Open Access

Comprehensive Analysis of NAC Domain Transcription Factor Gene Family in Populus trichocarpa

  • Ruibo Hu1,
  • Guang Qi1,
  • Yingzhen Kong1, 2,
  • Dejing Kong1,
  • Qian Gao1 and
  • Gongke Zhou1Email author
Contributed equally
BMC Plant Biology201010:145

DOI: 10.1186/1471-2229-10-145

Received: 8 February 2010

Accepted: 15 July 2010

Published: 15 July 2010

Abstract

Background

NAC (NAM, ATAF1/2 and CUC2) domain proteins are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. NAC transcription factors comprise of a large gene family represented by more than 100 members in Arabidopsis, rice and soybean etc. Recently, a preliminary phylogenetic analysis was reported for NAC gene family from 11 plant species. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling analysis has been presented thus far for the model tree species Populus.

Results

In the present study, a comprehensive analysis of NAC gene family in Populus was performed. A total of 163 full-length NAC genes were identified in Populus, and they were phylogeneticly clustered into 18 distinct subfamilies. The gene structure and motif compositions were considerably conserved among the subfamilies. The distributions of 120 Populus NAC genes were non-random across the 19 linkage groups (LGs), and 87 genes (73%) were preferentially retained duplicates that located in both duplicated regions. The majority of NACs showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses. However, the expression patterns of a majority of duplicate genes were partially redundant, suggesting the occurrence of subfunctionalization during subsequent evolutionary process. Furthermore, quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the tissue-specific expression patterns of 25 NAC genes.

Conclusion

Based on the genomic organizations, we can conclude that segmental duplications contribute significantly to the expansion of Populus NAC gene family. The comprehensive expression profiles analysis provides first insights into the functional divergence among members in NAC gene family. In addition, the high divergence rate of expression patterns after segmental duplications indicates that NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Taken together, our results presented here would be helpful in laying the foundation for functional characterization of NAC gene family and further gaining an understanding of the structure-function relationship between these family members.

Background

Transcriptional regulation of gene expression controls many important cellular processes in plants, such as cellular morphogenesis, signaling transduction and environmental stress responses [1]. Transcription factors (TFs) are types of proteins that regulate gene expression by binding to specific cis-acting promoter elements, thereby activating or repressing the transcriptional rates of their target genes [1, 2]. Thus, the identification and functional characterization of TFs is essential for the reconstruction of transcriptional regulatory networks [3]. In plants, totally more than 50 different families of TFs have been identified based on bioinformatics analysis so far [1, 3]. The Arabidopsis genome encodes at least 1550 TFs, accounting for about 6.2% of its estimated total number of gene [4]. As for Populus, about 6.4% of its genome was found to encode more than 2900 TFs [3, 5].

NAC (NAM, ATAF1/2 and CUC2) domain proteins comprise of one of the largest plant-specific TFs represented by ~105 genes in Arabidopsis [6], ~140 genes in rice [7] and ~101 genes in soybean genome [8]. NAC proteins commonly possess a conserved NAC domain at the N-terminus, which comprises nearly 160 amino acid residues that are divided into five subdomains (A-E) [6, 9, 10]. A sequence region of 60 residues within the NAC domain contains a unique TF fold consisting of a twisted-sheet bounded by a few helical elements [11]. NAC domain has been implicated in nuclear localization, DNA binding, and the formation of homodimers or heterodimers with other NAC domain proteins [1116]. In contrast, the C-terminal regions of NAC proteins are highly divergent [69, 14], and confer the regulation diversities of transcriptional activation activity [11, 13, 1522].

NAC proteins have been implicated to participate in a wide range of plant developmental processes, including shoot apical meristem development [10, 2326], floral morphogenesis [27], lateral root development [15, 28], leaf senescence [29, 30], stress inducible flowering induction [31, 32], embryo development [11], cell cycle control [3335], hormone signaling [15, 18, 33, 36], grain nutrient remobilization [30, 37] and shoot branching determination [38]. Particularly, numerous NAC domain proteins have also been implicated in plant abiotic stresses and defense responses such as drought, salinity, cold shock, mechanical wounding and viral infection [18, 20, 28, 36, 3947]. Recently, accumulating evidences indicated that a considerable portion of NAC domain proteins also play crucial roles in the processes of xylogenesis, fiber development, and wood formation in vascular plants [17, 19, 21, 4855].

In Arabidopsis, evidences indicated that a subset of closely related NAC domain proteins including NST1/ANAC043 (NAC Secondary Wall Thickening Promoting Factor 1), NST2/ANAC066, and NST3/SND1(Secondary Wall-associated NAC Domain Protein)/ANAC012 act as master transcriptional switches governing secondary cell wall biosynthesis in a partially redundant manner [5255]. NST1/ANAC043 and NST2/ANAC066 were shown to function redundantly in secondary cell wall thickening in anther endothecium and induced ectopic secondary wall thickenings in various tissues when expressed ectopically [52, 55], whereas NST1 and NST3/SND1/ANAC012 redundantly regulate the secondary wall thickening in inter-fascicular fiber of inflorescence stems and secondary xylem of hypocotyls in Arabidopsis [53, 54]. SND2/ANAC073 and SND3/ANAC010 have also been shown to function in the formation of secondary cell wall in fibers, and their dominant repression resulted in remarkable reduction in the secondary wall thickening [51]. Interestingly, both of SND2 and SND3 seem to function at downstream of NST1 and NST3/SND1, whereas SND2 was identified as a direct target of NST3/SND1 [51]. VND6 and VND7 (Vascular-related NAC-Domain) have been proposed to be regulators of the formation of vascular vessels in Arabidopsis [21, 56]. They act as key transcriptional switches regulating the differentiation of metaxylem and protoxylem, respectively, in primary roots [21, 56]. Consequently, NSTs may activate the secondary wall biosynthetic program in fibers, whereas VNDs are proposed to specifically regulate secondary wall biosynthesis in vascular vessels [51]. Another XND1/ANAC104 (Xylem NAC Domain 1) gene has been revealed to influence the differentiation of tracheary elements and xylem development in Arabidopsis by negatively regulating terminal secondary wall biosynthesis and programmed cell death in xylem vessel cells.

Although quite a few NAC TFs have been functionally characterized in model plants Arabidopsis and rice, the functions of majority of NAC members remain unknown. Especially in Populus, the typical model tree species, there are only very limited reports on the functional characterization of NAC TFs. Recently, Zhong et al (2009) reported the molecular cloning and functional characterization of six NAC genes in Populus [19]. Among the six NAC genes, WND2B (Wood-Associated NAC Domain Transcription Factors) and WND6B effectively complemented the secondary wall defects in snd1/nst1 double mutant, and when ectopically over-expressed in Arabidopsis, they induced ectopic deposition of secondary walls. These findings demonstrated that WND2B and WND6B genes are functional orthologs of Arabidopsis SND1 and master switches activating secondary wall biosynthesis during wood formation in Populus [19]. Shen et al (2009) carried out a genome-wide bioinformatics survey on plant NAC domain TFs and identified a total of 1,232 NAC proteins from 11 different plant species including 148 NAC TFs from Populus [57]. However, only sequence phylogeny analysis of Populus NAC TFs was performed in their report and no detailed analysis including genome organization, gene structure and expression compendium have been conducted [57]. In the present study, we further performed a genome-wide identification of NAC domain TFs in Populus and revealed an expanded NAC family with totally 163 members. Detailed analysis including sequence phylogeny, genome organization, gene structure, conserved motifs and expression profiling was performed. It is noteworthy that a subset of more than thirty NAC genes showed the highest level of transcript abundance in differentiating xylem and cambium tissues. Among them, twenty-five genes were further investigated for their tissue-specific expressions by quantitative real-time RT-PCR (RT-qPCR) analysis. Our results may provide a subset of potential candidate NAC genes for future engineering modification of lignocellulosic biomass characteristics in Populus.

Results and Discussion

Identification of NAC gene family in Populus

The NAC domain genes are plant-specific TFs presented as one of the largest multigene families in Arabidopsis, rice, soybean, maize, grape and sorghum etc [3, 6, 8, 57]. Hidden Markov Model (HMM) profile of the Pfam NAC domain (PF02365) was exploited as query to identify the NAC genes in Populus genome (release 2.0, http://www.phytozome.net/poplar). Initially, a total of 170 nonredundant putative NAC genes were identified. In an attempt to demonstrate the reliability of the identified genes, keyword search with NAM against NCBI nucleotide database was performed, resulting in 153 members which were all included in the NACs identified above. In comparison to the NAC gene family in PlnTFDB http://plntfdb.bio.uni-potsdam.de/v3.0/[3] and DPTF http://dptf.cbi.pku.edu.cn/[58], in which 167 and 172 members were proposed respectively, the 170 gene loci revealed in the present study were roughly in agreement with the former. Then, the discrepancy loci identified in our studies with the two databases mentioned above were further scrutinized to see if any misannotations were inferred. Among them, sixteen genes may represent putative pseudogenes or incorrect annotations, and manual reannotation was performed to correct and reannotate the putative NACs using online web server FGENESH http://linux1.softberry.com/berry.phtml[59]. In this endeavor, seven sequences encoding only truncated proteins were excluded from further analysis. In addition, we combined the newly released genome annotations with the results inferred by FGENESH to make the annotations more convincing. Finally, all the putative 163 NAC genes were further manually analyzed using InterProScan program http://www.ebi.ac.uk/Tools/InterProScan/ to confirm the presence of NAM domain [60]. In a recently published report, a total of 148 NAC genes were identified in Populus by a genome-wide bioinformatics survey [57]. In this study, we further revealed an expanded NAC family in Populus with totally 163 members. We designated Populus NAC genes as PNAC following the nomenclature proposed in the previous study [6]. The identified NAC genes in Populus encode proteins ranging from 117 to 718 amino acids (aa) in length with an average of 342 aa. Remarkably, in most cases, two or more Populus NAC genes were found for every ortholog in Arabidopsis. The detailed information of NAC family genes in Populus, including accession numbers and similarities to their Arabidopsis orthologs as well as nucleotide and protein sequences was listed in Table 1 and Additional file 1.
Table 1

NAC gene family in Populus.

