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  • Research article
  • Open Access

Genome-wide identification, phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon

Contributed equally
BMC Genomics201617:636

https://doi.org/10.1186/s12864-016-2968-8

  • Received: 20 January 2016
  • Accepted: 26 July 2016
  • Published:

Abstract

Background

The AP2/ERF transcription factor is one of the most important gene families in plants, which plays the vital role in regulating plant growth and development as well as in response to diverse stresses. Although AP2/ERFs have been thoroughly characterized in many plant species, little is known about this family in the model plant Brachypodium distachyon, especially those involved in the regulatory network of stress processes.

Results

In this study, a comprehensive genome-wide search was performed to identify AP2/ERF gene family in Brachypodium and a total of 141 BdAP2/ERFs were obtained. Phylogenetic analysis classified them into four subfamilies, of which 112 belonged to ERF, four to RAV and 24 to AP2 as well as one to soloist subfamily respectively, which was in accordance with the number of AP2 domains and gene structure analysis. Chromosomal localization, gene structure, conserved protein motif and cis-regulatory elements as well as gene duplication events analysis were further performed to systematically investigate the evolutionary features of these BdAP2/ERF genes. Furthermore, the regulatory network between BdAP2/ERF and other genes were constructed using the orthology-based method, and 39 BdAP2/ERFs were found to be involved in the regulatory network and 517 network branches were identified. The expression profiles of BdAP2/ERF during development and under diverse stresses were investigated using the available RNA-seq and microarray data and ten tissue-specific and several stress-responsive BdAP2/ERF genes were identified. Finally, 11 AP2/ERF genes were selected to validate their expressions in different tissues and under different stress treatments using RT-PCR method and results verified that these AP2/ERFs were involved in various developmental and physiological processes.

Conclusions

This study for the first time reported the characteristics of the BdAP2/ERF family, which will provide the invaluable information for further evolutionary and functional studies of AP2/ERF in Brachypodium, and also contribute to better understanding the molecular basis for development and stresses tolerance in this model species and beyond.

Keywords

  • Abiotic stress
  • AP2/ERF
  • Brachypodium
  • Expression profiles
  • Gene family
  • Transcription factor

Background

Plant growth, development and productivity are adversely affected by numerous abiotic stresses, such as drought, salt and heat. To survive and flourish under these environmental stresses, plants have developed a complicated response mechanism by repressing or inducing the expression of a series of genes with diverse functions. Transcription factors (TF), as an important group of regulatory proteins, play the central roles in regulation network and signaling pathways of plant development and in response to abiotic stresses. Among them, AP2/ERF (APETALA2/Ethylene Responsive Factor) superfamily is one of the biggest plant TFs, which distinguished by one or two highly conserved ethylene-responsive element-binding factor domains that consisted of 50–60 amino acids [1, 2]. Based on sequence similarities and repetitions of AP2 DNA-binding domains, it can be classified into AP2, ERF and RAV families [3]. The members of AP2 family proteins contain two AP2/ERF domains and are further divided into AP2 and AINTEGUMENTA (ANT) monophyletic groups [4, 5], while the members of ERF subfamily possesses a AP2/ERF domain with the specific WLG motif and are subdivided into ten group [3], of which Group I to IV belong to the DREB subfamily and group V to X belong to the ERF subfamily. The ERF subfamily is characterized by an additional cis-acting element AGCCCGCC of the GCC-box in the promoter regions [6], whereas the DREB subfamily typically binds to dehydration-responsive element-binding factor, which has a core motif of CCGAC [7]. The RAV family members containing the single AP2/ERF domain and a specific B3 DNA-binding motif [8]. In addition, other members with an AP2-like domain but lacking additional motifs are often defined as Soloist.

Extensive studies have revealed the crucial role of the AP2/ERF genes playing in plant growth, development and stress responses [4, 911]. Generally, the AP2 subfamily members were the main factors involving in regulating organ architecture and development, such as leaf epidermal cell determinacy, spikelet meristem differentiation and floral organ patterning [12] as well as seed mass and seed yield [13, 14], while the RAV subfamily showed the important functions in plant hormone signal transduction, such as ethylene [15], Brassinosteroid [16], and also involved in response to biotic and aboitic stresses [17, 18]. Additionally, the DREB, together with other members in ERF subfamily mainly involved in response to biotic and abiotic stresses, such as water deficit [19], low and high temperature [20, 21] and high salinity [22].

B. distachyon, belong to Brachypodium tribe Poaceae family which has a close phylogentic relationships with the major cereal crops, including wheat, barley and rye. It has many favorable features, such as small genome (~300 Mb), diploid accessions, self-fertility, a short lifecycle and easy transformation, which make it an ideal model organism for functional genomic studies of temperate grasses, cereals and biofuel crops [23, 24] and now its genome has been completely sequenced [25]. The available genome data facilitated the studies to reveal the gene function and regulation network in this species, and the study of B. distachyon will provide the vital clue for better understand the molecular mechanism of stress response and subsequently improve the abiotic stress tolerance of other cereal crops. So far, the AP2/ERF family has been identified in Arabidopsis [1], Bamboo [26], grapevine [27], maize [28], peach [29] and rice [30]. However, to the best of our knowledge, the systematic identification of AP2/ERF family has not been performed in B. distachyon, limiting the further function analysis of this important gene family.

In this study, a genome-wide bioinformatics analysis was conducted to investigate the genomic organization, phylogenetic relationship and expression profiles of AP2/ERF genes in B. distachyon. The chromosomal localization, gene structures, cis-elements in the promoter region as well as gene duplication and evolutionary mechanisms were subsequently analyzed. By using RNA-seq and microarray expression data, the expression profiling of these identified AP2/ERF genes in different tissue as well as under cold and drought stresses was further investigated. Our study provided a basis for further study on the regulation roles of the AP2/ERF family playing in B. distachyon development and in response to biotic and abiotic stresses, which will not only provide the helpful information on the evolutionary mechanism of this TFs family in plant, but also contribute to revealing the molecular mechanism of development and stresses response in B. distachyon and other cereal crops.

Methods

Identification of AP2/ERF gene family in Brachpodium genome

The whole genome data of B. distachyon was available at Ensemble plants database (http://plants.ensembl.org/index.html). The predicted protein sequences were downloaded as the dataset for downstream analysis (v1.0.29). The AP2/ERF domain (PF00847) obtained from PFAM database (http://pfam.xfam.org/) was used as the query for Hidden Markov Model (HMM) search using HMMER 3.0 program with a pre-defined threshold of E <1e−5. Furhtermore, the AP2/ERF protein sequences ofArabidopsis and rice were obtained from the plant transcription factor database (http://plntfdb.bio.uni-potsdam.de/v3.0/) and then used as query to search against the Brachpodium protein dataset using the BLASTP program with an e-value of 1e-5 and identity of 50 % as the threshold. Furthermore, HMMER and BLAST hits were compared and parsed and then a self-blast of these sequences was performed to remove the redundancy and no any alternative splice variants were considered. After manual correcting, the putative BdAR2/ERF proteins were obtained. Then, the NCBI-CDD web server (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/Structure/cdd/wrpsb.cgi) and SMART database (http://smart.embl-heidelberg.de/webcite) were used to further confirm the predicted BdAR2/ERF genes. The theoretical isoelectric point (PI) and molecular weight (MW) of the obtained proteins were conducted by the compute pI/Mw tool in the ExPASy server (http://www.expasy.org/). The subcellular localization prediction of each gene was predicted using the cello web server (http://cello.life.nctu.edu.tw/).

