# Gene Structures, Classification, and Expression Models of theDREB Transcription Factor Subfamily in Populus trichocarpa

Authors: Yunlin Chen; Jingli Yang; Zhanchao Wang; Haizhen Zhang; Xuliang Mao; Chenghao Li
Journal: The Scientific World Journal
 (2013)

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## Abstract

We identified 75 dehydration-responsive element-binding (DREB) protein  genes in Populus trichocarpa. We analyzed gene structures, phylogenies, domain duplications, genome localizations, and expression profiles. The phylogenic construction suggests that the PtrDREB gene subfamily can be classified broadly into six subtypes (DREB A-1 to A-6) in Populus. The chromosomal localizations of the PtrDREB genes indicated 18 segmental duplication events involving 36 genes and six redundant PtrDREB genes were involved in tandem duplication events. There were fewer introns in the PtrDREB subfamily. The motif composition of PtrDREB was highly conserved in the same subtype. We investigated expression profiles of this gene subfamily from different tissues and/or developmental stages. Sixteen genes present in the digital expression analysis had high levels of transcript accumulation. The microarray results suggest that 18 genes were upregulated. We further examined the stress responsiveness of 15 genes by qRT-PCR. A digital northern analysis showed that the PtrDREB17, 18, and 32 genes were highly induced in leaves under cold stress, and the same expression trends were shown by qRT-PCR. Taken together, these observations may lay the foundation for future functional analyses to unravel the biological roles of Populus’ DREB genes.


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## Body

## 1. Introduction

Environmental stresses, such as drought, high salt, and low temperature, have adverse effects on plant growth and development. During evolution, plants established physiological and metabolic defense system responses to adverse conditions. In essence, stress induced the expression of specific genes and their products play a role in the plant’s stress defense mechanism. Transcription factors (TFs) can upregulate a set of genes under their control by interacting with cis-elements present in the promoter region of targeted genes. The products act as regulatory proteins, consequently enhancing the stress tolerance of the plant. The dehydration-responsive element-binding (DREBs) protein TFs play an important role in regulating plant growth and the response to external environmental stresses.In the plant kingdom,DREB is a large subfamily belonging to the APETALA2/ethylene-responsive element-binding protein (AP2/EREBP) family. DREB genes contain the highly conserved AP2/ERF DNA-binding domain [1]. The AP2/ERF domains, which consist of 50 to 60 amino acids, are found in proteins involved in a variety of regulatory mechanisms throughout the plant life cycle. The DREB subfamily play an important role in the resistance of plants to abiotic stresses by recognizing the dehydration-responsive element (DRE), which has a core motif of A/GCCGAC [2], and some members of this gene subfamily recognize the cis-acting element AGCCGCC, known as the GCC box [3]. The DREB subfamily TFs have been identified in various plant species, including mangrove[4], soybean[5], and potato[6]. The roles of DREBproteins in the plant’s response to abiotic stress have also been extensively documented [7]. In the genomes of Arabidopsis [3], Vitis vinifera[8], and rice[9], 56, 36, and 57 AP2/ERF-related proteins, respectively, are encoded. Genetic and molecular approaches have been used in combination to characterize a series of DREBfamily regulatory genes involved in many different pathways, including genes related to cold, drought, high salinity, heavy metals, and abscisic acid (ABA) [4].The characterization of theDREBsubfamily of genes in Populus can aid the understanding of the molecular mechanisms of stress resistance and thus aid in the development of Populusvarieties, using transgenic technology, with a greater tolerance to many adverse environments. Some DREBsubfamily genes have been isolated from rice, Arabidopsis, and other plants [10], but they have not been isolated from Populus. Thus, their functions remain to be determined in Populus. The completion of the high-quality sequencing of the Populus genome [11] has provided an excellent opportunity for genome-wide analysis of genes belonging to specific gene families. Therefore, we present a comprehensive and specific analysis of gene structure, chromosome localization, and expression of the Populus’ DREB subfamily for the first time. Here, we identified 75 PtrDREBgenes in Populususing database searches and classified these genes according to their homology with known genes. We describe DREB subtypes more specifically and present novel information from different tissues and/or developmental stages. Some subtypes of this gene subfamily were differentially expressed under abiotic stress conditions. PtrDREBgenes play an important role in the cross-talk of signaling pathways responding to different kinds of stress. We analyzed the phylogenetic relationships of the DREBgenes in Populus and attempted the complete alignment of the subtypes. We examined gene structure and conserved motifs of DREBgenes. Taken together, our results will be helpful in determining the functions of each DREBgene.


## 2. Materials and Methods

### 2.1. Database Search and Sequence Retrieval

TheP. trichocarpa genome DNA database was downloaded from Phytozome (http://www.phytozome.net/). The database of the A. thaliana DREBsubfamily was downloaded from the ArabidopsisInformation Resource (TAIR, http://www.arabidopsis.org/, release 10.0). A local BLAST search was performed using the amino acid sequences of the AP2/ERF domains from Arabidopsisas the queries for the identification of the DREBgenes from Populus. All of the located sequences were further manually analyzed to confirm the presence of an AP2 domain using the InterProScan program (http://www.ebi.ac.uk/Tools/InterProScan/). The Arabidopsis At4g13040was used as an outlier.


### 2.2. Phylogenetic Analysis

A phylogenetic analysis was initially performed using all theDREBgenes from Arabidopsisand Populus. To construct the phylogenetic trees, full-length Arabidopsisand Populusamino acid sequences were aligned using ClustalX 1.83 software [12] and manually edited using Jalview to reduce gaps [13]. The phylogenetic analysis was performed by the maximum parsimony method with 1,000 bootstrap replicates using MEGA 4 software [14].


### 2.3. Chromosome Localization

Each of theDREB genes’ chromosomal position in Populus was identified and plotted using the Phytozome (http://www.phytozome.net/) and Joint Genome Institute (http://genome.jgi-psf.org/pages/blast.jsf?db=Poptr1_1) websites. This information is provided in Table 2. A schematic view of the chromosomes was reorganized by the most recent whole-genome duplication in Populus.


### 2.4. Exon/Intron Structure and Motif Analysis

The exon/intron organization for individual DREB genes was illustrated using the Gene structure display server (GSDS) program (http://gsds.cbi.pku.edu.cn/). The CDS and genome sequences of the P. trichocarpa genes were obtained from NCBI (http://www.ncbi.nlm.nih.gov/). The program MEME (v4.3.0) (http://meme.sdsc.edu/) was used to deduce 75 Populus DREB protein sequences.


### 2.5. EST Profiling and Microarray Analysis

The expression profile for each gene was obtained by a Digital Northern tool at PopGenIE (http://www.popgenie.org/) that generated a digital northern expression profile heat map based on the EST representations of 19 cDNA libraries derived from different tissues and/or developmental stages [15]. The heat map was visualized using the Heatmapper Plus tool at the Bio-Array Resource for Plant Functional Genomics (http://bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper_plus.cgi/) [16]. The microarray data for various tissues/organs available at NCBI Gene Expression Omnibus (GEO) database under the series accession number GSE6422 were used for the tissue-specific expression analysis.


