Mol Genet Genomics DOI 10.1007/s00438-014-0849-x

Original Paper

Transcriptome‑wide identification of bread wheat WRKY transcription factors in response to drought stress Sezer Okay · Ebru Derelli · Turgay Unver 

Received: 13 February 2014 / Accepted: 27 March 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  The WRKY superfamily of transcription factors was shown to be involved in biotic and abiotic stress responses in plants such as wheat (Triticum aestivum L.), one of the major crops largely cultivated and consumed all over the world. Drought is an important abiotic stress resulting in a considerable amount of loss in agronomical yield. Therefore, identification of drought responsive WRKY members in wheat has a profound significance. Here, a total of 160 TaWRKY proteins were characterized according to sequence similarity, motif varieties, and their phylogenetic relationships. The conserved sequences of the TaWRKYs were aligned and classified into three main groups and five subgroups. A novel motif in wheat, WRKYGQR, was identified. To putatively determine the drought responsive TaWRKY members, publicly available RNA-Seq data were analyzed for the first time in this study. Through in silico searches, 35 transcripts were detected having an identity to ten known TaWRKY genes. Furthermore, relative expression levels of TaWRKY16/TaWRKY16A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY59, TaWRKY61, and TaWRKY82 were measured in root and leaf tissues of drought-tolerant Sivas 111/33 and susceptible Atay 85 cultivars. All of the quantified TaWRKY transcripts were found to be up-regulated in root tissue of Sivas 111/33. Differential expression of TaWRKY16, TaWRKY24, Communicated by S. Hohmann. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-014-0849-x) contains supplementary material, which is available to authorized users. S. Okay (*) · E. Derelli · T. Unver  Department of Biology, Faculty of Science, Çankırı Karatekin University, 18100 Çankırı, Turkey e-mail: [email protected]

TaWRKY59, TaWRKY61 and TaWRKY82 genes was discovered for the first time upon drought stress in wheat. These comprehensive analyses bestow a better understanding about the WRKY TFs in bread wheat under water deficit, and increased number of drought responsive WRKYs would contribute to the molecular breeding of tolerant wheat cultivars. Keywords  Drought stress · Transcriptome · WRKY transcription factor · Wheat Abbreviations HMM Hidden Markov model NCBI National Center for Biotechnology Information PlantTFDB Plant Transcription Factor Database qRT-PCR Quantitative reverse transcription PCR SRA Sequence Read Archive TF Transcription factor

Introduction Hexaploid bread wheat (Triticum aestivum L.) is one of the three staple crops, together with rice and maize, being an essential food source for a large part of the world’s population covering 18.8 % of daily caloric intake in human diet and its global production is about 670.8 million tonnes per annum in 215.4 million Ha land (FAO, 2012). Since it can be grown in diverse climate conditions as well as the unique properties of its flour in food processing, the cultivation of wheat is widespread all over the world (Shewry and Tatham 1997). The need for wheat is estimated to increase 60 % by the year 2050, however, the production is expected to decrease 29 % due to the environmental stress factors

13



(Manickavelu et al. 2012). Therefore, comprehension of the molecular mechanisms, such as involvement of transcription factors, underlying the stress tolerance in wheat has a great importance for improving tolerant cultivars. Being sessile organisms, the plants encounter a number of stress factors in their environment (Zhu et al. 2013). Drought is one of the major abiotic stress factors due to the limited water resources resulting in huge yield loss in the production of crops (Saad et al. 2013). Therefore, an understanding of plant tolerance mechanisms against water deficit at the molecular level provides great promise for the improvement of more productive crop plants (Tripathi et al. 2014). The plants possess various mechanisms to cope with stress conditions. One of them is transcription factors (TFs), the molecules in regulation of gene expression through binding to either promoter or enhancer region of a gene. TFs up/down regulate the gene expression by a modular structure with one or more DNA-binding domains (Tombuloglu et al. 2013). The WRKY proteins form a superfamily of plant TFs included in the regulation of plant growth processes as well as biotic and abiotic stress responses (Zhu et al. 2013). The WRKY members are characterized by their WRKY domain of approximately 60 amino acids, which is composed of highly conserved WRKYGQK sequence followed by a zinc-finger motif (Rushton et al. 2010) showing high binding affinity to a DNA cis-acting element designated as the W box, (C/T)TGAC(T/C) (Ulker and Somssich 2004). WRKYGQK motif may have slight modifications such as WRKYGEK or WRKYGKK (Wang et al. 2013). The members of WRKY TFs were grouped into three distinct groups depending on the number and type of their WRKY domains as well as zinc-finger structure. WRKY proteins with two WRKY domains were related to group I. The members of group II and group III have only one WRKY domain. In general, the WRKY domains of group I and group II members have the same type of finger motif C2H2 (C–X4–5–C–X22–23–H–X1–H), whereas in group III, the WRKY domain contains a C2HC (C–X7–C–X23–H–X1–C) motif (Eulgem et al. 2000). Genome-wide identification of WRKY family members in Arabidopsis (Eulgem et al. 2000), rice (Xie et al. 2005), canola (Yang et al. 2009), coffee (Ramiro et al. 2010), cucumber (Ling et al. 2011), tomato (Huang et al. 2012), and poplar (He et al. 2012) has been reported. The participation of WRKY TFs in abiotic stress tolerance was shown in various plants. Overexpression of several WRKY genes belonging to Arabidopsis (Chen et al. 2010), rice (Song et al. 2010), soybean (Zhou et al. 2008), and wheat (Wang et al. 2013) conferred tolerance to different abiotic stresses in various transgenic host plant species. Expression profiles of 15 TaWRKY genes in different wheat tissues upon various abiotic stresses and senescence

13

Mol Genet Genomics

were shown by Wu et al. (2008). Niu et al. (2012) identified forty-three TaWRKYs through analysis of wheat ESTs and reported that transgenic Arabidopsis plants overexpressing TaWRKY2 and TaWRKY19 exhibited increased tolerance to various abiotic stresses. Similarly, Wang et al. (2013) identified ten TaWRKYs and showed that TaWRKY10 enhances the tolerance to multiple abiotic stresses in transgenic tobacco plants. Recently, a more comprehensive study on wheat identifying ninety-two TaWRKY members was conducted by Zhu et al. (2013). Here, we provide a more extensive knowledge on TaWRKYs by characterization of 160 members and their expression profiling in RNA-Seq libraries. Some of the drought stress responsive TaWRKY members discovered via in silico analyses were validated in leaf and root tissues of tolerant and susceptible wheat cultivars upon water deficit.

