Plant Molecular Biology

, Volume 59, Issue 4, pp 533–551 | Cite as

Structural, Functional, and Phylogenetic Characterization of a Large CBF Gene Family in Barley

  • Jeffrey S. Skinner
  • Jarislav von  Zitzewitz
  • Péter Szűcs
  • Luis Marquez-Cedillo
  • Tanya Filichkin
  • Keenan Amundsen
  • Eric J. Stockinger
  • Michael F. Thomashow
  • Tony H.H. Chen
  • Patrick M. Hayes


CBFs are key regulators in the Arabidopsis cold signaling pathway. We used Hordeum vulgare (barley), an important crop and a diploid Triticeae model, to characterize the CBF family from a low temperature tolerant cereal. We report that barley contains a large CBF family consisting of at least 20 genes (HvCBFs) comprising three multigene phylogenetic groupings designated the HvCBF1-, HvCBF3-, and HvCBF4-subgroups. For the HvCBF1- and HvCBF3-subgroups, there are comparable levels of phylogenetic diversity among rice, a cold-sensitive cereal, and the cold-hardy Triticeae. For the HvCBF4-subgroup, while similar diversity levels are observed in the Triticeae, only a single ancestral rice member was identified. The barley CBFs share many functional characteristics with dicot CBFs, including a general primary domain structure, transcript accumulation in response to cold, specific binding to the CRT motif, and the capacity to induce cor gene expression when ectopically expressed in Arabidopsis. Individual HvCBF genes differed in response to abiotic stress types and in the response time frame, suggesting different sets of HvCBF genes are employed relative to particular stresses. HvCBFs specifically bound monocot and dicot cor gene CRT elements in vitro under both warm and cold conditions; however, binding of HvCBF4-subgroup members was cold dependent. The temperature-independent HvCBFs activated cor gene expression at warm temperatures in transgenic Arabidopsis, while the cold-dependent HvCBF4-subgroup members of three Triticeae species did not. These results suggest that in the Triticeae – as in Arabidopsis – members of the CBF gene family function as fundamental components of the winter hardiness regulon.

Key words

barley CBF cereal HvCBF low temperature tolerance Triticeae 





dehydration response element


expressed sequence tag


genomic DNA


low temperature


polymerase chain reaction


quantitative trait locus


untranslated region


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  1. Amundsen, K.L. 2004. Conservation of the CBF low temperature response pathway in cereals. M.S. Thesis, Michigan State University, East Lansing, Michigan, U.S.AGoogle Scholar
  2. Benedict, C., Skinner, J.S., Meng, R., Chang, Y., Bhalerao, R., Finn, C., Chen, T.H.H., Hurry, V. 2005

    The role of the CBF-dependent signaling pathway in woody perennials

    Chen, T.H.H.Uemura, M.Fujikawa, S. eds. Cold Hardiness in Plants: Molecular Genetics, Cell Biology and PhysiologyCAB InternationalOxon, UK167180
    Google Scholar
  3. Cattivelli, L., Baldi, P., Crosatti, C., Di Fonzo, N., Faccioli, P., Grossi, M., Mastrangelo, A.M., Pecchioni, N., Stanca, A.M. 2002Chromosome regions and stress-related sequences involved in resistance to abiotic stress in TriticeaePlant Mol. Biol.48649665CrossRefPubMedGoogle Scholar
  4. Chang, S., Puryear, J., Cairney, J. 1993A simple and efficient method for isolating RNA from pine treesPlant Mol. Biol. Rep.11113116Google Scholar
  5. Choi, D.-W., Zhu, B., Close, T.J. 1999The barley (Hordeumvulgare L.) dehydrin multigene family: sequences, allele types, chromosome assignments, and expression characteristics of 11 Dhn genes of cv. DicktooPlant Physiol.9813241247Google Scholar
  6. Choi, D.-W., Koag, M.C., Close, T.J. 2000Map locations of barley Dhn genes determined by gene-specific PCRTheor. Appl. Genet.101350354CrossRefGoogle Scholar
  7. Choi, D.-W., Rodriguez, E.M., Close, T.J. 2002Barley Cbf3 gene identification, expression pattern, and map locationPlant Physiol.12917811787CrossRefPubMedGoogle Scholar
  8. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M., Zhu, J.K. 2003ICE1: a regulator of cold-induced transcriptome and freezing tolerance in ArabidopsisGenes Dev.1710431054CrossRefPubMedGoogle Scholar
  9. Clough, S.J., Bent, A.F. 1998Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thalianaPlant J.16735743CrossRefPubMedGoogle Scholar
  10. Dal Bosco, C., Busconi, M., Govoni, C., Baldi, P., Stanca, A.M., Crosatti, C., Bassi, R., Cattivelli, L. 2003cor gene expression in barley mutants affected in chloroplast development and photosynthetic electron transportPlant Physiol.131793802CrossRefPubMedGoogle Scholar
  11. Dubcovsky, J., Lijavetzky, D., Appendino, L., Tranquilli, G. 1998Comparative RFLP mapping of Triticum monococcum genes controlling vernalization requirementTheor. Appl. Genet.97968975CrossRefGoogle Scholar
  12. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K. 2003OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expressionPlant J.33751763CrossRefPubMedGoogle Scholar
  13. Foster, R., Gasch, A., Kay, S., Chua, N.H. 1992

