Mol Genet Genomics DOI 10.1007/s00438-015-1061-3

ORIGINAL PAPER

Genome‑wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress Meng‑Yao Li1 · Zhi‑Sheng Xu1 · Ying Huang1 · Chang Tian1 · Feng Wang1 · Ai‑Sheng Xiong1 

Received: 16 March 2015 / Accepted: 2 May 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  AP2/ERF is a large transcription factor family that regulates plant physiological processes, such as plant development and stress response. Carrot (Daucus carota L.) is an important economical crop with a genome size of 480 Mb; the draft genome sequencing of this crop has been completed by our group. However, little is known about the AP2/ERF factors in carrot. In this study, a total of 267 putative AP2/ERF factors were identified from the whole-genome sequence of carrot. These AP2/ERF proteins were phylogenetically clustered into five subfamilies based on their similarity to the amino acid sequences from Arabidopsis. The distribution and comparative genome analysis of the AP2/ERF factors among plants showed the AP2/ERF factors had expansion during the evolutionary process, and the AP2 domain was highly conserved during evolution. The number of AP2/ERF factors in land plants expanded during their evolution. A total of 60 orthologous and 145 coorthologous AP2/ERF gene pairs between carrot and Arabidopsis were identified, and the interaction network of orthologous genes was constructed. The expression patterns of eight AP2/ERF family genes from each subfamily (DREB, ERF, AP2, and RAV) were related to abiotic stresses. Yeast one-hybrid and β-galactosidase activity

Communicated by S. Hohmann. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-015-1061-3) contains supplementary material, which is available to authorized users. * Ai‑Sheng Xiong [email protected] 1



State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

assays confirmed the DRE and GCC box-binding activities of DREB subfamily genes. This study is the first to identify and characterize the AP2/ERF transcription factors in carrot using whole-genome analysis, and the findings may serve as references for future functional research on the transcription factors in carrot. Keywords  Genome-wide analysis · AP2/ERF transcription factor · Evolution · Abiotic stress · Yeast one-hybrid · Carrot

Introduction Plant growth and development are significantly affected by abiotic stresses, such as high or low temperature, drought, salt, and metal ions. The continuously changing global climate may exacerbate these stresses. Plants have developed multiple strategies to survive (Singh et al. 2002; Zhu 2002; Katagiri 2004). Therefore, understanding the response patterns of plants against various stresses is indispensable. Transcription factors (TFs) participate in the stress regulatory network (Chinnusamy et al. 2004; Mizoi et al. 2012). TFs can directly respond to stresses or regulate the expression of downstream target genes (Shinozaki et al. 2003). TFs can be grouped into families; members of the same family have similar functions (Riechmann et al. 2000; Xu et al. 2011). A complete genome sequence has enabled the genome-wide analysis of TF families. A total of 1533 genes in Arabidopsis have been identified and classified into 34 families (Riechmann et al. 2000), whereas 1611 TF genes in rice have been classified into 37 gene families (Xiong et al. 2005). Carrot, a family member of Apiaceae, is an important economical crop worldwide. However, only a few TF members have been identified and

