Plant Molecular Biology

, Volume 80, Issue 6, pp 571–585 | Cite as

Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice

  • Shuxin Zhang
  • Imran Haider
  • Wouter Kohlen
  • Li Jiang
  • Harro Bouwmeester
  • Annemarie H. Meijer
  • Henriette Schluepmann
  • Chun-Ming LiuEmail author
  • Pieter B. F. OuwerkerkEmail author


Oshox22 belongs to the homeodomain-leucine zipper (HD-Zip) family I of transcription factors, most of which have unknown functions. Here we show that the expression of Oshox22 is strongly induced by salt stress, abscisic acid (ABA), and polyethylene glycol treatment (PEG), and weakly by cold stress. Trans-activation assays in yeast and transient expression analyses in rice protoplasts demonstrated that Oshox22 is able to bind the CAAT(G/C)ATTG element and acts as a transcriptional activator that requires both the HD and Zip domains. Rice plants homozygous for a T-DNA insertion in the promoter region of Oshox22 showed reduced Oshox22 expression and ABA content, decreased sensitivity to ABA, and enhanced tolerance to drought and salt stresses at the seedling stage. In contrast, transgenic rice over-expressing Oshox22 showed increased sensitivity to ABA, increased ABA content, and decreased drought and salt tolerances. Based on these results, we conclude that Oshox22 affects ABA biosynthesis and regulates drought and salt responses through ABA-mediated signal transduction pathways.


Rice Transcription factor HD-Zip Drought stress Regulation Abiotic stress 



Homeodomain-leucine zipper


Abscisic acid


Polyethylene glycol


Green fluorescent protein


Reverse transcription


Polymerase chain reaction





We acknowledge the support from projects CEDROME (INCO-CT-2005-015468) for PBFO and CML, the National Natural Science Foundation of China (30821007) and the CAS/SAFEA International Partnership Program for Creative Research Teams (20090491019) for SZ and CML, TF-STRESS (QLK3-CT-2000-00328) for AHM, RNA Seed from the Royal Netherlands Academy of Arts and Sciences (KNAW-CEP 08CDP036) for SZ, PBFO and HS, from the Higher Education Commission (HEC) Pakistan to IH and the Netherlands Organization for Scientific Research (NWO; VICI-grant to HB). We thank Francel Verstappen for his technical support at WUR in The Netherlands.

Supplementary material

11103_2012_9967_MOESM1_ESM.doc (33 kb)
Supplementary material 1 (DOC 33 kb)
11103_2012_9967_MOESM2_ESM.tif (669 kb)
Fig. S1 The copy number of the T-DNA insertion in oshox22-1. Genomic DNA was digested by EcoRI and the HPT cDNA fragment was used as probe in the Southern blot experiment (TIFF 669 kb)
11103_2012_9967_MOESM3_ESM.tif (5.9 mb)
Fig. S2 Northern blot analysis of Oshox22 expression in transgenic Oshox22 over-expression plants (middle panel). Equal loading of the RNAs was verified by ethidium bromide staining (lower panel). Upper panel: left: phenotypes of the Oshox22-OX plants; right: wild type Zhonghua 11 (ZH11) plant (TIFF 6074 kb)