### 2.6. Plant Treatment and qRT-PCR Analysis

For expression pattern analysis of thePopulus DREB gene subfamily under abiotic stresses, plants were exposed to 42°C for 0, 0.5, and 1 h; 4°C for 0, 12, and 24 h; 200 mM NaCl for 0, 4, and 8 h; 100 μM ABA for 0, 2, 4, and 6 h; and 4% PEG6000 for 0, 4, and 8 h. Young leaves were harvested at various time points. All samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA isolation.Total RNA from leaf was extracted using the CTAB method. Synthesized cDNAs were used for qRT-PCR, which was performed using the TaKaRa ExTaq RT PCR Kit and SYBR green dye (TaKaRa, Dalian, China) in 96-well optical reaction plates (Applied Biosystems, USA). The results obtained for the different stages were standardized to the levels of theactin gene using the 2-ΔΔCT method. We selected 15 DREB genes to observe tolerance under stress conditions in Populus via EST profiling and microarray analysis. We designed the primers for gene expression analysis using Primer Premier 5 to produce amplified lengths of 180 to 200 bp (Table 1).Table 1
The primers ofDREB genes were generated in Primer 5 for qRT-PCR.
Name
Sequence (5′-3′)
PtrDREB4F
GTATTGAGGGGAGAAATGGATGG
PtrDREB4R
CATATCATGGTCGGAAGACAAGC
PtrDREB16F
AATCTTGCTACCACCACATCACAGT
PtrDREB16R
ATGCCTCCGCCTGACTCCTCTAT
PtrDREB17F
ACTCTGGCTTGGCACATTTGAC
PtrDREB17R
GGCTTGTATTCGCCGATGTAGGA
PtrDREB18F
GGCTCCAAAACCTGTCCCTATGA
PtrDREB18R
CCCAATGTCTCTGCCTCACTCCT
PtrDREB19F
GAGGAGGCGGCTTTGGCTTAT
PtrDREB19R
AACCGAGGAATGGAGAGGCTTG
PtrDREB28F
CAGTCAAAAAAGTTCAGAGGGGT
PtrDREB28R
CTCTTCTGCTGTTTCAAATGTGC
PtrDREB30F
GCATGTAACGGTAGAAAGGAGGGGG
PtrDREB30R
AGATTGGCGGTAGATCAAGAGTG
PtrDREB32F
AGAAGGAAGTCATCAACAAGGGG
PtrDREB32R
ATTTGGTGCAGGCTGAGGCAA
PtrDREB38F
GTGAGAGGCAATACAAGGGGA
PtrDREB38R
CGCTACTGGTGTTGAGTAGGAA
PtrDREB51F
TGACCCGACCTCAAACTCTCCAG
PtrDREB51R
TCAGACACCCATTTCCCCCACCT
PtrDREB55F
GATTCTCAACCAACCAAAACCTC
PtrDREB55R
GGCTCTCTAATTTCAGACACCCA
PtrDREB60F
GAAGAAGAACAAAGCGGGAAGGA
PtrDREB60R
CATTTCTGGGCTCTTGAAGGTCC
PtrDREB61F
GCAGGAAGGAAGAAGTTCAAGGA
PtrDREB61R
GGCTAGTGAAGGTCCCTAACCAAAT
PtrDREB62F
TCTTCTTTCTCCGATAGCAGCAC
PtrDREB62R
CACCCTATTGTTACCATTCCTCT
PtrDREB68F
TCTAAGCGAAACCAAGACCCGAA
PtrDREB68R
TTTGCCCCATTGACGCATTCT
ActinF
CATCAAAGCATCGGTGAGGTC
ActinR
GTTGCCATCCAGGCTGTCCTable 2
TheDREB genes identified from the P.  trichocarpa genome.
Gene  symbol
Gene  Locus
PF00847 AP2 domain
Arabidopsisortholog locus
PtrDREB1
POPTR_0008s07120
102–151
AT1G12610.1
PtrDREB2
POPTR_0010s19370
147–196
AT1G63030.1
PtrDREB3
POPTR_0006s10510
67–125
AT4G25470.1
PtrDREB4
POPTR_0016s13380
74–132
AT4G25480.1
PtrDREB5
POPTR_0006s05320
28–78
AT4G25490.1
PtrDREB6
POPTR_0016s05360
29–78
AT5G51990.1
PtrDREB7
POPTR_0010s19100
78–128
AT1G75490.1
PtrDREB8
POPTR_0008s07360
78–128
AT2G38340.1
PtrDREB9
POPTR_0005s25470
43–92
AT2G40340.1
PtrDREB10
POPTR_0002s03090
40–89
AT2G40350.1
PtrDREB11
POPTR_0001s32250
285–333
AT3G11020.1
PtrDREB12
POPTR_0017s08250
276–324
AT3G57600.1
PtrDREB13
POPTR_0003s13910
107–155
AT5G05410.1
PtrDREB14
POPTR_0019s13330
232–281
AT5G18450.1
PtrDREB15
POPTR_0013s13920
232–281
AT2G40220.1
PtrDREB16
POPTR_0005s07900
173–222
AT1G01250.1
PtrDREB17
POPTR_0002s09480
163–211
AT1G12630.1
PtrDREB18
POPTR_0007s05690
173–222
AT1G33760.1
PtrDREB19
POPTR_0005s16690
120–168
AT1G63040.1
PtrDREB20
POPTR_0003s17830
7–56
AT1G71450.1
PtrDREB21
POPTR_0001s14720
46–95
AT1G77200.1
PtrDREB22
POPTR_0018s02280
7–56
AT2G25820.1
PtrDREB23
POPTR_0006s27710
7–56
AT2G35700.1
PtrDREB24
POPTR_0018s01680
5–55
AT2G36450.1
PtrDREB25
POPTR_0006s26990
21–71
AT2G44940.1
PtrDREB26
POPTR_0003s02750
5–55
AT3G16280.1
PtrDREB27
POPTR_0006s06870
5–55
AT3G60490.1
PtrDREB28
POPTR_0018s13130
5–55
AT4G16750.1
PtrDREB29
POPTR_0006s08000
22–71
AT4G32800.1
PtrDREB30
POPTR_0007s10750
22–71
AT5G11590.1
PtrDREB31
POPTR_0005s18430
22–71
AT5G25810.1
PtrDREB32
POPTR_0002s12550
35–84
AT5G52020.1
PtrDREB33
POPTR_0014s02530
35–84
AT1G19210.1
PtrDREB34
POPTR_0018s00700
14–63
AT1G21910.1
PtrDREB35
POPTR_0006s14110
14–63
AT1G22810.1
PtrDREB36
POPTR_0005s15830
31–80
AT1G44830.1
PtrDREB37
POPTR_0002s08610
26–76
AT1G46768.1
PtrDREB38
POPTR_0019s10220
29–78
AT1G71520.1
PtrDREB39
POPTR_0013s10420
20–70
AT1G74930.1
PtrDREB40
POPTR_0018s08320
18–68
AT1G77640.1
PtrDREB41
POPTR_0006s23480
23–73
AT2G23340.1
PtrDREB42
POPTR_0006s14090
16–66
AT3G50260.1
PtrDREB43
POPTR_0006s14100
16–66
AT4G06746.1
PtrDREB44
POPTR_0001s18180
15–65
AT4G31060.1
PtrDREB45
POPTR_0013s10340
14–64
AT4G36900.1
PtrDREB46
POPTR_0019s10420
11–62
AT5G21960.1
PtrDREB47
POPTR_0003S05300
16–66
AT5G67190.1
PtrDREB48
POPTR_0013s10330
49–99
AT1G36060.1
PtrDREB49
POPTR_0019s10430
15–65
AT1G64380.1
PtrDREB50
POPTR_0019s09530
36–84
AT1G78080.1
PtrDREB51
POPTR_0014s09540
38–88
AT2G22200.1
PtrDREB52
POPTR_0855s00200
20–68
AT4G13620.1
PtrDREB53
POPTR_0002s17330
20–68
AT4G28140.1
PtrDREB54
POPTR_0006s02180
44–93
AT4G39780.1
PtrDREB55
POPTR_0016s02010
50–98
AT5G65130.1
PtrDREB56
POPTR_0015s13840
84–132
AT1G22190.1
PtrDREB57
POPTR_0012s13880
115–163
AT4G13040.1
PtrDREB58
POPTR_0003s12120
14–61
PtrDREB59
POPTR_0001s08740
14–61
PtrDREB60
POPTR_0009s14990
57–108
PtrDREB61
POPTR_0004s19820
59–110
PtrDREB62
POPTR_0001s08710
66–116
PtrDREB63
POPTR_0001s08720
62–111
PtrDREB64
POPTR_0012s13870
85–135
PtrDREB65
POPTR_0015s13830
79–129
PtrDREB66
POPTR_0002s14210
94–143
PtrDREB67
POPTR_0003s07700
96–146
PtrDREB68
POPTR_0001s15550
71–122
PtrDREB69
POPTR_0003s04920
87–137
PtrDREB70
POPTR_0001s18800
54–103
PtrDREB71
POPTR_0018s09270
69–119
PtrDREB72
POPTR_0006s17670
67–117
PtrDREB73
POPTR_0006s25500
59–109
PtrDREB74
POPTR_0018s00270
59–109
PtrDREB75
POPTR_0014s05500
90–139


