Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 132, Issue 2, pp 279–294 | Cite as

Functional analysis of different promoter haplotypes of the coffee (Coffea canephora) CcDREB1D gene through genetic transformation of Nicotiana tabacum

  • Sinara Oliveira de Aquino
  • Fernanda de Araújo Carneiro
  • Erica Cristina Silva Rêgo
  • Gabriel Sergio Costa Alves
  • Alan Carvalho Andrade
  • Pierre Marraccini
Original Article


Previous results showed that the three promoter haplotypes (HP15, HP16 and HP17) of the CcDREB1D gene (encoding the dehydration responsive element binding transcription factor) found in the drought-tolerant (HP15/HP16) and drought-sensitive (HP15/HP17) clones of Coffea canephora, diverged by several single nucleotide polymorphisms and insertions/deletions. In order to compare the activities and regulation of these haplotypes in response to abiotic stresses, these sequences were cloned in front of the uidA and analyzed in transgenic tobacco (Nicotiana tabacum) for their ability to regulate the expression of this reporter gene by monitoring GUS histochemical activity under drought (mimicked by dehydration), heat shock and cold treatments. Under unstressed condition, GUS staining was mainly observed in leaf and root vascular tissues of young tobacco plants transformed by the longest sequences of CcDREB1D promoter haplotypes. These GUS activities were not observed in the same tissues of older plants as well as in plants transformed by shorter proximal regions, suggesting a developmentally-regulated activity of CcDREB1D promoters in tobacco and the existence of cis-regulatory elements essential for their regulation in distal regions. Under dehydration and heat shock conditions, GUS staining detected in leaf midribs and secondary veins of pHP17L-transformed plants was correlated with up-regulated expression of uidA reporter gene while no GUS activities were observed in pHP16L-transformed plants. However, all CcDREB1D promoter haplotypes were positively regulated by cold stress in transgenic tobacco. These results showed that these coffee promoters were recognized by the tobacco transcriptional machinery but were regulated in different manners in response to abiotic stress.


Abiotic stress Coffee DREB1D Promoter haplotypes Tobacco 



Abscisic acid


Dehydration responsive element binding transcription factor





This work was carried out as part of the Brazilian Agricultural Research Corporation (Embrapa, Brazil)—Centre for International Cooperation in Agricultural Research for Development (CIRAD) Scientific Cooperation Project “Genetic determinism of drought tolerance in coffee”. PM acknowledges UMR-AGAP ( from CIRAD for financial support. ACA acknowledges financial support from the Brazilian Coffee R&D Consortium, Brazilian Innovation Agency (FINEP) and INCT-café (Brazilian National Council for Scientific and Technological Development). SOA and FAC received doctoral fellowships from Coordination for the Improvement of Higher Education Personnel (CAPES), and GSCA a fellowship in connection with the CAPES/Cofecub Project 407-2012 developed between the University of Lavras (Brazil) and Montpellier SupAgro (France). We are also grateful to Peter Biggins (CIRAD) for its critical reading of the manuscript.

Author contributions

SOA performed tobacco genetic transformation, DH/HS/CS treatments of transformed tobacco plants, histochemical GUS assays, microscopy analyses and RT-qPCR experiments with the technical help of FAC and RECS. GSCA performed vector constructions. SOA, ACA and PM designed the study, drew up the experimental design and its execution. PM drafted the manuscript. All the authors read and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2017_1328_MOESM1_ESM.tif (66 kb)
Supplementary Figure 1— Schematic representation of CcDREB1D promoter constructions. (TIF 66 KB)