  1. Agalou A, Purwantomo S, Overnäs E, Johannesson H, Zhu X, Estiati A, de Kam RJ, Engström P, Slamet-Loedin IH, Zhu Z, Wang M, Xiong L, Meijer AH, Ouwerkerk PBF (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
  2. Aso K, Kato M, Banks JA, Hasebe M (1999) Characterization of homeodomain-leucine zipper genes in the fern Ceratopteris richardii and the evolution of the homeodomain-leucine zipper gene family in vascular plants. Mol Biol Evol 16:544–552PubMedCrossRefGoogle Scholar
  3. Bray EA (2004) Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J Exp Bot 55:2331–2341PubMedCrossRefGoogle Scholar
  4. Chan RL, Gago GM, Palena CM, Gonzalez DH (1998) Homeoboxes in plant development. Biochim Biophys Acta 1442:1–19PubMedCrossRefGoogle Scholar
  5. Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice. Mol Plant Pathol 7:417–427PubMedCrossRefGoogle Scholar
  6. Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330PubMedCrossRefGoogle Scholar
  7. Cooper B, Clarke JD, Budworth P, Kreps J, Hutchison D, Park S, Guimil S, Dunn M, Luginbuhl P, Ellero C, Goff SA, Glazebrook J (2003) A network of rice genes associated with stress response and seed development. Proc Natl Acad Sci USA 100:4945–4950PubMedCrossRefGoogle Scholar
  8. Deng X, Phillips J, Meijer AH, Salamini F, Bartels D (2002) Characterization of five novel dehydration-responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum. Plant Mol Biol 49:601–610PubMedCrossRefGoogle Scholar
  9. Deng X, Phillips J, Bräutigam A, Engström P, Johannesson H, Ouwerkerk PBF, Ruberti I, Salinas J, Vera P, Iannacone R, Meijer AH, Bartels D (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
  10. Derelle R, Lopez P, Le Guyader H, Manuel M (2007) Homeodomain proteins belong to the ancestral molecular toolkit of eukaryotes. Evol Dev 9:212–219PubMedCrossRefGoogle Scholar
  11. Dezar CA, Gago GM, Gonzalez DH, Chan RL (2005a) HAHB-4, a sunflower homeobox-leucine zipper gene, confers drought tolerance to Arabidopsis thaliana plants. Transgenic Res 14:429–440PubMedCrossRefGoogle Scholar
  12. Dezar CA, Fedrigo GV, Chan RL (2005b) The promoter of the sunflower HD-Zip protein gene HAHB4 directs tissue-specific expression and is inducible by water stress, high salt concentrations and ABA. Plant Sci 169:447–459CrossRefGoogle Scholar
  13. Frank W, Phillips J, Salamini F, Bartels D (1998) Two dehydration inducible transcripts from the resurrection plant Craterostigma plantagineum encode interacting homeodomain-leucine zipper proteins. Plant J 15:413–421PubMedCrossRefGoogle Scholar
  14. Gago GM, Almoguera C, Jordano J, Gonzales DH, Chan RL (2002) Hahb-4, a homeobox-leucine zipper gene potentially involved in abscisic acid-dependent responses to water stress in sunflower. Plant, Cell Environ 25:633–640CrossRefGoogle Scholar
  15. 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
  16. Himmelbach A, Hoffmann T, Leube M, Höhner 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
  17. Hjellström M, Olsson ASB, Engström P, Söderman EM (2003) Constitutive expression of the water deficit-inducible homeobox gene Athb7 in transgenic Arabidopsis causes a suppression of stem elongation growth. Plant, Cell Environ 26:1127–1136CrossRefGoogle Scholar
  18. Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong LZ (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA 103:12987–12992PubMedCrossRefGoogle Scholar
  19. Huang XY, Chao DY, Gao JP, Zhu MZ, Shi M, Lin HX (2009) A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev 23:1805–1817PubMedCrossRefGoogle Scholar
  20. Huang G-T, Ma S-L, Bai L-P, Zhang L, Ma H, Jia P, Liu J, Zhong M, Guo Z-F (2012) Signal transduction during cold, salt, and drought stresses in plants. Mol Biol Rep 39:969–987PubMedCrossRefGoogle Scholar
  21. Jain M, Tyagi AK, Khurana JP (2008) Genome-wide identification, classification, evolutionary expansion and expression analyses of homeobox genes in rice. FEBS J 275:2845–2861PubMedCrossRefGoogle Scholar
  22. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  23. Jeong DH, An S, Park S, Kang HG, Park GG, Kim SR, Sim J, Kim YO, Kim MK, Kim SR, Kim J, Shin M, Jung M, An G (2006) Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J 45:123–132PubMedCrossRefGoogle Scholar
  24. Ji X, Dong B, Shiran B, Talbot MJ, Edlington JE, Hughes T, White RG, Gubler F, Dolferus R (2011) Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals. Plant Physiol 156:647–662PubMedCrossRefGoogle Scholar