## 2.1. Database Search and Sequence Retrieval

TheP. trichocarpa genome DNA database was downloaded from Phytozome (http://www.phytozome.net/). The database of the A. thaliana DREBsubfamily was downloaded from the ArabidopsisInformation Resource (TAIR, http://www.arabidopsis.org/, release 10.0). A local BLAST search was performed using the amino acid sequences of the AP2/ERF domains from Arabidopsisas the queries for the identification of the DREBgenes from Populus. All of the located sequences were further manually analyzed to confirm the presence of an AP2 domain using the InterProScan program (http://www.ebi.ac.uk/Tools/InterProScan/). The Arabidopsis At4g13040was used as an outlier.


## 2.2. Phylogenetic Analysis

A phylogenetic analysis was initially performed using all theDREBgenes from Arabidopsisand Populus. To construct the phylogenetic trees, full-length Arabidopsisand Populusamino acid sequences were aligned using ClustalX 1.83 software [12] and manually edited using Jalview to reduce gaps [13]. The phylogenetic analysis was performed by the maximum parsimony method with 1,000 bootstrap replicates using MEGA 4 software [14].


## 2.3. Chromosome Localization

Each of theDREB genes’ chromosomal position in Populus was identified and plotted using the Phytozome (http://www.phytozome.net/) and Joint Genome Institute (http://genome.jgi-psf.org/pages/blast.jsf?db=Poptr1_1) websites. This information is provided in Table 2. A schematic view of the chromosomes was reorganized by the most recent whole-genome duplication in Populus.


## 2.4. Exon/Intron Structure and Motif Analysis

The exon/intron organization for individual DREB genes was illustrated using the Gene structure display server (GSDS) program (http://gsds.cbi.pku.edu.cn/). The CDS and genome sequences of the P. trichocarpa genes were obtained from NCBI (http://www.ncbi.nlm.nih.gov/). The program MEME (v4.3.0) (http://meme.sdsc.edu/) was used to deduce 75 Populus DREB protein sequences.


## 2.5. EST Profiling and Microarray Analysis

The expression profile for each gene was obtained by a Digital Northern tool at PopGenIE (http://www.popgenie.org/) that generated a digital northern expression profile heat map based on the EST representations of 19 cDNA libraries derived from different tissues and/or developmental stages [15]. The heat map was visualized using the Heatmapper Plus tool at the Bio-Array Resource for Plant Functional Genomics (http://bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper_plus.cgi/) [16]. The microarray data for various tissues/organs available at NCBI Gene Expression Omnibus (GEO) database under the series accession number GSE6422 were used for the tissue-specific expression analysis.


