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Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. don

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Abstract

Pinus radiata is the most important conifer species for commercial forestry in countries such as Australia, New Zeeland and Chile. Nowadays, SE (somatic embryogenesis) is considered the most promising in vitro method for large scale vegetative propagation of woody plants. The understanding of the molecular basis of SE is in its very beginning and a number of embryogenesis-related genes have been identified in conifers. Among the molecular mechanisms involved in regulation of SE, DNA methylation, which is an epigenetic modification associated with transcriptional silencing, has shown to be a pivotal factor controlling gene expression. In this work, we studied the morphological and molecular differences between cell lines previously characterized in terms of their embryogenic potential as embryogenic (E) and non embryogenic (NE), obtained from immature zygotic embryos of P. radiata. In contrast to E lines, NE lines were composed of multicellular aggregates lacking polarity, and they were characterized by the presence of significantly lower transcript levels of embryogenesis-related genes and higher global DNA methylation. Furthermore, the detection of vibrational markers of DNA conformation indicated that DNA samples obtained from E lines presented the common B-DNA conformation, while NE samples presented Z-conformation. Taken together, our results highlight the role of epigenetic mechanisms such as DNA methylation in regulating the expression of embryogenesis- related genes, having impact on the embryo patterning and cell differentiation.

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Acknowledgements

This work was supported by Universidad Andres Bello (DI-537-14/R) and Forestal Mininco S. A. (EMP6). The authors are also grateful to the Center of biotechnology of CMPC Mininco, Los Angeles, Chile, especially to Adelaida Poblete, Valeria Jara and Rebeca Sanhueza, for providing plant material, knowledge and experience.

Authors contributions

S. B. and R. H. planned and designed the research, analyzed and interpreted the results and wrote the manuscript. A. B did the DNA methylation experiments. A.T. extracted DNA and acquired FT-IR data. F.S. was responsible for conventional RT-PCR assays. P.J. performed qRT-PCR analysis. M. J. P. obtained micro-morphological data. All the authors participated in drafting or revising the work and approved the final manuscript in agreement with all its aspects.

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Authors and Affiliations

  1. Facultad de Ciencias Biológicas, Centro de Biotecnología Vegetal, Universidad Andres Bello, campus Concepción, Autopista Concepción – Talcahuano 7100, Talcahuano, Chile

    Soraya Bravo, Ariana Bertín, Aileen Turner, Francisco Sepúlveda & Paz Jopia

  2. Facultad de Ciencia, Universidad San Sebastián, Lientur 1457, Concepción, Chile

    María José Parra

  3. Departamento de Análisis Instrumental, Facultad de Farmacia, Universidad de Concepción, Edmundo Larenas s/n, Concepción, Chile

    Rosario Castillo

  4. Laboratorio de Epigenética Vegetal, Departamento de Silvicultura, Facultad de Ciencias Forestales, Universidad de Concepción, Victoria 631, Concepción, Chile

    Rodrigo Hasbún

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  1. Soraya Bravo
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  2. Ariana Bertín
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  3. Aileen Turner
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Correspondence to Soraya Bravo.

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Communicated by Jose M. Segui-Simarro.

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Supplementary material 1 (DOCX 15 KB)

11240_2017_1242_MOESM2_ESM.tif

Supplementary Figure S1—Somatic embryos at cotyledonary stage derived from E lines. The arrow indicates a cotyledonary embryo (TIF 15318 KB)

11240_2017_1242_MOESM3_ESM.tif

Supplementary Figure S2—FT-IR spectra of E and NE DNA samples. Comparison of second derivate spectra obtained by FT-IR microspectroscopic analysis from E (continued line) and NE (dashed line) DNA samples. The bigger differences between them were indicated by encircle the wavenumber beneath the corresponding peak (TIF 2723 KB)

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Bravo, S., Bertín, A., Turner, A. et al. Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. don. Plant Cell Tiss Organ Cult 130, 521–529 (2017). https://doi.org/10.1007/s11240-017-1242-3

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