Materials and methods Identification and phylogenetic analysis of WRKY superfamily in bread wheat For identification of WRKY proteins in bread wheat, the amino acid sequences in Plant Transcription Factor Database v3.0 (PlantTFDB, http://planttfdb.cbi.pku.edu.cn/ index.php) (Jin et al. 2014) and in GenBank (release 199.0) (http://www.ncbi.nlm.nih.gov/protein) were used. The sequences were blast searched either at NCBI (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) or at PlantTFDB (http:/ /planttfdb.cbi.pku.edu.cn/blast.php) browsers to find out their identity. Hidden Markov models (HMMs) of the TaWRKYs were determined using the Pfam database (http://pf am.sanger.ac.uk) (Punta et al. 2012). TaWRKY sequences were aligned by ClustalW using MEGA v6.0 (MEGA6) program (Tamura et al. 2013). The unrooted phylogenetic tree was constructed by the Neighbor-Joining method with a bootstrap test (1,000 replicates) using MEGA6. The MEME Suite tool v4.9.1 (http://meme.nbcr.net/meme) (Bailey et al. 2009) was utilized for analysis of the conserved motifs of TaWRKY sequences. Transcriptome‑wide expression analysis of TaWRKY genes The bread wheat (T. aestivum cv. Chinese Spring) transcriptome data were downloaded from the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra) database with the accession numbers of ERX101745 for drought-stressed samples and ERX101747 for untreated samples. BLASTX (DNA vs protein) search for T. aestivum in PlantTFDB was used with default parameters to identify the transcripts encoding for a WRKY motif. Amino acid translations of the nucleotide sequences of transcripts were

Mol Genet Genomics

determined using ExPASy portal (http://web.expasy.org/ translate).

Table 1. All reactions were repeated three times with triple biological replicates. The expression levels were calculated as the mean-signal intensity across the three replicates.

Plant materials, growth and stress conditions Hexaploid bread wheat (T. aestivum L.) cv. Sivas 111/33 and cv. Atay 85, drought tolerant and drought susceptible, respectively, were kindly provided by Research Institute of Field Crops (TARM) in Ankara, Turkey. Seeds were surface-sterilized in 70 % (v/v) ethanol for 2 min, and in 10 % (v/v) sodium hypochlorite for 10 min, then rinsed three times with sterile dH2O. The sterilized seeds were germinated in Petri dishes on two layers of filter papers at room temperature. After 4 days, the germinated seedlings were transferred to a Murashige and Skoog (MS) medium, pH 5.7, containing 0.3 % agar and 3 % sucrose, and grown under controlled conditions (16/8 h photoperiod, 25 ± 1 °C, 60 % relative humidity and photon flux density of 200 μmol m−2s−1). Ten-day-old plants were exposed to dehydration stress in plastic pots containing MS medium containing 20 % polyethylene glycol (PEG) 6000 for 24 h. Control plants were kept in fresh MS medium. 15 seedlings were included in each treatment and control group. RNA isolation, cDNA synthesis and qRT‑PCR Total RNA from flag leaves and roots of the bread wheat was isolated using TRIzol (Ambion, TX, USA) according to the manufacturer’s instructions. The RNA quality was checked on 1.5 % agarose gel, and the concentration of the RNA was determined using a NanoDrop ND2000c spectrophotometer (Thermo Scientific, KS, USA). Total RNA samples were used to synthesize first strand cDNA primed with oligo(dT) in a 20 μl reaction mix using M-MuLV Reverse Transcriptase (Thermo Scientific) following the manufacturer’s instructions. To determine the relative expression levels of nine WRKY transcription factors in root and leaf tissues of bread wheat, qRTPCR was conducted as previously reported (Turktas et al. 2013) using SYBR Green I Master Kit (Roche Germany) on LightCycler 480 Instrument II (Roche, Germany). The qRT-PCR was carried out in 96-well optical plates, and PCR reactions were performed in a total volume of 20 μl containing 0.1 μl each of the primers (100 pmol), 2 μl of cDNA, 10 μl FastStart SYBR Green I Master Mix and nuclease-free water was added up to 20 μl. The 18S rRNA gene was used as the internal control (Wang et al. 2010; Budak et al. 2013). The qRT-PCR conditions were set up as follows: preheating at 95 °C for 5 min; followed by 50 cycles of 95 °C for 10 s; 53 °C or 55 °C for 20 s; and 72 °C for 10 s. The melting curves were adjusted to 95 °C for 5 s and 55 °C for 1 min and then cooled to 40 °C for 30 s. A list of the primers used in qRT-PCR is presented in Suppl.