## 2.6. Plant Treatment and qRT-PCR Analysis

For expression pattern analysis of thePopulus DREB gene subfamily under abiotic stresses, plants were exposed to 42°C for 0, 0.5, and 1 h; 4°C for 0, 12, and 24 h; 200 mM NaCl for 0, 4, and 8 h; 100 μM ABA for 0, 2, 4, and 6 h; and 4% PEG6000 for 0, 4, and 8 h. Young leaves were harvested at various time points. All samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA isolation.Total RNA from leaf was extracted using the CTAB method. Synthesized cDNAs were used for qRT-PCR, which was performed using the TaKaRa ExTaq RT PCR Kit and SYBR green dye (TaKaRa, Dalian, China) in 96-well optical reaction plates (Applied Biosystems, USA). The results obtained for the different stages were standardized to the levels of theactin gene using the 2-ΔΔCT method. We selected 15 DREB genes to observe tolerance under stress conditions in Populus via EST profiling and microarray analysis. We designed the primers for gene expression analysis using Primer Premier 5 to produce amplified lengths of 180 to 200 bp (Table 1).Table 1
The primers ofDREB genes were generated in Primer 5 for qRT-PCR.
Name
Sequence (5′-3′)
PtrDREB4F
GTATTGAGGGGAGAAATGGATGG
PtrDREB4R
CATATCATGGTCGGAAGACAAGC
PtrDREB16F
AATCTTGCTACCACCACATCACAGT
PtrDREB16R
ATGCCTCCGCCTGACTCCTCTAT
PtrDREB17F
ACTCTGGCTTGGCACATTTGAC
PtrDREB17R
GGCTTGTATTCGCCGATGTAGGA
PtrDREB18F
GGCTCCAAAACCTGTCCCTATGA
PtrDREB18R
CCCAATGTCTCTGCCTCACTCCT
PtrDREB19F
GAGGAGGCGGCTTTGGCTTAT
PtrDREB19R
AACCGAGGAATGGAGAGGCTTG
PtrDREB28F
CAGTCAAAAAAGTTCAGAGGGGT
PtrDREB28R
CTCTTCTGCTGTTTCAAATGTGC
PtrDREB30F
GCATGTAACGGTAGAAAGGAGGGGG
PtrDREB30R
AGATTGGCGGTAGATCAAGAGTG
PtrDREB32F
AGAAGGAAGTCATCAACAAGGGG
PtrDREB32R
ATTTGGTGCAGGCTGAGGCAA
PtrDREB38F
GTGAGAGGCAATACAAGGGGA
PtrDREB38R
CGCTACTGGTGTTGAGTAGGAA
PtrDREB51F
TGACCCGACCTCAAACTCTCCAG
PtrDREB51R
TCAGACACCCATTTCCCCCACCT
PtrDREB55F
GATTCTCAACCAACCAAAACCTC
PtrDREB55R
GGCTCTCTAATTTCAGACACCCA
PtrDREB60F
GAAGAAGAACAAAGCGGGAAGGA
PtrDREB60R
CATTTCTGGGCTCTTGAAGGTCC
PtrDREB61F
GCAGGAAGGAAGAAGTTCAAGGA
PtrDREB61R
GGCTAGTGAAGGTCCCTAACCAAAT
PtrDREB62F
TCTTCTTTCTCCGATAGCAGCAC
PtrDREB62R
CACCCTATTGTTACCATTCCTCT
PtrDREB68F
TCTAAGCGAAACCAAGACCCGAA
PtrDREB68R
TTTGCCCCATTGACGCATTCT
ActinF
CATCAAAGCATCGGTGAGGTC
ActinR
GTTGCCATCCAGGCTGTCCTable 2
TheDREB genes identified from the P.  trichocarpa genome.
Gene  symbol
Gene  Locus
PF00847 AP2 domain
Arabidopsisortholog locus
PtrDREB1
POPTR_0008s07120
102–151
AT1G12610.1
PtrDREB2
POPTR_0010s19370
147–196
AT1G63030.1
PtrDREB3
POPTR_0006s10510
67–125
AT4G25470.1
PtrDREB4
POPTR_0016s13380
74–132
AT4G25480.1
PtrDREB5
POPTR_0006s05320
28–78
AT4G25490.1
PtrDREB6
POPTR_0016s05360
29–78
AT5G51990.1
PtrDREB7
POPTR_0010s19100
78–128
AT1G75490.1
PtrDREB8
POPTR_0008s07360
78–128
AT2G38340.1
PtrDREB9
POPTR_0005s25470
43–92
AT2G40340.1
PtrDREB10
POPTR_0002s03090
40–89
AT2G40350.1
PtrDREB11
POPTR_0001s32250
285–333
AT3G11020.1
PtrDREB12
POPTR_0017s08250
276–324
AT3G57600.1
PtrDREB13
POPTR_0003s13910
107–155
AT5G05410.1
PtrDREB14
POPTR_0019s13330
232–281
AT5G18450.1
PtrDREB15
POPTR_0013s13920
232–281
AT2G40220.1
PtrDREB16
POPTR_0005s07900
173–222
AT1G01250.1
PtrDREB17
POPTR_0002s09480
163–211
AT1G12630.1
PtrDREB18
POPTR_0007s05690
173–222
AT1G33760.1
PtrDREB19
POPTR_0005s16690
120–168
AT1G63040.1
PtrDREB20
POPTR_0003s17830
7–56
AT1G71450.1
PtrDREB21
POPTR_0001s14720
46–95
AT1G77200.1
PtrDREB22
POPTR_0018s02280
7–56
AT2G25820.1
PtrDREB23
POPTR_0006s27710
7–56
AT2G35700.1
PtrDREB24
POPTR_0018s01680
5–55
AT2G36450.1
PtrDREB25
POPTR_0006s26990
21–71
AT2G44940.1
PtrDREB26
POPTR_0003s02750
5–55
AT3G16280.1
PtrDREB27
POPTR_0006s06870
5–55
AT3G60490.1
PtrDREB28
POPTR_0018s13130
5–55
AT4G16750.1
PtrDREB29
POPTR_0006s08000
22–71
AT4G32800.1
PtrDREB30
POPTR_0007s10750
22–71
AT5G11590.1
PtrDREB31
POPTR_0005s18430
22–71
AT5G25810.1
PtrDREB32
POPTR_0002s12550
35–84
AT5G52020.1
PtrDREB33
POPTR_0014s02530
35–84
AT1G19210.1
PtrDREB34
POPTR_0018s00700
14–63
AT1G21910.1
PtrDREB35
POPTR_0006s14110
14–63
AT1G22810.1
PtrDREB36
POPTR_0005s15830
31–80
AT1G44830.1
PtrDREB37
POPTR_0002s08610
26–76
AT1G46768.1
PtrDREB38
POPTR_0019s10220
29–78
AT1G71520.1
PtrDREB39
POPTR_0013s10420
20–70
AT1G74930.1
PtrDREB40
POPTR_0018s08320
18–68
AT1G77640.1
PtrDREB41
POPTR_0006s23480
23–73
AT2G23340.1
PtrDREB42
POPTR_0006s14090
16–66
AT3G50260.1
PtrDREB43
POPTR_0006s14100
16–66
AT4G06746.1
PtrDREB44
POPTR_0001s18180
15–65
AT4G31060.1
PtrDREB45
POPTR_0013s10340
14–64
AT4G36900.1
PtrDREB46
POPTR_0019s10420
11–62
AT5G21960.1
PtrDREB47
POPTR_0003S05300
16–66
AT5G67190.1
PtrDREB48
POPTR_0013s10330
49–99
AT1G36060.1
PtrDREB49
POPTR_0019s10430
15–65
AT1G64380.1
PtrDREB50
POPTR_0019s09530
36–84
AT1G78080.1
PtrDREB51
POPTR_0014s09540
38–88
AT2G22200.1
PtrDREB52
POPTR_0855s00200
20–68
AT4G13620.1
PtrDREB53
POPTR_0002s17330
20–68
AT4G28140.1
PtrDREB54
POPTR_0006s02180
44–93
AT4G39780.1
PtrDREB55
POPTR_0016s02010
50–98
AT5G65130.1
PtrDREB56
POPTR_0015s13840
84–132
AT1G22190.1
PtrDREB57
POPTR_0012s13880
115–163
AT4G13040.1
PtrDREB58
POPTR_0003s12120
14–61
PtrDREB59
POPTR_0001s08740
14–61
PtrDREB60
POPTR_0009s14990
57–108
PtrDREB61
POPTR_0004s19820
59–110
PtrDREB62
POPTR_0001s08710
66–116
PtrDREB63
POPTR_0001s08720
62–111
PtrDREB64
POPTR_0012s13870
85–135
PtrDREB65
POPTR_0015s13830
79–129
PtrDREB66
POPTR_0002s14210
94–143
PtrDREB67
POPTR_0003s07700
96–146
PtrDREB68
POPTR_0001s15550
71–122
PtrDREB69
POPTR_0003s04920
87–137
PtrDREB70
POPTR_0001s18800
54–103
PtrDREB71
POPTR_0018s09270
69–119
PtrDREB72
POPTR_0006s17670
67–117
PtrDREB73
POPTR_0006s25500
59–109
PtrDREB74
POPTR_0018s00270
59–109
PtrDREB75
POPTR_0014s05500
90–139


## 3. Results and Discussion

### 3.1. Identification ofDREB Subfamily TFs in Populus

To identify putativeDREB genes in Populus, we performed a BLASTP search against Populusgenome release v2.1 using DREBprotein sequences from Arabidopsis. By removing the redundant sequences, 75 DREBgenes were identified in the Populusgenome. All DREBcandidates were analyzed using the smart database (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) to verify the presence of AP2/ERF domains. Seventy-five DREBgenes were used for the analysis of bioinformatics and gene expression profiling. The overall strategy used in this study was depicted in Figure 1 and presented in detail below. We designated Populus DREBgenes as PtrDREBfollowing the nomenclature proposed in a previous study [17]. A. thaliana At4g13040was used as a query sequence and it includes an AP2/ERF-like domain sequence; however, its homology appears quite low in comparison with the other AP2/ERF genes. Therefore, this gene was also designated as an outlier. Detailed information on the DREBsubfamily of genes in Populusand Arabidopsisare listed in Table 1.Figure 1
Integrated systems analysis workflow for elucidation of the role ofDREBsubfamily in the bioinformatics and data analysis in Populus. A: A phylogenetic analysis was performed using all the DREB subfamily amino acid sequences from Arabidopsis and Populus by MEGA 4 software. B: Each of the DREB genes’ chromosomal position in Populus was using the Phytozome and Joint Genome Institute websites. C: The exon/intron organization for individualDREB genes was using the gene structure display server (GSDS) program and motif analysis was performed using the program MEME (v4.3.0). D: Gene expression profiling of Populus DREBsubfamily was used to characterize differentially expressed genes.


### 3.2. Phylogenetic Relationships and Alignments of theDREB Subfamily in Populus

Based on the alignment of the AP2/ERF coding region protein sequences of allPopulus and Arabidopsis DREBsubfamily genes, 75 DREBsubfamily genes of Populuswere classified into six groups, A1, A2, A3, A4, A5, and A6, containing six, 17, two, 26, 15, and nine members, respectively. The phylogenetic trees of the DREBsubfamilies of Populusand Arabidopsisare shown in Figure 2. The alignment analysis indicates that DREBs share high homology in the AP2/ERF domain (see Supplementary Figure 1; available online at http://dx.doi.org/10.1155/2013/954640) and contain a conserved WLG motif in the AP2/ERF domain of Populus. In the proteins encoded by DREBs, position 14 is normally valine and position 19 is glutamic acid. This region may play an important role in the recognition of different DNA-binding sites by the DRE and GCC box cis-elements of the DREBsubfamily [3]. Also, the DREBscontain alanine at position 319 in the β-sheet. Group A-1 possesses a conserved C/SEV/LR amino acid sequence between V319 and E324, and this region is converted into an AEIR amino acid sequence in groups A-2, A-3, and A-6. In Group A-4, Ser-324 is crucial for the specific binding of the ERE element, and Ser-324/Ala-324 is crucial in Group A-5.Figure 2
Relationships amongPopulus DREBs proteins after alignment with ClustalW. Proteins were allocated to six distinct subgroups of DREB, A-1 to A-6.