Multiple sequence alignment and phylogenetic analysis

Multiple sequence alignment was performed using Clustal X v2.0 [31] with the default parameters. An un-rooted neighbor joining (NJ) tree with 1000 bootstrap replications was constructed using MEGA 6.0 [32] based on the full-length protein alignment.

Chromosome distribution, gene structure and conserved motif analysis

The chromosome distribution of these genes were obtained from the genome annotation information, and then validated by BLASTN search. The exon-intron organizations and splicing phase of these predicted AR2/ERF genes were also investigated based on the annotation file of B. distachyon genome, and then graphically displayed by the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). Conserved motifs or domains were predicted using the MEME Suite web server (http://meme-suite.org/), with the following parameters: maximum number of motifs set at 25 and optimum with of motifs set from 5 to 200 amino acids.

Promoter analysis and identification of miRNAs targets

The upstream 2 kb genomic DNA sequences of each predicted AR2/ERF genes were extracted from the B. distachyon genome, and then submitted to PLACE database (http://www.dna.affrc.go. jp/PLACE/) to identify the putative cis-regulatory elements in the promoter regions. Furthermore, all the identified AP2/ERF transcripts were searched against the published B. distachyon miRNAs in the miRBase using psRNATarget tool (http://plantgrn.noble.org/psRNATarget/) to predict the AR2/ERF targeted by miRNA.

Gene duplication and synteny analysis

Gene duplication events were identified manually using the method as described by Chen et al. [33]. The segmental duplication events were characterized as copying the whole blocks of genes from one chromosome to another, while contiguous homologous genes with the original duplication on a single chromosome were defined as tandem duplications [34]. For synteny analysis, duplications between B. distachyon AP2/ERF genes, as well as the synteny block of this family among B. distachyon and other 5 grass species (rice, maize, sorghum, foxtail millet and switchgrass) were obtained from the Plant Genome Duplication Database (http://chibba.pgml.uga.edu/duplication/) and the diagrams were visualized using the program Circos v0.67 [35].

Gene expression and network interaction analysis

Microarray data of B. distachyon were obtained from Gene Expression Omnibus (GEO) (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/geo/) and EBI ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) databases, and then used to detect the expression of the AR2/ERFs in different tissue and in response to abiotic stresses. Additionally, high throughput RNA sequencing data were also retrieved and downloaded from the SRA database (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/sra) and then used to detect the differential expression of the AR2/ERF genes by FPKM analysis. A total of 9 RNA data of different tissues at different development stages were used, including anther, pistil, leaves (20 days), seed (5 and 10 days after pollination), endosperm(25 days after pollination), embryo(25 days after pollination), and inflorescence (early and emerging time). Finally, the interaction network which these putative AR2/EFR genes involved in were investigated based on the orthogous genes between B. distachyon and Arabidopsis using the AraNet V2 tool (http://www.inetbio.org/aranet/) [36].

Plant growth, stress treatment and RT-PCR analysis

Roots, stems, leaves and spikes were collected from two-months-old Bd21 genotype for RNA extraction and then used for organ-specific expression analysis. The 3 weeks old seedling were subjected to 4 °C, 20 % PEG, 150 mM NaCl conditions as cold,drought and salt treatments. After 24 hours treatment, the leaves of plant under these 3 stresses were collected for RNA isolation, respectively. Total RNA was isolated using RNAiso Reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Semi-quantitative RT-PCR was employed to determine the transcript levels of 11 randomly selected BdAP2/ERF genes following the method as described by Chen et al. [33]. The primers are listed in Additional file 1: Table S1.

Results

Identification of AP2/ERF family in Brachypodium

Using the method as described above, a total of 141 genes were identified as putative AP2/ERF genes in the Brachypodium genome, accounting for approximately 0.45 % of all annotated Brachypodium genes. Previous study has reported there were 146 AP2/ERF genes in Brachypodium through exploration of genes encoding TF domains to construct TF database [37]. The difference between them were further compared and results found that previous study considered the alternative splices transcripts encoded by the same gene into different AP2/ERF members, which resulted in the increase of the gene number. Since there is no standard nomenclature, the predicted BdAP2/ERF genes were then designated as BdAP2/ERF001 to BdAP2/ERF141 based on their chromosome location and family classification (Table 1). The detailed sequence information including genomic, transcript, CDS and protein sequence as well as 2 kb upstream has been listed in Additional file 2. Among them, 24 genes containing two repeated AP2/ERF domains were assigned to the AP2 family, and 4 genes possessed a single AP2/ERF DNA binding motif together with a B3 type domain were grouped into the RAV family. The remaining 113 genes with a single AP2/ERF domain were assigned to the ERF superfamily and further divided into ERF and DREB subfamilies. Additionally, a special AP2/ERF gene, namely BdAP2/ERF091 showed little similarity to other AP2/ERF genes, which was grouped into Soloist subfamily (Table 2 and Additional file 1: Table S2).
Table 1

Characteristic features of AP2/ERF Transcription factor gene family identified in B. distachyon

Gene Name

Ggene id

Physical position

Properties of AP2/ERF proteins

Subcell location

EST valadation

Chrom no

Start position (bp)

End Position (bp)

Protein length (aa)

pI

Molecular weight (Da)

