance in soybean plants. J Exp Bot 66:695–707 [DOI] [PMC free article] [PubMed]
  66. Wen F, Zhu H, Li P, Jiang M, Mao WQ, Ong C, Chu ZQ. Genome-wide evolutionary characterization and expression analyses of WRKY family genes in Brachypodium distachyon. DNA Res. 2014;21:327–339. doi: 10.1093/dnares/dst060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Xie Z, Zhang ZL, Zou XL, Huang J, Ruas P, Thompson D, Shen QJ. Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol. 2005;137:176–189. doi: 10.1104/pp.104.054312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Xie Z, Zhang ZL, Hanzlik S, Cook E, Shen QXJ. Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol Biol. 2007;64:293–303. doi: 10.1007/s11103-007-9152-0. [DOI] [PubMed] [Google Scholar]
  69. Yan JH, Wang BA, Jiang YN, Cheng LJ, Wu TL. GmFNSII-controlled soybean flavone metabolism responds to abiotic stresses and regulates plant salt tolerance. Plant Cell Physiol. 2014;55:74–86. doi: 10.1093/pcp/pct159. [DOI] [PubMed] [Google Scholar]
  70. Yu YC, Hu RB, Wang HM, Cao YP, He G, Fu CX, Zhou GK. MlWRKY12, a novel Miscanthus transcription factor, participates in pith secondary cell wall formation and promotes flowering. Plant Sci. 2013;212:1–9. doi: 10.1016/j.plantsci.2013.07.010. [DOI] [PubMed] [Google Scholar]
  71. Yu YC, Liu ZH, Wang L, Kim SG, Seo PJ, Qiao M, Wang N, Li S, Cao XF, Park CM, Xiang FN (2016) WRKY71 accelerates flowering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana. Plant J 85:96–106 [DOI] [PubMed]
  72. Zentella R, Zhang ZL, Park M, Thomas SG, Endo A, Murase K, Fleet CM, Jikumaru Y, Nambara E, Kamiya Y, Sun TP (2007) Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis. Plant Cell 19:3037–3057 [DOI] [PMC free article] [PubMed]
  73. Zhai Y, Wang Y, Li Y, Lei T, Yan F, Su L, Li X, Zhao Y, Sun X, Li J, Wang Q (2013) Isolation and molecular characterization of GmERF7, a soybean ethylene-response factor that increases salt stress tolerance in tobacco. Gene 513:174–183 [DOI] [PubMed]
  74. Zhou QY, Tian AG, Zou HF, Xie ZM, Lei G, Huang J, Wang CM, Wang HW, Zhang JS, Chen SY (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol J 6:486–503 [DOI] [PubMed]
  75. Zhou GA, Chang RZ, Qiu LJ. Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis. Plant Mol Biol. 2010;72:357–367. doi: 10.1007/s11103-009-9575-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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