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DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.)

Abstract

We isolated a DREB orthologue, MtDREB1C, from Medicago truncatula. Its deduced protein contains an AP2 domain of 57 amino acids. Yeast one-hybrid assay revealed that MtDREB1C specifically bound to the dehydration-responsive element (DRE) and activated the expression of HIS3 and LacZ reporter genes. In a transcriptional activation assay, coexpression of the MtDREB1C cDNA resulted in much higher (21.2 times) transactivation of the LacZ reporter gene than experiments performed without MtDREB1C. Transformation of Medicago revealed that overexpression of MtDREB1C suppressed shoot growth, and enhanced the freezing tolerance of M. truncatula. The MtDREB1C gene was transformed into China Rose (Rosa chinensis Jacq.) driven by Arabidopsis rd29A promoter. Southern-blot analysis showed that the target gene was integrated into the genome of a surviving transgenic rose plant. Northern-blot analysis illustrated that robust expression of MtDREB1C was only activated under stress conditions, and the expressed MtDREB1C mRNA reached maximum accumulation 10 h following freezing treatment. The performance of the transgenic line under freezing stress was superior to untransformed controls. This transgenic plant continued to grow, flowered under unstressed conditions, and was phenotypically normal. These facts indicate that the MtDREB1C gene, isolated from Medicago truncatula and driven by the Arabidopsis rd29A promoter, enhanced freezing tolerance in transgenic China Rose significantly without any obvious morphological or developmental abnormality.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxy acetic acid

BD:

binding domain

CaMV35S:

Cauliflower mosaic virus 35S promoter

CBF:

C-repeat/DRE binding factor

CRT:

C-repeat

DRE:

Dehydration-responsive element

DREB:

Dehydration-responsive element binding

EST:

Expressed sequence tag

EREBP:

Ethylene-responsive element binding protein

ERF:

Ethylene-responsive-element-binding factor

ICE1:

Inducer of CBF expression 1

MtDREB1C:

Medicago truncatula DREB1C

ONPG:

o-nitrophenyl β-D-galactopyranoside

rd29A:

Responsive drought 29A

SD medium:

Synthetic dextrose medium

References

  • Baker SS, Wilhelm KS, Thomashow MF (1994) The 59-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24:701–713

    Article  CAS  PubMed  Google Scholar 

  • Behnam B, Kikuchi A, Celebi-Toprak F, Yamanaka S, Kasuga M, Yamaguchi-Shinozaki K, Watanabe KN (2006) The Arabidopsis DREB1A gene driven by the stress-inducible rd29A promoter increases salt-tolerance in proportion to its copy number in tetrasomic tetraploid potato (Solanum tuberosum). Plant Biotechnol 23:169–177

    CAS  Google Scholar 

  • Behnam B, Kikuchi A, Celebi-Toprak F, Kasuga M, Yamaguchi-Shinozaki K, Watanabe KN (2007) Arabidopsis rd29A:DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Rep 26:1275–1282

    Article  CAS  PubMed  Google Scholar 

  • Bray EA (1997) Plant responses to water deficit. Trends Plant Sci 2:48–54

    Article  Google Scholar 

  • Chabaud M, Larsonneau C, Marmouget C, Huguet T (1996) Transformation of barrel medic (Medicago truncatula Gaertn) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep 15:305–310

    CAS  Google Scholar 

  • Chen JQ, Dong Y, Wang YJ, Liu Q, Zhang JS, Chen SY (2003) An AP2/EREBP-type transcription-factor gene from rice is cold-inducible and encodes a nuclear-localized protein. Theor Appl Genet 107:972–979

    Article  CAS  PubMed  Google Scholar 

  • Chen JR, Liu R, Wang HF (2006) Plant regeneration of transgenic China Rose (Rosa chinesis Jacq.) from organogenic callus. For Stud China 8:92–97

    Article  Google Scholar 

  • Chen JR, Lü JJ, Wang HF (2008) Rapid and efficient gene splicing using megaprimer-based protocol. Mol Biotechnol 40:224–230