BdAP2/ERF001

Bradi1g00670

1

521659

522548

192

8.13

20.2498

Nuclear

17

BdAP2/ERF002

Bradi1g03880

1

2601482

2604919

451

7.23

48.97473

Nuclear

6

BdAP2/ERF003

Bradi1g04110

1

2788453

2789893

302

10.05

32.70946

Nuclear

31

BdAP2/ERF004

Bradi1g07290

1

5105505

5109153

635

6.54

67.00742

Nuclear

-

BdAP2/ERF005

Bradi1g18580

1

14895085

14896008

308

6.13

32.25646

Nuclear

8

BdAP2/ERF006

Bradi1g18870

1

15104782

15107266

260

9.9

27.76687

Mitochondrial

-

BdAP2/ERF007

Bradi1g23756

1

19130000

19131273

299

9.05

32.04507

Nuclear

5

BdAP2/ERF008

Bradi1g30337

1

25719025

25721056

379

9.07

40.85655

Nuclear

1

BdAP2/ERF009

Bradi1g31337

1

26832687

26835421

467

5.81

50.91159

Nuclear

2

BdAP2/ERF010

Bradi1g33550

1

29119163

29120322

189

5.17

20.18015

Nuclear

2

BdAP2/ERF011

Bradi1g35400

1

30927335

30927973

213

9.16

22.94622

Chloroplast

-

BdAP2/ERF012

Bradi1g35410

1

30934707

30935330

208

8.33

22.09309

Nuclear

-

BdAP2/ERF013

Bradi1g35420

1

30939140

30939700

187

5.11

20.32711

Cytoplasmic

-

BdAP2/ERF014

Bradi1g36590

1

32262325

32263730

226

9.3

23.87795

Nuclear

-

BdAP2/ERF015

Bradi1g38110

1

34253209

34254003

265

4.85

27.79697

Chloroplast

1

BdAP2/ERF016

Bradi1g45470

1

43685130

43686821

352

7.79

37.90218

Nuclear

7

BdAP2/ERF017

Bradi1g46120

1

44406118

44407133

236

4.62

24.42913

Chloroplast

2

BdAP2/ERF018

Bradi1g46690

1

45270114

45272465

352

4.78

38.51971

Nuclear

53

BdAP2/ERF019

Bradi1g47480

1

46027554

46028362

154

6.97

16.7786

Nuclear

5

BdAP2/ERF020

Bradi1g48320

1

46956726

46957256

177

9.99

18.74815

Nuclear

23

BdAP2/ERF021

Bradi1g49560

1

48258020

48259205

221

5.32

23.59387

Nuclear

5

BdAP2/ERF022

Bradi1g49570

1

48261395

48262309

225

5.62

23.88046

Nuclear

8

BdAP2/ERF023

Bradi1g53650

1

51957465

51961449

415

6.14

45.29458

Nuclear

9

BdAP2/ERF024

Bradi1g54450

1

52809017

52809787

257

9.32

27.56207

Nuclear

3

BdAP2/ERF025

Bradi1g57560

1

56305875

56308647

605

7.17

63.09426

Nuclear

-

BdAP2/ERF026

Bradi1g57970

1

56789254

56789982

243

4.7

26.05227

Chloroplast

9

BdAP2/ERF027

Bradi1g64240

1

63443708

63448081

395

9.09

42.75122

Nuclear

-

BdAP2/ERF028

Bradi1g67350

1

65990605

65991651

247

5.25

25.45989

Nuclear

4

BdAP2/ERF029

Bradi1g69207

1

67693295

67697013

628

6.98

67.31695

Nuclear

2

BdAP2/ERF030

Bradi1g71740

1

69679681

69681013

288

6.17

31.22614

Nuclear

7

BdAP2/ERF031

Bradi1g72450

1

70182711

70183727

339

4.78

35.80922

Nuclear

63

BdAP2/ERF032

Bradi1g72457

1

70186902

70188241

308

5.82

32.56157

Chloroplast

10

BdAP2/ERF033

Bradi1g72890

1

70503793

70507456

526

7.82

55.3123

Nuclear

10

BdAP2/ERF034

Bradi1g72990

1

70580747

70581774

331

6.41

34.74665

Nuclear

4

BdAP2/ERF035

Bradi1g75040

1

72026796

72027191

132

6.61

14.16364

Nuclear

7

BdAP2/ERF036

Bradi1g77120

1

73494001

73494771

257

5.43

27.522

Chloroplast

8

BdAP2/ERF037

Bradi2g02100

2

1434535

1436793

337

4.77

36.11855

Nuclear

14

BdAP2/ERF038

Bradi2g02710

2

1905801

1907344

364

7.16

39.28463

Nuclear

17

BdAP2/ERF039

Bradi2g02720

2

1921269

1922752

365

9.96

38.9569

Chloroplast

14

BdAP2/ERF040

Bradi2g04000

2

2818870

2820722

280

5.97

30.81911

Nuclear

1

BdAP2/ERF041

Bradi2g06180

2

4632942

4633697

252

4.8

26.7444

Nuclear

-

BdAP2/ERF042

Bradi2g07357

2

5714188

5715258

357

4.85

38.60973

Nuclear

9

BdAP2/ERF043

Bradi2g09434

2

7714796

7719951

1338

5.98

148.56946

Nuclear

2

BdAP2/ERF044

Bradi2g11890

2

10206158

10207184

198

6.19

21.45306

Nuclear

57

BdAP2/ERF045

Bradi2g15847

2

14025073

14025927

285

9.51

30.25695

Nuclear

7

BdAP2/ERF046

Bradi2g17610

2

15668356

15669979

409

9.64

43.71991

Nuclear

29

BdAP2/ERF047

Bradi2g18570

2

16501499

16503701

454

8.62

48.82772

Chloroplast

-

BdAP2/ERF048

Bradi2g21060

2

18434490

18435619

237

8.87

23.84977

Nuclear

19

BdAP2/ERF049

Bradi2g21067

2

18444903

18445651

196

10.25

20.59425

Nuclear

4

BdAP2/ERF050

Bradi2g24170

2

22011630

22012545

228

6.51

23.49464

Nuclear

15

BdAP2/ERF051

Bradi2g25050

2

22846421

22847117

180

9.63

19.10829

Nuclear

-

BdAP2/ERF052

Bradi2g26987

2

25743726

25748213

394

7.6

43.00918

Chloroplast

-

BdAP2/ERF053

Bradi2g27920

2

26951955

26953071

169

6.63

17.67988

Chloroplast

10

BdAP2/ERF054

Bradi2g29960

2

29508803

29511680

382

5

41.65054

Nuclear

15

BdAP2/ERF055

Bradi2g31480

2

31231590

31233055

272

8.54

29.35912

Nuclear

5

BdAP2/ERF056

Bradi2g37800

2

38179562

38184091

494

6.48

53.7679

Nuclear

12

BdAP2/ERF057

Bradi2g45530

2

45915509

45916627

301

4.44

31.9448

Nuclear

9

BdAP2/ERF058

Bradi2g47220

2

47558425

47559938

404

9.4

42.57902

Chloroplast

32

BdAP2/ERF059

Bradi2g48130

2

48444901

48447985

349

9.12

39.41296

Mitochondrial

-

BdAP2/ERF060

Bradi2g52370

2

51766250

51767401

244

9.57

25.15542

Nuclear

9

BdAP2/ERF061

Bradi2g52380

2

51772580

51773238

172

10.25

18.47806

Nuclear

-

BdAP2/ERF062

Bradi2g53070

2

52290848

52293688

436

8.91

47.4695

Nuclear

-

BdAP2/ERF063

Bradi2g56140

2

54552519

54554079

252

7.28

26.8432

Nuclear

19

BdAP2/ERF064