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their functions have been rarely investigated (Kimura et al. 2008). AP2/ERF, one of the largest TF families in plants, has received considerable attention in recent years. Several AP2/ERF genes have been successfully identified in many plants since the first AP2 gene has been reported from the model plant A. thaliana (Jofuku et al. 1994; Haake et al. 2002; Sasaki et al. 2007). Previous studies showed that AP2/ERF genes play important roles in plant development, pathogen defense, and stress response (Xu et al. 2011; Mizoi et al. 2012). AP2/ERF TFs were originally believed to be plant specific. However, extensive research revealed that AP2/ERF genes also exist in nonplants, such as the ciliate Tetrahymena thermophile (Wuitschick et al. 2004) and the viruses Bacteriophage Felix 01 and Enterbacteria phage RB49 (Magnani et al. 2004). AP2/ERF TFs are characterized by the presence of an AP2/ERF domain that binds to the GCC-box (GCCGCC) and/or DRE/C-repeat element to regulate downstream genes (Fujimoto et al. 2000; Xu et al. 2011). AP2/ERFs can be classified according to their sequence similarity into the following subfamilies: ERF (one AP2 domain and ethylene-responsive factor), DREB (one AP2 domain and dehydration-responsive element-binding protein), RAV (one AP2 domain and one B3 domain), AP2 (two AP2 domains), and Soloist (AP2-like domain) (Sakuma et al. 2002). AP2/ERF TFs serve important functions in plant growth, development, and stress response. Some AP2 subfamily members in Arabidopsis regulate the development of flowers, ovules, petals, and sepals (Kunst et al. 1989; Jofuku et al. 1994; Krizek 2009). The expression of SlAP2a in tomato is upregulated during fruit development and ripening (Chung et al. 2010). The rice AP2 gene CRL5 is highly expressed in the stem region where crown root initiation occurs, whereas the crl5 mutant demonstrates an impaired initiation of crow root (Kitomi et al. 2011). The two major subfamilies (DREB and ERF) are associated with many abiotic and biotic stress responses. ERF subfamily members directly regulated pathogenesis-related (PR) by binding the DNA with the GCC-box, can increase plant resistance to fungi, bacteria, and viruses (Park et al. 2001; Zhang et al. 2009; Pan et al. 2010). Overexpressing ERF genes confer tolerance against salt and cold stresses in transgenic plants. TaERF1 overexpression improves salt and freezing tolerance in Arabidopsis, and GmERF3 overexpression increases stress tolerance in transgenic tobacco (Zhang et al. 2009, 2010). DREB TFs activate a series of target gene expression by binding to the DRE/CRT cis-acting element. Transforming 35S:DREB1A into Arabidopsis induces a minimum of 40 stress-response genes and thus enhances stress tolerance in the plant (Seki et al. 2001). The maize DREB-type gene ZmDREB1A was isolated using the yeast one-hybrid system and was confirmed to be involved in

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Mol Genet Genomics

cold-responsive gene expression. ZmDREB1A overexpression provides transgenic plants with high tolerance against drought and freezing stresses (Qin et al. 2004). The present study identified AP2/ERFs using the completed genome sequence of carrot. This study has the following objectives: (1) to identify AP2/ERF TFs in the carrot genome; (2) to analyze the phylogenetic relationships of AP2/ERFs among species; (3) to analyze the expression patterns of AP2/ERFs under abiotic stresses; and (4) to verify the transcriptional activation activity of DREB proteins. This study provided insights into the functions of the AP2/ ERF family in carrot.

Materials and methods Database search and AP2/ERF gene sequences retrieval The whole-genome sequencing of carrot has been completed in our laboratory (Lab of Apiaceae Plant Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China). All nucleotide and protein sequences of Daucus carota L. cv. Kuroda were downloaded from the website of the Carrot Genome Project (http://apiaceae.njau.edu.cn/carrot/). The AP2/ERF family domain model (AP2, PF00847) was downloaded from the Sanger database (http://pfam.sanger.ac.uk/family/AP2). All putative carrot AP2/ERF family genes were obtained by screening the carrot genome sequence by HMMER3.0 software (http://hmmer.janelia.org/) using default parameters. All putative sequences were submitted to the National Center for Biotechnology Information database (http:// ncbi.nlm.nih.gov/) to search for the AP2 domain. The AP2/ ERF family databases of Arabidopsis and other species were downloaded from the plant transcription factor database (http://planttfdb.cbi.pku.edu.cn/). Multiple sequence alignments and phylogenetic analyses Sequence alignments of the AP2/ERF proteins in carrot and Arabidopsis were performed with ClustalW (Thompson et al. 1994) using default parameters. A phylogenetic tree was constructed with MEGA 5.0 (Tamura et al. 2011) using the neighbor-joining method, and tree reliability was set to 1000 bootstrap replicates. Identification of conserved motifs The deduced amino acid sequences of carrot AP2/ERF genes were analyzed with MEME Suite (version 4.9.0) (http://meme.nbcr.net/meme/) (Bailey et al. 2009) using default parameters. However, the values of minimum and

Mol Genet Genomics

maximum width of each motif were set to 10 and 200, respectively.