Gene symbol

Gene locus

Arabidopsisortholog locus

Arabidopsislocus description

Score

E-value

PNAC001

POPTR_0009s05700.1

AT1G01720.1

ANAC002, ATAF1

179

3e-045

PNAC002

POPTR_0018s10310.1

AT1G01720.1

ANAC002, ATAF1

187

8e-048

PNAC003

POPTR_0017s09630.1

AT3G04070.2

ANAC002, ATAF1

146

2e-048

PNAC004

POPTR_0005s20240.1

AT1G01720.1

ANAC002, ATAF1

392

1e-109

PNAC005

POPTR_0005s07060.1

AT1G01720.1

ANAC002, ATAF1

357

5e-099

PNAC006

POPTR_0002s08150.1

AT1G01720.1

ANAC002, ATAF1

416

1e-117

PNAC007

POPTR_0007s04780.1

AT1G01720.1

ANAC002, ATAF1

366

1e-101

PNAC008

POPTR_0015s14770.1

AT1G12260.1

ANAC007, VND4

374

1e-104

PNAC009

POPTR_0003s11250.1

AT1G12260.1

ANAC007, VND4

436

1e-122

PNAC010

POPTR_0012s14660.1

AT1G12260.1

ANAC007, VND4

340

7e-094

PNAC011

POPTR_0001s00220.1

AT1G12260.1

ANAC007, VND4

449

1e-127

PNAC012

POPTR_0010s13980.1

AT1G25580.1

ANAC008

476

1e-134

PNAC013

POPTR_0008s11550.1

AT1G25580.1

ANAC008

509

1e-144

PNAC014

POPTR_0017s11830.1

AT1G26870.1

ANAC009

299

2e-081

PNAC015

POPTR_0004s12850.1

AT1G26870.1

ANAC009

310

2e-084

PNAC016

POPTR_0004s12850.1

AT1G26870.1

ANAC009

100

9e-022

PNAC017

POPTR_0001s45250.1

AT1G32770.1

ANAC012, SND1, NST3

294

6e-080

PNAC018

POPTR_0011s15230.1

AT1G33060.1

ANAC014

420

1e-117

PNAC019

POPTR_0011s15230.1

AT1G33060.2

ANAC014

431

1e-121

PNAC020

POPTR_0014s07220.1

AT1G33060.2

ANAC014

103

3e-022

PNAC021

POPTR_0002s15550.1

AT1G33060.1

ANAC014

135

9e-032

PNAC022

POPTR_0014s07220.1

AT1G33060.1

ANAC014

109

4e-024

PNAC023

POPTR_0002s15530.1

AT1G33060.2

ANAC014

154

1e-037

PNAC024

POPTR_0012s06340.1

AT1G34190.1

ANAC017

117

2e-027

PNAC025

POPTR_0043s00220.1

AT1G34190.1

ANAC017

140

2e-034

PNAC026

POPTR_0013s10770.1

AT1G34190.1

ANAC017

140

2e-034

PNAC027

POPTR_0043s00220.1

AT1G34190.1

ANAC017

180

4e-046

PNAC028

POPTR_0005s22180.1

AT1G34190.1

ANAC017

441

1e-124

PNAC029

POPTR_0002s06210.1

AT1G34190.1

ANAC017

432

1e-121

PNAC030

POPTR_0014s07210.1

AT4G35580.2

ANAC018, NAM

157

4e-039

PNAC031

POPTR_0014s10350.1

AT4G35580.2

ANAC018, NAM

180

9e-046

PNAC032

POPTR_0014s10350.1

AT4G35580.2

ANAC018, NAM

181

5e-046

PNAC033

POPTR_0002s18310.1

AT4G35580.2

ANAC018, NAM

184

3e-047

PNAC034

POPTR_0002s18320.1

AT4G35580.2

ANAC018, NAM

144

2e-034

PNAC035

POPTR_0002s17700.1

AT4G35580.2

ANAC018, NAM

130

2e-030

PNAC036

POPTR_0002s15560.1

AT4G35580.2

ANAC018, NAM

171

4e-093

PNAC037

POPTR_0014s10320.1

AT4G35580.2

ANAC018, NAM

138

8e-033

PNAC038

POPTR_0014s10320.1

AT4G35580.2

ANAC018, NAM

129

9e-031

PNAC039

POPTR_0010s18180.1

AT1G54330.1

ANAC020

290

9e-079

PNAC040

POPTR_0008s08100.1

AT1G54330.1

ANAC020

316

1e-086

PNAC041

POPTR_0005s10100.1

AT1G56010.2

ANAC021/22, AtNAC1

323

7e-089

PNAC042

POPTR_0007s08420.1 POPTR_0007s08430.1

AT1G56010.2

ANAC021/22, AtNAC1

337

5e-093

PNAC043

POPTR_0004s04920.1

AT1G61110.1

ANAC025

232

3e-061

PNAC044

POPTR_0016s05580.1

AT1G61110.1

ANAC025

137

2e-032

PNAC045

POPTR_0016s08970.1

AT1G61110.1

ANAC025

205

2e-053

PNAC046

POPTR_0006s13140.1

AT1G61110.1

ANAC025

247

7e-066

PNAC047

POPTR_0011s04650.1

AT1G61110.1

ANAC025

339

1e-093

PNAC048

POPTR_0004s03820.1

AT1G61110.1

ANAC025

332

3e-091

PNAC049

POPTR_0017s02020.1

AT1G65910.1

ANAC028

464

1e-130

PNAC050

POPTR_0004s07910.1

AT1G65910.1

ANAC028

465

1e-131

PNAC051

POPTR_0011s12190.1

AT1G65910.1

ANAC028

68

1e-011

PNAC052

POPTR_0014s04080.1

AT1G65910.1

ANAC028

73

1e-013

PNAC053

POPTR_0010s17350.1

AT1G69490.1

ANAC029, ATNAP, NAP

305

2e-083

PNAC054

POPTR_0003s10250.1

AT1G69490.1

ANAC029, ATNAP, NAP

119

3e-027

PNAC055

POPTR_0014s02580.1

AT1G69490.1

ANAC029, ATNAP, NAP

89

4e-018

PNAC056

POPTR_0019s11330.1

AT1G71930.1

ANAC030, VND7

249

9e-067

PNAC057

POPTR_0015s00520.1

AT1G71930.1

ANAC030, VND7

71

1e-012

PNAC058

POPTR_0013s11740.1

AT1G71930.1

ANAC030, VND7

305

2e-083

PNAC059

POPTR_0005s27720.1

AT1G76420.1

ANAC031, CUC3

282

2e-076

PNAC060

POPTR_0002s00730.1

AT1G76420.1

ANAC031, CUC3

319

2e-087

PNAC061

POPTR_0001s22770.1

AT1G77450.1

ANAC032

65

8e-011

PNAC062

POPTR_0008s07950.1

AT1G79580.3

ANAC033

333

1e-091

PNAC063

POPTR_0010s18420.1

AT1G79580.3

ANAC033

344

5e-095

PNAC064

POPTR_0019s09130.1

AT1G79580.3

ANAC033

56

2e-008

PNAC065

POPTR_0003s03670.1

AT2G02450.2

ANAC034, ANAC035

318

2e-087

PNAC066

POPTR_0004s23970.1

AT2G02450.2

ANAC034, ANAC035

310

1e-084

PNAC067

POPTR_0005s10610.1

AT2G17040.1

ANAC036

249

1e-066

PNAC068

POPTR_0004s18860.1

AT2G17040.1

ANAC036

321

4e-088

PNAC069

POPTR_0009s14370.1

AT2G17040.1

ANAC036

315

2e-086

PNAC070

POPTR_0007s13910.1

AT2G18060.1

ANAC037, VND1

428

1e-120

PNAC071

POPTR_0007s13910.1

AT2G18060.1

ANAC037, VND1

432

1e-121

PNAC072

POPTR_0006s29180.1

AT2G24430.2

ANAC038, ANAC039

348

3e-096

PNAC073

POPTR_0018s03910.1

AT2G24430.2

ANAC038, ANAC039

289

2e-078

PNAC074

POPTR_0016s02780.1

AT2G24430.2

ANAC038, ANAC039

65

4e-011

PNAC075

POPTR_0097s00280.1

AT2G24430.2

ANAC038, ANAC039

65

7e-011

PNAC076

POPTR_0009s16290.1

AT2G27300.1

ANAC040, NTL8

272

2e-073

PNAC077

POPTR_0015s00940.1

AT2G33480.1

ANAC041

100

9e-022

PNAC078

POPTR_0017s06000.1

AT2G33480.1

ANAC041

89

3e-018

PNAC079

POPTR_0009s07640.1

AT2G33480.1

ANAC041

59

3e-009

PNAC080

POPTR_0001s11660.1

AT2G43000.1

ANAC042

245

2e-065

PNAC081

POPTR_0005s22710.1

AT2G43000.1

ANAC042

268

2e-072

PNAC082

POPTR_0003s14950.1

AT2G43000.1

ANAC042

245

3e-065

PNAC083

POPTR_0002s05820.1

AT2G43000.1

ANAC042

296

7e-081

PNAC084

POPTR_0014s10060.1

AT2G46770.1

ANAC043, NST1

381

1e-106

PNAC085

POPTR_0002s17950.1

AT2G46770.1

ANAC043, NST1

370

1e-103

PNAC086

POPTR_0011s15640.1

AT2G46770.1

ANAC043, NST1

298

6e-081

PNAC087

POPTR_0001s35490.1

AT3G01600.1

ANAC044

259

2e-069

PNAC088

POPTR_0019s13130.1

AT3G03200.1

ANAC045

87

3e-018

PNAC089

POPTR_0001s26380.1

AT3G04070.2

ANAC047

189

3e-048

PNAC090

POPTR_0013s05080.1

AT3G04070.1

ANAC047

338

4e-093

PNAC091

POPTR_0019s04690.1

AT3G04070.1

ANAC047

332

2e-091

PNAC092

POPTR_0017s05970.1

AT3G04070.2

ANAC047

87

8e-018

PNAC093

POPTR_0006s04990.1

AT3G04070.2

ANAC047

122

3e-028

PNAC094

POPTR_0010s23630.1

AT3G10480.1

ANAC050

418

1e-117

PNAC095

POPTR_0006s02930.1

AT3G10480.2

ANAC050

75

5e-014

PNAC096

POPTR_0097s00260.1

AT3G10480.2

ANAC050

70

1e-012

PNAC097

POPTR_0302s00220.1

AT3G10480.2

ANAC050

74

2e-013

PNAC098

POPTR_0013s07720.1

AT3G10480.2

ANAC050

64

6e-011

PNAC099

POPTR_0006s02910.1

AT3G10480.2

ANAC050

74

2e-013

PNAC100

POPTR_0011s12420.1

AT3G15510.1

ANAC056, AtNAC2

322

2e-088

PNAC101

POPTR_0001s41490.1

AT3G15510.1

ANAC056, AtNAC2

345

4e-095

PNAC102

POPTR_0011s05760.1

AT3G15510.1

ANAC056, AtNAC2

221

1e-058

PNAC103

POPTR_0015s05450.1

AT3G17730.1

ANAC057

372

1e-103

PNAC104

POPTR_0012s03510.1

AT3G17730.1

ANAC057

350

4e-097

PNAC105

POPTR_0015s03700.1

AT3G18400.1

ANAC058

325

3e-089

PNAC106

POPTR_0012s05280.1

AT3G18400.1

ANAC058

315

4e-086

PNAC107

POPTR_0009s02430.1

AT3G44350.2

ANAC061

256

8e-069

PNAC108

POPTR_0001s22630.1

AT3G44350.2

ANAC061

277

5e-075

PNAC109

POPTR_0006s19350.1

AT3G44350.1

ANAC061

81

8e-016

PNAC110

POPTR_0015s00640.1

AT3G49530.1

ANAC062

307

9e-084

PNAC111

POPTR_0012s00760.1

AT3G49530.1

ANAC062

306

2e-083

PNAC112

POPTR_0013s09280.1

AT4G10350.1

ANAC070

436

1e-123

PNAC113

POPTR_0019s09400.1

AT4G10350.1

ANAC070

451

1e-127

PNAC114

POPTR_0003s08830.1

AT4G17980.1

ANAC071

296

1e-080

PNAC115

POPTR_0001s02710.1

AT4G17980.1

ANAC071

296

1e-080

PNAC116

POPTR_0019s13140.1

AT4G17980.1

ANAC071

89

5e-018

PNAC117

POPTR_0009s05690.1

AT4G27410.2

ANAC072, RD26

189

5e-048

PNAC118

POPTR_0011s12400.1

AT4G27410.2

ANAC072, RD26

353

9e-098

PNAC119

POPTR_0007s02050.1

AT4G27410.2

ANAC072, RD26

87

1e-017

PNAC120

POPTR_0001s41460.1

AT4G27410.2

ANAC072, RD26

375

1e-104

PNAC121

POPTR_0017s04800.1

AT4G28500.1

ANAC073

344

4e-095

PNAC122

POPTR_0007s01350.1

AT4G28500.1

ANAC073

337

6e-093

PNAC123

POPTR_0011s05740.1

AT4G28500.1

ANAC073

345

2e-095

PNAC124

POPTR_0004s04900.1

AT4G28500.1

ANAC073

355

2e-098

PNAC125

POPTR_0005s24750.1

AT4G28530.1

ANAC074

271

4e-073

PNAC126

POPTR_0002s03830.1

AT4G28530.1

ANAC074

276

7e-075

PNAC127

POPTR_0006s15740.1

AT4G29230.1

ANAC075

511

1e-145

PNAC128

POPTR_0006s15740.1

AT4G29230.1

ANAC075

486

1e-137

PNAC129

POPTR_0010s23650.1

AT5G04410.1

ANAC078, NAC2

483

1e-136

PNAC130

POPTR_0008s03170.1

AT5G04410.1

ANAC078, NAC2

499

1e-141

PNAC131

POPTR_0007s03840.1

AT5G09330.3

ANAC082

265

4e-071

PNAC132

POPTR_0005s06040.1

AT5G09330.3

ANAC082

256

2e-068

PNAC133

POPTR_0015s11420.1

AT5G13180.1

ANAC083

238

2e-063

PNAC134

POPTR_0001s33260.1

AT5G13180.1

ANAC083

162

1e-040

PNAC135

POPTR_0017s09030.1

AT5G13180.1

ANAC083

151

3e-037

PNAC136

POPTR_0003s16490.1

AT5G13180.1

ANAC083

310

4e-085

PNAC137

POPTR_0001s13380.1

AT5G13180.1

ANAC083

309

1e-084

PNAC138

POPTR_0012s10530.1

AT5G13180.1

ANAC083

228

3e-060

PNAC139

POPTR_0004s11840.1

AT5G17260.1

ANAC086

89

7e-019

PNAC140

POPTR_0012s03100.1

AT5G17260.1

ANAC086

87

4e-018

PNAC141

POPTR_0012s03130.1

AT5G17260.1

ANAC086

95

1e-020

PNAC142

POPTR_0014s10370.1

AT5G17260.1

ANAC086

139

2e-033

PNAC143

POPTR_0012s03120.1

AT5G17260.1

ANAC086

68

6e-019

PNAC144

POPTR_0019s04710.1

AT5G18270.2

ANAC087

267

1e-071

PNAC145

POPTR_0013s05110.1

AT5G18270.2

ANAC087

248

6e-066

PNAC146

POPTR_0016s07680.1

AT5G22380.1

ANAC090