Bradi2g57200

2

55409637

55410303

147

6.61

15.52733

Nuclear

6

BdAP2/ERF065

Bradi2g57747

2

55829067

55832804

628

6.17

66.83063

Nuclear

1

BdAP2/ERF066

Bradi2g60331

2

57699689

57700420

244

5.18

25.33294

PlasmaMembrane

18

BdAP2/ERF067

Bradi2g60340

2

57707368

57708090

241

4.9

25.10965

Chloroplast

21

BdAP2/ERF068

Bradi2g61630

2

58640558

58642834

331

11.63

35.95634

Nuclear

17

BdAP2/ERF069

Bradi3g04370

3

2981846

2982121

92

10.92

9.77404

Nuclear

-

BdAP2/ERF070

Bradi3g04380

3

2988281

2989361

118

9.69

12.80229

Nuclear

-

BdAP2/ERF071

Bradi3g04410

3

3030811

3031468

194

6.58

19.75096

Nuclear

9

BdAP2/ERF072

Bradi3g06562

3

4743747

4744779

195

5.32

20.50215

Nuclear

3

BdAP2/ERF073

Bradi3g07450

3

5592374

5593517

212

9.07

22.84071

Nuclear

20

BdAP2/ERF074

Bradi3g08790

3

6915336

6916085

250

5.5

26.42149

Nuclear

2

BdAP2/ERF075

Bradi3g12565

3

11243949

11244320

124

8.85

13.81715

Nuclear

14

BdAP2/ERF076

Bradi3g12680

3

11374939

11375580

214

8.74

22.85931

Nuclear

-

BdAP2/ERF077

Bradi3g15880

3

14106918

14109546

288

5.41

31.29551

Nuclear

2

BdAP2/ERF078

Bradi3g18070

3

16466074

16466826

171

10.58

17.59787

Nuclear

8

BdAP2/ERF079

Bradi3g24000

3

23546847

23547533

229

8.73

24.80911

Nuclear

4

BdAP2/ERF080

Bradi3g27690

3

28745318

28745683

122

5.57

12.79902

Cytoplasmic

2

BdAP2/ERF081

Bradi3g31600

3

33875837

33876445

203

5.26

21.76436

Chloroplast

8

BdAP2/ERF082

Bradi3g33355

3

35731449

35732636

264

4.9

27.94319

Nuclear

3

BdAP2/ERF083

Bradi3g33670

3

36046660

36047028

123

10.08

13.23904

Nuclear

-

BdAP2/ERF084

Bradi3g35560

3

37854781

37856423

275

9.46

29.16776

Nuclear

38

BdAP2/ERF085

Bradi3g36820

3

39192052

39195892

376

7.09

41.41683

Nuclear

-

BdAP2/ERF086

Bradi3g37544

3

40042914

40043558

215

4.89

22.74426

Nuclear

-

BdAP2/ERF087

Bradi3g38140

3

40608825

40610019

280

5.69

30.34868

Nuclear

24

BdAP2/ERF088

Bradi3g41543

3

43477691

43479816

236

6.71

25.05387

Nuclear

4

BdAP2/ERF089

Bradi3g41546

3

43481400

43482857

234

8.44

24.97712

Chloroplast

-

BdAP2/ERF090

Bradi3g42627

3

44115159

44119544

487

8.75

53.34822

Chloroplast

-

BdAP2/ERF091

Bradi3g43822

3

45506144

45512571

276

9.2

31.0089

Nuclear

21

BdAP2/ERF092

Bradi3g44470

3

46345345

46346091

249

5.07

26.56773

Nuclear

13

BdAP2/ERF093

Bradi3g45997

3

47939264

47939779

172

5.2

18.7198

Nuclear

-

BdAP2/ERF094

Bradi3g47610

3

49227063

49228217

308

6.97

32.78146

Nuclear

1

BdAP2/ERF095

Bradi3g48697

3

50080298

50084464

690

6.11

73.30758

Nuclear

-

BdAP2/ERF096

Bradi3g49810

3

51052812

51054767

437

5.72

47.32396

Nuclear

22

BdAP2/ERF097

Bradi3g50490

3

51687040

51688418

297

6.32

30.96065

Nuclear

24

BdAP2/ERF098

Bradi3g50620

3

51763593

51764381

263

5.44

27.54566

Nuclear

2

BdAP2/ERF099

Bradi3g50630

3

51784618

51785497

238

5.35

24.99462

Nuclear

7

BdAP2/ERF100

Bradi3g51610

3

52662982

52664420

286

4.77

29.4364

Nuclear

-

BdAP2/ERF101

Bradi3g51630

3

52685121

52686098

228

5.87

24.17896

Nuclear

5

BdAP2/ERF102

Bradi3g54160

3

54676859

54677475

169

8.8

18.11231

Nuclear

6

BdAP2/ERF103

Bradi3g57360

3

57003839

57004549

237

5.39

25.75201

Chloroplast

5

BdAP2/ERF104

Bradi3g57867

3

57514740

57522455

545

6.05

58.32911

Chloroplast

49

BdAP2/ERF105

Bradi3g58015

3

57607422

57608473

259

5.54

27.34452

Extracellular

9

BdAP2/ERF106

Bradi3g58980

3

58281872

58283733

316

7.02

33.64749

Nuclear

33

BdAP2/ERF107

Bradi3g59300

3

58524999

58529801

373

5.87

40.81537

Nuclear

8

BdAP2/ERF108

Bradi3g60120

3

59149405

59152513

307

4.76

33.5793

Chloroplast

10

BdAP2/ERF109

Bradi4g21265

4

24609196

24609797

193

5

20.80231

Cytoplasmic

-

BdAP2/ERF110

Bradi4g27850

4

33135019

33136560

315

4.61

34.73159

Nuclear

3

BdAP2/ERF111

Bradi4g29010

4

34421980

34423859

283

6.09

30.38067

Nuclear

25

BdAP2/ERF112

Bradi4g30617

4

36372483

36375685

394

7.19

42.82752

Chloroplast

-

BdAP2/ERF113

Bradi4g31040

4

36772794

36775742

402

4.71

43.41933

Nuclear

18

BdAP2/ERF114

Bradi4g35570

4

40939109

40939843

245

5.63

26.34075

Chloroplast

8

BdAP2/ERF115

Bradi4g35580

4

40942477

40943253

259

5.89

27.62901

Nuclear

2

BdAP2/ERF116

Bradi4g35590

4

40947469

40948215

249

4.99

26.7398

Nuclear

9

BdAP2/ERF117

Bradi4g35600

4

40952136

40952876

247

4.72

26.38781

Chloroplast

4

BdAP2/ERF118

Bradi4g35610

4

40959730

40960464

245

5.58

25.92315

Nuclear

6

BdAP2/ERF119

Bradi4g35620

4

40963208

40963969

254

4.94

26.84695

Chloroplast

4

BdAP2/ERF120

Bradi4g35630

4

40965956

40967004

255

5.09

26.88492

Chloroplast

7

BdAP2/ERF121

Bradi4g35650

4

40976869

40977919

239

4.66

25.76346

Nuclear

36

BdAP2/ERF122

Bradi4g38930

4

43634131

43635066

312

6.66

33.31991

Nuclear

14

BdAP2/ERF123

Bradi4g43877

4

47462247

47467299

421

5.46

45.50805

Nuclear

2

BdAP2/ERF124

Bradi5g08380

5

11092852

11094953

286

9.92

30.21775

Nuclear

9

BdAP2/ERF125

Bradi5g14960

5

18387279

18391400

687

5.99

71.21576

Nuclear

-

BdAP2/ERF126

Bradi5g16450

5

19777125

19778588

488

8.34

51.67208

Nuclear

11

