, Volume 220, Issue 3, pp 424–433 | Cite as

Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot Dc3 gene

  • Hwa-Jee Chung
  • Hong Yong Fu
  • Terry L. ThomasEmail author
Original Article


The carrot (Daucus carota L.) lea-class gene Dc3 is expressed in developing seeds and in vegetative tissues subject to drought and treatment with exogenous abscisic acid (ABA). Cis regulatory elements involved in seed-specific expression and in response to ABA were identified in transgenic tobacco (Nicotiana tabacum L.) using β-glucuronidase (GUS) reporter gene constructs containing a series of deletion and orientation mutants of the Dc3 promoter. These experiments demonstrated that the Dc3 promoter is comprised of a proximal promoter region (PPR) and a distal promoter region (DPR). TCGTGT motifs in the DPR in combination with the PPR comprise a novel, bipartite ABA module in the Dc3 gene promoter. The PPR contains cis-acting elements responsible for the developmental regulation of Dc3 expression in seeds. Five similar sequence motifs with the consensus ACACgtGCa were identified in the PPR. Both DPR and PPR interact with common nuclear proteins that are present in embryos and are inducible by ABA in vegetative tissues.


Abscisic acid Cis regulatory element Daucus Seed development Transcription factor 



Abscisic acid


ABA-response element


Basic leucine zipper


Distal ABA-response region


Dc3 promoter-binding factor


Distal promoter region


Gel mobility-shift assay




Late embryogenesis abundant


Proximal promoter region



This work was financially supported in part by USDA NRICGP grants (Nos. 94373041228, 97353044552, and 013530410940).