Results Identification of WRKY superfamily in bread wheat The amino acid sequences of 187 WRKY family proteins for T. aestivum found in PlantTFDB (111 sequences) and GenBank (76 sequences) were selected for the analyses. Initially, TaWRKY sequences found in PlantTFDB were searched for their identity to the TaWRKYs in GenBank using BLASTP program (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). 27 of the TaWRKY sequences in GenBank and PlantTFDB were found to be identical; therefore, a total of 160 TaWRKY sequences, 49 from GenBank and 111 from PlantTFDB, were used in the characterization of WRKY TFs in T. aestivum. Three different accession numbers for the same amino acid sequence were detected in GenBank, ABC65847, AFM91580, and AFM91581 for TaWRKY1B. On the other hand, a couple of different sequences and accession numbers were found for the same TaWRKY TFs, TaWRKY8, TaWRKY10, TaWRKY13, TaWRKY28, and TaWRKY41, some of them including the partial sequences. These repetitive accessions and the ones without a WRKY name in GenBank as well as the sequences in PlantTFDB were defined and organized by BLASTP search. The resulting similar sequences were named with a letter suffix. Eventually, 160 TaWRKY sequences were named from TaWRKY1 to TaWRKY91 (Table 1). HMMs of these 160 TaWRKYs were determined using the Pfam database. Since 72 of the proteins have 2 WRKY domains, a total of 182 HMM profiles are represented in Table 1. Multiple sequence alignment, structure and phylogenetic analysis of TaWRKY proteins A total of 160 TaWRKY sequences were aligned using MEGA6 program and the conserved WRKY and zinc-fingerlike domains were defined manually (Fig. 1). The WRKY TFs in T. aestivum were categorized into seven groups and subgroups (I, IIa, IIb, IIc, IId, IIe, and III), according to the features specified for the WRKY family TFs in A. thaliana (Eulgem et al. 2000) (Fig. 1). Group I members have highly conserved amino acid sequence with the C2H2 type zincfinger structure of C–X4–C–X23–H–X1–H and seventeen out of twenty of the members contain two WRKY domains. Eighty-one of the TaWRKY sequences belong to group II, generally with a C2H2 type zinc-finger motif of C–X4-5–C– X23–H–X1–H. Fifty-seven of the TaWRKY members were identified in group III, generally carrying the C2HC type

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Mol Genet Genomics

Table 1  A catalog of WRKY proteins in T. aestivum with their HMM profiles TaWRKY name*

GenBank accession no.

