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Hypomethylation and transcriptional reactivation of retrotransposon-like sequences in ddm1 transgenic plants of Brassica rapa

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Abstract

DNA methylation and histone modification play important roles in regulating gene expression. The DDM1 gene in Arabidopsis thaliana (AtDDM1) is required for the maintenance of DNA methylation level and histone H3 methylation pattern. We isolated DDM1 homologs of Brassica rapa, BrDDM1a and BrDDM1b, which have 84.4% and 84.1% deduced amino acid sequence identities with AtDDM1, respectively. Both the BrDDM1a and BrDDM1b genes were found to be expressed in vegetative and reproductive tissues. B. rapa ddm1-RNAi transgenic plants with reduced levels of BrDDM1a/BrDDM1b expression showed genome-wide and non-tissue-specific demethylation. These results suggest that the BrDDM1a and BrDDM1b genes are orthologs of AtDDM1 and are required for the maintenance of DNA methylation as is AtDDM1. Despite genome-wide demethylation, developmental abnormalities were not found in the ddm1-RNAi transgenic plants. Dominance relationships of SP11/SCR alleles, the determinant of pollen recognition specificity in Brassica self-incompatibility, in S heterozygotes in B. rapa were not influenced by the low level of the BrDDM1 expression. Transcriptional reactivation of retrotransposon-like sequences observed in the ddm1-RNAi transgenic plants indicates that BrDDM1a and BrDDM1b participate in silencing of retrotransposons. Hypomethylation states of the ddm1-RNAi transgenic plants were inherited by plants of the next generation even by plants which had lost the RNAi construct by segregation. Remethylation was observed in a few progenies. Efficiencies of remethylation in the progenies without the RNAi construct were different between 18S rDNA, BoSTF12a/15a, and BrTto1 sequences.

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Abbreviations

CMT3:

Chromomethylase 3

DDM1:

Decrease in DNA methylation 1

GUS:

β-Glucuronidase

LTR:

Long terminal repeat

Lsh:

Lymphocyte-specific helicase

NosT:

Nopaline synthase gene

MET1:

Methyltransferase 1

rDNA:

Ribosomal DNA

RNAi:

RNA interference

SCR:

S-locus cysteine-rich

SP11:

S-locus protein 11

SSC:

Saline-sodium citrate

STF:

S-locus retrotransposon family

Tto1:

Tobacco retrotransposon 1

WT:

Wild type

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Acknowledgements

We are grateful to Dr. T. Kakutani for his helpful suggestions on this manuscript, Dr. Y. Sato for his technical advice, and Dr. Y. Kuginuki for providing a doubled haploid line.

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  1. Ryo Fujimoto

    Present address: National Institute of Genetics, Yata 1111, Mishima, Shizuoka, 411-8540, Japan

Authors and Affiliations

  1. Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, 981-8555, Japan

    Ryo Fujimoto, Taku Sasaki, Hisashi Inoue & Takeshi Nishio

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  1. Ryo Fujimoto
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  2. Taku Sasaki
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Correspondence to Takeshi Nishio.

Electronic supplementary material

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11103_2007_9285_MOESM1_ESM.TIF.tif

Supplementary Fig. 1. Alignment of the amino acid sequences of BrDDM1a, BrDDM1b, and AtDDM1 (AAD28303). Boxes outlined by a dotted line and a solid line indicate the SNF2 family N-terminal domain and the helicase-conserved C-terminal domain, respectively, both of which were identified by P fam analysis. (TIF 4850 kb)

11103_2007_9285_MOESM2_ESM.TIF.tif

Supplementary Fig. 2. Analysis of DNA methylation levels of repetitive sequences and retrotransposon-like sequences in the transgenic plants by Southern blot analysis using methylation-sensitive and methylation-insensitive restriction enzymes. (A) After electrophoresis, genomic DNAs of T0-2 and WT digested with Msp I (M) and Hpa II (H) were hybridized with the probes of the promoter region of 25S rDNA, the coding region of 5S rDNA, and putative centromeric repeat. (B) Genomic DNAs of T0-2 and WT digested with Hind III/Msp I (M) and Hind III/Hpa II (H) were probed with gag regions of BoSTF7a and BoSTF12b, an rvt region of BrSTF60a, and an MuDR region of MuDR transposon. (TIF 4497 kb)

11103_2007_9285_MOESM3_ESM.TIF.tif

Supplementary Fig. 3. Analysis of DNA methylation levels in genomic DNAs extracted from various tissues of T0-2 by Southern blot analysis. DNAs digested with Msp I (M) and Hpa II (H) were probed with the promoter region of 18S rDNA. L, leaves; FB, flower buds; ST, stamens; PI, pistils. 0-1, 1-3, 3-5, and 5-8 indicate the lengths of flower buds (mm). (TIF 4669 kb)

11103_2007_9285_MOESM4_ESM.TIF.tif

Supplementary Fig. 4. Southern-blot analysis of genomic DNAs of T0 plants. After electrophoresis, genomic DNAs digested with Eco RI were hybridized with the probes of rvt regions of BoSTF12a/15a and BrSTF60a, rnaseH region of BrTto1, and MuDR region of MuDR transposon. (TIF 4316 kb)

11103_2007_9285_MOESM5_ESM.TIF.tif

Supplementary Fig. 5. Analysis of DNA methylation and transcripts of the transposon-like sequences in the T1 plants derived from T0-2. (A, B) Southern blot analysis of genomic DNAs of the T1 and F1 plants of T0-2. Genomic DNAs digested with Hind III/Msp I and Hind III/Hpa II were probed with an rvt region of BoSTF12a/15a (A) and an rnaseH region of BrTto1 (B). (C) Detection of transcripts in leaves of BoSTF12a/15a and BrTto1. The actin gene was used as a positive control. RNAi+ and RNAi- indicate plants having the RNAi construct and those without the RNAi construct, respectively. (TIF 4533 kb)

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Fujimoto, R., Sasaki, T., Inoue, H. et al. Hypomethylation and transcriptional reactivation of retrotransposon-like sequences in ddm1 transgenic plants of Brassica rapa . Plant Mol Biol 66, 463–473 (2008). https://doi.org/10.1007/s11103-007-9285-1

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