  1. Almoguera C, Jordano J (1992) Developmental and environmental, concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs. Plant Mol Biol 19:781–792PubMedGoogle Scholar
  2. Baker SS, Wilhelm KS, Thomashow MF (1994) The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24:701–713PubMedGoogle Scholar
  3. Beaudoin N, Serizet C, Giraudat J (2000) Interactions between abscisic acid and ethylene signaling cascades. Plant Cell 12:1103–1115CrossRefPubMedGoogle Scholar
  4. Bensmihen S, Rippa S, Lambert G, Jublot D, Pautot V, Granier F, Giraudat J, Parcy F (2002) The homologous ABI5 and EEL transcription factors function antagonistically to fine-tune gene expression during late embryogenesis. Plant Cell 14:1391–1403CrossRefPubMedGoogle Scholar
  5. Block A, Dangl JL, Hahlbrock K, Schulze-Lefert P (1990) Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proc Natl Acad Sci USA 87:5387–5391PubMedGoogle Scholar
  6. Busk PK, Pagès M (1998) Regulation of abscisic acid induced transcription. Plant Mol. Biol 37:425–435CrossRefPubMedGoogle Scholar
  7. Colmenero-Flores JM, Moreno LP, Smith CE, Covarrubias AA (1999) Pvlea-18, a member of a new late-embryogenesis-abundant protein family that accumulates during water stress and in the growing regions of well- irrigated bean seedlings. Plant Physiol 120:93–104CrossRefPubMedGoogle Scholar
  8. Chern M-S, Bobb AJ, Bustos MM (1996) The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated expression. Plant Cell 8:305–321CrossRefPubMedGoogle Scholar
  9. Chung H-J (1996) Analysis of the 5′ upstream region of the carrot Dc3 gene: bipartite structure of the Dc3 promoter for embryo-specific and ABA-inducible expression. Ph.D. Dissertation, Texas A&M University, College StationGoogle Scholar
  10. Donald RGK, Cashmore AR (1990) Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1a promoter. EMBO J 9:1717–1726PubMedGoogle Scholar
  11. Dure L, Crouch M, Harada J, Ho TD, Mundy J, Quatrano R, Thomas T, Sung ZR (1989) Common amino acid sequence domains among the lea proteins of higher plants. Plant Mol Biol 12:475–486Google Scholar
  12. Espelund M, Saebøe-Larssen S, Wayne H, Galau GA, Larsen F, Jakobsen KS (1992) Late embryogenesis-abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress. Plant J 2:241–252.CrossRefPubMedGoogle Scholar
  13. Finkelstein RR, Lynch TJ (2000) The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 12:599–609CrossRefPubMedGoogle Scholar
  14. Finkelstein RR, Gampala SSL, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell [Suppl 2002] 15–45Google Scholar
  15. Gaubier P, Raynal M, Hull G, Huestis GM, Grellet F, Arenal C, Pagès M, Delseny M (1993) Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation. Mol Gen Genet 238:409–418PubMedGoogle Scholar
  16. Ghassemian M, Nambara E, Cutler S, Kawaids H, Kamiya Y, McCourt P (2000) Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. Plant Cell 12:1117–1126CrossRefPubMedGoogle Scholar
  17. Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR (1988) An evolutionarily conserved protein binding sequence upstream of a plant light regulated gene. Proc Natl Acad Sci USA 85:7089–7093PubMedGoogle Scholar
  18. Goldberg RB, de Paiva G, Yadegari R (1994) Plant embryogenesis: zygote to seed. Science 266:605–614Google Scholar
  19. Goldsbrough A, Bevan M (1991) New patterns of gene activity in plants detected using an Agrobacterium vector. Plant Mol Biol 16:263–269PubMedGoogle Scholar
  20. Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250:267–271PubMedGoogle Scholar
  21. Hobo T, Asada M, Kowyarna Y, Hattori T (1999) ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J 19:679–689CrossRefPubMedGoogle Scholar
  22. Jakoby M, Weisshaar B, geLaser W, Vincente-Carbajosa J, Yiedemann J, Kroj T, Parcy F (2002) bZIP transcription factors in Arabidopsis. Trends Plant Sci 7:106–111CrossRefPubMedGoogle Scholar
  23. 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
  24. Kerppola TK (1994) DNA bending specificity among bZIP family proteins. In: Conaway RC, Conaway JW (eds) Transcription: mechanisms and regulation. Raven Press, New York, pp 387–424Google Scholar
  25. Kim SY, Thomas TL (1998) A family of basic leucine zipper proteins binds to seed specification elements in the carrot Dc3 gene promoter. J Plant Physiol 152:607–613Google Scholar
  26. Kim SY, Chung H-J, Thomas TL (1997) Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system. Plant J 11:1237–1251CrossRefPubMedGoogle Scholar
  27. Kim SY, Ma J, Li Z, Thomas TL (2002) Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities. Plant Physiol 130:688–697CrossRefPubMedGoogle Scholar
  28. Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49:199–222CrossRefPubMedGoogle Scholar
  29. Liu Z-B, Ulmasov T, Shi X, Hagen G, Guilfoyle TJ (1994) Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell 6:645–657CrossRefPubMedGoogle Scholar
  30. Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acid-responsive sequences from the Em gene of wheat. Plant Cell 1:969–976CrossRefPubMedGoogle Scholar