  1. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alves GSC (2015) Characterization of a candidate gene for drought tolerance in Coffea: the CcDREB1D gene, in contrasting genotypes of Coffea canephora and related species. PhD Thesis Montpellier SupAgro - France / Federal University of Lavras (UFLA), BrazilGoogle Scholar
  3. Alves GSC, Torres LF, Déchamp E, Breitler J-C, Joët T, Gatineau F, Andrade AC, Bertrand B, Marraccini P, Etienne H (2017) Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit. J Exp Bot 68:3017–3031. CrossRefPubMedGoogle Scholar
  4. Assad ED, Pinto HS, Zullo J Jr, Ávila AMH (2004) Impacto das mudanças climáticas no zoneamento agroclimático do café no Brasil. Pesqui Agropecu Bras 39:1057–1064. CrossRefGoogle Scholar
  5. Benfey PN, Chua NH (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966. CrossRefPubMedGoogle Scholar
  6. Bhattacharjee P, Das R, Mandal A, Kundu P (2017) Functional characterization of tomato membrane-bound NAC transcription factors. Plant Mol Biol 93:511–532. CrossRefPubMedGoogle Scholar
  7. Bunn C, Läderach P, Pérez Jimenez JG, Montagnon C, Schilling T (2015) Multiclass classification of agro-ecological zones for Arabica coffee: an improved understanding of the impacts of climate change. PLoS ONE 10:e0140490. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen M, Xu Z, Xia L, Li L, Cheng X, Dong J, Wang Q, Ma Y (2009) Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.). J Exp Bot 60:121–135. CrossRefPubMedGoogle Scholar
  9. Chen H, Je J, Song C, Hwang JE, Lim CO (2012) A proximal promoter region of Arabidopsis DREB2C confers tissue-specific expression under heat stress. J Integr Plant Biol 54:640–651. CrossRefPubMedGoogle Scholar
  10. Chu Y, Huang Q, Zhang B, Ding C, Su X (2014) Expression and molecular evolution of two DREB1 genes in black poplar (Populus nigra). PLoS ONE 9:e98334. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cominelli E, Galbiati M, Albertini A, Fornara F, Conti L, Coupland G, Tonelli C (2011) DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter. BMC Plant Biol 11:162. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Costa TS (2014) Análise do perfil transcriptômico e proteômico de raízes de diferentes clones de Coffea canephora em condições de déficit hídrico. PhD dissertation, Federal University of Lavras, BrazilGoogle Scholar
  13. Cotta MG, Barros LMG, de Almeida JD, de Lamotte F, Barbosa EA, Vieira NG, Alves GSC, Vinecky F, Andrade AC, Marraccini P (2014) Lipid transfer proteins in coffee: isolation of Coffea orthologs, Coffea arabica homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants. Plant Mol Biol 85:11–31. PubMedGoogle Scholar
  14. Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38:366–379. CrossRefPubMedGoogle Scholar
  15. DaMatta FM, Ramalho JC (2006) Impact of drought and temperature stress on coffee physiology and production: a review. Braz J Plant Physiol 18:55–81. CrossRefGoogle Scholar
  16. Davis AP, Gole TW, Baena S, Moat J (2012) The impact of climate change on indigenous Arabica coffee (Coffea arabica): predicting future trends and identifying priorities. PLoS ONE 7:e47981. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Ding Z, Li S, An X, Liu X, Qin H, Wang D (2009) Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana. J Genet Genomics 36:17–29. CrossRefPubMedGoogle Scholar
  18. Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763. CrossRefPubMedGoogle Scholar
  19. Fang ZW, Xu XY, Gao JF, Wang PK, Liu ZX, Feng BL (2015) Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum). Genet Mol Res 14:7990–8000. CrossRefPubMedGoogle Scholar
  20. Freire LP, Marraccini P, Rodrigues GC, Andrade AC (2013) Análise da expressão do gene da manose 6 fosfato redutase em cafeeiros submetidos ao déficit hídrico no campo. Coffee Sci 8:17–23Google Scholar
  21. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9:436–442. CrossRefPubMedGoogle Scholar
  22. Gutha LR, Reddy AR (2008) Rice DREB1B promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance. Plant Mol Biol 68:533–555. CrossRefPubMedGoogle Scholar
  23. Guttikonda SK, Valliyodan B, Neelakandan AK, Tran LS, Kumar R, Quach TN, Voothuluru P, Gutierrez-Gonzalez JJ, Aldrich DL, Pallardy SG, Sharp RE, Ho TH, Nguyen HT (2014) Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean. Mol Biol Rep 41:7995–8008. CrossRefPubMedGoogle Scholar
  24. Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol 130:639–648. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jaramillo J, Muchugu E, Vega FE, Davis A, Borgemeister C, Chabi-Olaye A (2011) Some like it hot: the influence and implications of climate change on coffee berry borer (Hypothenemus hampei) and coffee production in east Africa. PLoS ONE 6:e24528. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lashermes P, Andrade AC, Etienne H (2008) Genomics of coffee, one of the world’s largest traded commodities. In: Moore H, Ming R (eds) Genomics of tropical crop plants. Springer, New York, pp 203–226CrossRefGoogle Scholar