DRBE subfamily gene cloning, yeast one‑hybrid assay, and β‑galactosidase activity assay

Protein structure analysis and subcellular localization prediction

Basing on the whole-genome sequence of carrot, we designed a pair of special primers with Bam HI and Sac I enzymes at the end of the primers to clone four DREB genes (Additional file: Table S1). PCR amplification was performed under the following conditions: 95 °C for 5 min; 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 60 s; and an extension at 72 °C for 10 min. The PCR product was ligated into the pMD-19 simple-T vector (TaKaRa, Dalian, China) for sequencing identification and enzyme digestion. The coding region of each DREB was fused into the BamHI-SacI site of the pPC86 vector that carries the Trp synthesis gene and contains a galactose-inducible proteinactivating domain under the control of the yeast alcohol dehydrogenase promoter. The recombinant plasmids were transformed into two types of the yeast strain EGY48, which synthesizes Ura and contains the reporter gene LacZ under the control of the GCC or DRE element, respectively. The yeast cells were cultured on SD/-Ura/-Trp plates for 3 days at 30 °C. The β-galactosidase colony-lift filter assay was used to screen the colonies with X-Gal. Three blue clones were selected and cultured in liquid SD/-Ura/Trp medium to conform the β-galactosidase relative activity. o-Nitrophenyl-β-D-galactopyranoside served as the substrate.

The composition and physical/chemical characterization of the carrot AP2/ERFs were analyzed. Protein statistics were analyzed using the Sequence Manipulation Suite (http:// www.bio-soft.net/sms/); the value of theoretical pI was calculated with ExPASy (http://web.expasy.org/compute_pi/), and the solubility of recombinant proteins was predicted using the RPSP program (http://biotech.ou.edu) (Wilkinson and Harrison 1991; Gasteiger et al. 2003). The subcellular locations of the deduced AP2/ERF proteins were predicted using WoLF PSORT (http://wolfpsort.org) (Horton et al. 2007) and TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP) (Emanuelsson et al. 2007). Identification of orthologous and paralogous AP2/ERFs The orthologous and paralogous AP2/ERF factors in alga (Chlamydomonas reinhardtii), moss (Physcomitrella patens), fern (Selaginella moellendorffii), Picea abies, Arabidopsis, carrot, grape, and rice were identified using OrthoMCL (http://orthomcl.org/orthomcl/) (Li et al. 2003) with default settings. The interaction network associated with the Arabidopsis AP2/ERF orthologous genes was constructed using the Arabidopsis Interactions Viewer (http:// bar.utoronto.ca/interactions/cgi-bin/arabidopsis_interactions_viewer.cgi). Plant materials, growth conditions, and abiotic stress treatments Two carrot varieties (D. carota L. cvs. ‘Kurodagosun’ and ‘Junchuanhong’) were used for experiments. ‘Kurodagosun’, a variety introduced from Japan, matures early and resists heat environment; ‘Junchuanhong’ was bred in China, grows fast and resists bolting. The seedlings were grown in pots in a controlled-environment growth chamber under a photoperiod of 16 h with 300 μmol m−2 s−1 light intensity at 25 °C and 8 h dark condition at 16 °C. After 2 months, the plants were transferred to different growth chambers under the same growth conditions but different abiotic stress treatments. The temperature was set at 4 or 38 °C for cold and heat treatments, respectively. The plants were irrigated with 200 mM NaCl and 20 % PEG6000 for salt and drought treatments, respectively. The plants were harvested under a continuous time course (0, 1, 2, 4, 8, and 24 h) after the treatments. The samples were immediately frozen in liquid nitrogen and then stored at −80 °C.

RNA isolation and expression analysis Carrot RNA was extracted using the total RNA kit (Tiangen, Beijing, China) and then treated with DNase I (TaKaRa, Dalian, China) to eliminate genomic DNA contamination. RNA (1.0 μg) was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, Dalian, China) in 20 μL of reaction volume. The cDNA reaction mixture was diluted to 1:20 using double-distilled water for quantitative real-time PCR (qRT-PCR). The PCR reactions included 2.0 μL of the diluted cDNA, 0.8 μL of each primer, 10 μL of SYBR GreenI mix (TaKaRa, Dalian, China), and 7.2 μL of ddH2O. qRT-PCR was performed using the MyiQ Single color RT-PCR detection system (Bio-rad, Hercules, CA, USA). The qRT-PCR conditions were as follows: at 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 58 °C for 30 s. A melting curve (65–95 °C, at increments of 0.5 °C) was generated to check the specificity of amplification. Each sample was processed in triplicate, and DcActin was used as an internal control. The relative gene expression was calculated with the 2−ΔΔCT method (Pfaffl 2001). The gene-specific primers are shown in Additional file: Table S1.