TaWRKY1

ACD80356

TaWRKY1-A

ABC65846

TaWRKY1-B

ABC65847

PTFDB ID

Envelope

Alignment

HMM

HMM Bit length score

E-value

Start

End

Start

End From To

120

179

120

178

1

59

60

105.5

8.6e-31

Tae005206

118

180

119

179

2

59

60

88.8

1.4e-25

Tae069013

119

181

120

180

2

59

60

88.5

1.8e-25

TaWRKY1-C

Tae038955

119

181

120

180

2

59

60

86.7

6.3e-25

TaWRKY1-D

Tae000532

AFM91580 AFM91581

TaWRKY2

ACD80357

TaWRKY2-A TaWRKY3

ACD80364

TaWRKY3-A

335

278

334

2

59

60

103.0

5.3e-30

549

490

548

1

59

60

104.0

2.6e-30

183

242

183

242

1

60

60

103.5

3.8e-30

337

396

337

395

1

59

60

105.7

7.6e-31

Tae036246

182

241

182

241

1

60

60

79.6

1.0e-22

Tae062841

151

210

151

209

1

59

60

101.9

1.1e-29

Tae003027

Tae035093

TaWRKY3-B

Tae066453

TaWRKY4

ACD80365

TaWRKY5

ACD80366

TaWRKY5-A

Tae057333

TaWRKY5-B TaWRKY6

277 490

Tae006014

210

151

209

1

59

60

94.6

2.3e-27

107

48

106

1

59

60

103.6

3.4e-30

48

107

48

106

1

59

60

103.6

3.4e-30

231

291

231

290

1

59

60

96.0

8.0e-28

156

218

157

216

2

58

60

82.7

1.1e-23

124

186

124

184

1

58

60

83.9

4.8e-24

90

125

95

123

30

58

60

23.8

2.8e-05

99

158

99

156

1

58

60

89.8

6.8e-26

Tae026938

129

188

129

186

1

58

60

89.5

9.0e-26

Tae004539

167

228

168

227

2

59

60

84.7

2.8e-24

ACD80367

TaWRKY6-A

151 48

TaWRKY7

ACD80368

TaWRKY8

ACD80369

195

255

195

254

1

59

60

96.2

6.9e-28

TaWRKY8-A

ABC61128

80

135

81

128

2

48

60

76.5

9.8e-22

TaWRKY9

ACD80370

253

309

255

307

3

54

60

51.7

5.5e-14

TaWRKY10

ABN43182

128

187

128

185

1

58

60

90.4

4.6e-26

TaWRKY10-A

ADY80578

129

188

129

186

1

58

60

88.9

1.3e-25

TaWRKY10-B

ACD80371

117

179

118

178

2

59

60

71.4

3.9e-20

TaWRKY10-C

Tae045759

129

188

129

186

1

58

60

88.9

1.3e-25

TaWRKY10-D

Tae009575

128

187

128

185

1

58

60

90.4

4.6e-26

51

113

51

112

1

59

60

75.2

2.6e-21

TaWRKY11

ACD80372

TaWRKY11-A TaWRKY12

Tae056045 ACD80373

TaWRKY12-A

21

68

25

67

20

59

60

38.6

6.9e-10

62

87

63

83

2

22

60

24.8

1.3e-05

Tae040732

32

83

33

70

2

36

60

50.8

1.0e-13

TaWRKY13

ACD80358

Tae052024

41

104

42

103

2

59

60

70.8

6.0e-20

TaWRKY13-A

ABO15543

Tae045911

80

138

83

137

6

59

60

82.1

1.8e-23

Tae068120

78

138

80

137

3

59

60

94.1

3.3e-27

TaWRKY13-B TaWRKY14

ACD80359

TaWRKY14-A TaWRKY15

Tae002472 Tae039559

320

261

320

1

60

60

103.2

4.6e-30

468

409

467

1

59

60

103.1

5.0e-30

186

216

189

215

4

30

60

40.7

1.5e-10

122

184

123

183

2

59

60

81.7

2.3e-23

TaWRKY15-A

Tae053170

99

161

100

160

2

59

60

84.3

3.7e-24

TaWRKY15-B

Tae048565

97

156

97

144

1

45

60

54.7

6.3e-15

13

ACD80374

261 409

Mol Genet Genomics Table 1  continued TaWRKY16

ACD80360

TaWRKY16-A TaWRKY17 TaWRKY18

ACD80361

Tae041763

281

341

283

340

3

59

60

100.2

4.0e-29

Tae044379

176

236

178

235

3

59

60

101.0

2.2e-29

Tae058598

ACD80375

TaWRKY18-A

Tae006125

TaWRKY19

ACD80362

TaWRKY19-A

ABN43183

TaWRKY19-B

ABO15545

229

287

230

287

2

60

60

99.5

6.6e-29

403

462

403

461

1

59

60

107.3

2.4e-31

1

56

1

56

5

60

60

96.0

8.4e-28

151

210

151

209

1

59

60

104.5

1.9e-30

3

62

6

62

4

60

60

95.6

1.1e-27

157

216

157

215

1

59

60

104.4

1.9e-30

198

255

199

254

2

59

60

86.4

8.0e-25

362

421

362

420

1

59

60

102.2

9.3e-30

104

166

105

165

2

59

60

77.4

5.2e-22

Tae007418

101

163

102

162

2

59

60

79.4

1.2e-22

TaWRKY19-C

Tae049406

167

224

168

223

2

59

60

85.5

1.6e-24

331

390

331

389

1

59

60

102.4

8.3e-30

TaWRKY19-D

Tae064536

129

191

130

190

2

59

60

77.2

6.1e-22

TaWRKY19-E

Tae036526

110

164

110

163

1

59

60

52.3

3.6e-14

TaWRKY19-F

Tae036237

33

95

34

94

2

59

60

84.4

3.4e-24

TaWRKY20

ACD80377

TaWRKY21

ACD69416

Tae017653

39

99

41

98

3

59

60

101.6

1.4e-29

45

105

46

104

2

59

60

100.8

2.5e-29

TaWRKY22

ACD80376

TaWRKY23

ACD80379

Tae026140

32

91

32

90

1

59

60

103.5

3.7e-30

Tae009934

35

94

36

93

2

59

60

105.0

1.2e-30

TaWRKY24

ACD80380

Tae056885

TaWRKY25

ACD80381

TaWRKY26

ACD69417

TaWRKY27

ACD80363

Tae002328

50

109

50

108

1

59

60

106.7

3.7e-31

10

53

11

52

2

40

60

64.7

4.7e-18

40

61

40

61

1

22

60

39.2

4.5e-10

216

274

217

273

2

59

60

100.6

3.0e-29

382

441

382

440

1

59

60

104.9

1.4e-30

TaWRKY27-A

Tae058579

17

68

18

67

11

59

60

23.3

4.1e-05

TaWRKY27-B

Tae067528

219

275

220

268

2

52

60

75.5

2.1e-21

TaWRKY27-C

Tae029023

222

280

223

279

2

59

60

99.7

5.5e-29

TaWRKY28

ACD69418

81

141

82

140

2

59

60

97.3

3.2e-28

TaWRKY28-A

ABC61127

73

133

74

132

2

59

60

97.4

3.0e-28

TaWRKY29

ACD69419

TaWRKY30

ACD80378

TaWRKY31

ACD69420

TaWRKY32

ACD69421

TaWRKY33 TaWRKY34

304

364

305

363

2

59

60

96.8

4.6e-28

22

74

23

65

2

43

60

73.2

1.1e-20

133

174

134

174

2

40

60

55.6

3.4e-15

132

199

134

198

3

59

60

67.2

7.7e-19

ACD69422

47

63

48

62

2

16

60

18.3

0.0015

ACD69423

100

119

100

118

1

19

60

27.2

2.4e-06

TaWRKY35

ACD80382

22

48

23

43

2

22

60

31.9

8.6e-08

TaWRKY36

ACD80383

58

117

59

116

2

59

60

104.1

2.3e-30

Tae054892

40

99

41

98