BdAP2/ERF127

Bradi5g17480

5

20740745

20742261

290

6.34

30.56629

Nuclear

33

BdAP2/ERF128

Bradi5g17490

5

20752451

20753777

365

5.04

38.57791

Nuclear

1

BdAP2/ERF129

Bradi5g17610

5

20857144

20858031

296

5.4

31.03794

Nuclear

2

BdAP2/ERF130

Bradi5g17620

5

20862925

20863756

252

5.23

26.4537

Chloroplast

3

BdAP2/ERF131

Bradi5g17630

5

20867545

20868171

209

5.96

22.66944

Chloroplast

-

BdAP2/ERF132

Bradi5g17640

5

20872897

20873850

212

5.21

22.43383

Nuclear

-

BdAP2/ERF133

Bradi5g18850

5

21957497

21958102

202

5.22

20.90808

Nuclear

-

BdAP2/ERF134

Bradi5g21250

5

23939710

23940850

217

9.69

22.12031

Nuclear

53

BdAP2/ERF135

Bradi5g24100

5

25842335

25845739

467

6.71

49.18327

Nuclear

2

BdAP2/ERF136

Bradi5g24110

5

25859990

25861523

250

9.55

26.88308

Nuclear

16

BdAP2/ERF137

Bradi5g24360

5

26013512

26017700

522

6.23

55.54319

Nuclear

-

BdAP2/ERF138

Bradi5g24700

5

26290017

26290721

143

6.29

15.30613

Nuclear

-

BdAP2/ERF139

Bradi5g24710

5

26293257

26294078

163

8.89

17.6381

Mitochondrial

-

BdAP2/ERF140

Bradi5g24720

5

26295256

26295857

144

6.09

15.98271

Nuclear

-

BdAP2/ERF141

Bradi5g25570

5

26828492

26829400

188

8.35

19.37177

Nuclear

11

Table 2

Summary of the abundance of each group of the AP2/ERF superfamily in B. distachyon, Arabidopsis and rice

Family

Subfamily

Group

B. distachyon

Arabidopsis

Rice

AP2

  

24

18

29

ERF

  

112

122

139

DREB

 

52

57

56

I

9

10

9

II

7

16

15

III

32

22

26

IV

4

9

6

ERF

 

53

65

76

V

9

12

11

VI

11

8

6

VII

7

5

15

VIII

14

15

13

IX

8

18

18

X

6

7

13

a single group

 

5

 

7

RAV

  

4

6

5

Soloist

  

1

1

1

Total AP2/ERF genes

  

141

147

174

genome size (Mbp)

  

355

125

430

The average number of AP2/ERF family genes per Mb (gene/MB)

  

0.3972

1.1760

0.4047

The percentage of AP2/ERF family genes (%)

  

0.45

0.55

0.43

Chromosome distribution analysis found that the BdAP2/ERF genes were unevenly distributed on all of the five chromosomes of Brachypodium. In detail, 40 AP2/ERF genes located on the chromosome 3, representing the most abundant regions, followed by the chromosome 1, 2 and 5, with the number of 36, 32 and 18 respectively, while there were only 15 genes on the chromosome 4, which have the minimum number of AP2/ERFs. Interestingly, all the 4 RAV genes located on the chromosome 2, which may be a Brachypodium-specific feature. The putative proteins of BdAP2/ERFs ranged from 92 to 1338 amino acids in length, with molecular weights (Mw) ranging from 9.8 to 148.6 kDa and theoretical isoelectric points (PI) ranging from 4.33 to 11.63. Subcellular localization analysis indicated that majority of BdAP2/ERFs (108 out of 141, 76.5 %) localized in the nucleus, while 25 genes were predicted to be located in the chloroplast and the remaining 7 genes located in cytoplasmic, mitochondrial, plasma membrane and extra-celluar (Table 1). To further assess the actual existence of these genes identified in this study, all the available Brachypodium expressed sequence tags (EST) were used to search against these genes using the BlastN program. Results showed that most of the AP2/ERFs were supported by EST hits, only 36 genes (25.5 %, 36/111) showed no EST hits. In light of the limit of available ESTs, the not-supported BdAP2/ERF gene might not express under any the used conditions or express with very low level that cannot be detected experimentally.

Phylogenetic relationship, conserved motif and gene structure analysis

To evaluate the evolutionary relationships of BdAP2/ERF genes, phylogenetic analysis was further conducted based on multiple sequence alignment of all of the BdAP2/ERF together with rice and Arabidopsis AP2/ERF genes. The phylogenetic tree clustered all the AP2/ERF genes into three major clades (ERF, AP2 and RAV) depending on their domain composition as described above (Fig. 1). Furthermore, the ERF clades further divided into ten groups. According to the classification criteria in Arabidopsis and rice [3], the ERF superfamily could be further divided into DREB and ERF subfamily. Four groups (group I-IV) of the ERF clades belonged to ERF subfamily, containing 9, 7, 32 and 4 members while the remaining six groups (V-X) were DREB subfamily, having 9, 11, 7, 14, 8 and 6 members, respectively (Table 2). It’s established that DREB subfamily were major factors involved in plant abiotic stress responses and many stress-inducible DREBs have been isolated from numerous plants to date [21–22, 25,]. The identified DREB genes of B. distachyon provided the valuable resource to characterize the stress-responsive genes. Additionally, the bootstrapping values of the nodes in this phylogenetic tree were not very high in every clade, which was consistent with previous studies [3, 38]. NJ-tree reliability was certified by generating another phylogenetic tree by Maximum Parsimony (MP) analysis (Additional file 3: Figure S1), and it was found that nearly all the BdAP2/ERF members were placed within the same topological clusters.
Fig. 1
Fig. 1

Phylogenetic analysis of AP2/ERF proteins in B. distachyon, Arabidopsis and rice. The phylogenetic tree was constructed using the NJ (Neighbor-joining) method with 1000 bootstrap replications