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Results Identification of AP2/ERF family TFs in carrot To obtain the AP2/ERF family TFs in carrot, we used the AP2/ERF family domain model (AP2, PF00847) as a query for multiple searches against the carrot genome sequences using HMMER and BLAST. A total of 267 AP2/ERFs were identified from the whole genome and were renumbered from Dc001 to Dc267 (Additional file: Table S2). A total of 214 genes were predicted and described with a single AP2/ ERF domain. Following the classification method in the AP2/ERF factors of Arabidopis (Sakuma et al. 2002), we further classified the 214 factors with only one domain into two subfamilies (ERF and DREB) based on the similarity of their amino acid sequences. The ERF subfamily factors were subdivided into six groups: B1–B6. The DREB subfamily factors were also subdivided into six groups: A1– A6. A total of 38 factors were grouped into the AP2 subfamily, which contained double AP2/ERF domains. Twelve factors that were identified with a single AP2/ERF domain and a B3 domain were clustered to the RAV subfamily. The remaining three factors (Dc026, Dc027, and Dc043) with an AP2-like domain were not classified to the ERF or DREB subfamily but were designated as Soloist members. The group distribution of the carrot AP2/ERFs is shown in Fig. 1, and the annotation IDs are shown in Additional file: Table S2. Phylogenetic relationship and gene structure of AP2/ ERF TFs To confirm the subfamily classification and analyze the evolutionary relationship between carrot and Arabidopsis, we used their AP2/ERF amino acid sequences to generate

Fig. 1  Distribution of carrot AP2/ERF transcription factors in subfamilies and subgroups. Each color represents different subgroups. The number and proportion of each subgroup are also presented (color figure online)

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a phylogenetic tree (Fig. 2). All AP2/ERFs were classified into five subfamilies: DREB, ERF, AP2, RAV, and Soloist. The differences within the subfamilies were further analyzed by examining the conserved motifs using MEME. The members in each subfamily had high sequence similarities (Fig. S1 and Additional file: Table S2). All members of the DREB subfamily and the ERF family contained a WLG motif; however, only a few ERF factors contained WIG. Characterization and subcellular location of the AP2/ ERF proteins To elucidate the physical and chemical characteristics of the carrot AP2/ERFs, we analyzed the number and composition of amino acids, as well as the theoretical Mw and pI, using ExPASy (http://cn.expasy.org/) and RPSP (http://biotech. ou.edu) (Additional file: Table S3). The recombinant protein solubility of AP2/ERFs was also predicted. Protein solubility is important in determining the structure and stability of proteins. A subcellular location analysis of the deduced AP2/ ERF proteins was performed with WoLF PROST and TargetP. Most AP2/ERFs were predicted to be located in the nucleus. Evolution and distribution of AP2/ERF family factors among plants Several AP2/ERF factors were identified in plants at the whole-genome level. We counted the number of AP2/ERF factors in the completed genome sequences of plants, ranging from Bryophyta to vascular plants. The AP2/ERF transcription factors in carrot have relatively higher members compared with those in other species (Fig. 3). Chinese cabbage, apple, potato, and P. abies also contain more than 200 family members, but Volvox carteri and C. reinhardtii contain 17 and 24 AP2/ERFs, respectively. It seems that the

Mol Genet Genomics Fig. 2  Phylogenetic tree of all AP2/ERF transcription factors from carrot and Arabidopsis. Sequences of the AP2/ERF proteins in carrot and Arabidopsi were aligned by ClustalW and the phylogenetic tree was constructed using MEGA 5.0 by the neighbor-joining method with 1000 bootstrap replicates. Each subfamily is represented by a specific color (color figure online)

Fig. 3  Schematic of plant phylogenetic relationships. The phylogenetic relationships of 16 representative species were drew according to the evolution of plant. The sizes of the AP2/ERF subgroups in different species are compared

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Table 1  The numbers of paralogous, orthologous, and coorthologous gene pair among species Cre