### 3.3. Chromosomal Locations ofDREB Subgroups

To examine the genomic distribution ofDREB genes on Populuschromosomes, we identified their positions by a Phytozome database search. In silico mapping of the gene loci showed that 75 Populus DREBgenes were mapped to linkage groups (LG) (Figure 3). Previous studies revealed that the Populusgenome has undergone genome-wide duplications followed by multiple segmental duplications, tandem duplications, and transposition events [18]. It was very clear that three pairs of genes were arranged in tandem repeats, LG I (PtrDREB63and 64), LG II (PtrDREB52and 53), and LG VI (PtrDREB43and 42), and 18 pairs of genes were duplicated. The dN/dS ratios from the 18 segmental duplication pairs were less than 0.5. About 56% (42 of 75) of Populus DREBswere preferentially retained tandem duplications and multiple segmental duplication events. Tandem duplications and segmental duplications were relatively underrepresented in groups A-1 to A-6 with rates of 67% (4 of 6), 47% (8 of 17), 100% (2 of 2), 53% (14 of 26), 40% (5 of 15), and 89% (8 of 9), respectively. This finding corroborates previous findings that genes involved in transcriptional regulation and signal transduction are preferentially retained following duplications [19].Figure 3
Locations ofP. trichocarpa DREB genes on the chromosomes LGI-XIX. A schematic view of chromosome reorganization by recent whole-genome duplication in Populusis shown (adapted from [21]). Regions that are assumed to correspond to homologous genome blocks are shaded in the same color and connected with lines.


### 3.4. Gene Structure and Conserved Motifs ofPopulus DREB Genes

To gain further insights into the structural diversity ofPopulus DREBgenes, we constructed a phylogenetic tree using the full-length DREBprotein sequences of Populus(Figure 4(a)). We compared the exon/intron organization in the coding sequences of each Populus DREBgene (Figure 4(b)). All but nine Populus DREBmembers had no introns in their coding regions and the nine Populus DREB genes had one intron (PtrDREB60, 61, 62, 24, 25, 26, 27, 28,and 3). We predicted conserved motifs using MEME motif detection software that revealed the diversification of the P. trichocarpa DREBgenes (Figure 4(c)), and 15 distinct motifs were identified (Table 3). The AP2/ERF domain consists of three β-sheet and one α-helix at the N termini [20, 21]. In this study, motif 3, specifying β-sheet strand 1; motif 1, specifying β-sheet strand 2 and 3; and motif 2, corresponding to the α-helix, were present in all of the Populus’ DREBsubfamily members. The CBF signature sequences (motif 14) were found in DREBGroup A-1 [20, 21]. Alignment of the deduced amino acid sequences of Arabidopsis DREBGroup A-1 TFs demonstrated significant similarity in the AP2/ERF binding domain and the CBF signature sequences [22]. These results suggest that the P. trichocarpa DREBGroup A-1 share remarkable similarities at the amino acid sequence level with known CBF/DREB proteins of Arabidopsis and carry critical amino acids that are needed for binding to the CRT elements in the target genes. The CMIV domain (motif 7) was found in the DREBGroup A-2. Alignment of the deduced amino acid sequences of Arabidopsis DREB Group A-2 TFs in the N-terminal region included the conserved motif CMIV-1 and the DNA-binding domain [23]. These results suggest that most of the closely related members in the phylogenetic tree shared a common motif composition with each other, suggesting functional similarities among the DREB proteins within the same subfamily.Table 3
Motif sequences ofDREB genes identified in P. trichocarpa by MEME tools.
Motif
Width
Best possible match
1
21
WGKWVCEIREPRKKSRIWLGT
2
24
FPTPEMAARAHDVAALCIKGDSAI
3
11
KHPVYRGVRMR
4
21
LPVPASTSPRDIQAAAASAAA
5
8
LNFPDLVH
6
25
EEALFDMPNLLVDMAGGMLLSPPRI
7
29
GDGGNKPVRKVPAKGSKKGCMKGKGGPEN
8
15
EDHHIEQMIEELLDR
9
18
YKPLHSSVDAKLQAICQS
10
25
HIGVWQKKAGSRSSSNWVMKVELGN
11
15
GPITVRLSPSQIQAI
12
30
DMSAASIRKRATEVGAHVDAIETALNHHHH
13
29
STSSLTSLVSLMDLSSQEEELCEIVELPS
14
21
EVMLASRNPKKRAGRKKFRET
15
21
FESGNFMLQKYPSYEIDWASIFigure 4
Phylogenomic analysis of 75DREBgenes in P. trichocarpa(a) with the integration of exon/intron structures (b) and MEME motifs (c). Exon/intron structure was obtained from the Gene Structure Display Server. Motifs were identified with the MEME software using the complete amino acid sequences of the DREB genes.


### 3.5. EST Profiling and Microarray Analysis

The expression profile for each gene was obtained by the Digital Northern tool, which generates a digital northern expression profile heat map based on the ESTs, and is a useful additional means of inferring gene function by examining expression patterns based on the frequency of ESTs in libraries prepared from various tissues [15] (Figure 5). Such analysis yielded 32 Populus DREB genes in the available cDNA libraries. Of the 32 DREBsexamined, 16 genes in the digital expression analysis had high transcript accumulation. A comparison of the digital northern expression analysis revealed that PtrDREB16and63 had high transcript accumulation in flower buds, PtrDREB51in apical shoots, PtrDREB24,25,and51 in petioles, PtrDREB39and 65 in dormant buds, PtrDREB33and69 in senescing leaves, PtrDREB11and30 in roots, PtrDREB37 in active cambium, PtrDREB71 in shoot meristem, PtrDREB13,16,and49 in female catkins, PtrDREB36in bark, and PtrDREB13in imbibed seeds. On the whole, the remaining genes had low transcript accumulation in the different libraries examined. The low-abundance TFs had relatively low EST frequencies [24] and most of the DREBswere represented by only one single EST in the cDNA libraries. Nevertheless, this expression analysis demonstrated that most of the DREBshave a broad expression pattern across different tissues.Figure 5
In silico EST analysis ofPopulus DREB genes. Color bar at bottom represents the frequencies of EST counts. FB: flower buds, MC: male catkins, CZ: cambial zone, TW: tension wood, AS: apical shoot, R: roots, CSL: cold stressed leaves, DB: dormant buds, DC: dormant cambium, SL: senescing leaves, P: petioles, AC: active cambium, YL: young leaves, FC: female catkins, SM: shoot meristem, B: bark, IS: imbibed seeds. Gene names are shown on the left.To gain more insights into the expression profiles ofDREBgenes, we then reanalyzed the previously published microarray data in Populus. We first investigated the global expression profiles of DREBgenes by examining Affymetrix (GSE6422) [25] microarray data from Gene Expression Omnibus. Seventy-five DREBgenes were included in GSE6422, and the DREBgenes showed a distinct tissue-specific expression pattern (Figure 6). A comparison of the different tissues revealed that PtrDREB13,15,33,51,and53 were overrepresented, howeverPtrDREB23 and 28 were under-represented in mature leaves. PtrDREB13,27,28,67,and69 were over-represented, howeverPtrDREB30 was under-represented in young leaves. PtrDREB20,30,49,70,and74 were over-represented in internodes. PtrDREB28,73,and74 were over-represented in nodes. PtrDREB7,11,and18 were over-represented, howeverPtrDREB28 was under-represented in roots. On the whole, the remaining genes showed low-abundance transcription levels in the different tissues. EST profiling and microarray analysis showed that PtrDREB28,73,74,37,and71 had high-abundance transcripts in the cambium. Previous studies using genome-wide transcriptional profiling in Arabidopsis revealed that stress-related and touch-inducible genes are upregulated in stem regions where secondary growth takes place [26].Figure 6
Expression profiles ofPopulus DREBgenes across different tissues. Heat map showing hierarchical clustering of 75 PtrDREB genes across various tissues analyzed. The Affymetrix microarray data were obtained from NCBI Gene Expression Omnibus (GEO) database under the series accession number GSE16422. ML: mature leaf; YL: young leaf; R: root; I: internodes; N: nodes. Color scale represents log2 expression values, green represents low level, and red indicates high level of transcript abundances.


