Plant Cell Reports

, Volume 35, Issue 1, pp 43–51 | Cite as

CaMV-35S promoter sequence-specific DNA methylation in lettuce

  • Azusa Okumura
  • Asahi Shimada
  • Satoshi Yamasaki
  • Takuya Horino
  • Yuji Iwata
  • Nozomu Koizumi
  • Masahiro Nishihara
  • Kei-ichiro Mishiba
Original Article


Key message

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery.


We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.


DNA methylation Lettuce 35S promoter Transgene silencing Gentian De novo methylation 



Cauliflower mosaic virus


Synthetic green fluorescent protein


Transfer DNA



We thank Ms. Ayumi Sakei (Osaka Prefecture University) for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (Grant Number, 23780027) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2015_1865_MOESM1_ESM.tif (4.7 mb)
Supplementary material 1 (TIFF 4811 kb)
299_2015_1865_MOESM2_ESM.docx (144 kb)
Supplementary material 2 (DOCX 144 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Azusa Okumura
    • 1
    • 3
  • Asahi Shimada
    • 1
  • Satoshi Yamasaki
    • 1
    • 4
  • Takuya Horino
    • 1
  • Yuji Iwata
    • 1
  • Nozomu Koizumi
    • 1
  • Masahiro Nishihara
    • 2
  • Kei-ichiro Mishiba
    • 1
  1. 1.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversitySakaiJapan
  2. 2.Iwate Biotechnology Research CenterKitamiJapan
  3. 3.Miyoshi Co., Ltd.HokutoJapan
  4. 4.Tempstaff People Co., LtdNagoyaJapan

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