Ppa

Smo

Pab

Ath

Dca

Vvi

Cre Ppa Smo Pab Ath Dca Vvi

3 0/0 4/0 2/0 0/0 0/0 0/0

312 15/51 9/25 7/17 6/11 6/6

2 18/56 15/13 20/23 15/3

1279 19/82 24/201 17/86

51 60/145 43/43

264 54/107

76

Osa

0/0

5/7

15/7

10/85

26/34

32/64

24/28

Osa

41

The numbers on the diagonal represent the paralogous genes of each species, the numbers located before ‘/’ represent the orthologous genes, the numbers located after ‘/’ represent the coorthologous genes The abbreviations represent the species as follows: Cre Chlamydomonas reinhardtii, Ppa Physcomitrella patens, Smo Selaginella moellendorffii, Pab Picea abies, Ath Arabidopsis thaliana, Dca Daucus carota, Vvi Vitis vinifera, Osa Oryza sativa

species which has a bigger genome contains a larger number of AP2/ERF factors. The densities of AP2/ERF factors in A. thaliana (1.2352 number/Mb) were the biggest, followed by Brassica rapa (0.5959 number/Mb) and D. carota (0.5563 number/Mb), and were more than those in algae, P. abies and Amborella trichopoda. The evolutionary analysis showed the number of AP2/ERF factors in land plants expanded during their evolution. The number of AP2/ERF subfamily members varies among land plants. ERF is consistently the largest subfamily, followed by DREB, AP2, RAV, and Soloist. However, only two subfamilies (AP2 and ERF) were identified in V. carteri and C. reinhardtii, while these two subfamilies exited in all land plants. This result also suggesting that the AP2 and ERF subfamilies might represent the oldest form of AP2/ERF protein. Identification of orthologous and paralogous AP2/ERFs among plants Comparative analysis was performed to identify the orthologous and paralogous AP2/ERF factors in alga (C. reinhardtii), moss (P. patens), fern (S. moellendorffii), P. abies, Arabidopsis, carrot, grape, and rice by OrthoMCL software. The relationships between the orthologous and paralogous genes among the eight species are represented in Table 1 and Additional file: Table S4. A total of 156 homologous gene groups were found among eight species, containing 2028 paralogous, 446 orthologous, and 1094 coorthologous gene pairs. We identified 60 orthologous gene pairs and 145 coorthologous gene pairs between carrot and Arabidopsis. Between carrot and the rest of species, the numbers of orthologous gene pairs were relatively small except Arabidopsis and grape, which also belong to dicotyledons. A large number of paralogous genes were found in P. abies (1297), followed by P. patens (312) and carrot (264).

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The interaction network of AP2/ERF genes in carrot Plant growth is a developmental process that multiple genes and their interaction genes are involved in. The Arabidopsis Interactions Viewer is a database that provides complex biomolecular and pathway information of thousands of interactions between proteins. In order to further understand the interactions between AP2/ERFs and other genes in carrot, an interaction network was constructed according to the orthologs in Arabidopsis (Fig. 4). There were 38 AP2/ERF genes that showed interactions with other genes in carrot genome. A total of 101 gene pairs with the value of Pearson correlation coefficient (PCC) over than zero were positive correlations, whereas 37 gene pairs with the value of PCC less than zero were negative correlations. Moreover, 22 gene pairs could not be calculated. The transcription factor Dc248 was potentially interact with at least 40 proteins, indicating that it was very important in transcriptional level regulation. However, twelve AP2/ERF factors were regulated by a carrot protein Dck19436. DRE and GCC element‑binding activity of DREB factors DREB factors bind to the DRE or/and GCC-box (Hao et al. 2002; Sun et al. 2008). The yeast one-hybrid system is an effective method to analyze whether or not the transcription factor can bind to the cis elements and induce the expression of target genes (Ye et al. 2004; Ou et al. 2011). In the present study, four genes encoding to DREB proteins were selected and cloned from the two carrot cultivars, ‘Kurodagosun’ and ‘Junchuanhong’. Dc080 and Dc144 belonged to the same subgroup DREB-A1, whereas Dc156 and Dc239 belonged to DREB-A6. The results of the yeast one-hybrid and β-galactosidase activity assays showed that the DREBs can bind to the DREs and/or GCC