2

59

60

103.7

3.3e-30

Tae003568

127

185

128

184

2

59

60

94.7

2.0e-27

294

353

294

352

1

59

60

102.5

7.7e-30

62

84

63

82

2

21

60

30.9

1.7e-07

206

229

206

226

1

21

60

19.7

0.00053

43

106

44

105

2

59

60

65.2

3.4e-18

TaWRKY36-A TaWRKY37

ACD80384

TaWRKY38

ACD80385

TaWRKY39

ACD80386

TaWRKY40

ACD80387

Tae002244

Tae035958

Tae023961

13



Mol Genet Genomics

Table 1  continued TaWRKY40-A TaWRKY41 TaWRKY41-A

Tae034729

32

95

33

94

2

59

60

65.5

2.6e-18

130

192

131

191

2

59

60

77.4

5.1e-22

246

308

247

307

2

59

60

83.8

5.4e-24

Tae042379

81

131

83

110

3

30

60

53.3

1.8e-14

Tae028511

163

225

164

224

2

59

60

72.3

2.0e-20

279

341

280

340

2

59

60

81.7

2.4e-23

AGW22214 ACD69424

TaWRKY41-B TaWRKY42

ACD80388

145

207

146

206

2

59

60

86.4

8.2e-25

TaWRKY43

ACD69425

207

230

207

226

1

20

60

23.9

2.7e-05

TaWRKY44

Tae026732

TaWRKY45

ABO15542

TaWRKY45-A

BAK53494

TaWRKY45-B

BAK53495

TaWRKY45-C TaWRKY45-D

Tae062497 Tae049480

BAK53496

TaWRKY45-E

Tae049336

TaWRKY45-F

Tae056509

TaWRKY46

173

117

172

3

59

60

101.9

1.2e-29

315

277

314

4

41

60

65.0

3.7e-18

107

169

108

168

2

59

60

70.8

5.8e-20

105

167

106

166

2

59

60

70.8

5.8e-20

105

167

106

166

2

59

60

70.1

9.6e-20

103

156

104

153

2

48

60

56.0

2.4e-15

103

165

104

164

2

59

60

70.1

1.0e-19

103

165

104

164

2

59

60

70.2

9.4e-20

37

83

37

58

1

22

60

26.9

3.1e-06

109

156

113

155

20

59

60

37.1

2.0e-09

41

104

42

103

2

59

60

68.6

3.0e-19

TaWRKY47

Tae060773

38

98

39

97

2

59

60

99.3

7.5e-29

TaWRKY48

Tae031570

44

102

45

100

2

58

60

69.5

1.5e-19

TaWRKY49

Tae042751

97

156

98

154

2

58

60

99.1

8.7e-29

TaWRKY49-A

Tae031786

79

138

81

136

3

58

60

102.9

5.5e-30

TaWRKY50

Tae052852

59

118

59

116

1

58

60

98.5

1.4e-28

TaWRKY51

ABN43186

115 274

241

301

242

300

2

59

60

98.0

1.9e-28

TaWRKY51-A

AFR66647 Tae020528

107

167

108

166

2

59

60

99.5

6.8e-29

TaWRKY51-B

Tae059162

254

314

255

313

2

59

60

96.8

4.6e-28

TaWRKY52

Tae045704

168

228

168

227

1

59

60

95.5

1.2e-27

Tae004973

166

226

166

225

1

59

60

93.8

4.0e-27

Tae050174

178

237

178

237

1

60

60

103.6

3.5e-30

TaWRKY52-A TaWRKY53

AGF90798

323

382

323

381

1

59

60

103.5

3.8e-30

TaWRKY53-A

ABN43178

186

245

186

245

1

60

60

103.4

3.8e-30

334

393

334

392

1

59

60

103.3

4.2e-30

TaWRKY53-B

ABN43185

178

237

178

237

1

60

60

103.6

3.5e-30

323

382

323

381

1

59

60

103.5

3.8e-30

TaWRKY54

Tae029529

195

255

195

252

1

57

60

83.4

7.1e-24

TaWRKY55

Tae041977

128

195

130

194

3

59

60

66.7

1.2e-18

TaWRKY56

Tae047299

115

175

116

174

2

59

60

94.5

2.4e-27

TaWRKY57

Tae005376

143

202

144

201

2

59

60

104.2

2.2e-30

TaWRKY58

Tae048976

175

234

175

233

1

59

60

101.3

1.8e-29

TaWRKY59

Tae064350

234

294

235

293

2

59

60

98.7

1.2e-28

TaWRKY60

Tae049971

17

77

17

76

1

59

60

87.4

3.8e-25

TaWRKY61

Tae039246

91

149

92

148

2

59

60

100.7

2.7e-29

248

307

248

306

1

59

60

108.7

9.0e-32

TaWRKY62

Tae062072

102

166

103

164

2

58

60

66.0

1.9e-18

13

Mol Genet Genomics Table 1  continued TaWRKY63

Tae030194

556

618

557

616

2

TaWRKY64

Tae064762

81

143

81

141

TaWRKY65

Tae043568

42

100

42

99

TaWRKY66

Tae024146

237

295

238

TaWRKY67

Tae022115

209

243

210

TaWRKY68

Tae025576

TaWRKY68-A

ABN43181

TaWRKY68-B

ABO15546

58

60

84.5

3.2e-24

1

58

60

86.0

1.1e-24

1

58

60

109.8

4.0e-32

294

2

59

60

98.2

1.7e-28

242

2

34

60

61.9

3.7e-17

234

294

236

293

3

59

60

89.3

1.0e-25

234

294

236

293

3

59

60

94.8

1.9e-27

221

281

223

280

3

59

60

98.5

1.4e-28

TaWRKY68-C

Tae025758

221

281

223

280

3

59

60

76.6

9.5e-22

TaWRKY68-D

Tae016948

48

103

50

102

9

59

60

65.4

2.9e-18

TaWRKY69

Tae002930

228

288

229

287

2

59

60

98.7

1.2e-28

TaWRKY69-A

Tae057008

77

137

78

136

2

59

60

99.6

6.3e-29

TaWRKY69-B

Tae047995

1

45

2

44

18

59

60

61.2

6.2e-17

TaWRKY70 TaWRKY71

Tae026500 ABN43177

TaWRKY72

Tae067719

TaWRKY72-A

ABN43179

TaWRKY72-B

ABN43184

111

146

111

144

1

34

60

32.8

4.5e-08

195

255

196

254

2

59

60

96.7

4.8e-28

113

172

113

171

1

59

60

105.4

9.5e-31

113

172

113

171

1

59

60

100.4

3.5e-29

113

172

113

171

1

59

60

105.4

9.5e-31

TaWRKY72-C

Tae050030

112

171

112

170

1

59

60

105.4

9.4e-31

TaWRKY73

Tae036520

123

188

124

187

2

59

60

65.7

2.4e-18

TaWRKY74

Tae043325

281

341

283

340

3

59

60

96.0

8.0e-28

TaWRKY74-A

ABN43180

124

186

125

184

2

58

60

83.0

9.6e-24

TaWRKY74-B

ABO15544

124

186

125

184

2

58

60

83.5

6.3e-24

TaWRKY74-C

ABB90551

124

186

125

184

2

58

60

83.0

9.6e-24

TaWRKY74-D

Tae000688

124

186

125

184

2

58

60

83.0

9.6e-24

TaWRKY75

Tae002848

239

299

239

298

1

59

60

93.3

5.8e-27

TaWRKY76

Tae025448

137

196

138

183

2

44

60

50.3

1.5e-13

Tae060129

TaWRKY77

166

211

167

204

2

38

60

64.5

5.4e-18

TaWRKY78

ADF28625

187

245

188

245

2

60

60

99.6

6.0e-29

360

419

360

418

1

59

60

107.4

2.2e-31

TaWRKY78-A

AAQ63878

1

34

1

34

26

60

60

36.8

2.5e-09

150

206

150

206

1

57

60