### 3.6. Expression ofDREB Genes under Abiotic Stress Conditions in Populus

Studies have previously been conducted to evaluate the expression of theDREB Populus genes in response to stresses such as low temperature, high temperature, salt, and dehydration [7]. However, the genes that were evaluated in our study were predicted to be candidate genes in Populusvia EST profiling and microarray analysis. We have quantified the expression levels of the genes in leaf tissue after exposure to different abiotic stress conditions. The results provide an abundant set of information regarding the expression of these genes in Populus in response to low temperature, high temperature, ABA, salt, and dehydration (Figure 7). Among 15 selected DREB genes, there was evidence of induced expression under different abiotic stresses conditions, with the exception of PtrDREB30.Expression analysis of fifteen selectedP. trichocarpa DREB genes in mature leaves under ABA (a), drought stresses (b), salinity (c), high temperature (d), and low temperature (e) by qRT-PCR. The data were normalized using the P. trichocarpa actin gene. Standard deviations were derived from three replicates of each experiment.
(a)
(b)
(c)
(d)
(e)PtrDREB60, 61, and 62, which belong to the A-1 subgroup, were stress-inducible by low temperature, salt, and dehydration (Figure 7). In addition, PtrDREB62 was stress-inducible by high temperature and PtrDREB60 was stress-inducible by ABA. These findings are consistent with those of Dubouzet et al. (2003), who reported the increased expression of an A-1 subgroup gene (OsDREB1) of rice [10]. It was a major regulator of cold-stress responses in the DREB1/CBF (A-1) subgroup [7]. In a recent study, they indicated that ethylene signaling plays a negative role in the adaptation of Arabidopsis to freezing stress [26]. Additionally, some studies indicated that the AmCBF2 was inducible by heavy metals (Pb2+ or Zn2+) [4].PtrDREB4 and PtrDREB28 belong to the A-2 subgroup (Figure 7), and they were stress-inducible by ABA, salt, and dehydration. In addition, PtrDREB28 was stress-inducible by high temperature and low temperature. This finding is consistent with those of Dubouzet et al. (2003) [10], who reported increased expression of an A-2 subgroup gene (MsDREB2C) ofMalus sieversii Roem[27]. It was a major regulator of dehydration and heat shock responses in the DREB2 subgroup [7]. This indicated that ethylene signaling plays a negative role in the adaptation of Arabidopsis to freezing stress [26]. The oxidative stress tolerance of DREB2C-overexpressing transgenic Arabidopsis plants was regulated by heat shock factor A3 (HsfA3) and HsfA3 is regulated at the transcriptional level by DREB2 [28].PtrDREB51, 55, and 68 belong to the A-4 subgroup, and they were stress-inducible by salt and dehydration. However, PtrDREB51, 55, and 68were down represented by high temperature PtrDREB68 was down represented by low temperature. Genes PtrDREB30, 32, and 38 belonged to subgroup A-5. PtrDREB32 was stress-inducible by cold and ABA, whereas PtrDREB38 was induced by hot and cold temperatures. PtrDREB30 was down represented by high temperature, whereas PtrDREB38 was down represented by salt. PtrDREB16, 17, 18,and 19 belonged to the A-6 subgroup. PtrDREB16was induced by drought and high salt, and PtrDREB17was induced by drought, high temperature, low temperature, and ABA treatment. PtrDREB18was induced by high temperature, low temperature, and ABA. In addition, PtrDREB19was only induced by low temperature. PtrDREB18 was down represented by by salt, whereas PtrDREB16 was down represented by high temperature and cold temperature (Figure 7). In our study, a digital northern analysis showed that the genes PtrDREB17, 18,and 32were expressed in cold-stressed leaves, and showed the same expression trends based on qRT-PCR. The expression patterns ofPopulus DREBgenes detected by qRT-PCR are generally consistent with microarray analyses and digital northern analyses.Some studies indicate that theHARDY(At2g36450) gene belongs to the A-4 subgroup. The overexpression of the HRD gene for the improvement of water-use efficiency is coincident with drought resistance in rice. The analogous genes At2g36450and PtrDREB68 were stress-inducible in drought [29]. The homologous genes, RAP2.4 (At1g78080) and PtrDREB19 and RAP2.4B(AT1G22190) and PtrDREB17, were expressed in response to dehydration, high salinity, and heat [30, 31]. Overexpression or mutation of RAP2.4 and RAP2.4B in Arabidopsisacts at or downstream of a point of convergence for light and ethylene signaling pathways that coordinately regulates multiple developmental processes and stress responses [30]. It is noteworthy that the expression of several other genes associated with lipid transport was altered in the RAP2.4 and RAP2.4B overexpression lines, further supporting the link between DREBTFs and adaptive alterations in lipid metabolism [31]. Hence, we think that the PtrDREB16and 17TFs are probably associated with enhanced drought tolerance by modulating the wax biosynthetic pathway.


## 3.1. Identification ofDREB Subfamily TFs in Populus

To identify putativeDREB genes in Populus, we performed a BLASTP search against Populusgenome release v2.1 using DREBprotein sequences from Arabidopsis. By removing the redundant sequences, 75 DREBgenes were identified in the Populusgenome. All DREBcandidates were analyzed using the smart database (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) to verify the presence of AP2/ERF domains. Seventy-five DREBgenes were used for the analysis of bioinformatics and gene expression profiling. The overall strategy used in this study was depicted in Figure 1 and presented in detail below. We designated Populus DREBgenes as PtrDREBfollowing the nomenclature proposed in a previous study [17]. A. thaliana At4g13040was used as a query sequence and it includes an AP2/ERF-like domain sequence; however, its homology appears quite low in comparison with the other AP2/ERF genes. Therefore, this gene was also designated as an outlier. Detailed information on the DREBsubfamily of genes in Populusand Arabidopsisare listed in Table 1.Figure 1
Integrated systems analysis workflow for elucidation of the role ofDREBsubfamily in the bioinformatics and data analysis in Populus. A: A phylogenetic analysis was performed using all the DREB subfamily amino acid sequences from Arabidopsis and Populus by MEGA 4 software. B: Each of the DREB genes’ chromosomal position in Populus was using the Phytozome and Joint Genome Institute websites. C: The exon/intron organization for individualDREB genes was using the gene structure display server (GSDS) program and motif analysis was performed using the program MEME (v4.3.0). D: Gene expression profiling of Populus DREBsubfamily was used to characterize differentially expressed genes.