Mol Genet Genomics

Dck22141 Dck14683

Dck41028

carrot AP2/ERF gene

Dck15962 Dc158

Dck19179

carrot gene

Dc074

Dc220

Dc112

Dc240

Dc224

Dc123

PCC > 0

Dck78756

Dck19176

Dc064

Dc242 Dck06364

Dck40834

Dc149

Dc094

Dc078

Dck35360

Dck00910

Dck19436

Dck06213

Dck18162

Dc217 Dc244

Dck65507

PCC < 0 PCC not caculated

Dc110

Dck00311

Dck58368

Dc119 Dck24047

Dck64004

Dck28050

Dck68474

Dck06627 Dck51266

Dc018

Dc147

Dck28117

Dc122

Dck30926

Dc028

Dck57254 Dck74284

Dc039

Dck44466 Dck14909

Dck58695

Dck17841 Dck09182

Dck16791

Dck72590

Dck12512

Dck25253

Dc043

Dc036

Dc248

Dck37576

Dck17842

Dc104

Dck62769 Dck29316

Dck56555

Dc073

Dck36708 Dck18293

Dck08379

Dck17740

Dck74097

Dck24396 Dck20736 Dck04345

Dck51355 Dck04961

Dc146

Dck73230 Dc156

Dck16369

Dck72879

Dck21001

Dc230

Dck03417 Dck18112

Dck77443

Dck13874 Dck56378

Dck21758

Dck21056

Dck31461 Dck03518

Dck00855 Dck35990

Dck72513 Dck03358

Dck39267

Dc145

Dck21198

Dck06909

Dc225

Dck61950

Dc193

Dck03470

Dc260

Dck35335

Dc185

Dck73778

Dck18255 Dc143

Dck27572

Dck78928

Dck49134 Dck02341

Dck02004

Dck22912

Dc219

Dck00716

Dck02298

Dck21772

Dc202

Dck12865

Dck28511

Dc155

Dck31688 Dck19544

Dck19611 Dck63984

Dck15211

Dck09006

Dck00586

Dck60591

Dck73049

Fig. 4  The interaction network of AP2/ERF genes in carrot according to the orthologs in Arabidopsis. PCC Pearson correlation coefficient. Different graphicals represent the subcelluar location of dif-

ferent proteins. Ellipse nucleus, rectangle unclear, triangle plastid, parallelogram peroxisome, diamond vacuole

cis elements (Fig. 5). Overall, the binding activities of the DREB proteins were higher to the DRE element than to the GCC-box. Of all the four proteins, Dc156 showed the strongest binding activity to the DRE and GCC elements. Dc239 had no or weak binding to promote the expression of the LacZ reporter gene. Dc080 exhibited relatively weak binding to the two cis elements, but Dc144 only bound to the DRE element. The amino acid sequence alignments of the four DREB genes in the two varieties were performed using MEGA 5.0. Some different residues were observed in the AP2 domain (Fig. 6). Several residues differed in the Dc156 and Dc239 sequences, whereas only one site differed in the Dc080 and Dc144 proteins. Previous studies showed that the two sites Val14 and Glu19 in the AP2 domain of DREBs are essential for the binding of DRE element (Cao et al. 2001; Sakuma et al. 2002). As shown in Fig. 6, the 14th amino acid in AP2 domain was valine in three factors (Dc080, Dc144, and Dc156,) which can bind the DRE element, while the 14th amino acid was alanine in Dc239 sequence. Dc156 showed

the strongest binding affinity to the cis element, but the 19th amino acid was leucine acid and not glutamic acid. Apparently, the valine residue seems more important. This result suggests that Lys9 plays a key role in GCC box-binding activity. In addition, the binding activities of the DREB factors were consistent in the two varieties and were possibly related to their similar sequences. However, one or two sites were different in each DREB between the two varieties (Fig. S2). Expression profiles of the selected AP2/ERF genes under abiotic stress treatments in different carrot cultivars The groups of ERF and DREB have a large family respectively, and the members are involved in adversity stress. qRT-PCR was used to analyze the AP2/ERF gene expression patterns under different abiotic stresses (cold, heat, salt, and drought). The eight AP2/ERF genes selected from the subfamilies were as follows: Dc100 from the

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Mol Genet Genomics

Fig. 5  Yeast one-hybrid and β-galactosidase activity assays of DREBs in yeast cells. a DRE cis-binding activity of four DREB proteins. The binding affinities of DREBs were examined using the yeast one-hybrid assay. The images were captured after 12 h of incubation on SD/-Ura/-Trp medium with 20 μg/mL X-gal. b Relative β-galactosidase activity of the DREBs that bind to the DRE cis ele-

ment. This assay was repeated three times with independent clones; error bars indicate standard deviations. The data are presented as the mean  ± SD of three replicates. c GCC box-binding activity of four DREB proteins. d Relative β-galactosidase activity of the DREBs that bind to the GCC-box