    Analysis of protein/DNA interaction

    Koncz, C.Chua, N.-H.Schell, J. eds. Methods in Arabidopsis ResearchWorld ScientificSingapore378392
    Google Scholar
  14. Fowler, D.B., Breton, G., Limin, A.E., Mahfoozi, S., Sarhan, F. 2001Photoperiod and temperature interactions regulate low-temperature-induced gene expression in barleyPlant Physiol.12716761681CrossRefPubMedGoogle Scholar
  15. Francia, E., Rizza, F., Cattivelli, L., Stanca, A.M., Galiba, G., Tóth, B., Hayes, P.M., Skinner, J.S., Pecchioni, N. 2004Two loci on chromosome 5H determine low-temperature tolerance in a ‘Nure’ (winter) x ‘Tremois’ (spring) barley mapTheor. Appl. Genet.108670680CrossRefPubMedGoogle Scholar
  16. Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M., Thomashow, M.F. 1998Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expressionPlant J.16433442CrossRefPubMedGoogle Scholar
  17. Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., Thomashow, M.F. 2000Overexpression of the ArabidopsisCBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimationPlant Physiol.12418541865CrossRefPubMedGoogle Scholar
  18. Goff, S.A., Ricke, D., Lan, T.-H., Presting, G., Wang, R.,  et al. 2002A draft sequence of the rice genome (Oryza sativa L. ssp. japonica)Science23692100CrossRefGoogle Scholar
  19. Haake, V., Cook, D., Riechmann, J.L., Pineda, O., Thomashow, M.F., Zhang, J.Z. 2002Transcription factor CBF4 is a regulator of drought adaptation in ArabidopsisPlant Physiol.130639648CrossRefPubMedGoogle Scholar
  20. Hayes, P.M., Chen, F.Q., Corey, A., Pan, A., Chen, T.H.H., Baird, E., Powell, W., Thomas, W., Waugh, R., Bedő, Z., Karsai, I., Blake, T., Oberthur, L. 1997

    The Dicktoo×Morex population: a model for dissecting components of winter hardiness in barley

    Li, P.H.Chen, T.H.H. eds. Plant Cold HardinessPlenum PressNew York, U.S.A.7787
    Google Scholar
  21. Hayes, P.M., Castro, A., Marquez-Cedillo, L., Corey, A., Henson, C., Jones, B.L., Kling, J., Mather, D., Matus, I., Rossi, C., Sato, K. 2003

    Genetic diversity for quantitatively inherited agronomic and malting quality traits

    von , R.Knupffer, H.van , T.Sato, K. eds. Diversity in BarleyElsevier Science PublishersAmsterdam Netherlands201226
    Google Scholar
  22. Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O., Thomashow, M.F. 1998Arabidopsis CBF1 overexpression induces COR genes and enhances freezing toleranceScience280104106CrossRefPubMedGoogle Scholar
  23. Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang, J.Z., Deits, T., Thomashow, M.F. 2001Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassicanapus and other plant speciesPlant Physiol.127910917CrossRefPubMedGoogle Scholar
  24. Karsai, I., Mészáros, K., Hayes, P.M., Bedö, Z. 1997

    QTL analysis of winter hardiness-related traits in a doubled haploid population of barley developed from a cross between Dicktoo × Plaisant

    Sutka, J.Tischner, T.Veisz, O. eds. Proceedings of the International Symposium on Cereal Adaptation to Low Temperature Stress in Controlled EnvironmentsMartonvásárHungary9296
    Google Scholar
  25. Karsai, I., Mészáros, K., Lang, L., Hayes, P.M., Bed˝, Z. 2001Multivariate analysis of traits determining adaptation in cultivated barleyPlant Breed.120217222CrossRefGoogle Scholar
  26. Karsai, I., SzŐcs, P., Mészáros, K., Filichkin, T., Hayes, P.M., Skinner, J.S., Lang, L., Bedő, Z. 2005The Vrn-H2 locus is a major determinant of flowering time in a facultative×winter growth habit barley (Hordeum vulgare L.) mapping populationTheor. Appl. Genet.11014581466CrossRefPubMedGoogle Scholar
  27. Kolar, S.C., Hayes, P.M., Chen, T.H.H., Linderman, R.G. 1991Genotypic variation for cold tolerance in winter and facultative barleyCrop Sci.3111491152Google Scholar
  28. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., Shinozaki, K. 1998Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in ArabidopsisPlant Cell1013911406CrossRefPubMedGoogle Scholar
  29. Mahfoozi, S., Limin, A.E., Hayes, P.M., Hucl, P., Fowler, D.B. 2000Influence of photoperiod response on the expression of cold hardiness in wheat and barleyCan. J. Plant Sci.80721724Google Scholar
  30. Pan, A., Hayes, P.M., Chen, F., Chen, T.H.H., Blake, T., Wright, S., Karsai, I., Bedő, Z. 1994Genetic analysis of the components of winter hardiness in barley (Hordeum vulgare L.)Theor. Appl. Genet.89900910CrossRefGoogle Scholar
  31. Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K., Yamaguchi-Shinozaki, K. 2002DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expressionBiochem. Biophys. Res. Comm.2909981009CrossRefPubMedGoogle Scholar
  32. Schowalter, D.B., Sommer, S.S. 1989The generation of radiolabeled DNA and RNA probes with polymerase chain reactionAnal. Biochem.1779094CrossRefPubMedGoogle Scholar
  33. Skinner, J.S., Timko, M.P. 1998Loblolly pine (Pinustaeda L.) contains multiple expressed genes encoding light-dependent NADPH:protochlorophyllide oxidoreductase (POR)Plant Cell Physiol.39795806PubMedGoogle Scholar
  34. Stockinger, E.J., Gilmour, S.J., Thomashow, M.F. 1997Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficitProc. Natl. Acad. Sci. USA9410351040CrossRefPubMedGoogle Scholar
  35. Stockinger, E.J., Cheng, H., Skinner, J.S. 2005