    Article  CAS  PubMed  Google Scholar 

  • Chen JR, Lü JJ, Wang TX, Chen SY, Wang HF (2009a) Activation of a DRE-binding transcription factor from Medicago truncatula by deleting a Ser/Thr-rich region. In Vitro Cell Dev Biol -Pl 45:1–11

    Article  CAS  Google Scholar 

  • Chen JR, Xiong XY, Wang TX, Lü JJ, Chen SY, Wang HF (2009b) Rapid construction of a plant RNA interference expression vector for hairpin RNA–mediated targeting using a PCR-based method. DNA Cell Biol 28(12):605–613

    Article  CAS  PubMed  Google Scholar 

  • Chinnusamy V, Ohta M, Kanrar S, Lee B-H, HongX AgarwalM, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17:1043–1054

    Article  CAS  PubMed  Google Scholar 

  • Cook D, Dreyer D, Bonne D, Howell M, Nony E, VandenBosch K (1995) Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti. Plant Cell 7:43–55

    Article  CAS  PubMed  Google Scholar 

  • Crawford EJ, Lake AW, Boyce KG (1989) Breeding annual: medicago species for semiarid conditions in southern Australia. Adv Agron 42:399–437

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Firoozabady E, Moy Y, Courtney-Gutterson N, Robinson K (1994) Regeneration of transgenic rose (Rosa hybrida) plants from embryogenic tissue. Biotechnology 12:609–613

    Article  CAS  Google Scholar 

  • Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16:433–442

    Article  CAS  PubMed  Google Scholar 

  • Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Over expression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol 124:1854–1865

    Article  CAS  PubMed  Google Scholar 

  • Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403

    Article  CAS  PubMed  Google Scholar 

  • Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 over-expression induces COR genes and enhances freezing tolerance. Sci 280:104–106

    Article  CAS  Google Scholar 

  • Jiang C, Iu B, Singh J (1996) Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus. Plant Mol Biol 30:679–684

    Article  CAS  PubMed  Google Scholar 

  • Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotech 17:287–291

    Article  CAS  Google Scholar 

  • 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 45:346–350

    Article  CAS  PubMed  Google Scholar 

  • Kim CK, Chung JD, Park SH, Burrell AM, Kamo KK, Byrne DH (2004) Agrobacterium tumefaciens-mediated transformation of Rosa hybrida using the green fluorescent protein (GFP) gene. Plant Cell Tiss Org Cult 78:107–111

    Article  CAS  Google Scholar 

  • Li XQ, Krasnyanski SF, Korban SS (2002) Optimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens. Plant Physiol Biochem 40:453–459

    Article  CAS  Google Scholar 

  • Li XP, Tian AG, Luo GZ, Gong ZZ, Zhang JS, Chen SY (2005) Soybean DRE-binding transcription factors that are responsive to abiotic stresses. Theor Appl Genet 110:1355–1362

    Article  CAS  PubMed  Google Scholar 

  • Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two 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 Arabidopsis. Plant Cell 10:1391–1406

    Article  CAS  PubMed  Google Scholar 

  • Marchant R, Davey MR, Lucas JA, Power JB (1996) Somatic embryogenesis and plant regeneration in Floribunda rose (Rosa hybrida L.) cvs. Trumpeter and Glad Tidings. Plant Sci 120:95–105

    Article  Google Scholar 

  • Marchant R, Power JB, Lucas JA, Davey MR (1998a) Biolistic transformation of rose (Rosa hybrida L.). Ann Bot 81:109–114

    Article  Google Scholar 

  • Marchant R, Michael RD, John AL, Chris JL, Rechard AD, Power JB (1998b) Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf). Mol Breed 4:187–194

    Article  CAS  Google Scholar 

  • Medina J, Catalá R, Salinas J (2001) Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of Arabidopsis encoding highly conserved hydrophobic proteins. Plant Physiol 125:1655–1656

    Article  CAS  PubMed  Google Scholar 

  • Novillo F, Alonso JM, Ecker JR, Salinas J (2004) CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc Natl Acad Sci USA 101(11):3985–3990

    Article  CAS  PubMed  Google Scholar 

  • Oh SJ, Song SI, Kim YS, Jang HJ, Kim SY, Kim M, Kim YK, Nahm BH, Kim JK (2005) Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol 138:341–351