RAV subfamily; Dc096 from the AP2 subfamily; Dc010 and Dc219 from the ERF subfamily; and Dc080, Dc144, Dc156, and Dc239 from the DREB subfamily. qRT-PCR analysis revealed that AP2/ERFs had a broad expression pattern under various stresses (Fig. 7). A comparison of gene expression patterns indicated that some genes rapidly responded to abiotic stress. Moreover, the expression levels of the eight genes varied under different abiotic stresses between the two carrot cultivars. The Dc090 gene from ‘Junchuanhong’ exhibited three expression patterns. In cold treatment, Dc090 expression initially decreased and then increased. In heat and salt treatment, Dc090 expression initially increased, peaked, and then slightly decreased. In drought treatment, Dc090 expression increased, peaked at 1 h, and then decreased. Several genes were also differently expressed between the two carrot varieties. The Dc080 gene of the DREB subfamily showed similar expression patterns between the two varieties, but ‘Kurodagosun’ seemed to be more sensitive to stress. In ‘Kurodagosun’, Dc080 was upregulated by more than 40-fold in heat treatment and was

down regulated by at least 25-fold in drought treatment. Dc239 was up-regulated and remained highly expressed in ‘Kurodagosun’ under heat treatment but was down regulated in ‘Junchuanhong’. Interestingly, Dc010 was highly expressed in ‘Junchuanhong’ but was relatively low in ‘Kurodagosun’. Dc219 from the same subfamily as Dc010 expressed a similar trend in the two varieties.

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Discussion Since the first AP2 TF was identified in Arabidopsis by Jofuku et al. (1994), more AP2/ERF TFs have been identified in many plants but not outside the plant kingdom over the following 10 years. The availability of genome sequence data and the development of bioinformatics have facilitated the identification of AP2/ERF genes in many species, such as Arabidopsis, Chinese cabbage, apple, poplar, and soybean (Zhang et al. 2008; Zhuang et al. 2008; Zhao et al. 2012; Li et al. 2013). This family was originally

Mol Genet Genomics

Fig. 6  Multiple sequence alignment of amino acid sequences of four DREB factors from two varieties using MEGA 5.0. The putative AP2 domains are noted by red box. The asterisks represent the key amino acid for determining the DRE binding affinity (Seki et al. 2001;

Atkinson et al. 2013). The down arrows show the differences between Dc080 and Dc144. The up arrows show the differences between Dc156 and Dc239 (color figure online)

believed to be plant specific (Riechmann and Meyerowitz 1998; Krizek 2003). In recent years, AP2/ERF TFs have been isolated in the ciliate T. thermophile (Wuitschick et al. 2004) and the viruses Bacteriophage Felix01 and Enterbacteria phage RB49 (Magnani et al. 2004). (Magnani et al. 2004) inferred that a lateral transfer of the HNH-AP2 factor from bacteria or viruses into plants may have originated the AP2/ERF transcription factor family and that animals may lose this factor through evolution. However, a comparative analysis of AP2/ERF factors among plants, bacteria, and viruses showed that the AP2 domain has been highly conserved during evolution. In the current study, we compared the AP2/ERF factors from some representative species in the evolution of the plant kingdom. Land plants have many AP2/ERF genes; this finding indicates that the number of AP2/ERFs in these plants expanded through evolution. Gene duplication plays important roles in gene family expansion, causing some genes to have similar structures and functions (Pickett and Meeks-Wagner 1995; Taylor and Raes 2004). As illustrated in Fig. 3, more than 200 AP2/ ERF factors have been identified in some plants, such as apple, Chinese cabbage, potato, carrot, and P. abies. Thus,

gene duplication is widespread during evolution. This process enriches AP2/ERF family members and serves other functions. The paralogous and orthologous AP2/ERF factors were also identified among these representative species in the plants evolution process. A large number of paralogous genes were found in P. abies, P. patens and carrot, meant that these species undergo gene duplication events. The number of orthologous gene pairs and coorthologous gene pairs were both greatest in carrot and Arabidopsis, followed by carrot and grape. There were 275 AP2/ERF genes identified in P. abies, but only 24 orthologous gene pairs exist between P. abies and carrot. This result suggested that the closer genetic relationship is, the more orthologous genes exist in the two species. TFs can regulate the expression of downstream genes at particular time, tissue, or conditions, such as stress, by binding the cis elements in the promoters. DREB is a TF involved in environment stress-signaling pathways (Shinozaki and Yamaguchi-Shinozaki 2000; Schramm et al. 2008). The DRE cis element has been identified in the promoter of abiotic stress-related genes (Stockinger et al. 1997; Maruyama et al. 2004, 2012). In the present study, the yeast one-hybrid

