Plant Molecular Biology

, Volume 80, Issue 4–5, pp 405–418 | Cite as

The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities

  • Ana Elisa Valdés
  • Elin Övernäs
  • Henrik Johansson
  • Alvaro Rada-Iglesias
  • Peter Engström


Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.


HD-Zip PP2C ABA SnRK2 ABA receptors Drought stress response 



This work was supported by the European commission (TF-STRESS), The Royal Swedish Academy of Sciences, and the Lars Hiertas Minne and Nils and Dorthi Troedsson Foundations. A.E.V. was supported by postdoctoral fellowships from the Spanish Ministry of Education and Science (EX2006-1690) and the Alfonso Martín Escudero Foundation. The authors would like to thank Pedro Rodríguez for the donation of the pp2c mutants, and Anna Olsson and Kerstin Nordin-Henriksson for their contribution to athb7-athb12 transgenic lines.

Supplementary material

11103_2012_9956_MOESM1_ESM.pdf (364 kb)
Supplementary material 1 (PDF 364 kb)


  1. Ades SE, Sauer RT (1994) Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50. Biochem 33:9187–9194CrossRefGoogle Scholar
  2. Agalou A, Purwantomo S, Övernäs E, Johannesson H, Zhu X, Estiati A, de Kam RJ, Engström P, Slamet-Loedin IH, Zhu Z et al (2008) A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol 66:87–103PubMedCrossRefGoogle Scholar
  3. Barrero JM, Millar AA, Griffiths J, Czechowski T, Scheible WR, Udvardi M, Reid JB, Ross JJ, Jacobsen JV, Gubler F (2010) Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds. Plant J 61:611–622PubMedCrossRefGoogle Scholar
  4. Bowler C, Benvenuto G, Laflamme P, Molino D, Probst AV, Tariq M, Paszkowski J (2004) Chromatin techniques for plant cells. Plant J 39:776–789PubMedCrossRefGoogle Scholar
  5. Bray EA (2004) Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J Exp Bot 55:2331–2341PubMedCrossRefGoogle Scholar
  6. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17PubMedCrossRefGoogle Scholar
  7. Deng X, Phillips J, Bräutigam A, Engström P, Johannesson H, Ouwerkerk PBF, Ruberti I, Salinas J, Vera P, Iannacone R et al (2006) A homeodomain leucine zipper gene from Craterostigma plantagineum regulates abscisic acid responsive gene expression and physiological responses. Plant Mol Biol 61:469–489PubMedCrossRefGoogle Scholar
  8. Dezar CA, Gago GM, González DH, Chan RL (2005) Hahb-4, a sunflower homeobox-leucine zipper gene, is a developmental regulator and confers drought tolerance to Arabidopsis thaliana plants. Transgenic Res 14:429–440PubMedCrossRefGoogle Scholar
  9. Fujii H, Zhu JK (2009) Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. PNAS 106:8380–8385PubMedCrossRefGoogle Scholar
  10. Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park S-Y, Cutler SR, Sheen J, Rodríguez PL, Zhu J-K (2009) In vitro reconstitution of an abscisic acid signalling pathway. Nature 462:660–664PubMedCrossRefGoogle Scholar
  11. Fujii H, Verslues PE, Zhu J-K (2011) Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo. Proc Natl Acad Sci 108:1717–1722PubMedCrossRefGoogle Scholar
  12. González DH, Valle EM, Chan GG (1997) Interaction between proteins containing homeodomains associated to leucine zippers from sunflower. Biochem Biophys Acta 1351:137–149PubMedCrossRefGoogle Scholar
  13. Haring M, Offermann S, Danker T, Horst I, Peterhansel C, Stam M (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:11–26PubMedCrossRefGoogle Scholar
  14. Henriksson E, Olsson ASB, Johannesson H, Johansson H, Hanson J, Engström P, Söderman E (2005) Homeodomain leucine zipper class I genes in Arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiol 139:509–518PubMedCrossRefGoogle Scholar
  15. Himmelbach A, Hoffmann T, Leube M, Höhener B, Grill E (2002) Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. EMBO J 21:3029–3038PubMedCrossRefGoogle Scholar
  16. Hjellström M, Olsson ASB, Engström P, Söderman E (2003) Constitutive expression of the water deficit-inducible homeobox gene ATHB7 in transgenic Arabidopsis causes a suppression of stem elongation growth. Plant Cell Env 26:1127–1136CrossRefGoogle Scholar
  17. Johannesson H, Wang Y, Engström P (2001) DNA-binding and dimerisation preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro. Plant Mol Biol 45:63–73PubMedCrossRefGoogle Scholar
  18. Johannesson H, Wang Y, Hanson J, Engström P (2003) The Arabidopsis thaliana homeobox gene ATHB5 is a potential regulator of abscisic acid responsiveness in developing seedlings. Plant Mol Biol 51:719–729PubMedCrossRefGoogle Scholar
  19. Kobayashi Y, Yamamoto S, Minami H, Kagaya Y, Hattori T (2004) Differential activation of the rice sucrose nonfermenting 1-related protein kinase 2 family by hyperosmotic stress and abscisic acid. Plant Cell 16:1163–1177PubMedCrossRefGoogle Scholar
  20. Kuhn JM, Boisson-Dernier A, Dizon MB, Maktabi MH, Schroeder JI (2006) The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA. Plant Physiol 140:127–139PubMedCrossRefGoogle Scholar
  21. Lechner E, Leonhardt N, Eisler H, Alioua M, Jacquet H, Leung J, Genschik P (2011) MATH/BTB CRL3 receptors target the homeodomain-leucine zipper ATHB6 to modulate abscisic acid signalling. Dev Cell 21:1116–1128PubMedCrossRefGoogle Scholar
  22. Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49:199–222PubMedCrossRefGoogle Scholar