    Article  CAS  PubMed  Google Scholar 

  • Pellegrineschi A, Reynolds M, Pacheco M, Brito RM, Almeraya R, Yamaguchi-Shinozaki K, Hoisington D (2004) Stress-induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions. Genome 47:493–500

    Article  CAS  PubMed  Google Scholar 

  • Pennycooke JC, Cheng H, Stockinger EJ (2008) Comparative genomic sequence and expression analyses of Medicago truncatula and Alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes. Plant Physiol 146:1242–1254

    Article  CAS  PubMed  Google Scholar 

  • Qin F, Li J, Zhang GY, Zhao J, Chen SY, Liu Q (2003) Isolation and structural analysis of DRE-binding transcription factor from Maize (Zea mays L.). Acta Bot Sin 45:331–339

    CAS  Google Scholar 

  • Reynolds A, Lundblad V (1999) Yeast vectors and assay for expression of cloned genes. In: Ausubel FM, Kingston RE, Seidman JG, Struhl K, Brent R, Moore DD, Smith JA (eds) Short protocols in molecular biology. 4th edn. (Trans by Ma XJ, Shu YL, et al.) New York: John Wiley &. Sons, p 576

  • Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-binding specificity the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. BBRC 290:998–1009

    CAS  PubMed  Google Scholar 

  • Sakuma Y, Maruyama K, Osakabe Y, Feng Q, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18:1292–1309

    Article  CAS  PubMed  Google Scholar 

  • Shen YG, Zhang WK, He SJ, Zhang JS, Liu Q, Chen SY (2003a) An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor Appl Genet 106:923–930

    CAS  PubMed  Google Scholar 

  • Shen YG, Zhang WK, Yan DQ, Du BX, Zhang JS, Liu Q, Chen SY (2003b) Characterization of a DRE-binding transcription factor from a halophyte Atriplex hortensis. Theor Appl Genet 107:155–161

    CAS  PubMed  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water stress response. Plant Physiol 115:327–334

    Article  CAS  PubMed  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3:217–223

    CAS  PubMed  Google Scholar 

  • Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 95:14570–14575

    Article  CAS  PubMed  Google Scholar 

  • Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis 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 deficit. Proc Natl Acad Sci USA 94:1035–1040

    Article  CAS  PubMed  Google Scholar 

  • Trieu AT, Harrison MJ (1996) Rapid transformation of Medicago truncatula: regeneration via shoot organogenesis. Plant Cell Rep 16:6–11

    Article  CAS  Google Scholar 

  • Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin H, Chiou T-J, Katagi H, Dewbre GR (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541

    Article  CAS  PubMed  Google Scholar 

  • van der Salm TPM, van der Toorn CJG, Hanischtencate CH, Dons HJM (1996) Somatic embryogenesis and shoot regeneration from excised adventitious roots of the root stock Rosa hybrida cv Money Way. Plant Cell Rep 15:522–526

    Article  Google Scholar 

  • Vergne P, Maene M, Gabant G, Chauvet A, Debener T, Bendahmane M (2009) Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush. Plant Cell Tiss Organ Cult 100:73–81

    Article  Google Scholar 

  • Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors gratefully acknowledge laboratory support provided by Professor Xiaofang Luo at Beijing Forestry University and Xingyao Xiong at Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization of Crops. We also thank Dr. Jingsong Zhang for guidance on yeast hybrid assay. This work is supported by China National “948” Program (Grant No. 2005-4-34, 2007-4-02); National High Technology Program (2006AA10Z182); Special Research Fund for Doctor’s Degree Dissertation in Chinese Universities (20060022012); Research Project in Hunan Universities (09C497); National Natural Science Foundation of China (30371148).

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Correspondence to Shou-Yi Chen or Hua-Fang Wang.

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Ji-Ren Chen and Jing-Jing Lü authors contributed equally to this work.

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Chen, JR., Lü, JJ., Liu, R. et al. DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.). Plant Growth Regul 60, 199–211 (2010). https://doi.org/10.1007/s10725-009-9434-4

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  • DOI: https://doi.org/10.1007/s10725-009-9434-4

Keywords

  • China rose
  • DRE-binding
  • Freezing tolerance
  • Medicago
  • Transcriptional factor