Skip to main content


Log in

Characterization of NAC domain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript


Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC domain transcription factors (NACs). We studied NACs with putative involvement as negative (XND1 from Arabidopsis and its poplar orthologs PopNAC118, PopNAC122, PopNAC128, PopNAC129), or positive (SND2 and SND3 from Arabidopsis and their poplar orthologs PopNAC105, PopNAC154, PopNAC156, PopNAC157) regulators of secondary cell wall synthesis. Using quantitative PCR and in situ hybridization, we evaluated expression of these Populus NACs in a developmental gradient and in association with reaction wood and found that representatives from both groups were associated with wood-forming tissue and phloem fibers. Additionally, XND1 orthologs were expressed in mesophyll cells of developing leaves. We prepared transgenic Arabidopsis and poplar plants for overexpression of selected NACs. XND1 overexpression in poplar resulted in severe stunting. Additionally, poplar XND1 overexpressors lacked phloem fibers and showed reductions in cell size and number, vessel number, and frequency of rays in the xylem. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Overexpression of PopNAC154 in poplar reduced height growth and increased the relative proportion of bark versus xylem.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others



Leaf plastochron index

3′ RACE:

3′ Rapid amplification of cDNA ends


Reverse transcriptase-quantitative PCR


Untranslated region


  • Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9:841–857

    Article  CAS  PubMed  Google Scholar 

  • Andersson-Gunneras S, Mellerowicz EJ, Love J, Segerman B, Ohmiya Y, Coutinho PM, Nilsson P, Henrissat B, Moritz T, Sundberg B (2006) Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis. Plant J 45:144–165

    Article  PubMed  Google Scholar 

  • Bhandari S, Fujino T, Thammanagowda S, Zhang D, Xu F, Joshi CP (2006) Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees. Planta 224:828–837

    Article  CAS  PubMed  Google Scholar 

  • Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14

    Article  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Dharmawardhana P, Brunner AM, Strauss SH (2010) Genome-wide transcriptome analysis of the transition from primary to secondary stem development in Populus trichocarpa. BMC Genomics 11:150

    Article  PubMed  Google Scholar 

  • Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  CAS  PubMed  Google Scholar 

  • Esau K (1965) Plant anatomy, 2nd edn. Wiley, New York

    Google Scholar 

  • Filichkin SA, Meilan R, Busov VB, Ma C, Brunner AM, Strauss SH (2006) Alcohol-inducible gene expression in transgenic Populus. Plant Cell Rep 25:660–667

    Article  CAS  PubMed  Google Scholar 

  • Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6:609–618

    Article  CAS  PubMed  Google Scholar 

  • Harding SA, Leshkevich J, Chiang VL, Tsai CJ (2002) Differential substrate inhibition couples kinetically distinct 4-coumarate: coenzyme a ligases with spatially distinct metabolic roles in quaking aspen. Plant Physiol 128:428–438

    Article  CAS  PubMed  Google Scholar 

  • Hellgren JM, Olofsson K, Sundberg B (2004) Patterns of auxin distribution during gravitational induction of reaction wood in poplar and pine. Plant Physiol 135:212–220

    Article  CAS  PubMed  Google Scholar 

  • Ko JH, Yang SH, Park AH, Lerouxel O, Han KH (2007) ANAC012, a member of the plant-specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana. Plant J 50:1035–1048

    Article  CAS  PubMed  Google Scholar 

  • Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19:1855–1860

    Article  CAS  PubMed  Google Scholar 

  • Larson PR, Isebrands JG (1971) The plastochron index as applied to developmental studies in cottonwood. Can J For Res 1:1–11

    Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Δ·ΔCt) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Maere S, De Bodt S, Raes J, Casneuf T, Van Montagu M, Kuiper M, Van de Peer Y (2005) Modeling gene and genome duplications in eukaryotes. Proc Natl Acad Sci USA 102:5454–5459

    Article  CAS  PubMed  Google Scholar 

  • Meilan R, Ma C (2006) Poplar (Populus spp.). Methods Mol Biol 344:143–151

    CAS  PubMed  Google Scholar 

  • Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M (2005) The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17:2993–3006

    Article  CAS  PubMed  Google Scholar 

  • Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270–280

    Article  CAS  PubMed  Google Scholar 

  • Olsen AN, Ernst HA, Leggio LL, Skriver K (2005) NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci 10:79–87