## 3.2. Phylogenetic Relationships and Alignments of theDREB Subfamily in Populus

Based on the alignment of the AP2/ERF coding region protein sequences of allPopulus and Arabidopsis DREBsubfamily genes, 75 DREBsubfamily genes of Populuswere classified into six groups, A1, A2, A3, A4, A5, and A6, containing six, 17, two, 26, 15, and nine members, respectively. The phylogenetic trees of the DREBsubfamilies of Populusand Arabidopsisare shown in Figure 2. The alignment analysis indicates that DREBs share high homology in the AP2/ERF domain (see Supplementary Figure 1; available online at http://dx.doi.org/10.1155/2013/954640) and contain a conserved WLG motif in the AP2/ERF domain of Populus. In the proteins encoded by DREBs, position 14 is normally valine and position 19 is glutamic acid. This region may play an important role in the recognition of different DNA-binding sites by the DRE and GCC box cis-elements of the DREBsubfamily [3]. Also, the DREBscontain alanine at position 319 in the β-sheet. Group A-1 possesses a conserved C/SEV/LR amino acid sequence between V319 and E324, and this region is converted into an AEIR amino acid sequence in groups A-2, A-3, and A-6. In Group A-4, Ser-324 is crucial for the specific binding of the ERE element, and Ser-324/Ala-324 is crucial in Group A-5.Figure 2
Relationships amongPopulus DREBs proteins after alignment with ClustalW. Proteins were allocated to six distinct subgroups of DREB, A-1 to A-6.


## 3.3. Chromosomal Locations ofDREB Subgroups

To examine the genomic distribution ofDREB genes on Populuschromosomes, we identified their positions by a Phytozome database search. In silico mapping of the gene loci showed that 75 Populus DREBgenes were mapped to linkage groups (LG) (Figure 3). Previous studies revealed that the Populusgenome has undergone genome-wide duplications followed by multiple segmental duplications, tandem duplications, and transposition events [18]. It was very clear that three pairs of genes were arranged in tandem repeats, LG I (PtrDREB63and 64), LG II (PtrDREB52and 53), and LG VI (PtrDREB43and 42), and 18 pairs of genes were duplicated. The dN/dS ratios from the 18 segmental duplication pairs were less than 0.5. About 56% (42 of 75) of Populus DREBswere preferentially retained tandem duplications and multiple segmental duplication events. Tandem duplications and segmental duplications were relatively underrepresented in groups A-1 to A-6 with rates of 67% (4 of 6), 47% (8 of 17), 100% (2 of 2), 53% (14 of 26), 40% (5 of 15), and 89% (8 of 9), respectively. This finding corroborates previous findings that genes involved in transcriptional regulation and signal transduction are preferentially retained following duplications [19].Figure 3
Locations ofP. trichocarpa DREB genes on the chromosomes LGI-XIX. A schematic view of chromosome reorganization by recent whole-genome duplication in Populusis shown (adapted from [21]). Regions that are assumed to correspond to homologous genome blocks are shaded in the same color and connected with lines.


## 3.4. Gene Structure and Conserved Motifs ofPopulus DREB Genes

To gain further insights into the structural diversity ofPopulus DREBgenes, we constructed a phylogenetic tree using the full-length DREBprotein sequences of Populus(Figure 4(a)). We compared the exon/intron organization in the coding sequences of each Populus DREBgene (Figure 4(b)). All but nine Populus DREBmembers had no introns in their coding regions and the nine Populus DREB genes had one intron (PtrDREB60, 61, 62, 24, 25, 26, 27, 28,and 3). We predicted conserved motifs using MEME motif detection software that revealed the diversification of the P. trichocarpa DREBgenes (Figure 4(c)), and 15 distinct motifs were identified (Table 3). The AP2/ERF domain consists of three β-sheet and one α-helix at the N termini [20, 21]. In this study, motif 3, specifying β-sheet strand 1; motif 1, specifying β-sheet strand 2 and 3; and motif 2, corresponding to the α-helix, were present in all of the Populus’ DREBsubfamily members. The CBF signature sequences (motif 14) were found in DREBGroup A-1 [20, 21]. Alignment of the deduced amino acid sequences of Arabidopsis DREBGroup A-1 TFs demonstrated significant similarity in the AP2/ERF binding domain and the CBF signature sequences [22]. These results suggest that the P. trichocarpa DREBGroup A-1 share remarkable similarities at the amino acid sequence level with known CBF/DREB proteins of Arabidopsis and carry critical amino acids that are needed for binding to the CRT elements in the target genes. The CMIV domain (motif 7) was found in the DREBGroup A-2. Alignment of the deduced amino acid sequences of Arabidopsis DREB Group A-2 TFs in the N-terminal region included the conserved motif CMIV-1 and the DNA-binding domain [23]. These results suggest that most of the closely related members in the phylogenetic tree shared a common motif composition with each other, suggesting functional similarities among the DREB proteins within the same subfamily.Table 3
Motif sequences ofDREB genes identified in P. trichocarpa by MEME tools.
Motif
Width
Best possible match
1
21
WGKWVCEIREPRKKSRIWLGT
2
24
FPTPEMAARAHDVAALCIKGDSAI
3
11
KHPVYRGVRMR
4
21
LPVPASTSPRDIQAAAASAAA
5
8
LNFPDLVH
6
25
EEALFDMPNLLVDMAGGMLLSPPRI
7
29
GDGGNKPVRKVPAKGSKKGCMKGKGGPEN
8
15
EDHHIEQMIEELLDR
9
18
YKPLHSSVDAKLQAICQS
10
25
HIGVWQKKAGSRSSSNWVMKVELGN
11
15
GPITVRLSPSQIQAI
12
30
DMSAASIRKRATEVGAHVDAIETALNHHHH
13
29
STSSLTSLVSLMDLSSQEEELCEIVELPS
14
21
EVMLASRNPKKRAGRKKFRET
15
21
FESGNFMLQKYPSYEIDWASIFigure 4
Phylogenomic analysis of 75DREBgenes in P. trichocarpa(a) with the integration of exon/intron structures (b) and MEME motifs (c). Exon/intron structure was obtained from the Gene Structure Display Server. Motifs were identified with the MEME software using the complete amino acid sequences of the DREB genes.