  27. Lata C, Prasad M (2011) Role of DREBs in regulation of abiotic stress responses in plants. J Exp Bot 62:4731–4748. CrossRefPubMedGoogle Scholar
  28. Li D, Zhang Y, Hu X, Shen X, Ma L, Su Z, Wang T, Dong J (2011) Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses. BMC Plant Biol 11:109. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lima EA, Carneiro FA, Costa TS, Rêgo ECS, Jorge A Jr, Furlanetto C, Marraccini P, Carneiro RMDG, Andrade AC (2014) Molecular characterization of resistance responses of C. canephora ‘Clone 14’ upon infection by Meloidogyne paranaensis. J Nematol 46:194Google Scholar
  30. Lima EA, Furlanetto C, Nicole M, Gomes ACMM, Almeida MRA, Jorge-Júnior A, Correa VR, Salgado SM, Ferrão MAG, Carneiro RMDG (2015) The multi-resistant reaction of drought-tolerant coffee ‘Conilon clone 14’ to Meloidogyne spp. and late hypersensitive-like response in Coffea canephora. Phytopathology 105:805–814. CrossRefPubMedGoogle Scholar
  31. Lindlöf A, Bräutigam M, Chawade A, Olsson O, Olsson B (2009) In silico analysis of promoter regions from cold-induced genes in rice (Oryza sativa L.) and Arabidopsis thaliana reveals the importance of combinatorial control. Bioinformatics 25:1345–1348. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Mandal A, Sarkar D, Kundu S, Kundu P (2015) Mechanism of regulation of tomato TRN1 gene expression in late infection with tomato leaf curl New Delhi virus (ToLCNDV). Plant Sci 241:221–237. CrossRefPubMedGoogle Scholar
  33. Mao D, Chen C (2012) Colinearity and similar expression pattern of rice DREB1s reveal their functional conservation in the cold-responsive pathway. PLoS ONE 7:e47275. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Marraccini P, Deshayes A, Pétiard V, Rogers WJ (1999) Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants. Plant Physiol Biochem 37:273–282. CrossRefGoogle Scholar
  35. Marraccini P, Courjault C, Caillet V, Lausanne F, Lepage B, Rogers WJ, Deshayes A (2003) Rubisco small subunit of Coffea arabica: cDNA sequence, gene cloning and promoter analysis in transgenic tobacco plants. Plant Physiol Biochem 41:17–25. CrossRefGoogle Scholar
  36. Marraccini P, Freire LP, Alves GSC., Vieira NG, Vinecky F, Elbelt S, Ramos HJO, Montagnon C, Vieira LGE, Leroy T, Pot D, Silva VA, Rodrigues GC, Andrade AC (2011) RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress. BMC Plant Biol 11:85. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Marraccini P, Vinecky F, Alves GSC, Ramos HJO, Elbelt S, Vieira NG, Carneiro FA, Sujii PS, Alekcevetch JC, Silva VA, DaMatta FM, Ferrão MAG, Leroy T, Pot D, Vieira LGE, da Silva FR, Andrade AC (2012) Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora. J Exp Bot 63:4191–4212. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Mofatto LS, Carneiro FA, Vieira NG, Duarte KE, Vidal RO, Alekcevetch JC, Cotta MG, Verdeil JL, Lapeyre-Montes F, Lartaud M, Leroy T, De Bellis F, Pot D, Rodrigues GC, Carazzolle MF, Pereira GAG, Andrade AC, Marraccini P (2016) Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars. BMC Plant Biol 16:94. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  40. Nair NR, Chidambareswaren M, Manjula S (2014) Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum. Mol Biotechnol 56:849–862. CrossRefPubMedGoogle Scholar
  41. Osakabe Y, Osakabe K, Shinozaki K, Tran LSP (2014) Response of plants to water stress. Front Plant Sci 5:86. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Peach C, Velten T (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60. CrossRefPubMedGoogle Scholar
  43. Plesch G, Ehrhardt T, Mueller-Roeber B (2001) Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression. Plant J 28:455–464. CrossRefPubMedGoogle Scholar
  44. Prado K, Boursiac Y, Tournaire-Roux C, Monneuse JM, Postaire O, Da Ines O, Schäffner AR, Hem S, Santoni V, Maurel C (2013) Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins. Plant Cell 25:1029–1039. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Quan W, Liu X, Wang H, Chan Z (2016) Physiological and transcriptional responses of contrasting alfalfa (Medicago sativa L.) varieties to salt stress. Plant Cell Tissue Organ Cult 126:105–115. CrossRefGoogle Scholar
  46. Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66. CrossRefPubMedGoogle Scholar
  47. Robert-Seilaniantz A, Grant M, Jones JD (2011) Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev Phytopathol 49:317–343. CrossRefPubMedGoogle Scholar