218

3e-057

PNAC147

POPTR_0016s07690.1

AT5G22380.1

ANAC090

213

7e-056

PNAC148

POPTR_0006s22580.1

AT5G22380.1

ANAC090

242

2e-064

PNAC149

POPTR_0097s00210.1

AT5G24590.2

ANAC091, TIP

56

3e-008

PNAC150

POPTR_0017s08490.1

AT5G39820.1

ANAC094

50

3e-006

PNAC151

POPTR_0011s11600.1

AT5G53950.1

ANAC098, CUC2

311

3e-085

PNAC152

POPTR_0001s40680.1

AT5G53950.1

ANAC098, CUC2

300

1e-081

PNAC153

POPTR_0017s12210.1

AT5G61430.1

ANAC100, ATNAC5

377

1e-105

PNAC154

POPTR_0015s02170.1

AT5G61430.1

ANAC100, ATNAC5

446

1e-125

PNAC155

POPTR_0012s01610.1

AT5G61430.1

ANAC100, ATNAC5

448

1e-126

PNAC156

POPTR_0006s24740.1

AT5G62380.1

ANAC101, VND6

61

1e-009

PNAC157

POPTR_0004s10720.1

AT5G62380.1

ANAC101, VND6

74

9e-014

PNAC158

POPTR_0005s08440.1

AT5G62380.1

ANAC101, VND6

74

2e-013

PNAC159

POPTR_0014s16160.1

AT5G62380.1

ANAC101, VND6

59

6e-009

PNAC160

POPTR_0007s04250.1

AT5G64530.1

ANAC104, XND1

185

2e-047

PNAC161

POPTR_0003s01720.1

AT5G64530.1

ANAC104, XND1

231

2e-061

PNAC162

POPTR_0001s21440.1

AT5G64530.1

ANAC104, XND1

215

1e-056

PNAC163

POPTR_0005s06510.1

AT5G64530.1

ANAC104, XND1

199

1e-051

Gene loci are from the Phytozome website http://www.phytozome.net/, release 2.0). A complete list of the coding sequences (CDS), deduced amino acid sequences and genomic DNA sequences is available in Additional file 1.

The NAC gene family in Populus is by far the largest one compared to the estimates for other plant species, which range from ~105 in Arabidopsis [6], ~140 in rice [7] and ~101 in soybean [8]. The member of NAC genes in Populus is roughly 1.58 fold than that of Arabidopsis, which is in consistency with the ratio of 1.4~1.6 putative Populus homologs for each Arabidopsis gene based on comparative genomics studies [5]. In comparison to its closest woody perennial grape (Vitis vinifera), which possesses only about 79 NAC genes [3], the NAC genes in Populus seems to be highly expanded [5]. It can be speculated that the presence of more NAC genes in Populus genome may reflect the great needs for these genes to involve in the complicated transcriptional regulations in this woody perennial species. This expansion appears to be arisen from multiple gene duplication events, including a whole-genome duplication event in the Populus lineage followed by multiple segmental and tandem duplication events [5].

Phylogenetic analysis of NAC gene family in Populus, Arabidopsisand rice

To examine the phylogenetic relationship among the NAC domain proteins in Populus, Arabidopsis and rice, an unrooted tree was constructed from alignments of the full-length NAC protein sequences (Figure 1). The phylogenetic tree was constructed using MEGA 4.0 by employing the Neighbor-Joining (NJ), Minimal Evolution (ME) and Maximum Parsimony (MP) methods, respectively. The tree topologies produced by the three algorithms were largely comparable with only minor modifications at interior branches (data not shown). Therefore, only the NJ phylogenetic tree was subject to further analysis in our study. Moreover, we constructed the phylogenetic tree with the conserved N-terminal NAC domains A to E using the same algorithm, which was largely consistent to the phylogenetic analyses performed by the previous studies [57] (see details in Additional file 2). As indicated in Figure 1, the phylogenetic tree divided the NAC family proteins into 18 distinct subgroups. PNAC109 was distinguished from other NAC members and formed an individual clade with a robust bootstrap value support (99%) (Figure 1 and Additional file 3). For simplicity, the subgroups were designated as alphabetical families (NAC-a to NAC-r) based on tree topologies. Although the bootstrap values were somewhat low due to the large number of sequences, which was also presented in previous studies [6165], more significant bootstrap values in the distal branches allowed us to group the Populus, Arabidopsis and rice NAC proteins into distinct families (Additional file 3). Moreover, we sought other evidences such as gene structure, motif compositions and expression patterns as described below to support the reliability of the subgroup classification.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig1_HTML.jpg
Figure 1

Joined phylogenetic tree of NAC domain-containing proteins from Populus , Arabidopsis and rice. The deduced full-length amino acid sequences of 163 Populus, 105 Arabidopsis and 140 rice NAC genes were aligned by Clustal X 1.83 and the phylogenetic tree was constructed using MEGA 4.0 by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. Each NAC subfamily was indicated in a specific color. Members of NAC protein from Populus were denoted in red.

In general, the NAC members demonstrated an interspersed distribution in majority of subfamilies, indicating that the expansion of NAC genes occurred before the divergence of Populus, Arabidopsis and rice. Noticeably, NAC genes with same functions showed a tendency to fall into one subgroup, similar to the previous reports [7, 8, 57]. For instances, subfamily NAC-a encompassed the NAC proteins involved in shoot organ boundary delimitation (eg. CUC1, 2) [25, 26], whereas subfamily NAC-d possessed all the NAC proteins that function in shoot apical meristem (SAM) establishment, pattern formation in embryos and flowers (eg. NAM) [11], stress responses (eg. ATAF1, RD26 and OsNAC6) [18, 47, 66] and leaf senescence (eg. NAP) [29]. This group also encompassed ANAC019 and ANAC055, which have been shown to be induced by abiotic stresses (ABA, drought and salinity) and to enhance tolerance to drought when ectopically over-expressed [40]. The membrane-associated NAC proteins that mediate either cytokinin signaling during cell division or endoplasmic reticulum stress responses were clustered into the NAC-i and NAC-k subfamilies. The members in these two subgroups are distinct with other NACs in that they harbor a transmembrane (TM) motif with a predicted α-helix in the far C-terminal region. In NAC-k subfamily, there were only two well functionally characterized members namely NTM1 and NTM2. The well-characterized genes in subfamily NAC-f included FEZ, which was demonstrated to be associated with the orientation of cell division in root stem cells [34].

Remarkably, subfamily NAC-j did not include any Arabidopsis and rice NAC proteins but only members from Populus, suggesting that they may have been either lost in Arabidopsis and rice or acquired in the Populus lineages after divergence from the last common ancestor. We speculate that these subsets of genes may also have specialized roles with respect to the woody perennial habit in Populus. Phylogenetic analysis also revealed a subgroup NAC-o that contained sequence representatives in Populus and Arabidopsis but not in rice, indicating that the NAC members in this subgroup were acquired or differentially retained in eudicots post-divergence from monocots. In contrast, the subgroup NAC-p was composed of 13 rice NAC proteins, but no Arabidopsis and Populus proteins, which suggests diversification and expansion of this subgroup after the monocot-eudicot radiation.

The NAC proteins associated with secondary wall formation in fiber and vascular vessel development were divided into three independent subfamilies. All NSTs (NST1, NST2, NST3/SND1) and VNDs (VND1-VND7) were clustered into one subfamily NAC-b, whereas SND2, SND3 and their counterparts were grouped into NAC-q. The other well-characterized NAC member XND1 in secondary wall formation was assigned to another different subfamily (NAC-e). All the six functional characterized NST3/SND1 orthologs (PtWND1B to 6B) in Populus fell into subfamily NAC-b [19]. Although enormous evidences exemplified that all these NAC genes performed as key transcriptional switches in the secondary cell wall formation process, their exact functional roles diversified [5255]. The phylogenetic analysis conducted herein may provide a potential supporting for their functional diversities. What's more, the tissue-specific expression profiling available on GENEVESTIGATOR lent further supports for this notion [67]. The expression pattern of XND1 in Arabidopsis was extremely different from that of NSTs and VNDs. Nevertheless, the expression pattern of SND2 and SND3 was somewhat more similar to that of SND1/NST3 and NST2 (Additional file 4). In accordance with the recent report, NST, VND, SND, and XND from 11 different species were also classified into three distinct phylogenetic subgroups conducted by Maximum Likelihood (ML) algorithm [57]. Another well-characterized gene namely SOMBRERO (SMB), which has been shown to control the reorientation and timing of cell division in root stem cells by negatively regulating FEZ activity [34], was clustered together with NSTs and VNDs into subfamily NAC-b. We inspected the expression patterns of SMB on GENESTIGATOR [67] but failed in an attempt to find any clues for their diversified roles in secondary wall formation. SMB was specifically highly expressed in callus and lateral root cap rather than secondary cell wall enriching tissues, suggesting a specific role in cell division and root development (Additional file 4).

It is noteworthy that the number of Populus genes was generally overrepresented than that of Arabidopsis and rice in almost all clades, especially within NAC-o subfamily, in which Populus NAC genes was particularly overrepresented and almost sixfold the number of genes with respect to Arabidopsis. Alternatively, in subfamily NAC-a and q, the number of Arabidopsis and rice genes nearly equaled that of Populus. In contrast, the number of rice NAC genes in subfamily NAC-r showed an overwhelming predominance with respect to that of Populus.