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Fig. 7  Expression patterns of AP2/ERF genes under different abiotic stresses. Each sample was processed in triplicate, the expression level was normalized to the reference gene DcActin. Each color repre-

sents a range of relative expression. K Kurodagosun, J Junchuanhong (color figure online)

system was used to demonstrate the DRE and/or GCC boxbinding activity of the DREB factors. Consistent with our expectation, the results showed that the DREB factors can bind to the DRE element. Many studies showed that ERF family members can bind to the GCC-box (Tournier et al. 2003; Jin et al. 2010). The DREBs in this study also showed a weak binding affinity to the GCC-box. The members of the DREB and ERF subfamilies exhibited high homology in the conserved domain; thus, some DREB genes can possibly

bind to the GCC-box. An ERF-type factor, Tsi1, can also bind to the GCC and DRE/CRT sequences with a stronger affinity to the GCC-box than to the DRE element (Park et al. 2001). Our group also obtained a modified ERF factor with increased binding activity to the GCC-box using the directed evolution methods; the modified ERF transcription factor from Brassica napus enhances cold tolerance in transgenic Arabidopsis (Xiong et al. 2013). Sequence alignment suggested that different amino acid sites might affect cis-binding

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activity. Previous studies showed that the two conserved amino acids Val14 and Glu19 in the AP2 domain are indispensable for the binding of DREBs to the DRE element (Cao et al. 2001; Sakuma et al. 2002). In our study, we found that the Val14 was might more important to the binding DRE element or GCC-box. We also found that Lys 9 might play a key role in GCC box-binding activity. However, other key residues that possibly affect the binding activity should be further studied. Previous studies showed that AP2/ERF genes have important roles in regulating plant responses to abiotic stresses (Shi et al. 2014; Zhuang et al. 2014). For example, an ERF protein from tomato, TSRF1, can improve drought tolerance in rice (Quan et al. 2010). Overexpression of LcERF054 increases salt stress tolerance in transgenic Arabidopsis plants (Sun et al. 2014). In the current study, eight AP2/ERF genes were involved in stress responses. Particularly, Dc080 showed the strongest upregulation to heat stress, whereas Dc219 showed an evident downregulation under heat treatment. The four DREB genes showed different sensitivities to stress conditions. Dc156 was induced by salt treatment. Dc080 expression was evidently changed by cold, heat, salt, and drought treatments. All these genes were shown to be involved in the stress response. Most stress-resistance traits are often controlled by multiple genes (Nakashima et al. 2009; Atkinson et al. 2013). With the rapid response of TFs to stress, many downstream genes were induced to participate in the resistance pathway. In our study, 38 AP2/ERF genes showed interactions with other genes in carrot genome. The interaction of multiple genes might lead to the different resistant pathways in different varieties. In summary, 267 AP2/ERF factors were identified and characterized in carrot based on genome sequences. The distribution and comparative genome analysis of AP2/ERF factor among plants provide a comprehensive insight into the evolution of AP2/ERF family. The expression analysis showed that AP2/ERF genes were related to abiotic stresses, and yeast one-hybrid confirmed that the DREBs can interact with both DRE element and GCC-box. However, more AP2/ERF genes and downstream stress-related genes, particularly those with unique functions, should be isolated and used in molecular breeding to improve plant tolerance against stress. Acknowledgments  The research was supported by the New Century Excellent Talents in University (NCET-11-0670); Jiangsu Natural Science Foundation (BK20130027); China Postdoctoral Science Foundation (2014M551609), the National Natural Science Foundation of China (31272175), Priority Academic Program Development of Jiangsu Higher Education Institutions. Conflict of interest  The authors declare that there are no competing interests.

Compliance with ethical standards  This article does not contain any studies with human participants or animal performed by any of the authors.

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