  25. 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
  26. Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol 45:346–350PubMedCrossRefGoogle Scholar
  27. Lee YH, Oh HS, Cheon CI, Hwang IT, Kim YJ, Chun JY (2001) Structure and expression of the Arabidopsis thaliana homeobox gene Athb-12. Biochem Biophys Res Commun 284:133–141PubMedCrossRefGoogle Scholar
  28. Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49:199–222PubMedCrossRefGoogle Scholar
  29. Leung J, Merlot S, Giraudat J (1997) The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant cell 9:759–771PubMedGoogle Scholar
  30. López-Ráez JA, Charnikhova T, Gómez-Roldán V, Matusova R, Kohlen W, De Vos R, Verstappen F, Puech-Pages V, Bécard G, Mulder P, Bouwmeester H (2008) Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation. New Phytol 178:863–874PubMedCrossRefGoogle Scholar
  31. López-Ráez JA, Kohlen W, Charnikhova T, Mulder P, Undas AK, Sergeant MJ, Verstappen F, Bugg TDH, Thompson AJ, Ruyter-Spira C (2010) Does abscisic acid affect strigolactone biosynthesis? New Phytol 187:343–354PubMedCrossRefGoogle Scholar
  32. Manavella PA, Arce AL, Dezar CA, Bitton F, Renou FP, Crespi M, Chan RL (2006) Cross-talk between ethylene and drought signaling pathways is mediated by the sunflower Hahb-4 transcription factor. Plant J 48:125–137PubMedCrossRefGoogle Scholar
  33. Meijer AH, Scarpella E, van Dijk EL, Qin L, Taal AJ, Rueb S, Harrington SE, McCouch SR, Schilperoort RA, Hoge JHC (1997) Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice. Plant J 11:263–276PubMedCrossRefGoogle Scholar
  34. Meijer AH, Ouwerkerk PBF, Hoge JHC (1998) Vectors for transcription factor isolation and target gene identification by means of genetic selection in yeast. Yeast 14:1407–1416PubMedCrossRefGoogle Scholar
  35. Meijer AH, Schouten J, Ouwerkerk PBF, Hoge JHC (2000a) Yeast as versatile tool in transcription factor research. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual (chap E3, 2nd edn, suppl IV). Kluwer, Dordrecht, pp 1–28Google Scholar
  36. Meijer AH, de Kam RJ, d’Ehrfurth I, Shen W, Hoge JHC (2000b) HD-Zip proteins of families I and II from rice: interactions and functional properties. Mol Gen Genet 263:12–21PubMedCrossRefGoogle Scholar
  37. Melcher K, Zhou XE, Xu HE (2010) Thirsty plants and beyond: structural mechanisms of abscisic acid perception and signaling. Curr Opin Struct Biol 20:722–729PubMedCrossRefGoogle Scholar
  38. Memelink J, Swords KMM, Staehelin LA, Hoge JHC (1994) Southern, northern and western blot analysis. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp F1–F23Google Scholar
  39. Morelli G, Ruberti I (2002) Light and shade in the photocontrol of Arabidopsis growth. Trends Plant Sci 7:399–404PubMedCrossRefGoogle Scholar
  40. Mukherjee K, Brocchieri L, Burglin TR (2009) A comprehensive classification and revolutionary analysis of plant homeobox genes. Mol Biol Evol 26:2775–2794PubMedCrossRefGoogle Scholar
  41. Ohgishi M, Oka A, Morelli G, Ruberti I, Aoyama T (2001) Negative autoregulation of the Arabidopsis homeobox gene Athb-2. Plant J 25:389–398PubMedCrossRefGoogle Scholar
  42. 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
  43. Osnato M, Stile MR, Wang Y, Meynard D, Curiale S, Guiderdoni E, Liu Y, Horner DS, Ouwerkerk PBF, Pozzi C, Müller KI, Salamini F, Rossini L (2010) Cross talk between the KNOX and ethylene pathways is mediated by intron-binding transcription factors in barley. Plant Physiol 154:1616–1632PubMedCrossRefGoogle Scholar
  44. Ouwerkerk PBF, Meijer AH (2001) Yeast one-hybrid screening for DNA-protein interactions. Curr Prot Mol Biol 12.12.1–12.12.22Google Scholar
  45. Ouwerkerk PBF, Meijer AH (2011) Yeast one-hybrid screens for detection of transcription factor DNA interactions. Methods Mol Biol 678:211–227PubMedCrossRefGoogle Scholar
  46. Palena CM, Gonzalez DH, Chan RL (1999) A monomer-dimer equilibrium modulates the interaction of the sunflower homeodomain leucine-zipper protein HaHB-4 with DNA. Biochem J 341:81–87PubMedCrossRefGoogle Scholar
  47. Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol 133:1755–1767PubMedCrossRefGoogle Scholar
  48. Ruberti I, Sessa G, Lucchetti S, Morelli G (1991) A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. EMBO J 10:1787–1791PubMedGoogle Scholar
  49. Rueb S, Leneman M, Schilperoort RA, Hensgens LAM (1994) Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.). Plant Cell Tiss Org Cult 36:259–264CrossRefGoogle Scholar