The phylogenetic tree obtained in this study is largely consistent with previous analyses [68, 57], although there are some discrepancies. The first systematic analysis of Arabidopsis and rice NAC proteins classified them into 18 subgroups [6]. However, another phylogenetic analysis of rice NAC proteins suggested that the NAC family can be divided into five groups and each subfamily was largely diversified [7]. In a report concerning soybean NAC family, the genes were clustered into 15 distinct subfamilies with robust bootstrap supporting [8]. More recently, systematic phylogenetic analyses of numerous NAC proteins from a wide range of plant species indicated that NAC proteins can be divided into eight subfamilies [57]. In our opinions, the main reason for the discrepancies of the phylogenetic trees reported may lie in the fact that all the previous NAC protein classifications were based on the conserved N-terminal NAC domains, either from sub-domain A to D or A to E, which did not take the highly divergent C-terminal sequences into consideration [68, 57]. In an attempt to gain better understanding of the phylogeny of NAC gene family, we performed the phylogenetic analysis with inclusion of the highly diverse C-terminal sequence. Moreover, different algorithms exploited in the phylogenetic analyses may lead to the inconsistent interpretations. In the previous analyses, different algorithms including Neighbor-Joining (NJ) [6, 8], Maximum likelihood (ML) [57] and Bayesian method [7] were implemented, which may make the results less comparable.

Inspection of the phylogenetic tree topology revealed several pairs of NAC proteins with a high degree of homology in the terminal nodes of each subfamily, suggesting that they are putative paralogous pairs (homologous genes within a species that diverged by gene duplication) (Figure 1 and Additional file 3). Totally, forty-nine pairs of putative paralogous NAC proteins were identified, accounting for more than 60% of the entire family, with sequence identities ranging from 71% to 97% (see Additional file 5 for details).

Chromosomal location and gene duplication of PopulusNAC genes

In silico mapping of the gene loci showed that 163 Populus NAC genes were distributed across all 19 Linkage Groups (LG). In the currently released sequences, totally 120 NAC genes were mapped to LGs, whereas 43 genes were remained on as yet unmapped scaffolds. The distributions of Populus NAC genes across the LGs appeared to be non-random (Figure 2). LG II encompassed the largest number of 12 NAC genes followed by 10 on LG V and LG I, respectively. In contrast, only one NAC gene was found on LG XVIII and two NAC genes were on LG XVII. Substantial clustering of Populus NAC genes was evident on several LGs, especially on those with high densities of NAC genes. For instances, PNAC020, 022 and 030 were cluster localized on a 17 kb segment on LG XIV, and three NACs (PNAC033, 034 and 035) were organized in another cluster within a 10 kb fragment on LG II, whereas PNAC140, 141 and 143 were arranged in a cluster localized to a 11 kb segment on LG XII (Figure 2).
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Figure 2

Chromosomal locations of Populus NAC genes. Only 120 NAC genes are mapped to the 19 Linkage Groups (LG), while the other 43 genes reside on unassembled scaffolds. The schematic diagram of genome-wide chromosome organization arisen from the salicoid genome duplication event in Populus was adapted from Tuskan et al., (2006) [5]. Segmental duplicated homeologous blocks are indicated with the same color. Only the duplicated regions containing NAC genes are connected with lines in shaded colors. Tandemly duplicated genes are indicated with red lines. Scale represents a 5 Mb chromosomal distance.

Previous studies revealed that Populus genome had undergone at least three rounds of genome-wide duplications followed by multiple segmental duplication, tandem duplication, and transposition events such as retroposition and replicative transposition [5, 68]. Among them, the segmental duplication associated with the salicoid duplication event that occurred 65 million years (Ma) ago significantly contribute to the amplification of many multi-gene families [61, 6971]. To determine the possible relationship between the NAC genes and potential segmental duplications, we mapped Populus NACs to the duplicated blocks established in the previous studies [5]. The distributions of NAC genes relative to the corresponding duplicate genomic blocks were illustrated in Figure 2. Within the 36 identified duplicated blocks associated with the recent salicoid duplication event, about 73% (87 of 120) of Populus NACs were preferentially retained duplicates that locate in both duplicated regions of 28 block pairs, whereas nine block pairs only harbored NACs on one of the blocks and lack the corresponding duplicates, suggesting that dynamic changes may have occurred following segmental duplication, leading to loss of some of the genes. In contrast, only a small number of 19 NAC genes were located outside of any duplicated blocks.

In addition, the tandem duplications might also have an impact on the expansion of the NAC gene family. Fine mapping analysis revealed the presence of 13 pairs of adjacent genes within a 20 kb distance possibly due to tandem duplication either in inverse or same orientation (Figure 2). It is intriguing that except for one pair (PNAC110 and 119), all the other putative tandem duplicates were represented within the recent segmental duplicate blocks. Similar results have also been reported in several other Populus gene families [71, 72]. These 26 NAC genes (16%) were represented in 12 distinct tandem duplicate gene clusters, with ten clusters containing two tandem genes and two clusters possessing three tandem genes. But further analysis of their similarities demonstrated that most of the tandem clustered NAC pairs shared relative low similarities (mostly below 50%), which may exclude them from tandem duplicate candidates. Therefore, ultimately only six pairs of NACs were conformed to the standards as tandem duplicates, which were represented in tandem clusters of two to three genes each. These tandem duplicated NAC genes were organized onto duplicated blocks, implying that the occurrence of local duplications was prior to the chromosomal segment duplication.

As a considerable proportion of the NAC proteins appeared to be paralogous pairs as revealed in the phylogenetic analysis, we further investigated whether traceable genome duplication events have contributed to the expansion of the NAC family. Nineteen out of 49 homologous pairs remained in conserved positions on segmental duplicated blocks (Figure 2), suggesting that these genes may be the result of segmental duplication event during the evolution. For 26 homologous pairs, no traceable duplication events could be inferred, because they are mapped to the yet non-assembled scaffolds. Among them, a total of 11 genes were located on segmental duplication blocks with their counterparts not mapped to LGs yet. Meanwhile, a subset of eight NAC genes were located outside of the segmental duplication blocks while their corresponding members mapped to non-assembled scaffolds. Taken together, nine genes of the homologous pairs were definitely not located on any duplicated blocks, and only three of them had the homologous counterparts located on the duplicated blocks. Duplicates of these genes appeared to have lost from the Populus genome. Interestingly, one homologous pair PNAC121 and 122 was located on two divergent rather than homologous duplicated blocks, thus we could not figure out any traceable duplication history for this pair of NACs even though they were covered by duplicated blocks.

Based primarily on the genomic organization of NAC genes, we could conclude that segmental duplications exclusively contributed to the complexity of NAC gene family in the Populus genome. Similarly, segmental duplications have also been shown to contribute to the expansion of other gene families in Populus [61, 7072]. Our studies indicated that Populus NAC genes have been preferentially retained at a rate of 73%, which is much higher than the average rate following the salicoid genome-wide duplication and rearrangement events [5]. On a genome-wide scale, approximately one third of predicted genes are retained in duplications resulting from the salicoid duplication event [5]. The high retention rate of duplicated genes was also previously reported in other Populus gene families [70, 71]. These findings are also in consistency with the previous studies demonstrating that genes involved in transcription regulation and signal transduction are preferentially retained [7375]. Another plausible explanation to the relatively high retention rate of duplicate genes in NAC gene family may lie in the fact that Populus genome has been indicated to evolve at a much slower rate compared to that of Arabidopsis [5]. The duplicated genes may undergo divergent fates during subsequent evolution such as nonfunctionalization (loss of original functions), neofunctionalization (acquisition of novel functions), or subfunctionalization (partition of original functions) [76, 77]. Whether the duplicated NAC genes correspond to genetic redundancy or have evolved divergent functions remains to be further functionally characterized.

The tandem duplication ratio of NAC genes in this study is relatively lower compared to some other gene families in Populus, which was represented in a considerable higher proportion [71, 72]. Interestingly, a similar low retention rate of tandem duplicates was also observed for Populus ARF gene family [71].

Gene structure and conserved motifs of PopulusNAC genes

It is well known that gene structural diversity is a possible mechanism for the evolution of multigene families. In order to gain further insights into the structural diversity of NAC genes, we compared the exon/intron organization in the coding sequences of individual NAC genes in Populus. A detailed illustration of the exon/intron structures was shown in Figure 3B. In addition, a separate phylogenetic tree was generated from complete protein sequences of all the NAC genes in Populus, which divided the NAC genes into ten subfamilies (Figure 3A). In general, most closely related members in same subfamilies shared similar exon/intron structure in terms of intron number and exon length (Figure 3B). For instances, the NAC genes in subfamily I, III, IV and VII contained two to three introns while those in subfamily IX all possessed no introns with exception of PNAC079, which harbored one intron. In contrast, the gene structure appeared to be more variable in subfamilies II and V, which had the largest number of exon/intron structure variants with striking distinctions. Interestingly, although the exon/intron organization of NACs varied significantly in terms of intron number, the intron phase was remarkably high conserved (Additional file 6), which was indicative of exon shuffling during the evolution process [78].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig3_HTML.jpg
Figure 3

Phylogenetic relationships, gene structure and motif compositons of Populus NAC genes. A. Multiple alignments of 163 full-length amino acids of NAC genes from Populus were executed by Clustal X 1.83 and the phylogenetic tree was constructed using MEGA 4.0 by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. The percentage bootstrap scores higher than 50% are indicated on the nodes. The ten major phylogenetic subfamilies designated as I to X are marked with different color backgrounds. B. Exon/intron structures of NAC genes from Populus. Exons and introns are represented by green boxes and black lines, respectively. The sizes of exons and introns can be estimated using the scale at bottom. C. Schematic representation of the conserved motifs in the NAC proteins from Populus elucidated by MEME. Each motif is represented by a number in the colored box. The black lines represent the non conserved sequences. Refer to Additional file 7 for the details of individual motif.

We further analyzed the exon/intron structure of the paralogous pairs in NAC gene family to obtain traceable intron gain or loss information about these genes. Of the 49 paralogous pairs, thirty-four pairs showed conserved exon/intron structure either in terms of intron numbers or gene length (Figure 3B). Remarkably, twenty-five pairs of genes exhibited a highly conserved distribution of exons and introns, with all members having three exons and two introns. Despite a majority of these genes have arisen from segmental duplication events, the others were located either on non-duplicated blocks or on not-yet assembled scaffolds. While the gene structure was conserved between some paralogous pairs, others exhibited some extent of variation. For example, eleven NAC genes out of the 49 paralogous pairs possessed three exons in their coding regions, whereas their homologous counterparts all contained four exons within nearly identical gene lengths (Figure 3B). We could firmly speculate that the differences in the last exons are probably derived from single intron loss or gain occurred during the process of structural evolution of NAC paralogs. The conserved exon/intron architecture shared by these homologous gene pairs may reciprocally lend supports for the results from the phylogenetic analysis and the duplication events.