  31. Mason HS, DeWald DB, Mullet JE (1993) Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. Plant Cell 5:241–251CrossRefPubMedGoogle Scholar
  32. McKendree WL, Ferl RJ (1990) Functional elements of the Arabidopsis Adh promoter include the G-box. Plant Mol Biol 19:859–862Google Scholar
  33. Michel D, Salamini F, Bartels D, Dale P, Baga M, Szalay A (1993) Analysis of a desiccation and ABA-responsive promoter from the resurrection plant Craterostigma plantagineum. Plant J 4:29–40CrossRefPubMedGoogle Scholar
  34. Mundy J, Chua N-H (1988) Abscisic acid and water-stress induce the expression of a novel rice gene. EMBO J 7:2279–2286PubMedGoogle Scholar
  35. Mundy J, Yamaguchi-Shinozaki K, Chua N-H (1990) Nuclear proteins bind conserved elements in the abscisic acid-responsive promoter of rice rab gene. Proc Natl Acad Sci USA 87:1046–1410Google Scholar
  36. Nunberg AN, Li Z, Bogue MA, Vivekananda J, Reddy AS, Thomas TL (1994) Developmental and hormonal regulation of sunflower helianthinin genes: proximal promoter sequences confer regionalized seed expression. Plant Cell 6:473–486CrossRefPubMedGoogle Scholar
  37. Pla M, Gómez J, Goday A, Pagés M (1991) Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants. Mol Gen Genet 230:394–400PubMedGoogle Scholar
  38. Pla M, Vilardell J, Guiltinan MJ, Marcotte WR, Niogret MF (1993) The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28. Plant Mol Biol 21:259–266PubMedGoogle Scholar
  39. Rogers JC, Rogers SW (1992) Definition and functional implications of gibberellin and abscisic acid cis-acting hormone response complexes. Plant Cell 4:1443–1451CrossRefPubMedGoogle Scholar
  40. Rogers JC, Lanahan MB, Rogers SW (1994) The cis-acting gibberellin response complex in high-pI α-amylase gene promoters. Plant Physiol 105:151–158CrossRefPubMedGoogle Scholar
  41. Schindler U, Beckmann H, Cashmore AR (1992) TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG. Plant Cell 4:1309–1319CrossRefPubMedGoogle Scholar
  42. Seffens WS, Almoguera C, Wilde HD, Vonder Haar RA, Thomas TL (1990) Molecular analysis of a phylogenetically conserved carrot gene: developmental and environmental regulation. Dev Genet 11:65–76PubMedGoogle Scholar
  43. Shen Q, Ho T-HD (1995) Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. Plant Cell 7:295–307CrossRefPubMedGoogle Scholar
  44. Shen Q, Zhang P, Ho T-HD (1996) Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. Plant Cell 8:1107–1119CrossRefPubMedGoogle Scholar
  45. Siddiqui NU, Chung H-J, Thomas TL, Drew MC (1998) Abscisic acid-dependent and -independent expression of carrot LEA-class gene Dc3 in transgenic tobacco seedlings. Plant Physiol 118:1181–1190CrossRefPubMedGoogle Scholar
  46. Straub PF, Shen Q, Ho T-HD (1994) Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1. Plant Mol Biol 26:617–630PubMedGoogle Scholar
  47. Suzuki M, Ketterling MG, Li QB, McCarty DR (2003) Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling. Plant Physiol 132:1664–1677CrossRefPubMedGoogle Scholar
  48. Thomas JC, Guiltinan MJ, Bustos S, Thomas T, Nessler C (1989) Carrot (Daucus carota) hypocotyl transformation using Agrobacterium tumefaciens. Plant Cell Rep 8:354–357Google Scholar
  49. Thomas TL, Chung H-J, Nunberg AN (1997) ABA signaling in plant development and growth. In: Adduci P (ed) Signal transduction in plants. Birkäuser, Basel, pp 23–43Google Scholar
  50. Velasco R, Salamini F, Bartels D (1998) Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst. Planta 204:459–471CrossRefPubMedGoogle Scholar
  51. Vilardell J, Goday A, Freire MA, Torrent M, Tartinez C, Torne JM, Pagès M (1990) Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize. Plant Mol Biol 14:423–432PubMedGoogle Scholar
  52. Vilardell J, Mundy J, Stilling B, Leroux B, Pla M, Freyssinet G, Pagès M (1991) Regulation of the maize rab17 gene promoter in transgenic heterologous systems. Plant Mol Biol 17:985–993PubMedGoogle Scholar
  53. Vivekananda J, Drew MC, Thomas TL (1992) Hormonal and environmental regulation of the carrot lea-class gene Dc3. Plant Physiol 100:576–581Google Scholar
  54. Weisshaar B, Armstrong GA, Block A, da Costa e Silva O, Hahlbrock K (1991) Light-inducible and constitutively expressed DNA-binding proteins recognizing a plant promoter element with functional relevance in light responsiveness. EMBO J 10:1777–1786PubMedGoogle Scholar
  55. Wilde HD, W.S. Nelson WS, Booij H. de Vries S, Thomas TL (1988) Gene expression programs in embryogenic carrot cultures. Planta 176:205–211Google Scholar
  56. Yao Z, Jones DH, Grose C (1992) Site-directed mutagenesis of herpes virus glycoprotein phosphorylation sites by recombination polymerase chain reaction. PCR Methods Appl 1:205–207PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Hwa-Jee Chung
    • 2
  • Hong Yong Fu
    • 3
  • Terry L. Thomas
    • 1
    Email author
  1. 1.Department of BiologyTexas A&M UniversityCollege StationUSA
  2. 2.Laboratory of Functional Genomics for Plant Secondary MetabolismEugentech Inc.DaejonKorea
  3. 3.Institute of BotanyAcademia SinicaTaipeiTaiwan, R.O.C.

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