Furthermore, the conserved motifs of BdAP2/ERFs were analyzed and compared. A total of 25 conserved motifs were characterized and named as motif1 to motif25 (Fig. 2 and Additional file 3: Figure S2). Among them, 8 motifs, including motif 1, 2, 3, 4, 6, 7, 16 and 22 were found to be located on the AP2/ERF domain region, while other 17 motifs were corresponded to the regions outside the DNA-binding domain, which was thought to contain either functionally factors, or domains relevant to nuclear localization and transcription regulation [39] (Additional file 1: Table S3). It is noteworthy that proteins within the same group shared one or more motifs that outside the AP2/ERF domain region. For example, motif 19 and 25 were shared by 9 members in the AP2 subfamily. Motifs 12, 15 and 20 were specifically shared by each member in the RAV subfamily, and the motif 11 was shared by ERF group I as well as motif 8, 9, 10, 14 and 18–23 were specifically presented within the group III members in the ERF subfamily. Finally, the motif 24 was shared by the group V in the DREB subfamily. The proteins within the same subfamilies contained the similar composition of conserved motifs, suggesting the similar function may be shared within each group.
Fig. 2
Fig. 2

Conserved motifs analysis of BdAP2/ERF genes according to the phylogenetic relationship. Each motif is represented by a number in a colored box. Box length corresponds to motif length

Gene structure analysis of B. distachyon AP2/ERF genes further showed that the member within the subfamily possessed the similar exon-intron structures. As a whole, the number of exon regions ranged from 1 to 12, with an average of 2.65. Most of the ERF subfamily genes (74.33 %) were observed to be intronless, which was consistent with the previous study [1]. In contrast, the AP2 subfamily members contained more intron than ERFs, which had at least four exons (Fig. 3). The highly diverse gene structure suggested that vast differentiation may occur during the B. distachyon genome formation and evolution.
Fig. 3
Fig. 3

Phylogenetic relationship and gene structure analysis of AP2/ERF genes in Brachypodium

Cis-elements and miRNA targets analysis

In order to understand the possible biological functions and regulation network of these AP2/BdERFs involved in, 2 kb genomic sequences upstream of the 5′-UTR of BdAP2/ERF genes were extracted and used to identify cis-regulatory elements. A total of 276 putative cis-elements were found to be presented in at least one BdAP2/ERF gene and only 7 (GT1CONSENSUS, DOFCOREZM, EBOXBNNAPA, MYCCONSENSUSAT, CAATBOX1, CACTFTPPCA1, WRKY71OS) out of them were presented in the promoter region of all BdAP2/ERF genes (Additional file 1: Table S4). In addition, 32 cis-elements were detected as gene-specific, such as S2FSORPL21, ABREDISTBBNNAPA and ABREDISTBBNNAPA were unique to Bradi5g24360, Bradi3g58980 and Bradi5g17620, respectively. The different numbers and types of cis-elements presenting in BdAP2/ERF genes indicated the differential regulatory network which the BdAP2/ERF genes may involve in. Further analysis found that hormones-response (e.g. abscisic acid, gibberellins, auxin, jasmonic acid and ethylene), abiotic stress-related (e.g., drought, extreme temperatures, high salinity, wounding, and disease) and organogenesis-related cis-elements were abundantly presented in the promoter regions of BdAP2/ERF (Additional file 1: Table S5), which indicated that these AP2/ERF genes might have potential functions involving in regulating a variety of stresses response and hormone signaling transduction.

Furthermore, the putative microRNAs (miRNAs) targeted BdAP2/ERF genes were also detected in this study and a total of 8 BdAP2/ERFs were predicted to be targeted by seven miRNAs (Additional file 1: Table S6). Although miRNA inhibition mostly involved the transcript cleavage, the BdAP2/ERF006 was predicted to be inhibited to translation. Most predicted microRNA target sites located into CDS region but outside the AP2 domain, whereas for gene BdAP2/ERF051 the cleavage site located in the 3′UTR region. The miRNAs-AP2/ERF complex identified in this study would be useful in interpreting the post-transcriptional control of gene expression during various stress-induced physiological and cellular processes in B. distachyon as well as other cereal crops.

Gene duplication and synteny analyses of AP2/ERFs between B. distachyon and other three grass species

The tandem and segmental duplication events of BdAP2/ERF genes were investigated through five B. distachyon chromosomes (Fig. 4). Four AP2/ERF gene clusters contained twelve tandem duplicated genes were identified, which located on chromosome 1, 2, 4, respectively. Each cluster had a pair of genes except the cluster located on chromosome 4, which contained six genes belonged to group III of ERF subfamily. Furthermore, 27 pairs of chromosomal segments duplication were also found (Fig. 4). Intriguingly, 3 out of 4 RAV family members showed orthologous relationship, suggesting they may share a common ancestor. To derive the origin and evolutionary relationships of AP2/ERF genes, the comparative syntenic analysis between B. distachyon with other three grass species (rice, sorghum and maize) was performed (Fig. 5a, b, c). Through whole genome-wide syntenic analysis, 44, 49 and 48 % of BdAP2/ERF were identified to be orthologous to rice, sorghum and maize, respectively. Most of BdAP2/ERF genes showed syntenic bias towards particular chromosomes of sorghum, maize, rice, which indicated that the chromosomal rearrangement events like duplication and inversion may predominantly shape the distribution and organization of AP2/ERF genes in these genomes.
Fig. 4
Fig. 4

Genomic locations of AP2/EFR genes and duplicated gene pairs in the B. distachyon genome

Fig. 5
Fig. 5

Comparative physical mapping showing the degree of orthologous relationships of BdAP2/ERF genes with (a) rice, (b) sorghum, (c) maize

The substitution rate of non-synonymous (Ka) versus synonymous (Ks) was an effective measure to examine the positive selection pressure after duplication, wherein Ka/Ks =1 means neutral selection, Ka/Ks <1 stands for purifying selection, and Ka/Ks >1 signifies accelerated evolution with positive selection [40]. Furthermore, the divergence rate of the tandem and segmental duplicated BdAP2/ERF genes was calculated to detect selection influence (Additional file 1: Table S7 and S8). The Ka/Ks ratio for tandem duplicated gene-pairs in B. distachyon AP2/ERF genes ranged from 0.23 to 0.51 with an average of 0.31, whereas Ka/Ks for segmental duplicated gene-pairs ranged from 0.19 to 0.85 with an average of 0.53. These results indicated that the duplicated BdAP2/ERF genes were under strong purifying selection pressure and had gone through substitution elimination and enormous selective constraint by natural selection during the process of evolution since their Ka/Ks ratios were estimated to be lower than one. In addition, the duplication event of these BdAP2/ERF tandem and segmental duplicated genes was estimated to have occurred around ~54 and ~61 Mya, respectively. Although the BdAP2/ERF gene-pairs of segmental (Ka/Ks = 0.53) and tandem duplication (Ka/Ks = 0.31) events are not under similar evolutionary positive selection pressure, both set of gene pairs revealed that these duplication events may take place simultaneously. Additionally, the Ka/Ks ratios of the orthologous gene-pairs between B. distachyon and other three grass species were also calculated (Additional file 1: Table S9, S10, S11). The average Ka/Ks value was maximum between B. distachyon and maize (0.47), followed by rice (0.44) and sorghum(0.43), suggesting the genes pairs between B. distachyon and those three grass species appeared to have undergone extensive intense purifying selection. The divergence time was about 47, 49 and 51Mya for rice, sorghum and maize, respectively. Therefore, it can be concluded that the segmental and tandem duplication events played a major role in evolution and functional divergent of AP2/ERF genes family in B. distachyon as well as other grass species.