    Structural organization of barley CBF genes coincident with QTLs for cold hardiness

    Chen, T.H.H.Uemura, M.Fujikawa, S. eds. Cold Hardiness in Plants: Molecular Genetics, Cell Biology and PhysiologyCAB InternationalOxon, UK5363
    Google Scholar
  36. Straub, P.F., Shen, Q., Ho, T.D. 1994Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1Plant Mol. Biol.26617630CrossRefPubMedGoogle Scholar
  37. Thomashow, M.F. 1999Plant cold acclimation: freezing tolerance genes and regulatory mechanismsAnn. Rev. Plant Physiol. Plant Mol. Biol.50571599CrossRefGoogle Scholar
  38. Vágújfalvi, A., Galiba, G., Cattivelli, L., Dubcovsky, J. 2003The cold regulated transcriptional activator Cbf3 is linked to the frost tolerance locus Fr-A2 on wheat chromosome 5AMol. Gen. Genom.2696067Google Scholar
  39. Zee, K., Chen, F.Q., Hayes, P.M., Close, T.J., Chen, T.H.H. 1995Cold-specific induction of a dehydrin gene family member in barleyPlant Physiol.10812331239PubMedGoogle Scholar
  40. Vazquez-Tello, A., Ouellet, F., Sarhan, F. 1998Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheatMol. Gen. Genet.257157166CrossRefPubMedGoogle Scholar
  41. Vlachonasios, K.E., Thomashow, M.F., Triezenberg, S.J. 2003Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expressionPlant Cell15626638CrossRefPubMedGoogle Scholar
  42. von Zitzewitz, J., SzŐcs, P., Dubcovsky, J., Yan, L., Pecchioni, N., Francia, E. Casas, A., Chen, T.H.H., Hayes, P.M., Skinner, J.S. 2005. Molecular and structural characterization of barley vernalization genes. Plant Mol. Biol. 59: 447–465Google Scholar
  43. Wang, Z., Triezenberg, S.J., Thomashow, M.F., Stockinger, E.J. 2005Multiple hydrophobic motifs in Arabidopsis CBF1 COOH-terminus provide functional redundancy in trans-activationPlant Mol. Biol.58543559CrossRefPubMedGoogle Scholar
  44. Xue, G.P. 2002Characterization of the DNA-binding profile of barley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activityNucl. Acids Res.30e77CrossRefPubMedGoogle Scholar
  45. Xue, G.P. 2003The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperaturePlant J.3337383CrossRefPubMedGoogle Scholar
  46. Yu, J., Hu, S., Wang, J., Wong, G.K.S., Li, S.,  et al. 2002A draft sequence of the rice genome (Oryza sativa L. ssp. indica)Science2367992CrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Jeffrey S. Skinner
    • 1
    • 2
  • Jarislav von  Zitzewitz
    • 2
  • Péter Szűcs
    • 2
    • 3
  • Luis Marquez-Cedillo
    • 2
  • Tanya Filichkin
    • 2
  • Keenan Amundsen
    • 4
    • 6
  • Eric J. Stockinger
    • 5
  • Michael F. Thomashow
    • 4
  • Tony H.H. Chen
    • 1
  • Patrick M. Hayes
    • 2
  1. 1.Department of Horticulture, College of Agriculture Oregon State UniversityCorvallisUSA
  2. 2.Department of Crop and Soil Science, College of Agriculture Oregon State UniversityCorvallisUSA
  3. 3.Agricultural Research Institute of the Hungarian Academy of SciencesMartonvásárHungary
  4. 4.Department of Crop and Soil SciencesMichigan State UniversityEast LansingUSA
  5. 5.Department of Horticulture and Crop ScienceThe Ohio State University/OARDCWoosterUSA
  6. 6.United States National ArboretumUSDA-ARSWashingtonUSA

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