To further reveal the diversification of NAC genes in Populus, putative motifs were predicted by the program MEME (Multiple Expectation Maximization for Motif Elicitation), and 20 distinct motifs were identified. As expected, most of the closely related members in the phylogenetic tree had common motif compositions, suggesting functional similarities among the NAC proteins within the same subfamily (Figure 3C). However, the biological significance of most of the putative motifs remains to be elucidated as they do not have homologs when searching against Pfam and SMART (Simple Modular Architecture Research Tool) databases [79, 80]. The details of the 20 putative motifs were referred in Additional file 7. As illustrated in the previous studies, most of the NAC proteins possessed A to E subdomains in the N termini that conferred the DNA-binding activities. In this study, motif 2, 5, 1, 3 and 6 specifying the NAM subdomain A to E respectively were present in most of the NAC family members in Populus, whereas a small subset of sequences did not have all five motifs in the typical NAC DNA-binding domains. Even though the C-terminal regions of NACs were highly divergent, we could also identify at least eight conserved motifs using the MEME motif search tool. Noticeably, some specific motifs were present in NACs from specific subfamilies, for instance, motif 16 for subfamily V, motif 9 for subfamily VIII and motif 11 for subfamily IX. Whether these motifs confer unique functional roles to NACs remains to be further investigated.

Of the 49 paralogous pairs, thirty-seven pairs of NACs shared conserved motif composition with each other, indicative of functional similarities. In contrast, specific motifs located in the C termini were observed for the rest 12 pairs of NACs, for example, motif 13 for PNAC041, 082, 113, 163, motif 14 for PNAC066, and motif 19 for PNAC120. We attempt to speculate that these specific motifs may by some extent attribute to the subfunctionalization or neofunctionalization of the duplicated genes during subsequent evolution processes.

The similar gene structures and conserved motifs of NAC genes in same subfamilies may provide additional supports to the phylogenetic analysis. On the other hand, the differences between gene organization and the divergences in motif compositions among different subfamilies may also indicate that Populus NACs are functionally diversified.

Differential expression profile of PopulusNAC genes

Publicly available Expressed Sequence Tags (ESTs) was considered as a useful means of studying gene expression profiles (digital northern) [81]. A scrutiny of the frequency of ESTs in different databases allows preliminary analysis of gene expression under various conditions and across tissues. The digital expression profiles of 81 Populus NAC genes were obtained from EST databases at NCBI (Oct., 2009), while the rest of other 82 NAC genes did not have corresponding EST sequences in the database. Noticeably, the frequency of ESTs was low with most of NACs represented by a single EST sequence, which is consistent with the commonly low transcript abundance feature of transcription factor genes [61]. Thus we postulate that the absent genes in EST database might either transcribe at relatively too low abundance to be detected or had special temporal and spatial expression patterns not examined in the libraries. Another possible explanation is that these genes might be pseudogenes, as they only possessed partial NAC domains as described previously (Figure 3C). In order to gain a better portrait of NAC gene expression profiles, we further largely divided the EST data into two groups, with one group contained all wood-forming tissues and the other derived from the other tissues. The EST digital expression profiles were illustrated in Figure 4, Figure 5 and Additional file 8. The expression profiles demonstrated that majority NACs have rather broad expression patterns with presence in diverse libraries. Not surprisingly, a considerable subset of genes was preferentially expressed in leaves (Figure 4). In addition to leaves, more than 40% (36 of 81) of the NAC genes were especially expressed during wood formation (in cambial zone and xylem) (Figure 5).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig4_HTML.jpg
Figure 4

In silico EST analysis of Populus NAC genes. EST frequency of 74 Populus NAC genes was obtained by screening the EST datasets from various libraries across a set of 14 tissues. Expression of NAC genes was plotted as counts of corresponding ESTs for particular gene in the database.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig5_HTML.jpg
Figure 5

EST profiles of Populus NAC genes in wood-forming tissues. EST distribution of 36 Populus NAC genes was obtained by searching the EST datasets originated from libraries of wood-forming tissues. Expression of NAC genes was plotted as counts of corresponding ESTs for particular gene in the database.

It is noteworthy that several putative NAC orthologs between Arabidopsis and Populus showed strikingly consistent expression patterns, which lend further supporting to the existence of functional conservation between these two species. For example, the expression profiles of homologous pair PNAC031 and 032, which had two corresponding ESTs derived from shoot meristem respectively (Figure 4), further reinforced the notion that they are the closest orthologs of NAM in Arabidopsis [11]. The EST data revealed that PNAC013 gene was specifically expressed in active cambium with two detectable ESTs and highly resembled the expression pattern of its closest ortholog ANAC009 in Arabidopsis, which was expressed predominantly in highly dividing and expanding tissues such as callus and root cap (Additional file 4).

Although the digital EST expression analyses provided a first glimpse of the patterns of NAC expression in Populus, we could not draw conclusive inferences regarding NAC gene expression due to the obvious limitations of EST data such as relative narrow sample coverage and biases towards highly expressed genes. Thus, we performed a more comprehensive microarray analyses for NAC gene expression profiles. To gain more insight into the expression patterns of NAC genes, a comprehensive expression analysis was further performed by utilizing the publicly available microarray data for Populus. Firstly, we re-analyzed the collections of microarray data from wood-formation series available at PopGenIE, a database repository of transcriptomics data available for Populus [82, 83]. Distinct expression profiles were identified for a total of 29 NAC genes (Figure 6 and Additional file 9), whereas no corresponding probe sets were available for the remaining NACs in the datasets. The relative low coverage of NACs in the datasets analyzed maybe partially due to the fact that these arrays were mainly derived from ESTs rather than offered a whole genome coverage [84].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig6_HTML.jpg
Figure 6

Expression profiles of Populus NAC genes in wood-forming tissues. Microarray data were obtained from PopGenIE ftp://aspnas.fysbot.umu.se/[84]. The expression data were gene-wise normalized and hierarchical clustered with average linkage. Each row corresponds to the relative expression levels normalized against the maximum value. Color scale at the top of each dendrogram represents relative expression levels: green represents low level and red indicates high level. A. Dynamic expression levels of 11 NAC genes in different states of cambiums. YR1 to YR9 represent the nine sampling time points from 20 April (before bud break) until 13 December (cambium dormant). B. Expression patterns of 19 NACs in active and dormant cambiums. C, D. Hierarchical clustering of expression profiles of 19 NAC genes in the cambial region. Phl, phloem; A, meristematic cells; B, early expansion; C, late expansion; D, secondary wall formation; E, late cell maturation. CS2A1, CS2A2, CS2B1, CS2B2 and CS2B4, phloem; CS2A3, CS2A4, CS2B6 and CS2B7, cambium region; CS2A5, CS2A7, CS2A8 and CS2B8, xylem. E. Expression pattern of three NAC genes in cambium regions. The tissues are the same as depicted in C and D. F. Expression profiles of 14 NACs in vascular tissues. Phl, phloem; C, cambium; EX, expanding xylem; MX0, initiation of secondary cell-wall deposition stage; MX1-3, maturing xylem. G. Deferential expressions of 21 NAC genes during the xylem differentiation. EX, early xylem; LX, late xylem.

Two subset of NAC genes exhibited specifically high transcript accumulation in active and dormant cambiums respectively, from two independent microarray datasets [85, 86], strongly indicating their specific roles in wood-formation (Figure 6A and 6B). In addition, by incorporating the different microarray data, we were capable to identify different subsets of NAC genes displaying specifically high expression levels in phloem, differentiating xylem and mature xylems, respectively. The expression levels of five NAC genes including PNAC070, 086, 121 and 122 were peaked exclusively in differentiating xylems supported by two independent microarray datasets (Figure 6C and 6G) [87, 88]. Among them, PNAC070 (PtWND5A), PNAC086 (PtWND1A) and PNAC008 (PtWND3A) were demonstrated in previous studies as putative redundant homologs involved in Populus wood formation [19]. Interestingly, several genes such as PNAC013, 020 and 120 were preferentially expressed in tissues enriched of secondary cell walls, whereas a subset of genes including PNAC028, 052, 055 and 023 showed constitutively high expressions in both secondary and primary cell wall enriching tissues (Figure 6A-E). Similarly, several genes such as PNAC118 and 136 displayed varied peak abundance of transcript across the same tissues tested among different microarray datasets (Figure 6A-E).

As the previous analyzed microarray are mainly derived from ESTs and only a small fraction of NACs is present in these datasets, we further seek to use microarray based on whole genome coverage to provide deep insights into the expression patterns of NACs. We have reanalyzed the Populus microarray data generated by Wilkins and coworkers [61]. Only six NAC genes (PNAC007, 018, 019, 052, 082 and 163) did not have their corresponding probe sets in the dataset, and the expression profiles of the rest of 157 NAC genes were analyzed as indicated in Figure 7. The majority of NAC genes showed distinct tissue-specific expression patterns across the nine tissues examined (Figure 7 and Additional file 9). It is notably that a relatively large fraction of NAC genes, accounting for more than 40% (67 of 163), were preferentially expressed in male and female catkins. Among these genes, thirty-four gene (50%) showed the highest transcript abundances in male catkins, twelve (18%) had the highest accumulation in female catkins, and the remaining 21 (32%) showed approximately equal transcript abundances in both male and female catkins. The number of NAC genes fell into this category is significantly higher compared to that of MYB transcription factor gene family analyzed by the same microarray data, in which, fifty (28%) R2R3-MYB genes showed the highest transcript accumulation in catkins [61]. The large proportion of NAC genes with the highest transcript abundance in catkins may probably attribute to the unique characteristic of sex determination in Populus lineage. The previous phylogenetic analysis revealed that these catkin-specific genes were dispersed among different subfamilies rather than divided into distinct subgroups (Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig7_HTML.jpg
Figure 7

Hierarchical clustering of expression profiles of Populus NAC gene across different tissues. The genome-wide microarray data generated by Wilkins and coworker were re-analyzes [61]. The expression data were gene-wise normalized and hierarchical clustered based on Pearson correlation. The genes marked in the same color indicate duplicated gene pairs. The relative expression level of particular gene in each row is normalized against the maximum value. Color scale at the top of each dendrogram represents log2 expression values, green represents low level and red indicates high level of transcript abundances. CL, continuous light-grown seedling; DL, etiolated dark-grown seedling transferred to light for 3h; DS, dark-grown seedlings; YL, young leaf; ML, mature leaf; R, root; DX, differentiating xylem; FC, female catkins; MC, male catkins.

Thirteen (8%) NAC genes showed the highest transcript abundances in differentiating xylems, including four xylem-specific NACs (PNAC070, 086, 121 and 122) identified in the previous microarray analysis (Figure 6C). The six well-characterized NAC genes involved in the secondary cell wall thickening in the previous studies were all included in this category [19]. The expression pattern of PNAC008 gene was inconsistent with the above microarray analysis (Figure 6). The current data revealed the highest expression of PNAC008 gene in young leaves and female catkins rather than in differentiating xylems. The number of NAC genes with the highest transcript abundances in differentiating xylems seems to be a little bit lower than that of R2R3 MYB gene family, which has 23 members accounting for about 12 percent of the total [61]. In addition, a considerable fraction of 24 (15%) NAC genes were specifically expressed in roots.

Interestingly, a subset of NAC genes demonstrated different expression patterns between dark-grown etiolated seedlings and continuous light grown seedlings, suggesting that these NACs may be subject to photoperiodic regulation. Strikingly, the majority of NAC genes did not show seemingly high abundances of transcript in either young or mature leaves as depicted by the previous ESTs digital profiles analysis (Figure 4 and Additional file 7). One possible explanation may lie in the fact of different sample size and coverage exemplified in the analyses.