  50. Sakakibara K, Nishiyama T, Kato M, Hasebe M (2001) Isolation of homeodomain-leucine zipper genes from the moss Physcomitrella patens and the evolution of homeodomain-leucine zipper genes in land plants. Mol Biol Evol 18:491–502PubMedCrossRefGoogle Scholar
  51. Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18:1292–1309PubMedCrossRefGoogle Scholar
  52. Sawa S, Ohgishi M, Goda H, Higuchi K, Shimada Y, Yoshida S, Koshiba T (2002) The HAT2 gene, a member of the HD-Zip gene family, isolated as an auxin inducible gene by DNA microarray screening, affects auxin response in Arabidopsis. Plant J 32:1011–1022PubMedCrossRefGoogle Scholar
  53. Scarpella E, Rueb S, Boot KJM, Hoge JHC, Meijer AH (2000) A role for the rice homeobox gene Oshox1 in provascular cell fate commitment. Development 127:3655–3669PubMedGoogle Scholar
  54. Schena M, Davis RW (1992) HD-Zip protein members of Arabidopsis homeodomain protein superfamily. Proc Natl Acad Sci USA 89:3894–3898PubMedCrossRefGoogle Scholar
  55. Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K, Carnici P, Hayashizaki Y, Shinozaki K (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell 13:61–72PubMedGoogle Scholar
  56. Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold, and high-salinity stresses using a full-length cDNA microarray. Plant J 31:279–292PubMedCrossRefGoogle Scholar
  57. 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
  58. Sessa G, Morelli G, Ruberti I (1997) DNA-binding specificity of the homeodomain leucine zipper domain. J Mol Biol 274:303–309PubMedCrossRefGoogle Scholar
  59. Shan H, Chen S, Jiang J, Chen F, Chen Y, Gu C, Li P, Song A, Zhu X Gao H Zhou G, Li T, Yang X (2011, in press) Heterologous expression of the Chrysanthemum R2R3-MYB transcription factor CmMYB2 enhances drought and salinity tolerance, increases hypersensitivity to ABA and delays flowering in Arabidopsis thaliana. Mol Biotech 51:160–173Google Scholar
  60. 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
  61. Söderman E, Hjellström M, Fahleson J, Engström P (1999) The HD-Zip gene Athb6 in Arabidopsis is expressed in developing leaves, roots and carpels and up-regulated by water deficit conditions. Plant Mol Biol 40:1073–1083PubMedCrossRefGoogle Scholar
  62. Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T, Morelli G, Ruberti I (1999) Shade avoidance responses are mediated by the Athb-2 HD-Zip protein, a negative regulator of gene expression. Development 126:4235–4245PubMedGoogle Scholar
  63. 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 USA 106:17588–17593PubMedCrossRefGoogle Scholar
  64. Wang Y, Henriksson E, Söderman E, Henriksson NK, Sundberg E, Engström P (2003) The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis. Dev Biol 264:228–239PubMedCrossRefGoogle Scholar
  65. Xiang Y, Tang N, Du H, Ye H, Xiong LZ (2008) Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice. Plant Physiol 148:1938–1952PubMedCrossRefGoogle Scholar
  66. Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803PubMedCrossRefGoogle Scholar
  67. Zhang J, Li C, Wu C, Xiong L, Chen G, Zhang Q, Wang S (2006) RMD: a rice mutant database for functional analysis of the rice genome. Nucl Acid Res 34:D745–D748CrossRefGoogle Scholar
  68. Zou MJ, Guan YC, Ren HB, Zhang F, Chen F (2007) Characterization of alternative splicing products of bZIP transcription factors OsABI5. Biochem Biophys Res Commun 360:307–313PubMedCrossRefGoogle Scholar
  69. Zou MJ, Guan YC, Ren HB, Zhang F, Chen F (2008) A bZIP transcription factor, OsABI5, is involved in rice fertility and stress tolerance. Plant Mol Biol 66:675–683PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Shuxin Zhang
    • 1
    • 2
    • 3
  • Imran Haider
    • 4
  • Wouter Kohlen
    • 4
  • Li Jiang
    • 1
    • 2
  • Harro Bouwmeester
    • 4
  • Annemarie H. Meijer
    • 3
  • Henriette Schluepmann
    • 5
  • Chun-Ming Liu
    • 1
    Email author
  • Pieter B. F. Ouwerkerk
    • 1
    • 3
    Email author
  1. 1.Key Laboratory of Plant Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingChina
  2. 2.Graduate School of Chinese Academy of SciencesBeijingChina
  3. 3.Institute of BiologyLeiden UniversityLeidenThe Netherlands
  4. 4.Laboratory of Plant PhysiologyWageningen UniversityWageningenThe Netherlands
  5. 5.Department of Molecular Plant PhysiologyUtrecht UniversityUtrechtThe Netherlands

Personalised recommendations