104.3

2.0e-30

Tae020712

186

246

186

245

1

59

60

93.7

4.4e-27

Tae055584

185

245

185

243

1

58

60

87.7

3.2e-25

TaWRKY79

AFN44008

TaWRKY79-A TaWRKY80

Tae004431

192

252

192

251

1

59

60

89.6

8.1e-26

TaWRKY80-A

AFW98256

Tae039387

192

252

192

251

1

59

60

91.3

2.3e-26

TaWRKY80-B

Tae004316

195

255

196

254

2

59

60

96.7

4.8e-28

TaWRKY81

Tae068163

50

110

51

109

2

59

60

69.2

1.9e-19

TaWRKY82

Tae058284

176

234

177

233

2

59

60

91.6

2.0e-26

344

403

345

402

2

59

60

100.9

2.5e-29

TaWRKY83

Tae039851

38

100

38

99

1

59

60

85.3

1.8e-24

TaWRKY84

Tae064675

17

79

17

77

1

58

60

87.1

4.8e-25

TaWRKY85

Tae034100

126

194

127

193

2

59

60

55.3

4.0e-15

TaWRKY86

Tae038427

12

71

13

70

2

59

60

104.5

1.8e-30

TaWRKY87

Tae045013

10

72

11

70

2

58

60

77.3

5.7e-22

13



Mol Genet Genomics

Table 1  continued TaWRKY88

Tae057908

124

186

125

185

2

59

60

63.1

1.5e-17

TaWRKY88-A

Tae007640

116

178

117

177

2

59

60

72.5

1.8e-20

TaWRKY89

Tae009095

129

188

130

185

2

54

60

65.9

2.1e-18

TaWRKY89-A

Tae009912

145

204

146

202

2

55

60

69.5

1.6e-19

TaWRKY90

Tae042230

51

110

52

109

2

59

60

106.5

4.3e-31

TaWRKY91

Tae065892

121

183

121

181

1

58

60

86.2

9.1e-25

* TaWRKY names in red were defined in this study. The ones in black were previously defined in GenBank. The ones in blue were repetitive names in GenBank which were re-defined with a suffix in this study The features same for all entries are i) family: WRKY, ii) description: WRKY DNA-binding domain, iii) entry type: domain, iv) clan: CL0274, v) predicted active sites: n/a (color figure online)

zinc-finger structure of C–X7–C–X23–H–X1–C. Two of the TaWRKY proteins could not be included in a group. The quantity of TaWRKY sequences belonging to each group and subgroup in comparison with different plant WRKY proteins is presented in Table 2. Alignment of 160 TaWRKY sequences identified 13 different WRKY motifs (WRKYGQK, WRKYGEK, WRKYGQE, WLKYGQK, WRKYGKK, LRKYGPK, WRNYGQN, WKKYGQK, WRKDGQK, WSKYGQK, WTKYGQK, GRKYGEK, and WMKYGQK). In this study, the WRKYGQR motif was additionally discovered in wheat transcriptome data. Phylogenetic relationship of 160 TaWRKY TFs was analyzed using MEGA6. The conserved amino acid sequences in Fig. 1 were aligned and the unrooted phylogenetic tree was constructed (Fig. 2). Group I members were found to be relatives of TaWRKYs in group II, especially the majority of TaWRKY proteins in group IIc, and TaWRKY82 in group I is a very close relative of group IIb members. TaWRKY proteins in groups IId and IIe were found to be relatives of each other. However, group IIc members were related to group I and group II subgroups. On the other hand, group III TaWRKY proteins were shown to be phylogenetically distinct to other groups and subgroups. TaWRKY5-B and TaWRKY27-A had a distance to all groups in the phylogenetic tree. Analysis of motif composition in TaWRKY proteins The amino acid sequences of TaWRKY proteins, in Fig. 1 were analyzed in MEME Suite tool and eight motifs were defined. Four of them (motifs 1, 2, 3, and 7) contain a WRKY domain and the zinc-finger structures are represented in motifs 4, 5, and 6 (Fig. 3). The TaWRKY sequences generally comprise a WRKY domain and a zincfinger structure together. Motif 1 is found in TaWRKY members in group III together with motif 4 or motif 6. Sixty of the TaWRKY sequences in group I and subgroup IIc, IId, and IIe carry motifs 2 and 5 together. Motif 7 is

13

mostly found in TaWRKY sequences lacking a zinc-finger structure. The TaWRKY members in subgroup IIa contain motifs 3 and 8 together with motif 4 or motif 5. The motif 3 is also found in subgroup IIb members with motif 4 or motif 6. Determination of drought responsive TaWRKY expression in wheat transcriptome To reveal the putative drought stress responsive WRKY TFs in wheat, the transcriptome data were downloaded from the SRA database with the accession number of ERX101745. This RNA-Seq dataset covers 454 Titanium normalized reads of T. aestivum cv. Chinese Spring (drought tolerant) cDNA from various tissues, drought-stressed and senescent leaves, and leaves over a circadian time-course. The libraries ERR125575, ERR125576, and ERR125577 contain 54.3 M, 71.6 M, and 39.9 M bases, respectively. The nucleotide sequences were subjected to BLASTX search in PlantTFDB browser for T. aestivum. A total of 35 transcripts, 12 from ERR125575, 16 from ERR125576, and 7 from ERR125577, were identified as coding for at least 1 WRKY domain. One of the transcripts encoding for WRKYGQR motif and three transcripts for WRKYGEK motif were identified, while thirty-one of the transcripts code for WRKYGQK motif. The identity of transcripts to the TaWRKYs in PlantTFDB was determined by BLASTX search (Fig. 4). A total of ten WRKY members, TaWRKY16, TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY37, TaWRKY40-A, TaWRKY59, TaWRKY61, and TaWRKY82 were identified. The highest number (11) of the transcripts showed an identity to TaWRKY61. The library ERR125579 with the SRA accession number of ERX101747 including 454 FLX + normalized reads of T. aestivum cv. Chinese Spring cDNA from roots, young flowers, young leaves, young shoots and immature seeds was used to determine the expression of TaWRKY genes in unstressed wheat. PlantTFDB browser