The high proportion of segmental duplication of NAC genes and the preferential retention of duplicates raises the question about their functional redundancy. Duplicate genes may have different evolutionary fates: nonfunctionalization, neofunctionalization, or subfunctionalization [76], which may be indicated with divergence in expression patterns. Of the 49 homologous pairs of NAC genes, six genes (PNAC007, 018, 019, 052, 082 and 163) did not have corresponding probe sets on Affymetrix microarray and were excluded for further analysis. Therefore, the remaining 44 homologous pairs were analyzed. Among them, nineteen pairs were located onto duplicated blocks and three pairs were tandem duplicates. Five pairs (PNAC004/006, PNAC012/013, PNAC084/085, PNAC129/130 and PNAC144/145) out of 19 segmental duplications shared the same expression patters with respect to the tissues examined (Figure 7). In addition, the expression pattern of homologous pair PNAC004/006 was also supported by a broad sample sets from the previously microarray and ESTs profiles (Figure 4). Although the expression patterns of the rest of 14 duplicate genes were partially redundant, distinct pattern shifts can be discerned, which suggested that they have undergone subfunctionalization. These findings indicated that expression patterns of NACs have diverged quickly after segmental duplications, thus the NAC genes in Populus are likely to have been retained by substantial subfunctionalization. For example, PNAC008 gene were mainly expressed in young leaves and male catkins, whereas its duplicate counterpart PNAC010 gene extended to a broader expression patterns in young and mature leaves, female and male catkins (Figure 7).

Surprisingly, two homologous pairs (PNAC049/050 and PNAC121/122) also exhibited almost identical patterns of transcript accumulation but were located onto different duplication blocks (Figure 7). The reasons underlying their highly identical expression profiles remain to be investigated since we could not draw any clues from the segmental duplication events. The rest ten homologous pairs also showed almost identical expression patterns, but we could not locate them onto duplicated blocks currently since some of them were mapped to not-yet assembled scaffolds.

As for the tandem duplicated clusters with three genes, the expression patterns of two of them (PNAC020/022, PNAC033/035, PNAC141/143) were almost identical, while significant diversification was observed for the third member, which may indicate neofunctionalization. For example, tandem duplicate PNAC020 and PNAC022 were predominantly expressed in male catkins followed by roots, while PNAC030 was largely expressed in root followed by female catkin (Figure 7). As for the tandem duplicated clusters with two genes, their expression patterns diversified significantly, indicative of substantial neofunctionalization during subsequent evolution processes.

Validation of NAC gene expressions by quantitative real-time RT-PCR

To verify the expression profiles of Populus NAC genes obtained by in silico EST and microarray data analysis, quantitative real-time RT-PCR (RT-qPCR) was performed on seven different tissues for 25 selected NAC genes. As illustrated in Figure 8, the genes showed very distinct tissue-specific expression patterns, which were in good agreement with the above microarray and EST profiles (Figure 6, 7). For example, in accordance with previous findings, a subset of eight genes among the 25 NACs tested were exclusively expressed in differentiating xylem, indicative of their putative roles in secondary cell wall formation. Three genes namely PNAC009 (PtWND4B), PNAC071 (PtWND1B) and PNAC085 (PtWND2B) have been functionally characterized in a recent report [19]. In the present study, PNAC071 gene was shown to primarily express in old root, while microarray profiles indicated the highest transcript abundance in differentiating xylems (Figure 7). Seven genes showed the highest transcript levels in phloem and four genes had the most abundant transcripts in cortex, which were both enriched in primary cell walls. These genes may have putative roles in primary cell wall biosynthesis and wood development. Consistent with the microarray profiles, four genes (PNAC004, 006, 130 and 162) were expressed specifically in leaves. Accordingly, PNAC004 gene had the largest number (up to 24) of corresponding EST sequences originated from leaves, which lent further supports to the above statement. All the 25 genes tested have constitutively weak expression levels in root meristem. Since only a few numbers of NAC genes have been functionally characterized up to date, our results presented here may provide some clues for the selection of candidate genes for further characterization.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-10-145/MediaObjects/12870_2010_Article_639_Fig8_HTML.jpg
Figure 8

Expression analysis of selected NAC genes using quantitative real-time RT-PCR (RT-qPCR). The relative mRNA abundance of 25 selected NAC genes was normalized with respect to reference gene UBQ10 in different tissues. The bars are standard deviations (SD) of three technical repeats. SA, shoot apices; L, leaf (4~6 internodes); Phl, phloem; DX, differentiating xylem; OR, old root; RT, root tip; C, cortex.

Conclusion

In the present study, a comprehensive analysis including phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling of NAC gene family in Populus was performed. We identified a sum of 163 full-length NAC genes in Populus genome, and they were phylogeneticly clustered into 18 distinct subfamilies. The exon/intron structure and motif compositions of NACs were highly conserved in each subfamily, indicative of their functional conservation. The NAC genes were non-randomly distributed across 19 LGs, and a high proportion of NACs were preferentially retained duplicates located on the duplicated blocks, which indicated that segmental duplications contribute significantly to the expansion of Populus NAC gene family. Although a majority of NAC genes showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses, the expression patterns of a considerable proportion of duplicate genes (14 of 44) were partially redundant, suggesting the occurrence of subfunctionalization during evolutionary process. In other words, a fraction of NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Furthermore, we identified a subset of Populus NAC genes with putative functional roles in wood-forming and secondary cell wall biosynthesis, and results presented here will be helpful for future functional studies to unravel their divergent roles.

Methods

Database search and sequence retrieval

The P. trichocarpa genome sequences were downloaded from Phytozome http://www.phytozome.net/poplar. Hidden Markov Model (HMM) profile of NAC domain (PF02365) downloaded from Protein family (Pfam) http://pfam.sanger.ac.uk/ was exploited for the identification of the NAC genes from Populus genome using HMMER (v 2.3.2) [89]. All non-redundant hits with expected values less than 1.0 were collected and then compared with the NAC family in PlnTFDB http://plntfdb.bio.uni-potsdam.de/v3.0/[3] and DPTF http://dptf.cbi.pku.edu.cn/[58]. As for the incorrectly predicted genes, manual reannotation was performed using online web server FGENESH http://linux1.softberry.com/berry.phtml[59]. The reannotated sequences were further manually analyzed to confirm the presence of NAM domain using InterProScan program http://www.ebi.ac.uk/Tools/InterProScan/[60]. Sequences of Arabidopsis and rice NAC domain proteins were downloaded from the Arabidopsis genome TAIR 9.0 release http://www.Arabidopsis.org/ and rice genome annotation database (http://rice.plantbiology.msu.edu/, release 5.0), respectively.

Phylogenetic analysis

Multiple sequence alignments of the full-length protein sequences, including the highly conserved N-terminal NAM domain and the more divergent C-terminal activation domain, were performed by Clustal X (version 1.83) program [90]. The unrooted phylogenetic trees were constructed with MEGA 4.0 using the Neighbor-Joining (NJ), Minimal Evolution (ME) and Maximum Parsimony (MP) methods and the bootstrap test carried out with 1000 iterations [91]. Pairwise gap deletion mode was used to ensure that the more divergent C-terminal domains could contribute to the topology of the NJ tree.

Genomic structure and chromosomal location

Gene structure display server (GSDS) program [92] was used to illustrate exon/intron organization for individual NAC genes by comparison of the cDNAs with their corresponding genomic DNA sequences from Phytozome http://www.phytozome.net/poplar. Identification of homeologous chromosome segments resulting from whole-genome duplication events was accomplished as described in Tuskan et al. (2006) [5]. Blocks of the same color represent the homologous chromosome segments. The tandem gene duplications in Populus were identified according to the same criteria described in rice [93]. Genes separated by five or fewer gene loci in a range of 100 kb distance were considered to be tandem duplicates.

Identification of conserved motifs

The program MEME version 4.3.0 was used for the elucidation of motifs in 163 deduced Populus NAC protein sequences http://meme.sdsc.edu[94]. MEME was run locally with the following parameters: number of repetitions - any, maximum number of motifs - 20, and the optimum motif widths were constrained to between 6 and 200 residues. Structural motif annotation was performed using the SMART http://smart.embl-heidelberg.de[95] and Pfam http://pfam.sanger.ac.uk databases [96].

EST profiling and microarray analysis

A total of 429,444 Populus EST sequences were downloaded from the GenBank database (http://www.ncbi.nlm.nih.gov/est/, release 121009). The coding regions of NAC genes were used as queries to perform a local BLASTN search against all of the ESTs. Matches above 96% identity and over an alignment of at least 100 bp were considered as corresponding sequences of the NAC genes. The entries identified were manually inspected for their tissue origin. In addition, the DigitalNorthen tool at the PopGenIE http://www.popgenie.org/ was utilized to produce the heat map with dendrograms of the NAC genes with their corresponding gene model IDs.

Populus microarray data were obtained from PopGenIE ftp://aspnas.fysbot.umu.se/, the expression data were gene-wise normalized and hierarchical clustered based on Pearson coefficients with average linkage in the Genesis (version 1.75) program [97].

The genome-wide microarray data performed by Wilkins and coworkers [61] were obtained from the NCBI Gene Expression Omnibus (GEO) [98] with accession number GSE13990. Probe sets corresponding to the putative Populus NACs were identified using an online Probe Match tool available at the NetAffx Analysis Center http://www.affymetrix.com/. For genes with more than one probe sets, the median of expression values were considered. When several genes have the same probe set, then they are considered to have same level of transcript abundance. the expression data were gene-wise normalized and hierarchical clustered based on Pearson coefficients with average linkage in the Genesis (version 1.75) program [97].

Plant material and growth conditions

One-year-old Populus deltoides was grown in the greenhouse under long day conditions (16 h light/8 h dark) at a temperature 25°C~28°C. Shoot apices (internodes 1~3 from top, same as below), leaf (from internodes 4~6), developing xylem (from the basal internodes), phloem (from the basal internodes), cortex (from the basal internodes), old root and root tip (terminal 3~5 mm) tissues were separately harvested. All samples were immediately frozen in liquid nitrogen and stored at -80°C until required.

RNA isolation and quantitative real-time RT-PCR (RT-qPCR)

Total RNA from the majority of the samples was extracted using TRIzol reagent (Invitrogen, Ca, USA) according to manufacturer's instructions. Alternatively, total RNA from cortex, old root and root tip were isolated by CTAB method [99] with minor modifications. RNA integrity was verified by 2% agar gel electrophoresis and SYBR Greenlstaining. Before cDNA synthesis, RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) according to the manufacturer's instructions to ensure no DNA contamination, and then the first-strand cDNA synthesis was carried out with approximately 2 μg RNA using the RevertAid First Strand cDNA Synthesis Kit (MBI, Fermentas) and oilgo-dT primers according to the manufacturer's procedure. Primers were designed using Beacon Designer v7.0 (Premier Biosoft International, Palo Alto, California, USA) with melting temperatures 58~60°C, primer lengths 20~24bp and amplicon lengths 51~199bp. Experimental details are given in additional file 10.

RT-qPCR was conducted on LightCycler® 480 Detection System (Roche, Penzberg, Germany) using SYBR Premix Ex Taq (TaKaRa, Toyoto, Japan). Reactions were prepared in a total volume of 20 μl containing: 2 μl of template, 10 μl of 2×SYBR Premix, 0.4 μl of each specific primer to a final concentration of 200 nM. The reactions were performed as the following conditions: initial denaturation step of 95°C for 10 s followed by two-step thermal cycling profile of denaturation at 95°C for 5 s, and combined primer annealing/extension at 60°C for 1 min for 40 cycles. No-template controls were included for each primer pair and each PCR reaction was completed in triplicate. To verify the specificity of the amplicon for each primer pair, a melting curve analysis was performed ranging from 60°C to 95°C with temperature increasing steps of 0.06°C/s (5 acquisitions per °C) at the end of each PCR run. Baseline and threshold cycles (Ct) were automatically determined using the LightCycler® 480 Software release 1.5.0. Relative gene expression with respect to internal reference gene UBQ10 was determined as described previously [100].