    Article  CAS  PubMed  Google Scholar 

  • 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 (2003) Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Res 10:239–247

    Article  CAS  PubMed  Google Scholar 

  • Prassinos C, Ko JH, Yang J, Han KH (2005) Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees. Plant Cell Physiol 46:1213–1225

    Article  CAS  PubMed  Google Scholar 

  • 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 (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110

    Article  CAS  PubMed  Google Scholar 

  • Souer E, van Houwelingen A, Kloos D, Mol J, Koes R (1996) 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 85:159–170

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  • Thornton CR, Talbot NJ (2006) Immunofluorescence microscopy and immunogold EM for investigating fungal infection of plants. Nat Protoc 1:2506–2511

    Article  CAS  PubMed  Google Scholar 

  • 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, Cunningham R, Davis J, Degroeve S, Dejardin A, Depamphilis C, Detter J, Dirks B, Dubchak I, Duplessis S, Ehlting J, Ellis B, Gendler K, Goodstein D, Gribskov M, Grimwood J, Groover A, Gunter L, Hamberger B, Heinze B, Helariutta Y, Henrissat B, Holligan D, Holt R, Huang W, Islam-Faridi N, Jones S, Jones-Rhoades M, Jorgensen R, Joshi C, Kangasjarvi J, Karlsson J, Kelleher C, Kirkpatrick R, Kirst M, Kohler A, Kalluri U, Larimer F, Leebens-Mack J, Leple JC, Locascio P, Lou Y, Lucas S, Martin F, Montanini B, Napoli C, Nelson DR, Nelson C, Nieminen K, Nilsson O, Pereda V, Peter G, Philippe R, Pilate G, Poliakov A, Razumovskaya J, Richardson P, Rinaldi C, Ritland K, Rouze P, Ryaboy D, Schmutz J, Schrader J, Segerman B, Shin H, Siddiqui A, Sterky F, Terry A, Tsai CJ, Uberbacher E, Unneberg P, Vahala J, Wall K, Wessler S, Yang G, Yin T, Douglas C, Marra M, Sandberg G, Van de Peer Y, Rokhsar D (2006) The genome of black cottonwood, Populus trichocarpa (Torr & Gray). Science 313:1596–1604

    Article  CAS  PubMed  Google Scholar 

  • van Engelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ (1995) pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res 4:288–290

    Article  PubMed  Google Scholar 

  • van Raemdonck D, Pesquet E, Cloquet S, Beeckman H, Boerjan W, Goffner D, El Jaziri M, Baucher M (2005) Molecular changes associated with the setting up of secondary growth in aspen. J Exp Bot 56:2211–2227

    Article  PubMed  Google Scholar 

  • Zhao C, Johnson BJ, Kositsup B, Beers EP (2000) Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases. Plant Physiol 123:1185–1196

    Article  CAS  PubMed  Google Scholar 

  • Zhao C, Craig JC, Petzold HE, Dickerman AW, Beers EP (2005) The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiol 138:803–818

    Article  CAS  PubMed  Google Scholar 

  • Zhao C, Avci U, Grant EH, Haigler CH, Beers EP (2008) XND1, a member of the NAC domain family in Arabidopsis thaliana, negatively regulates lignocellulose synthesis and programmed cell death in xylem. Plant J 53:425–436

    Article  CAS  PubMed  Google Scholar 

  • Zhong R, Demura T, Ye ZH (2006) SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18:3158–3170

    Article  CAS  PubMed  Google Scholar 

  • Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH (2008) A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20:2763–2782

    Article  CAS  PubMed  Google Scholar 

Download references


We thank Dr. Sarah Covert for providing her manual annotation and phylogenetic analysis of the P. trichocarpa NAC gene family, and Xiaoyan Sheng, Zhengxing Shen, and Shelley Moore for technical assistance. This work was supported by the Office of Science (BER and BES), U.S. Department of Energy, Grant Nos. DE-FG02-06ER64185, DE-FG02-04ER15627, and DE-FG02-07ER64449.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Amy M. Brunner.

Additional information

E. H. Grant and T. Fujino contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grant, E.H., Fujino, T., Beers, E.P. et al. Characterization of NAC domain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus . Planta 232, 337–352 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: