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Poplars with a PtDDM1-RNAi transgene have reduced DNA methylation and show aberrant post-dormancy morphology

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Abstract

The Arabidopsis thaliana DDM1 (Decreased DNA Methylation) gene is necessary for the maintenance of DNA methylation and heterochromatin assembly. In Arabidopsis, ddm1 mutants exhibit strong but delayed morphological phenotypes. We used RNA interference (RNAi) to suppress transcripts of two orthologous DDM1 paralogs in Populus trichocarpa and examined effects on whole plant phenotypes during perennial growth and seasonal dormancy. The RNAi-PtDDM1 transgenic poplars showed a wide range of DDM1 transcript suppression; the most strongly suppressed line had 37.5 % of the expression of the non-transgenic control. Genomic cytosine methylation (mC %) was 11.1 % in the non-transgenic control, compared with 9.1 % for the transgenic event with lowest mC %, a reduction of 18.1 %. An evaluation of greenhouse growth directly after acclimation of in vitro grown plants showed no developmental or growth rate abnormalities associated with the decrease in PtDDM1 expression. However, after a dormancy cycle and growth outdoors, a mottled leaf phenotype appeared in some of the transgenic insertion events that had strongly reduced PtDDM1 expression and DNA methylation. The phenotypic consequences of reduced DDM1 activity and DNA methylation appears to increase with cumulative plant propagation and growth.

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Acknowledgments

We thank Dr. Christopher Matthews for guidance on methylation analysis and the use of his HPLC equipment; Alain Delaunay and Julien Clémot for technical assistance on DNA extraction and HPLC analysis (University of Orléans); Liz Etherington for help in laboratory management; Stephane Verger for assisting with greenhouse measurements, DNA extraction, and HPLC; Kelly Vining for technical advice for qPCR experiments. This work was supported by grant DE-FG02-08ER64665 from the US Department of Energy, Joint USDA Program USDA on Feedstock Genomics.

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Authors and Affiliations

  1. Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR, 97331, USA

    Ruoqing Zhu, Olga Shevchenko, Cathleen Ma & Steven H. Strauss

  2. Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, USC1328 ARCHE INRA, rue de Chartres, BP 6759, Faculté des Sciences, Université d’Orléans, 45067, Orléans Cedex 2, France

    Stéphane Maury

  3. Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA

    Michael Freitag

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  1. Ruoqing Zhu
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Correspondence to Steven H. Strauss.

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Zhu, R., Shevchenko, O., Ma, C. et al. Poplars with a PtDDM1-RNAi transgene have reduced DNA methylation and show aberrant post-dormancy morphology. Planta 237, 1483–1493 (2013). https://doi.org/10.1007/s00425-013-1858-4

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