Co-expression network between AP2/ERFs and other genes in B. distachyon

To get the preliminary information about the interaction relationship between AP2/ERF and other genes in B. distachyon, we constructed the interaction network of them based on the orthology-based prediction followed the network in Arabidopsis (Fig. 6). A total of 39 AP2/ERFs, with 517 gene pairs of network interactions, were detected. The GO annotations of interacted genes were involved diverse biological process, cellular component and molecular function (Additional file 1: Table S12). For example, symbols BLH6, IAA16, IAA31, ZCW32, LBD41 and HAT3, which play an important role in organ development and response to osmotic stress, were identified as the most closed linked genes with AP2/ERFs. Furthermore, we found AP2/ERF61 and AP2/ERF100 regulated 50 downstream genes involved in multiple biological processes, including stress response, hormone, and light response. The co-expression network analysis of AP2/ERF genes may provide important information for the better understanding AP2/ERF transduction pathways in B. distachyon as well as in other species.
Fig. 6
Fig. 6

The interaction network of AP2/ERF genes in Brachypdium according to the orthologs in Arabidopsis

Expression profiles of BdAP2/ERF genes at different developmental stages and under stresses

The tissue-specific expression profiles of BdAP2/ERF genes at different developmental stages were investigated using RNA-Seq data based on the FPKM analysis. Results found there was high variance in the expression levels among BdAP2/ERF genes (Fig. 7). Several proteins showed relatively high expression in all the tissues, including BdAP2/ERF106, BdAP2/ERF018, BdAP2/ERF113, BdAP2/ERF108, BdAP2/ERF023, BdAP2/ERF048, BdAP2/ERF037, BdAP2/ERF003 and BdAP2/ERF111, suggesting they played the indispensable roles in regulating growth and development. However, three genes, including BdAP2/ERF119, BdAP2/ERF116 and BdAP2/ERF118 showed very low expression in all the tested organs. Furthermore, the tissue-specific expressed AP2/EFR genes were also identified. BdAP2/ERF083 and BdAP2/ERF064 were found to be predominantly expressed in pistil and leaf, respectively, while BdAP2/ERF005 and BdAP2/ERF006 showed preferential expression in the emerging inflorescence. In addition, six genes namely BdAP2/ERF092, BdAP2/ERF131, BdAP2/ERF011, BdAP2/ERF012, BdAP2/ERF013 and BdAP2/ERF139 were found to be mainly expressed during pollination, which may contribute to further study of the reproductive growth and seed formation in B. distachyon.
Fig. 7
Fig. 7

The expression profiles of BdAP2/ERF genes in different tissue and development stage

To study the roles of BdAP2/ERF genes in the response to abiotic stresses, the RNA-seq data of B. distachyon under cold treatment (4 °C, 24 h) [41] was first used to investigate their expression patterns. Based on the RNA-seq data, a total of 106 BdAP2/ERF genes were detected. Using the fold change method (log2-bias ratio) with more than one fold as criterion, 69 genes were identified as differentially expressed genes (Fig. 8). Among them, 34 genes were up-regulated whereas 35 were down-regulated. Remarkably, BdAP2/ERF122 presented 32 fold up-regulated, while BdAP2/ERF118 showed 122 times down-regulated. Furthermore, the expression profiles of BdAP2/ERF genes under drought stress were also analyzed using the available microarray data [42]. Results found that 16 BdAP2/ERF genes were differentially expressed under drought treatment (Fig. 9). In the expansion zone, five genes were identified as differentially expressed genes, of which one was up-regulated, the remaining four was down-regulated. In the mature zone, we detected eight differentially expressed genes, six genes showed up-regulated while the remaining two showed down-regulated. In the proliferation zone, we characterized three up-regulated genes and three down-regulated genes, respectively. Remarkably, AP2/ERF062 showed down-regulated in all three zones, whereas AP2/ERF022 showed up-regulated expansion in zone and mature zone.
Fig. 8
Fig. 8

Heatmap of expression profiles of BdAP2/ERF genes under cold stress

Fig. 9
Fig. 9

Heatmap of expression profiles of BdAP2/ERF genes under drought stress

Expression patterns of BdAP2/ERFs in various tissues and under stress treatment by semi-quantitative RT-PCR analysis

To further verify the expression of these identified AP2/ERF genes, 11 BdAP2/ERF genes were randomly selected to detect their expression levels in four tissues and under three stresses treatments through semi-quantitative RT-PCR analysis (Fig. 10). Results showed only one gene (BdAP2/ERF114) was not expressed in these four tissues and the other ten genes were detected to be expressed. Among them, seven genes were found to be expressed in all four tissues with different profiles. In addition, BdAP2/ERF014 was found to be specifically expressed in stems. BdAP2/ERF076 showed high expression level in stem and leaf, while BdAP2/ERF022 and BdAP2/ERF073 showed high expression level in leaf and spike. Under stress conditions, all of the 11 genes were detected to be expressed. BdAP2/ERF 014, BdAP2/ERF022 and BdAP2/ERF120 were down-regulated under all three stress conditions compared to control, while BdAP2/ERF045, BdAP2/ERF053 and BdAP2/ERF062 was up-regulated under all the treatments. Furthermore, BdAP2/ERF113 showed higher expression under drought treatment, while BdAP2/ERF076 and BdAP2/ERF114 showed high expression under cold and drought treatment respectively, which were consistent with that of RNA-seq and microarray analysis.
Fig. 10
Fig. 10

