Skip to main content
SpringerLink
Log in

Identification and Characterization of a T-DNA Promoter Trap Line of Arabidopsis thaliana Uncovers an Embryo Sac-Specific Bi-directional Promoter

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

The Arabidopsis thaliana promoter trap line GUS-650 showing uidA reporter gene expression specifically in mature embryo sacs was found to carry transfer DNA (T-DNA) insertion in the 5′ untranslated region (UTR) of At3g17150. At3g17140 and At3g17150 are a pair of head-to-head-oriented genes separated by a 668-bp intergenic region encoding for a putative pectin methyl esterase inhibitor protein. In the GUS-650 line, uidA reporter gene was inserted in head-to-head orientation to the At3g17140 gene. Presence of At3g17140 transcripts in the GUS-650 line indicated that T-DNA insertion did not affect this gene and the 668-bp intergenic region functions as a bi-directional promoter. The 672-bp fragment separating the uidA and At3g17140 genes was cloned and placed in forward or reverse orientation next to the uidA gene. Transgenic Arabidopsis plants carrying either of these constructs showed β-glucuronidase (GUS) activity in the ovules confirming that the 672-bp sequence is a bi-directional promoter specific to ovules. These results were also supported by the presence of cis-acting motifs such as TATA box, CAAT box, seed storage protein element (CAACAC), endosperm-specific elements (AACA), Skn-1 motif (GTCAT) and bZIP transcription factor binding element (ACGT) within the 672-bp sequence in both orientations. The significance of a promoter trap approach and the utility of such a promoter are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Banerjee J, Sahoo DK, Dey N, Houtz RL, Maiti IB (2013) An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic Arabidopsis and tobacco plants. PLoS ONE 8(11):e79622. doi:10.1371/journal.pone.0079622

    Article  PubMed Central  PubMed  Google Scholar 

  • Bhalothia P, Alok A, Mehrotra S (2013) AACA element negatively regulates expression of protein phosphatase 2c (pp2c) like promoter in Arabidopsis thaliana. Am J Plant Sci 4:549–554

    Article  Google Scholar 

  • Bondino HG, Valle EM (2009) A small intergenic region drives exclusive tissue-specific expression of the adjacent genes in Arabidopsis thaliana. BMC Mol Biol 10:1471–2199

    Article  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Coutu C, Brandle J, Brown D, Brown K, Miki B, Simmonds J, Hegedus DD (2007) pORE: a modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res 16:771–781

    Article  CAS  PubMed  Google Scholar 

  • Foster R, Izawa T, Chua NH (1994) Plant bZIP proteins gather at ACGT elements. FASEB J 8:192–200

    CAS  PubMed  Google Scholar 

  • Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M, Streubel M, Westhoff P (2004) cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell 16:1077–1090

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 13:3901–3907

    Google Scholar 

  • Kubo T, Fujita M, Takahashi H, Nakazono M, Tsutsumi N, Kurata N (2013) Transcriptome analysis of developing ovules in rice isolated by laser microdissection. Plant Cell Physiol 54:750–765

    Article  CAS  PubMed  Google Scholar 

  • Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Peer YV, Rouze P, Rombauts S (2002) Plant CARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Li ZT, Jayasankar S, Gray DJ (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res 13:143–154

    Article  CAS  PubMed  Google Scholar 

  • Lindsey K, Wei W, Clarke MC, McArdle HF, Rooke LM, Topping JF (1993) Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Res 2:33–47

    Article  CAS  PubMed  Google Scholar 

  • Mitra A, Han J, Zhang ZJ, Mitra A (2009) The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter. Planta 229:1015–1022

    Article  CAS  PubMed  Google Scholar 

  • Mollier P, Hoffmann B, Orsel M, Pelletier G (2000) Tagging of a cryptic promoter that confers root-specific GUS expression in Arabidopsis thaliana. Plant Cell Rep 19:1076–1083

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Okresz L, Mathe C, Horvath E, Schell J, Koncz C, Szabados L (1998) T-DNA trapping of a cryptic promoter identifies an ortholog of highly conserved SNZ growth arrest response genes in Arabidopsis. Plant Sci 138:217–228

    Article  CAS  Google Scholar 

  • Plesch G, Kamann E, Mueller RB (2000) Cloning regulatory sequences mediating guard cell-specific gene expression. Gene 249:83–89