Acknowledgements

This study was supported by grants from the National High-Tech Research and Development Program of China (to G.Z., 2009AA10Z101), the Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences (to G.Z., No. 428) and National Natural Science Foundation of China (No. 30901157).

Notes

Declarations

Authors’ Affiliations

(1)
Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences
(2)
Complex Carbohydrate Research Center, The University of Georgia

References

  1. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G: Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science. 2000, 290 (5499): 2105-2110. 10.1126/science.290.5499.2105.PubMedView ArticleGoogle Scholar
  2. Wray GA, Hahn MW, Abouheif E, Balhoff JP, Pizer M, Rockman MV, Romano LA: The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol. 2003, 20 (9): 1377-1419. 10.1093/molbev/msg140.PubMedView ArticleGoogle Scholar
  3. Riano-Pachon DM, Ruzicic S, Dreyer I, Mueller-Roeber B: PlnTFDB: an integrative plant transcription factor database. BMC Bioinformatics. 2007, 8: 42-10.1186/1471-2105-8-42.PubMedPubMed CentralView ArticleGoogle Scholar
  4. AtTFDB - Arabidopsis transcription factor database. [http://arabidopsis.med.ohio-state.edu/AtTFDB/]
  5. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP, Blaudez D, Boerjan W, Brun A, Brunner A, Busov V, Campbell M, Carlson J, Chalot M, Chapman J, Chen GL, Cooper D, Coutinho PM, Couturier J, Covert S, Cronk Q, et al: The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science. 2006, 313 (5793): 1596-1604. 10.1126/science.1128691.PubMedView ArticleGoogle Scholar
  6. Ooka H, Satoh K, Doi K, Nagata T, Otomo Y, Murakami K, Matsubara K, Osato N, Kawai J, Carninci P, Hayashizaki Y, Suzuki K, Kojima K, Takahara Y, Yamamoto K, Kikuchi S: Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Res. 2003, 10 (6): 239-247. 10.1093/dnares/10.6.239.PubMedView ArticleGoogle Scholar
  7. Fang Y, You J, Xie K, Xie W, Xiong L: Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NAC transcription factor family in rice. Mol Genet Genomics. 2008, 280 (6): 547-563. 10.1007/s00438-008-0386-6.PubMedView ArticleGoogle Scholar
  8. Pinheiro GL, Marques CS, Costa MD, Reis PA, Alves MS, Carvalho CM, Fietto LG, Fontes EP: Complete inventory of soybean NAC transcription factors: sequence conservation and expression analysis uncover their distinct roles in stress response. Gene. 2009, 444 (1-2): 10-23. 10.1016/j.gene.2009.05.012.PubMedView ArticleGoogle Scholar
  9. Kikuchi K, Ueguchi-Tanaka M, Yoshida KT, Nagato Y, Matsusoka M, Hirano HY: Molecular analysis of the NAC gene family in rice. Mol Gen Genet. 2000, 262 (6): 1047-1051. 10.1007/PL00008647.PubMedView ArticleGoogle Scholar
  10. Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M: Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell. 1997, 9 (6): 841-857. 10.1105/tpc.9.6.841.PubMedPubMed CentralView ArticleGoogle Scholar
  11. Duval M, Hsieh TF, Kim SY, Thomas TL: Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily. Plant Mol Biol. 2002, 50 (2): 237-248. 10.1023/A:1016028530943.PubMedView ArticleGoogle Scholar
  12. Ernst HA, Olsen AN, Larsen S, Lo Leggio L: Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors. EMBO Rep. 2004, 5 (3): 297-303. 10.1038/sj.embor.7400093.PubMedPubMed CentralView ArticleGoogle Scholar
  13. Hegedus D, Yu M, Baldwin D, Gruber M, Sharpe A, Parkin I, Whitwill S, Lydiate D: Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress. Plant Mol Biol. 2003, 53 (3): 383-397. 10.1023/B:PLAN.0000006944.61384.11.PubMedView ArticleGoogle Scholar
  14. Olsen AN, Ernst HA, Leggio LL, Skriver K: NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci. 2005, 10 (2): 79-87. 10.1016/j.tplants.2004.12.010.PubMedView ArticleGoogle Scholar
  15. Xie Q, Frugis G, Colgan D, Chua NH: Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev. 2000, 14 (23): 3024-3036. 10.1101/gad.852200.PubMedPubMed CentralView ArticleGoogle Scholar
  16. Jensen MK, Kjaersgaard T, Nielsen MM, Galberg P, Petersen K, O'Shea C, Skriver K: The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and determinants of ANAC019 stress signaling. Biochem J. 2009Google Scholar
  17. Ko JH, Yang SH, Park AH, Lerouxel O, Han KH: ANAC012, a member of the plant-specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana. Plant J. 2007, 50 (6): 1035-1048. 10.1111/j.1365-313X.2007.03109.x.PubMedView ArticleGoogle Scholar
  18. Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran LS, Yamaguchi-Shinozaki K, Shinozaki K: A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J. 2004, 39 (6): 863-876. 10.1111/j.1365-313X.2004.02171.x.PubMedView ArticleGoogle Scholar
  19. Zhong R, Lee C, Ye ZH: Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors. Plant Physiol. 2009Google Scholar
  20. Zheng X, Chen B, Lu G, Han B: Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochem Biophys Res Commun. 2009, 379 (4): 985-989. 10.1016/j.bbrc.2008.12.163.PubMedView ArticleGoogle Scholar
  21. Yamaguchi M, Kubo M, Fukuda H, Demura T: Vascular-related NAC-DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. Plant J. 2008, 55 (4): 652-664. 10.1111/j.1365-313X.2008.03533.x.PubMedView ArticleGoogle Scholar
  22. Taoka K, Yanagimoto Y, Daimon Y, Hibara K, Aida M, Tasaka M: The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins. Plant J. 2004, 40 (4): 462-473. 10.1111/j.1365-313X.2004.02238.x.PubMedView ArticleGoogle Scholar
  23. Souer E, van Houwelingen A, Kloos D, Mol J, Koes R: The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell. 1996, 85 (2): 159-170. 10.1016/S0092-8674(00)81093-4.PubMedView ArticleGoogle Scholar
  24. Weir I, Lu J, Cook H, Causier B, Schwarz-Sommer Z, Davies B: CUPULIFORMIS establishes lateral organ boundaries in Antirrhinum. Development. 2004, 131 (4): 915-922. 10.1242/dev.00993.PubMedView ArticleGoogle Scholar
  25. Hibara K, Takada S, Tasaka M: CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation. Plant J. 2003, 36 (5): 687-696. 10.1046/j.1365-313X.2003.01911.x.PubMedView ArticleGoogle Scholar
  26. Nikovics K, Blein T, Peaucelle A, Ishida T, Morin H, Aida M, Laufs P: The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. Plant Cell. 2006, 18 (11): 2929-2945. 10.1105/tpc.106.045617.PubMedPubMed CentralView ArticleGoogle Scholar
  27. Sablowski RW, Meyerowitz EM: A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA. Cell. 1998, 92 (1): 93-103. 10.1016/S0092-8674(00)80902-2.PubMedView ArticleGoogle Scholar
  28. He XJ, Mu RL, Cao WH, Zhang ZG, Zhang JS, Chen SY: AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 2005, 44 (6): 903-916. 10.1111/j.1365-313X.2005.02575.x.PubMedView ArticleGoogle Scholar
  29. Guo Y, Gan S: AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. 2006, 46 (4): 601-612. 10.1111/j.1365-313X.2006.02723.x.PubMedView ArticleGoogle Scholar
  30. Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J: A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science. 2006, 314 (5803): 1298-1301. 10.1126/science.1133649.PubMedView ArticleGoogle Scholar
  31. Yoo SY, Kim Y, Kim SY, Lee JS, Ahn JH: Control of flowering time and cold response by a NAC-domain protein in Arabidopsis. PLoS One. 2007, 2 (7): e642-10.1371/journal.pone.0000642.PubMedPubMed CentralView ArticleGoogle Scholar
  32. Kim SG, Kim SY, Park CM: A membrane-associated NAC transcription factor regulates salt-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Planta. 2007, 226 (3): 647-654. 10.1007/s00425-007-0513-3.PubMedView ArticleGoogle Scholar
  33. Kim YS, Kim SG, Park JE, Park HY, Lim MH, Chua NH, Park CM: A membrane-bound NAC transcription factor regulates cell division in Arabidopsis. Plant Cell. 2006, 18 (11): 3132-3144. 10.1105/tpc.106.043018.PubMedPubMed CentralView ArticleGoogle Scholar
  34. Willemsen V, Bauch M, Bennett T, Campilho A, Wolkenfelt H, Xu J, Haseloff J, Scheres B: The NAC domain transcription factors FEZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells. Dev Cell. 2008, 15 (6): 913-922. 10.1016/j.devcel.2008.09.019.PubMedView ArticleGoogle Scholar
  35. Kato H, Motomura T, Komeda Y, Saito T, Kato A: Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana. J Plant Physiol. 2009Google Scholar
  36. Jensen MK, Hagedorn PH, de Torres-Zabala M, Grant MR, Rung JH, Collinge DB, Lyngkjaer MF: Transcriptional regulation by an NAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis. Plant J. 2008, 56 (6): 867-880. 10.1111/j.1365-313X.2008.03646.x.PubMedView ArticleGoogle Scholar
  37. Waters BM, Uauy C, Dubcovsky J, Grusak MA: Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain. J Exp Bot. 2009, 60 (15): 4263-4274. 10.1093/jxb/erp257.PubMedView ArticleGoogle Scholar
  38. Mao C, Ding W, Wu Y, Yu J, He X, Shou H, Wu P: Overexpression of a NAC-domain protein promotes shoot branching in rice. New Phytol. 2007, 176 (2): 288-298. 10.1111/j.1469-8137.2007.02177.x.PubMedView ArticleGoogle Scholar
  39. Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L: Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA. 2006, 103 (35): 12987-12992. 10.1073/pnas.0604882103.PubMedPubMed CentralView ArticleGoogle Scholar
  40. Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K: Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell. 2004, 16 (9): 2481-2498. 10.1105/tpc.104.022699.PubMedPubMed CentralView ArticleGoogle Scholar
  41. Jiang Y, Deyholos MK: Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol. 2006, 6: 25-10.1186/1471-2229-6-25.PubMedPubMed CentralView ArticleGoogle Scholar
  42. Collinge M, Boller T: Differential induction of two potato genes, Stprx2 and StNAC, in response to infection by Phytophthora infestans and to wounding. Plant Mol Biol. 2001, 46 (5): 521-529. 10.1023/A:1010639225091.PubMedView ArticleGoogle Scholar
  43. Ren T, Qu F, Morris TJ: HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus. Plant Cell. 2000, 12 (10): 1917-1926. 10.1105/tpc.12.10.1917.PubMedPubMed CentralView ArticleGoogle Scholar
  44. Delessert C, Kazan K, Wilson IW, Van Der Straeten D, Manners J, Dennis ES, Dolferus R: The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. Plant J. 2005, 43 (5): 745-757. 10.1111/j.1365-313X.2005.02488.x.PubMedView ArticleGoogle Scholar
  45. Kim SG, Lee AK, Yoon HK, Park CM: A membrane-bound NAC transcription factor NTL8 regulates gibberellic acid-mediated salt signaling in Arabidopsis seed germination. Plant J. 2008, 55 (1): 77-88. 10.1111/j.1365-313X.2008.03493.x.PubMedView ArticleGoogle Scholar