## 3.5. EST Profiling and Microarray Analysis

The expression profile for each gene was obtained by the Digital Northern tool, which generates a digital northern expression profile heat map based on the ESTs, and is a useful additional means of inferring gene function by examining expression patterns based on the frequency of ESTs in libraries prepared from various tissues [15] (Figure 5). Such analysis yielded 32 Populus DREB genes in the available cDNA libraries. Of the 32 DREBsexamined, 16 genes in the digital expression analysis had high transcript accumulation. A comparison of the digital northern expression analysis revealed that PtrDREB16and63 had high transcript accumulation in flower buds, PtrDREB51in apical shoots, PtrDREB24,25,and51 in petioles, PtrDREB39and 65 in dormant buds, PtrDREB33and69 in senescing leaves, PtrDREB11and30 in roots, PtrDREB37 in active cambium, PtrDREB71 in shoot meristem, PtrDREB13,16,and49 in female catkins, PtrDREB36in bark, and PtrDREB13in imbibed seeds. On the whole, the remaining genes had low transcript accumulation in the different libraries examined. The low-abundance TFs had relatively low EST frequencies [24] and most of the DREBswere represented by only one single EST in the cDNA libraries. Nevertheless, this expression analysis demonstrated that most of the DREBshave a broad expression pattern across different tissues.Figure 5
In silico EST analysis ofPopulus DREB genes. Color bar at bottom represents the frequencies of EST counts. FB: flower buds, MC: male catkins, CZ: cambial zone, TW: tension wood, AS: apical shoot, R: roots, CSL: cold stressed leaves, DB: dormant buds, DC: dormant cambium, SL: senescing leaves, P: petioles, AC: active cambium, YL: young leaves, FC: female catkins, SM: shoot meristem, B: bark, IS: imbibed seeds. Gene names are shown on the left.To gain more insights into the expression profiles ofDREBgenes, we then reanalyzed the previously published microarray data in Populus. We first investigated the global expression profiles of DREBgenes by examining Affymetrix (GSE6422) [25] microarray data from Gene Expression Omnibus. Seventy-five DREBgenes were included in GSE6422, and the DREBgenes showed a distinct tissue-specific expression pattern (Figure 6). A comparison of the different tissues revealed that PtrDREB13,15,33,51,and53 were overrepresented, howeverPtrDREB23 and 28 were under-represented in mature leaves. PtrDREB13,27,28,67,and69 were over-represented, howeverPtrDREB30 was under-represented in young leaves. PtrDREB20,30,49,70,and74 were over-represented in internodes. PtrDREB28,73,and74 were over-represented in nodes. PtrDREB7,11,and18 were over-represented, howeverPtrDREB28 was under-represented in roots. On the whole, the remaining genes showed low-abundance transcription levels in the different tissues. EST profiling and microarray analysis showed that PtrDREB28,73,74,37,and71 had high-abundance transcripts in the cambium. Previous studies using genome-wide transcriptional profiling in Arabidopsis revealed that stress-related and touch-inducible genes are upregulated in stem regions where secondary growth takes place [26].Figure 6
Expression profiles ofPopulus DREBgenes across different tissues. Heat map showing hierarchical clustering of 75 PtrDREB genes across various tissues analyzed. The Affymetrix microarray data were obtained from NCBI Gene Expression Omnibus (GEO) database under the series accession number GSE16422. ML: mature leaf; YL: young leaf; R: root; I: internodes; N: nodes. Color scale represents log2 expression values, green represents low level, and red indicates high level of transcript abundances.


## 3.6. Expression ofDREB Genes under Abiotic Stress Conditions in Populus

Studies have previously been conducted to evaluate the expression of theDREB Populus genes in response to stresses such as low temperature, high temperature, salt, and dehydration [7]. However, the genes that were evaluated in our study were predicted to be candidate genes in Populusvia EST profiling and microarray analysis. We have quantified the expression levels of the genes in leaf tissue after exposure to different abiotic stress conditions. The results provide an abundant set of information regarding the expression of these genes in Populus in response to low temperature, high temperature, ABA, salt, and dehydration (Figure 7). Among 15 selected DREB genes, there was evidence of induced expression under different abiotic stresses conditions, with the exception of PtrDREB30.Expression analysis of fifteen selectedP. trichocarpa DREB genes in mature leaves under ABA (a), drought stresses (b), salinity (c), high temperature (d), and low temperature (e) by qRT-PCR. The data were normalized using the P. trichocarpa actin gene. Standard deviations were derived from three replicates of each experiment.
(a)
(b)
(c)
(d)
(e)PtrDREB60, 61, and 62, which belong to the A-1 subgroup, were stress-inducible by low temperature, salt, and dehydration (Figure 7). In addition, PtrDREB62 was stress-inducible by high temperature and PtrDREB60 was stress-inducible by ABA. These findings are consistent with those of Dubouzet et al. (2003), who reported the increased expression of an A-1 subgroup gene (OsDREB1) of rice [10]. It was a major regulator of cold-stress responses in the DREB1/CBF (A-1) subgroup [7]. In a recent study, they indicated that ethylene signaling plays a negative role in the adaptation of Arabidopsis to freezing stress [26]. Additionally, some studies indicated that the AmCBF2 was inducible by heavy metals (Pb2+ or Zn2+) [4].PtrDREB4 and PtrDREB28 belong to the A-2 subgroup (Figure 7), and they were stress-inducible by ABA, salt, and dehydration. In addition, PtrDREB28 was stress-inducible by high temperature and low temperature. This finding is consistent with those of Dubouzet et al. (2003) [10], who reported increased expression of an A-2 subgroup gene (MsDREB2C) ofMalus sieversii Roem[27]. It was a major regulator of dehydration and heat shock responses in the DREB2 subgroup [7]. This indicated that ethylene signaling plays a negative role in the adaptation of Arabidopsis to freezing stress [26]. The oxidative stress tolerance of DREB2C-overexpressing transgenic Arabidopsis plants was regulated by heat shock factor A3 (HsfA3) and HsfA3 is regulated at the transcriptional level by DREB2 [28].PtrDREB51, 55, and 68 belong to the A-4 subgroup, and they were stress-inducible by salt and dehydration. However, PtrDREB51, 55, and 68were down represented by high temperature PtrDREB68 was down represented by low temperature. Genes PtrDREB30, 32, and 38 belonged to subgroup A-5. PtrDREB32 was stress-inducible by cold and ABA, whereas PtrDREB38 was induced by hot and cold temperatures. PtrDREB30 was down represented by high temperature, whereas PtrDREB38 was down represented by salt. PtrDREB16, 17, 18,and 19 belonged to the A-6 subgroup. PtrDREB16was induced by drought and high salt, and PtrDREB17was induced by drought, high temperature, low temperature, and ABA treatment. PtrDREB18was induced by high temperature, low temperature, and ABA. In addition, PtrDREB19was only induced by low temperature. PtrDREB18 was down represented by by salt, whereas PtrDREB16 was down represented by high temperature and cold temperature (Figure 7). In our study, a digital northern analysis showed that the genes PtrDREB17, 18,and 32were expressed in cold-stressed leaves, and showed the same expression trends based on qRT-PCR. The expression patterns ofPopulus DREBgenes detected by qRT-PCR are generally consistent with microarray analyses and digital northern analyses.Some studies indicate that theHARDY(At2g36450) gene belongs to the A-4 subgroup. The overexpression of the HRD gene for the improvement of water-use efficiency is coincident with drought resistance in rice. The analogous genes At2g36450and PtrDREB68 were stress-inducible in drought [29]. The homologous genes, RAP2.4 (At1g78080) and PtrDREB19 and RAP2.4B(AT1G22190) and PtrDREB17, were expressed in response to dehydration, high salinity, and heat [30, 31]. Overexpression or mutation of RAP2.4 and RAP2.4B in Arabidopsisacts at or downstream of a point of convergence for light and ethylene signaling pathways that coordinately regulates multiple developmental processes and stress responses [30]. It is noteworthy that the expression of several other genes associated with lipid transport was altered in the RAP2.4 and RAP2.4B overexpression lines, further supporting the link between DREBTFs and adaptive alterations in lipid metabolism [31]. Hence, we think that the PtrDREB16and 17TFs are probably associated with enhanced drought tolerance by modulating the wax biosynthetic pathway.


## 4. Conclusions

Understanding the plantDREB subfamily is important for elucidating the mechanisms of a variety of stress responses. Therefore, we present a comprehensive and specific analysis of gene structure, chromosome localization, and expression of the Populus DREBsubfamily for the first time. We predicted P. trichocarpa DREBgene expression and function through comparisons with similar genes that have been well studied in model or other plants. The chromosomal localizations of the PtrDREBgenes involved in transcription regulation and signal transduction are preferentially retained following duplications. The conserved motif composition of PtrDREBgenes were highly conserved in the same subtype. EST profiling and microarray analysis of this gene subfamily from different tissues and/or developmental stages showed the same expression trends based on qRT-PCR. The results in the present study indicate that DREBs function as important transcriptional activators and may be useful in improving plant tolerance to abiotic stresses. Taken together, these observations may lay the foundation for future functional analyses of Populus DREBgenes to unravel their biological roles.



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