  23. Ma S, Gong Q, Bohnert HJ (2007) An Arabidopsis gene network based on the graphical Gaussian model. Genome Res 17:1614–1625PubMedCrossRefGoogle Scholar
  24. Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324:1064–1068PubMedGoogle Scholar
  25. Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J (2001) The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 25:295–303PubMedCrossRefGoogle Scholar
  26. Miyazono K, Mikayawa T, Sawano Y, Kubota K, Kang H-J, Asano A, Miyauchi Y, Takahashi M, Zhi Y, Fujita K et al (2009) Structural basis of abscisic acid signalling. Nature 462:609–614PubMedCrossRefGoogle Scholar
  27. Murashige T, Skoog F (1962) A revised medium for growth and bioassays with tobacco tissue culture. Physiol Plant 15:493–497CrossRefGoogle Scholar
  28. Nakashima K, Fujita Y, Kanamori N, Katagiri T, Umezawa T, Kidokiro S, Maruyama K, Yoshida T, Ishiyama K, Kobayashi M et al (2009) Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol 50:1345–1363PubMedCrossRefGoogle Scholar
  29. Olsson ASB (2004) HD-Zip I transcription factors in Arabidopsis thaliana: expression and function in relation to environmental stress conditions. Dissertation, Uppsala UniversityGoogle Scholar
  30. Olsson ASB, Engström P, Söderman E (2004) The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol 55:663–677PubMedCrossRefGoogle Scholar
  31. Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TF et al (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324:1068–1071PubMedGoogle Scholar
  32. Ré DA, Dezar CA, Chan RL, Baldwin IT, Bonaventure G (2011) Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, bezylacetone emission from flowers and the timing of bolting and flower transitions. J Exp Bot 62:155–166PubMedCrossRefGoogle Scholar
  33. Reyes D, Rodríguez D, González-García MP, Lorenzo O, Nicolás G, García-Martínez JL, Nicolás C (2006) Overexpression of a protein phosphatase 2C from beech seeds in Arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis. Plant Physiol 141:1414–1424PubMedCrossRefGoogle Scholar
  34. Robert N, Merlot S, N′Guyen V, Boisson-Dernier A, Schroeder J (2006) A hypermorphic mutation in the protein phosphatases 2C HAB1 strongly affects ABA signalling in Arabidopsis. FEBS Lett 580:4691–4696PubMedCrossRefGoogle Scholar
  35. Rubio S, Rodrigues A, Saez A, Dizon MB, Galle A, Kim T-H, Santiago J, Flexas J, Schroeder JI, Rodríguez PL (2009) Triple loss of function of protein phosphatases type 2C leads to partial constitutive response to endogenous abscisic acid. Plant Physiol 150:1354–1355CrossRefGoogle Scholar
  36. Saez A, Apostolova N, González-Guzmán M, González-García MP, Nicolás C, Lorenzo O, Rodríguez PL (2004) Gain-of-function and loss-of-function phenotypes of the protein phosphatases 2C HAB1 reveal its role as a negative regulator of abscisic acid signaling. Plant J 37:354–369PubMedCrossRefGoogle Scholar
  37. Santiago J, Rodrigues A, Saez A, Rubio S, Antoni R, Dupeux F, Park S-Y, Márquez JA, Cutler SR, Rodríguez PL (2009) Modulation of drought resistance by the abscisic acid-receptor PYL5 through inhibition of clade A PP2Cs. Plant J 60:575–588PubMedCrossRefGoogle Scholar
  38. Schweighofer A, Hirt H, Meskiene I (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci 9:236–243PubMedCrossRefGoogle Scholar
  39. Sessa G, Morelli G, Ruberti I (1993) The ATHB-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO J 12:3507–3517PubMedGoogle Scholar
  40. Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417PubMedCrossRefGoogle Scholar
  41. Söderman E, Mattsson J, Engström P (1996) The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. Plant J 10:375–381PubMedCrossRefGoogle Scholar
  42. Son O, Hur Y-S, Kim Y-K, Lee H-J, Kim S, Kim M-R, Nam KH, Lee M-S, Kim B-Y, Park J et al (2010) ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene. Plant Cell Physiol 51:1537–1547PubMedCrossRefGoogle Scholar
  43. Umezawa T, Yoshida R, Maruyama K, Yamaguchi-Shinozaki K, Shinozaki K (2004) SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc Natl Acad Sci 101:17306–17311PubMedCrossRefGoogle Scholar
  44. Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K (2009) Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci 106:17588–17593PubMedCrossRefGoogle Scholar
  45. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):research 0034.1–0034.11Google Scholar
  46. Xiong L, Shumaker KS, Zhu J-K (2002) Cell signaling during cold, drought and salt stress. Plant Cell 14(Suppl.):S165–S183PubMedGoogle Scholar
  47. Yoshida T, Nishimura M, Kitahata N, Kuromori T, Ito T, Asami T, Shinozaki K, Hirayama T (2006) ABA-Hypersensitive Germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs. Plant Physiol 140:115–126PubMedCrossRefGoogle Scholar
  48. Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEINVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Ana Elisa Valdés
    • 1
    • 2
  • Elin Övernäs
    • 1
  • Henrik Johansson
    • 1
  • Alvaro Rada-Iglesias
    • 3
  • Peter Engström
    • 1
    • 2
  1. 1.Physiological Botany, Uppsala BioCenterUppsala UniversityUppsalaSweden
  2. 2.Linnean Center for Plant BiologyUppsalaSweden
  3. 3.Department of Chemical and Systems BiologyStanford University School of MedicineStanfordUSA

Personalised recommendations