RT-PCR analysis of 11BdAP2/ERF genes

Discussion

AP2/ERF superfamily is one of the largest groups of plant-specific transcription factors, which has been widely studied in diverse plant species, such as Arabidopsis, soybean, rice, maize, foxtail millet and switchgrass [1, 28, 30, 43, 44]. In this study, we performed a comprehensive search for AP2/ERF genes throughout Brachypodium genome, and 141 BdAP2/ERF genes were found, accounting for 0.45 % of all the Brachypodium genes, which was similar with the result in rice (0.43 %), maize (0.44 %) and foxtail millet (0.44 %) [43]. While compared to other plants, the number of AP2/ERF in Brachpodium is much lower than that of rice (174), maize (184) as well as foxtail millet (171), and also slight lower than that of Arabidopsis (148) and grape (149). It has been revealed that the number of AP2/ERF gene family was mainly depending on the number of ERF family members [45]. It’s found that there are 112 members in ERF family in Brachypodium, while 122, 132 and 158 in Arabidopsis, rice and maize, respectively. In contrast, the number of AP2 and RAV family members showed no significantly difference among them, with the value of 28, 24, 34 and 25 in Brachypodium, Arabidopsis, rice and maize respectively. Thus, the lower AP2/ERF gene abundance in Brachpodium may also due to the lower number of ERF and DREB subfamily. Furthermore, the gene density is 0.3972 AP2/ERF genes per Mb in B. distachyon, while the value for rice and Arabidopsis is 0.4047 and 1.1760 respectively. B. distachyon shows closer AP2/ERF density with rice than Arabidopsis, suggesting the specific evolutionary events might occur to regulate the retention and disposition of this gene family between Monocots and Eudicots.

It has been widely revealed that AP2/ERF transcription factors played crucial roles in regulating plant growth, development and response to diverse stresses as well as signal transduction pathway in plants [46]. However, the function of BdAP2/ERFs is not well understood at present. In this study, the expression patterns of these genes in different tissues and under different stresses were systematically investigated to understand their potential function during development and stress response. Results found that a total of 138 BdAP2/ERFs were expressed in at least one tested tissue, indicating they widely involved in growth and development. Compared to AP2 family, the members in ERF family showed higher expression levels in these tissues. We found that the ERF family genes had less intron than AP2 family in Brachypodium, which may cause the quicker response and higher expression of ERF genes during development [45]. At the same time, BdAP2/ERF genes also showed obvious spatial and temporal expression profiles. For example, BdAP2/ERF064 is specifically expressed in leaf, and BdAP2/ERF005 showed preferential expression in the emerging inflorescence. In addition, six genes having significantly higher expressed during pollination were also identified, which may play the vital role in embryo and endosperm development. AP2/ERF proteins could bind to GCC-box or DRE motifs through the ERF domain, and then regulated the target gene expression under stress conditions [47, 48]. Compare to control, 69 BdAP2/ERFs showed differential expression under cold stress and 16 showed differential expression under drought stress, respectively. Among them, BdAP2/ERF120 (Bradi4g35630) which is a member of DREB subfamily, showed significantly up-regulated under both cold and drought stresses. Previous study have reported Bradi4g35630 encoding a C-repeat binding factor 3-like protein, is a cold-responsive gene and over-expression of this gene could improve the drought, salt and cold tolerance in Brachypodium [49]. Moreover, a total of 5 ACGTATERD1 (element of early responsive to dehydration), 1 DRE1COREZMRAB17 (element of responsive to drought) and 9 MYCCONSENSUSAT (element of responsive to dehydration and cold) cis-elements were identified in the promoter region of BdAP2/ERF120. In addition, BdAP2/ERF053 (Bradi2g27920) is found to be highly expressed in all three stress treatments, which contained 5 LTRECOREATCOR15 (core element of low temperature responsive), 6 EMBP1TAEM (element involving in ABA-mediated stress-signaling pathway) and 1 GT1GMSCAM4 (element required for salt-induced gene expression) cis-elements. We speculated that cis-elements were the vital regulators to control the spatial and temporal expression of the BdAP2/ERFs, which integrated other functional proteins with the AP2/ERF transcription factor to form the complex regulatory metabolic network during development and stress response processes [50]. These identified tissue-specific and stress-induced BdAP2/ERF provided the valuable candidates for further functional studies of AP2/ERF genes in B. distachyon as well as in other cereal crops.

Conclusions

Our current study identified and characterized the AP2/ERF transcription factors in the model grass B. distachyon. By performing a genome-wide search, a total of 141 BdAP2/ERF genes were obtained. EST hits or full-length cDNA sequences confirmed their actual existence. The chromosome location, exon-intron structure and conserved motif composition as well as phylogenetic relationship of these BdAP2/ERFs were systematically analyzed and compared. BdAP2/ERFs could be classified into four subgroups in accordance with the number of AP2 domains and putative functions. Co-expression network analysis found that 39 BdAP2/ERFs were involved in regulating other B. distachyon genes, and 517 network branches were found. The expression profiles of BdAP2/ERF genes in various tissues as well as under cold and drought stresses were investigated, and several tissue-specific or stress-induced BdAP2/ERF genes were identified, which could considered as the candidates for further study of their function in plant development and stress response. Our study for the first time reported the organization, structure, evolutionary and expression features of the BdAP2/ERF family, which will facilitate the future functional analysis of BdAP2/ERF genes, and lay the foundation for better understanding the molecular mechanism of plant development and stress physiological processes in B. distachyon and beyond.

Abbreviations

AP2/ERF, APETALA2/Ethylene responsive factor; DREB, dehydration responsive element binding protein; EST, expressed sequence tag; FPKM, fragments kilobase of exon model per millon mapped reads; Ka, substitution rate of non-synonymous; Ks, substitution rate of synonymous; MP, maximum parsimony; MW, molecular weight; NJ, neighbor joining; PI, isoelectric point; TF, transcription factors

Declarations

Acknowledgment

This work was mainly funded by the National Natural Science Foundation of China (Grant No. 31401373) and partially supported by the Open Project Program of State Key Laboratory of Crop Stress Biology in Arid Areas, China (CSBAA2014002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to Dr. Haifeng Li for providing the plant materials, and also grateful to Jianxin Bian and Le Wang for their constructive suggestion on data analysis as well as we would like to thank the anonymous reviewers for their constructive comments.

Availability of data and material

All of the datasets supporting the results of this article are included within the article and its Additional files. The deposited the phylogenetic data in our manuscript to Treebase to make it available publically with the accession NO. S19605. The access URL is http://purl.org/phylo/treebase/phylows/study/TB2:S19605.

Authors’ contributions

CLC and FKW collected the public dataset, perform bioinformatics analysis and also drafted the manuscript. WMX contributed to data collection. WM and DPC contributed to data analysis and manuscript preparation. SWN provided the experimental coordination and reviewed the manuscript. NXJ conceived this study and prepared the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Crop Stress Biology in Arid Areas, College of Agronomy and Yangling Branch of China Wheat Improvement Center, Northwest A&F University, Yangling, 712100, Shaanxi, China
(2)
Australia-China Joint Research Centre for Abiotic and Biotic Stress Management in Agriculture, Horticulture and Forestry, Yangling, 712100, Shaanxi, China

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Copyright

© The Author(s). 2016

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