    Article  CAS  PubMed  Google Scholar 

  • Pratibha P, Singh SK, Sharma I, Kumar R, Srinivasan R, Bhat SR, Ahuja PS, Sreenivasulu Y (2013) Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter. Gene 524:22–27

    Article  CAS  PubMed  Google Scholar 

  • Shin R, Kim MJ, Paek KH (2003) The CaTin1 (Capsicum annuum TMV-induced clone 1) and CaTin1–2 genes are linked head-to-head and share a bidirectional promoter. Plant Cell Physiol 44:549–554

    Article  CAS  PubMed  Google Scholar 

  • Sivanandan C, Sujatha TP, Prasad AM, Resminath R, Thakare D, Bhat SR, Srinivasan R (2005) T-DNA tagging and characterization of a cryptic root-specific promoter in Arabidopsis. Biochem Biophys Acta 1731:202–208

    CAS  PubMed  Google Scholar 

  • Stalberg K, Ellerstöm M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199:515–519

    Article  CAS  PubMed  Google Scholar 

  • Tang W, Perry SE (2003) Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J Biol Chem 278:28154–28159

    Article  CAS  PubMed  Google Scholar 

  • Trinklein ND, Aldred SF, Hartman SJ, Schroeder DI, Otillar RP, Myers RM (2004) An abundance of bidirectional promoters in the human genome. Genome Res 14:62–66

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Vedel V, Scotti I (2011) Promoting the promoter. Plant Sci 180:182–189

    Article  CAS  PubMed  Google Scholar 

  • Verk MCV, Dimitri P, Neeleman L, Bol JF, Linthorst HJM (2008) A novel WRKY transcription factor is required for induction of PR-1A gene expression by salicylic acid and bacterial elicitors. Plant Physiol 146:1983–1995

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang C, Ding D, Yan R, Yu X, Li W, Li M (2008) A novel bi-directional promoter cloned from melon and its activity in cucumber and tobacco. J Plant Biol 51:108–115

    Article  CAS  Google Scholar 

  • Wang Q, Wan L, Li D, Zhu L, Qian M, Deng M (2009) Searching for bidirectional promoters in Arabidopsis thaliana. BMC Bioinforma 10:S29

    Article  Google Scholar 

  • Zanotto E, Shah ZH, Jacobs HT (2007) The bidirectional promoter of two genes for the mitochondrial translational apparatus in mouse is regulated by an array of CCAAT boxes interacting with the transcription factor NF-Y. Nucleic Acids Res 35:664–677

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zheng H, Lei Y, Lin S, Zhang Q, Zhang Z (2011) Bidirectionalization of a methyl jasmonate-inducible plant promoter. Biotechnol Lett 33:387–393

    Article  CAS  PubMed  Google Scholar 

  • Zhu C, Yang Q, Ni X, Bai C, Sheng Y, Shi L, Capell T, Sandmann G, Christou P (2014) Cloning and functional analysis of the promoters that up regulate carotenogenic gene expression during flower development in Gentiana lutea. Physiol Plant 150:493–504

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank the Indian Council of Agricultural Research, New Delhi, India, for financial assistance through the National Agricultural Innovative Project (NAIP/C4/C1072/2006-07) and are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing infrastructural and financial support in the form of projects MLP-072 and BSC-0107. The manuscript represents CSIR-IHBT communication number 3700.

Author information

Authors and Affiliations

  1. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India

    Isha Sharma, Paramvir Singh Ahuja & Yelam Sreenivasulu

  2. National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India

    Ramamurthy Srinivasan & Shripad Ramachandra Bhat

Authors
  1. Isha Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ramamurthy Srinivasan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paramvir Singh Ahuja
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shripad Ramachandra Bhat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yelam Sreenivasulu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Shripad Ramachandra Bhat or Yelam Sreenivasulu.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Table 1

(DOC 38 kb)

Supplementary Table 2

(DOCX 50 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sharma, I., Srinivasan, R., Ahuja, P.S. et al. Identification and Characterization of a T-DNA Promoter Trap Line of Arabidopsis thaliana Uncovers an Embryo Sac-Specific Bi-directional Promoter. Plant Mol Biol Rep 33, 1404–1412 (2015). https://doi.org/10.1007/s11105-014-0829-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-014-0829-7

Keywords

Search

Navigation