  48. Santino A, Taurino M, De Domenico S, Bonsegna S, Poltronieri P, Pastor V, Flors V (2013) Jasmonate signaling in plant development and defense response to multiple (a)biotic stresses. Plant Cell Rep 32:1085–1098. CrossRefPubMedGoogle Scholar
  49. Shinozaki K, Yamaguchi-Shinozaki (2007) Gene networks involved in drought stress response and tolerance. J Exp Bot 58:221–227. CrossRefPubMedGoogle Scholar
  50. Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J 33:259–270. CrossRefPubMedGoogle Scholar
  51. Tang L, Cai H, Zhai H, Luo X, Wang Z, Cui L, Bai X (2014) Overexpression of Glycine soja WRKY20 enhances both drought and salt tolerance in transgenic alfalfa (Medicago sativa L.). Plant Cell Tissue Organ Cult 18:77–86. CrossRefGoogle Scholar
  52. Terzaghi WB, Cashmore AR (1995) Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol 46:445–474. CrossRefGoogle Scholar
  53. Thioune E-H, McCarthy J, Gallagher T, Osborne B (2017) A humidity shock leads to rapid, temperature dependent changes in coffee leaf physiology and gene expression. Tree Physiol 37:367–379. PubMedGoogle Scholar
  54. Tian Q, Chen J, Wang D, Wang H-L, Liu C, Wang S, Xia X, Yin W (2017) Overexpression of a Populus euphratica CBF4 gene in poplar confers tolerance to multiple stresses. Plant Cell Tissue Organ Cult 128:391–407. CrossRefGoogle Scholar
  55. Torres LF (2017) Expression of the CcDREB1D promoter in Coffea arabica: functional genomics and transcriptome analysis. PhD dissertation, Federal University of Lavras, BrazilGoogle Scholar
  56. van der Vossen H, Bertrand B, Charrier A (2015) Next generation variety development for sustainable production of arabica coffee (Coffea arabica L.): a review. Euphytica 204:243–256. CrossRefGoogle Scholar
  57. Vieira NG, Carneiro FA, Sujii PS, Alekcevetch JC, Freire LP, Vinecky F, Elbelt S, Silva VA, DaMatta FM, Ferrão MAG, Marraccini P, Andrade AC (2013) Different molecular mechanisms account for drought tolerance in Coffea canephora var. Conilon. Trop Plant Biol 6:181–190. CrossRefGoogle Scholar
  58. Vinecky F, da Silva FR, Andrade AC (2012) Análise in silico das bibliotecas de cDNA SH2 e SH3 para a identificação de genes responsivos à seca em cafeeiro. Coffee Sci 7:1–19Google Scholar
  59. Wagner GJ, Wang E, Shepherd RW (2004) New approaches for studying and exploiting an old protuberance, the plant trichome. Ann Bot 93:3–11. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Wang Q, Guan Y, Wu Y, Chen H, Chen F, Chu C (2008) Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice. Plant Mol Biol 67:589–602. CrossRefPubMedGoogle Scholar
  61. Wenzel CL, Hester Q, Mattsson J (2008) Identification of genes expressed in vascular tissues using NPA-induced vascular overgrowth in Arabidopsis. Plant Cell Physiol 49:457–468. CrossRefPubMedGoogle Scholar
  62. Werker E (2000) Trichome diversity and development. Adv Bot Res 31:1–35. CrossRefGoogle Scholar
  63. Xia Z, Su X, Liu J, Wang M (2013) The RING-H2 finger gene 1 (RHF1) encodes an E3 ubiquitin ligase and participates in drought stress response in Nicotiana tabacum. Genetica 141:11–21. CrossRefPubMedGoogle Scholar
  64. Xiao H, Siddiqua M, Braybrook S, Nassuth A (2006) Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid. Plant Cell Environ 29:1410–1421. CrossRefPubMedGoogle Scholar
  65. Xiao H, Tattersall EAR, Siddiqua MK, Cramer GR, Nassuth A (2008) CBF4 is a unique member of the CBF transcription factor family of Vitis vinifera and Vitis riparia. Plant Cell Environ 31:1–10. PubMedGoogle Scholar
  66. Zandkarimi H, Ebadi A, Salami SA, Alizade H, Baisakh N (2015) Analyzing the expression profile of AREB/ABF and DREB/CBF genes under drought and salinity stresses in grape (Vitis vinifera L.). PLoS ONE 10:e0134288. CrossRefPubMedPubMedCentralGoogle Scholar
  67. Zarka DG, Vogel JT, Cook D, Thomashow MF (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 133:910–918. CrossRefPubMedPubMedCentralGoogle Scholar
  68. Zhang X, Fowler SG, Cheng H, Lou Y, Rhee SY, Stockinger EJ, Thomashow MF (2004) Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant J 39:905–919. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Sinara Oliveira de Aquino
    • 1
    • 2
  • Fernanda de Araújo Carneiro
    • 1
  • Erica Cristina Silva Rêgo
    • 2
  • Gabriel Sergio Costa Alves
    • 1
    • 2
  • Alan Carvalho Andrade
    • 2
  • Pierre Marraccini
    • 2
    • 3
    • 4
  1. 1.Federal University of Lavras UFLALavrasBrazil
  2. 2.EMBRAPA Genetic Resources and BiotechnologyBrasiliaBrazil
  3. 3.CIRAD, UMR AGAPMontpellierFrance
  4. 4.CIRAD, UMR IPME (University Montpellier, CIRAD, IRD, Montpellier), Agricultural Genetics Institute, LMI RICE2HanoiVietnam

Personalised recommendations