Mol Genet Genomics Fig. 1  Comparison of conserved sequences of 160 TaWRKY members. The sequences were aligned by ClustalW in MEGA6 and the conserved amino acid residues were determined manually according to Eulgem et al. (2000). Gaps (dots) were inserted for optimal alignment. Residues that are highly conserved within each of the major groups are shown in red and potential zinc-finger structures are highlighted in black boxes (color figure online)

was used for BLASTX search of 46.5 Mb data, however, no transcripts coding for a WRKY motif could be identified. Tissue‑specific relative expression of drought responsive TaWRKY genes To determine the tissue-specific expression levels of the drought responsive TaWRKY genes, qRT-PCR

primers were designed specific to the transcripts coding for a WRKYGQK motif and having an identity to TaWRKY16/16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY37, TaWRKY59, TaWRKY61, and TaWRKY82 defined in transcriptome analysis. The nucleotide sequence of the single transcript for TaWRKY16 was highly identical to the sequence for TaWRKY16-A, therefore, specific primers could not be designed for each. Since a primer pair (Suppl. Table 1) detected both

13



Mol Genet Genomics

Fig. 1  continued

transcripts, expression of TaWRKY16 and TaWRKY16-A was evaluated together. The relative expressions of TaWRKY genes were determined by qRT-PCR in root and leaf tissues of T. aestivum cv. Atay 85 (drought susceptible) and cv. Sivas 111/33 (drought tolerant) upon 20 % PEG treatment for 24 h (Fig. 5). The expression level of TaWRKY37 was not significant in both cultivars upon stress application. Expression of the other seven TaWRKY genes was found to be

13

up-regulated in root tissue of cv. Sivas 111/33, the highest was TaWRKY59. Similarly, five of the TaWRKY genes were up-regulated in roots of cv. Atay 85, TaWRKY19-C had the highest expression, whereas expression of TaWRKY82 and TaWRKY24 did not change significantly. In leaf tissue of cv. Sivas 111/33, a significant up-regulation was only observed in TaWRKY19-C, while TaWRKY59 and TaWRKY82 were up-regulated in leaves of cv. Atay 85.

Mol Genet Genomics Fig. 1  continued

Table 2  The number of WRKY proteins belonging to different groups and subgroups in varying plants Plant

Group I Group IIa Group IIb Group IIc Group IId Group IIe Group III NG* Total References

T. aestivum

20

16

3

34

17

11

57

2

160

Brachypodium distachyon 15 34 Oryza sativa

3

6

21

6

9

23

3

86

4

8

7

11

–

36

–

100

This study Tripathi et al. (2012) Wu et al. (2005)

8

4

1

11

5

3

13

–

45

Mangelsen et al. (2008)

Arabidopsis thaliana

14

3

7

14

6

6

8

–

58

Eulgem et al. (2000)

Brassica napus

12

2

5

8

7

6

3

–

43

Yang et al. (2009)

Solanum lycopersicum

15

5

8

16

6

17

11

3

81

Huang et al. (2012)

Cucumis sativus

10

4

4

16

8

7

6

–

55

Ling et al. (2011)

Populus trichocarpa

50

5

9

13

13

4

10

–

104

Hordeum vulgare

He et al. (2012)

* NG non-grouped

13



Mol Genet Genomics

Fig. 2  Unrooted phylogenetic tree representing relationships among TaWRKY members. The tree was constructed using neighbor-joining method with bootstrap test (1,000 replicates) in MEGA6. The percentages of replicates in which the associated taxa clustered together in the bootstrap test are shown next to the main branches. The colors

show the groups and subgroups; red (group I), pink (subgroup IIa), turquoise (subgroup IIb), green (subgroup IIc), yellow (subgroup IId), purple (subgroup IIe), blue (group III), and black (non-grouped) (color figure online)

Discussion

acid sequences for their phylogenetic relationship and conserved WRKY motifs. Similarly, Niu et al. (2012) identified 43 putative TaWRKY sequences and classified them from TaWRKY1 to TaWRKY43, which were represented with the same names in this study. The conserved domains of these 43 TaWRKYs, as well as their groups, and phylogenetic relationships were determined. A more comprehensive study conducted by Zhu et al. (2013) presented the phylogeny, conserved motifs and groups of the 92 TaWRKY sequences retrieved from NCBI dbEST dataset and Dana-Farber Cancer Institute (DFCI, http://compbio.dfci. harvard.edu/tgi) gene index.

Characterization and phylogenetic relationships of WRKY proteins in bread wheat The WRKY superfamily of TFs constitutes one of the largest groups of the transcriptional regulators playing important roles in many plant processes (Rushton et al. 2010). The WRKY family in wheat has been investigated in a couple of studies but the number of TaWRKY members characterized was insufficient. Wu et al. (2008) studied 15 wheat WRKY genes and characterized corresponding amino

13

Mol Genet Genomics

Fig. 3  Conserved motifs of amino acid sequences of TaWRKY members. The motifs were discovered using MEME suite tool. a Schematic representation of the related motif at its position in the amino acid sequence, b logos showing the conserved residues

13



Mol Genet Genomics

Fig. 4  Identification of drought responsive TaWRKY members in RNA-Seq datasets. The y-axis shows the number of transcripts detected in transcriptome data of drought-stressed wheat tissues, and the x-axis shows the TaWRKY members having an identity to the transcripts defined

Fig. 5  Relative expression levels of TaWRKY genes in root and leaf tissues of droughtsusceptible T. aestivum cv. Atay 85 and tolerant T. aestivum cv. Sivas 111/33. The bars show the fold changes of 20 % PEG treated samples as compared to untreated samples. Positive values indicate an up-regulation and negative values indicate a down-regulation. AR Atay 85 root, AL Atay 85 leaf, SR Sivas 111/33 root, SL Sivas 111/33 leaf

The present study improves the knowledge on WRKY family TFs in bread wheat by characterization of 160 TaWRKY sequences in terms of their HMM profiles, conserved domains, distribution among WRKY groups, and phylogenetic relationships. Similar to the findings of Zhu et al. (2013), TaWRKY members in subgroup IIc were shown to be relatives of group I TaWRKYs. Moreover, the members of subgroups IIa and IIb as well as the ones in subgroups IId and IIe were found to be closely related to each other. The TaWRKYs in group III were separated from the members of group I and group II.