  46. Lu PL, Chen NZ, An R, Su Z, Qi BS, Ren F, Chen J, Wang XC: A novel drought-inducible gene, ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis. Plant Mol Biol. 2007, 63 (2): 289-305. 10.1007/s11103-006-9089-8.PubMedView ArticleGoogle Scholar
  47. Wang X, Basnayake BM, Zhang H, Li G, Li W, Virk N, Mengiste T, Song F: The Arabidopsis ATAF1, a NAC transcription factor, is a negative regulator of defense responses against necrotrophic fungal and bacterial pathogens. Mol Plant Microbe Interact. 2009, 22 (10): 1227-1238. 10.1094/MPMI-22-10-1227.PubMedView ArticleGoogle Scholar
  48. Zhao C, Avci U, Grant EH, Haigler CH, Beers EP: XND1, a member of the NAC domain family in Arabidopsis thaliana, negatively regulates lignocellulose synthesis and programmed cell death in xylem. Plant J. 2008, 53 (3): 425-436. 10.1111/j.1365-313X.2007.03350.x.PubMedView ArticleGoogle Scholar
  49. Zhao C, Craig JC, Petzold HE, Dickerman AW, Beers EP: The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiol. 2005, 138 (2): 803-818. 10.1104/pp.105.060202.PubMedPubMed CentralView ArticleGoogle Scholar
  50. Zhong R, Demura T, Ye ZH: SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell. 2006, 18 (11): 3158-3170. 10.1105/tpc.106.047399.PubMedPubMed CentralView ArticleGoogle Scholar
  51. Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH: A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell. 2008, 20 (10): 2763-2782. 10.1105/tpc.108.061325.PubMedPubMed CentralView ArticleGoogle Scholar
  52. Zhong R, Richardson EA, Ye ZH: Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. Planta. 2007, 225 (6): 1603-1611. 10.1007/s00425-007-0498-y.PubMedView ArticleGoogle Scholar
  53. Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M: NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell. 2007, 19 (1): 270-280. 10.1105/tpc.106.047043.PubMedPubMed CentralView ArticleGoogle Scholar
  54. Mitsuda N, Ohme-Takagi M: NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. Plant J. 2008, 56 (5): 768-778. 10.1111/j.1365-313X.2008.03633.x.PubMedView ArticleGoogle Scholar
  55. Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M: The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell. 2005, 17 (11): 2993-3006. 10.1105/tpc.105.036004.PubMedPubMed CentralView ArticleGoogle Scholar
  56. Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T: Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 2005, 19 (16): 1855-1860. 10.1101/gad.1331305.PubMedPubMed CentralView ArticleGoogle Scholar
  57. Shen H, Yin YB, Chen F, Xu Y, Dixon RA: A Bioinformatic Analysis of NAC Genes for Plant Cell Wall Development in Relation to Lignocellulosic Bioenergy Production. Bioenerg Res. 2009, 2: 217-232. 10.1007/s12155-009-9047-9.View ArticleGoogle Scholar
  58. Zhu QH, Guo AY, Gao G, Zhong YF, Xu M, Huang M, Luo J: DPTF: a database of poplar transcription factors. Bioinformatics. 2007, 23 (10): 1307-1308. 10.1093/bioinformatics/btm113.PubMedView ArticleGoogle Scholar
  59. Salamov AA, Solovyev VV: Ab initio gene finding in Drosophila genomic DNA. Genome Res. 2000, 10 (4): 516-522. 10.1101/gr.10.4.516.PubMedPubMed CentralView ArticleGoogle Scholar
  60. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R: InterProScan: protein domains identifier. Nucleic Acids Res. 2005, W116-120. 10.1093/nar/gki442. 33 Web Server
  61. Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM: Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol. 2009, 149 (2): 981-993. 10.1104/pp.108.132795.PubMedPubMed CentralView ArticleGoogle Scholar
  62. Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, Guo J, Liang W, Chen L, Yin J, Ma H, Wang J, Zhang D: Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol. 2006, 141 (4): 1167-1184. 10.1104/pp.106.080580.PubMedPubMed CentralView ArticleGoogle Scholar
  63. Lijavetzky D, Carbonero P, Vicente-Carbajosa J: Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol Biol. 2003, 3: 17-10.1186/1471-2148-3-17.PubMedPubMed CentralView ArticleGoogle Scholar
  64. Wang D, Guo Y, Wu C, Yang G, Li Y, Zheng C: Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice. BMC Genomics. 2008, 9: 44-10.1186/1471-2164-9-44.PubMedPubMed CentralView ArticleGoogle Scholar
  65. Zhang Y, Wang L: The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol. 2005, 5 (1): 1-10.1186/1471-2148-5-1.PubMedPubMed CentralView ArticleGoogle Scholar
  66. Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K: Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 2007, 51 (4): 617-630. 10.1111/j.1365-313X.2007.03168.x.PubMedView ArticleGoogle Scholar
  67. Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W: GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 2004, 136 (1): 2621-2632. 10.1104/pp.104.046367.PubMedPubMed CentralView ArticleGoogle Scholar
  68. Brunner AM, Busov VB, Strauss SH: Poplar genome sequence: functional genomics in an ecologically dominant plant species. Trends Plant Sci. 2004, 9 (1): 49-56. 10.1016/j.tplants.2003.11.006.PubMedView ArticleGoogle Scholar
  69. Cannon SB, Mitra A, Baumgarten A, Young ND, May G: The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4: 10-10.1186/1471-2229-4-10.PubMedPubMed CentralView ArticleGoogle Scholar
  70. Barakat A, Bagniewska-Zadworna A, Choi A, Plakkat U, DiLoreto DS, Yellanki P, Carlson JE: The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression. BMC Plant Biol. 2009, 9: 26-10.1186/1471-2229-9-26.PubMedPubMed CentralView ArticleGoogle Scholar
  71. Kalluri UC, Difazio SP, Brunner AM, Tuskan GA: Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biol. 2007, 7: 59-10.1186/1471-2229-7-59.PubMedPubMed CentralView ArticleGoogle Scholar
  72. Lan T, Yang ZL, Yang X, Liu YJ, Wang XR, Zeng QY: Extensive Functional Diversification of the Populus Glutathione S-Transferase Supergene Family. Plant Cell. 2009Google Scholar
  73. Blanc G, Wolfe KH: Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell. 2004, 16 (7): 1679-1691. 10.1105/tpc.021410.PubMedPubMed CentralView ArticleGoogle Scholar
  74. Blanc G, Wolfe KH: Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. Plant Cell. 2004, 16 (7): 1667-1678. 10.1105/tpc.021345.PubMedPubMed CentralView ArticleGoogle Scholar
  75. Maere S, De Bodt S, Raes J, Casneuf T, Van Montagu M, Kuiper M, Van de Peer Y: Modeling gene and genome duplications in eukaryotes. Proc Natl Acad Sci USA. 2005, 102 (15): 5454-5459. 10.1073/pnas.0501102102.PubMedPubMed CentralView ArticleGoogle Scholar
  76. Prince VE, Pickett FB: Splitting pairs: the diverging fates of duplicated genes. Nat Rev Genet. 2002, 3 (11): 827-837. 10.1038/nrg928.PubMedView ArticleGoogle Scholar
  77. Vandepoele K, Simillion C, Van de Peer Y: Evidence that rice and other cereals are ancient aneuploids. Plant Cell. 2003, 15 (9): 2192-2202. 10.1105/tpc.014019.PubMedPubMed CentralView ArticleGoogle Scholar
  78. Kolkman JA, Stemmer WP: Directed evolution of proteins by exon shuffling. Nat Biotechnol. 2001, 19 (5): 423-428. 10.1038/88084.PubMedView ArticleGoogle Scholar
  79. Pfam. [http://pfam.sanger.ac.uk/search]
  80. SMART. [http://smart.embl-heidelberg.de/]
  81. Ohlrogge J, Benning C: Unraveling plant metabolism by EST analysis. Curr Opin Plant Biol. 2000, 3 (3): 224-228.PubMedView ArticleGoogle Scholar
  82. PopGenIE. [http://www.popgenie.org/]
  83. Sjodin A, Bylesjo M, Skogstrom O, Eriksson D, Nilsson P, Ryden P, Jansson S, Karlsson J: UPSC-BASE--Populus transcriptomics online. Plant J. 2006, 48 (5): 806-817. 10.1111/j.1365-313X.2006.02920.x.PubMedView ArticleGoogle Scholar
  84. Sjodin A, Street NR, Sandberg G, Gustafsson P, Jansson S: The Populus Genome Integrative Explorer (PopGenIE): a new resource for exploring the Populus genome. New Phytol. 2009Google Scholar
  85. Druart N, Johansson A, Baba K, Schrader J, Sjodin A, Bhalerao RR, Resman L, Trygg J, Moritz T, Bhalerao RP: Environmental and hormonal regulation of the activity-dormancy cycle in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks. Plant J. 2007, 50 (4): 557-573. 10.1111/j.1365-313X.2007.03077.x.PubMedView ArticleGoogle Scholar
  86. Schrader J, Moyle R, Bhalerao R, Hertzberg M, Lundeberg J, Nilsson P, Bhalerao RP: Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome. Plant J. 2004, 40 (2): 173-187. 10.1111/j.1365-313X.2004.02199.x.PubMedView ArticleGoogle Scholar
  87. Moreau C, Aksenov N, Lorenzo MG, Segerman B, Funk C, Nilsson P, Jansson S, Tuominen H: A genomic approach to investigate developmental cell death in woody tissues of Populus trees. Genome Biol. 2005, 6 (4): R34-10.1186/gb-2005-6-4-r34.PubMedPubMed CentralView ArticleGoogle Scholar
  88. Schrader J, Nilsson J, Mellerowicz E, Berglund A, Nilsson P, Hertzberg M, Sandberg G: A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity. Plant Cell. 2004, 16 (9): 2278-2292. 10.1105/tpc.104.024190.PubMedPubMed CentralView ArticleGoogle Scholar
  89. HMMER. [http://hmmer.janelia.org/]
  90. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25 (24): 4876-4882. 10.1093/nar/25.24.4876.PubMedPubMed CentralView ArticleGoogle Scholar
  91. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007, 24 (8): 1596-1599. 10.1093/molbev/msm092.PubMedView ArticleGoogle Scholar
  92. Guo AY, Zhu QH, Chen X, Luo JC: [GSDS: a gene structure display server]. Yi Chuan. 2007, 29 (8): 1023-1026.PubMedView ArticleGoogle Scholar
  93. TIGR Rice Genome Annotation. [http://compbio.dfci.harvard.edu/tgi/]
  94. Bailey TL, Williams N, Misleh C, Li WW: MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 2006, W369-373. 10.1093/nar/gkl198. 34 Web Server
  95. Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP, Bork P: SMART 4.0: towards genomic data integration. Nucleic Acids Res. 2004, D142-144. 10.1093/nar/gkh088. 32 Database
  96. Finn RD, Mistry J, Schuster-Bockler B, Griffiths-Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer EL, Bateman A: Pfam: clans, web tools and services. Nucleic Acids Res. 2006, D247-251. 10.1093/nar/gkj149. 34 Database
  97. Sturn A, Quackenbush J, Trajanoski Z: Genesis: cluster analysis of microarray data. Bioinformatics. 2002, 18 (1): 207-208. 10.1093/bioinformatics/18.1.207.PubMedView ArticleGoogle Scholar
  98. Gene Expression Omnibus. [http://www.ncbi.nlm.nih.gov/geo/]
  99. Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter. 1993, 11: 113-116. 10.1007/BF02670468.View ArticleGoogle Scholar
  100. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25 (4): 402-408. 10.1006/meth.2001.1262.PubMedView ArticleGoogle Scholar

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