13

The WRKY proteins with two WRKY domains have been described in group I in different plants such as Arabidopsis (Eulgem et al. 2000), coffee (Ramiro et al. 2010), tomato (Huang et al. 2012) and wheat (Zhu et al. 2013). However, according to our phylogenetic analysis, TaWRKY41, TaWRKY41-B, and TaWRKY45-F were identified in group III as well as TaWRKY3-A and TaWRKY44 were found to be close relatives of group IIc members, all of which have two WRKY domains. On the other hand, TaWRKY1, TaWRKY26, and TaWRKY65 with a single WRKY domain were identified in the group I. Hence, we

Mol Genet Genomics

showed that the number of WRKY domains may not show the WRKY class. Zhu et al. (2013) identified 5 members in group IIa, 12 members in subgroup IIc, and 33 members in group III while 13, 34, and 57 members, respectively, were defined in the present study, furthermore, the number of WRKY members in other subgroups was increased was well. The higher number of WRKY proteins in hexaploid bread wheat as compared to the ones in other plants (Table 2) is probably due to its large genome (17 Gbp) with three (A, B and D) component genomes (Brenchley et al. 2012) because polyploidy in plants results in gene redundancy (Comai 2005). Expression profiles of TaWRKY genes under drought stress Transcriptome analysis is one of the tools for the identification of WRKY genes playing roles in stress response. Nuruzzaman et al. (2014) compared transcriptome profiles of the WRKY gene family in rice under stress conditions and identified 42 genes up-regulated upon drought stress in the root and panicle in the tolerant line. The number of studies on transcriptome-wide analysis of WRKY genes in wheat is limited. Gregersen and Holm (2007) identified the TaWRKY genes participated in senescence from the transcriptome of flag leaves. Ergen et al. (2009) analyzed the transcriptome of emmer wheat (T. turgidum) upon water deficit and reported three transcripts having an identity to TaWRKY29, TaWRKY40, and TaWRKY90 which were differentially expressed. In the present study, for the first time, TaWRKY members responsive to drought stress were identified in a transcriptome data. Ten of the TaWRKY genes (TaWRKY16, TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY37, TaWRKY40-A, TaWRKY59, TaWRKY61, and TaWRKY82) were found in RNA-Seq datasets of droughtstressed wheat tissues. The involvement of TaWRKY TFs in biotic and abiotic stress responses as well as development processes has been reported (Wu et al. 2008; Niu et al. 2012; Wang et al. 2013; Zhu et al. 2013). Expression patterns of TaWRKY1, TaWRKY2, TaWRKY14, TaWRKY17, TaWRKY19, and TaWRKY27 were found to be induced in wheat leaf tissue upon drought stress (Niu et al. 2012). Wu et al. (2008) examined the effects of 15 TaWRKY genes on different biotic and abiotic stress responses, hormone responses and development in bread wheat, and reported that TaWRKY13, TaWRKY19-b, TaWRKY45, TaWRKY53a, TaWRKY68-b, TaWRKY72-a and TaWRKY74-a were not responsive to any of the abiotic stresses, whereas expression of TaWRKY10, TaWRKY46, TaWRKY68-a and TaWRKY72-b was up-regulated in leaves after 20 % PEG treatment for 4 h. Moreover, Zhu et al. (2013)

investigated tissue-specific relative expression levels of eighteen TaWRKY genes under different stress conditions in the bread wheat somatic hybridization derivative cv. SR3 and its parent cv. JN177. Regulation of the 12 genes did not show a significant change, while the expression of TaWRKY7 and TaWRKY12 (homologs of TaWRKY6-A and TaWRKY68, respectively, since the WRKY members were named differently) was induced in leaves, whereas TaWRKY20, TaWRKY32, TaWRKY34 and TaWRKY60 (homologs of TaWRKY13, TaWRKY80, TaWRKY1-B and TaWRKY1-A, respectively) were found to be up-regulated in root upon 18 % PEG treatment (Zhu et al. 2013). Besides, expression of TaWRKY10 gene in wheat cv. Chinese Spring was induced and reached a maximum level at 1 h after 20 % PEG treatment and TaWRKY10 overexpression enhanced the tolerance to drought stress in transgenic tobacco (Wang et al. 2013). The leaf and root tissues of two different cultivars, drought-tolerant Sivas 111/33 and susceptible Atay 85 were used here for determination of relative expression levels of TaWRKY genes upon drought stress. The findings are controversial with Niu et al. (2012). Although no change in gene expression was reported for TaWRKY16 in leaves of wheat cv. Xifeng 20, it was shown to be upregulated in roots and down-regulated in leaves of both cultivars. Similarly, no gene expression was reported for TaWRKY24, however, it was up-regulated in root tissue of Sivas 111/33. Moreover, Niu et al. (2012) reported that TaWRKY17 expression was increased in leaf tissue, however, it was found to be decreased in leaves yet increased in roots. Likewise, TaWRKY19 expression was reported to be up-regulated in leaves; this finding was similar for TaWRKY19-C in Sivas 111/33 albeit it was down-regulated in leaves of Atay 85. These discrepancies may be aroused due to the utilization of wheat cultivars with different genetic backgrounds and additional investigation of root tissue here. Relative expression levels of TaWRKY59, TaWRKY61 and TaWRKY82 upon drought stress conditions were reported for the first time in this study. As a conclusion, the knowledge on WRKY TFs in wheat was extended in the present study via analysis of 160 TaWRKY members in terms of HMM profiles, groups, conserved motifs and phylogenetic relationships. A transcriptome-wide identification of drought responsive TaWRKYs was carried out and ten members were detected. Moreover, differential expression of five genes, TaWRKY16, TaWRKY24, TaWRKY59, TaWRKY61 and TaWRKY82, was shown in root and leaf tissues of drought-tolerant and susceptible cultivars, for the first time. These findings will pave the way for further studies on the molecular breeding of drought-tolerant wheat cultivars.

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Acknowledgments  The authors thank Bianka Martinez (International Language Learning Center, Cankiri Karatekin University) for revising the manuscript.

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