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Expression analysis of epigenetic and abscisic acid-related genes during maturation of Quercus suber somatic embryos

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Abstract

Clonal propagation of Quercus suber via somatic embryogenesis is an alternative to conventional tree propagation methods; however, complete maturation of somatic embryos is considered the major bottleneck for mass propagation of Quercus species. During somatic embryogenesis, embryo development and maturation are controlled by signaling pathways that integrate information from genetic and epigenetic programs as well as hormonal signals. Therefore, in this study genes were identified related to epigenetic regulation and the abscisic acid (ABA) pathway during development and maturation of cork oak somatic embryos. A total of eight expressed sequence tags were obtained of genes encoding a 9-cis-epoxycarotenoid dioxygenase (NCED), two histone deacetylases (HDA6 and HDA19), two histone monoubiquitinases (HUB1 and HUB2), a histone H3 kinase (AUR3) as well as genes related to chromatin remodeling processes PICKLE and VP1/ABSCISIC ACID INSENSITIVE 3-LIKE 1 (VAL1). The analysis of the expression patterns of selected genes during different developmental stages indicated that QsNCED3 may play a role in ABA synthesis during embryogenesis. The change in the expression levels for all seven genes associated with epigenetic regulation showed that QsHUB1 and QsHUB2 may have a role in ABA signalling while QsHDA6 and QsHDA19 could act in different pathways than in Arabidopsis. Furthermore, expression levels of QsAUR3 indicated that histone phosphorylation is an early epigenetic mark in Q. suber somatic embryos while QsPICKLE and QsVAL1 may be necessary for the correct development of cork oak somatic embryos.

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Acknowledgments

We acknowledge Victor Granda (University of Oviedo) for his design of degenerated primers of NCED3. This work was supported by Spanish national projects AGL2007-62907/FOR and AGL2010-22351-C03-01. FICYT foundation supported the fellowship of M. Pérez.

Conflict of Interest

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

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Authors and Affiliations

  1. Laboratorio de Fisiología Vegetal, Dpto B.O.S., Facultad de Biología, Universidad de Oviedo, C/Catedrático Rodrigo Uría s/n, 33071, Oviedo, Asturias, Spain

    Marta Pérez & Maria Jesús Cañal

  2. Intituto Universitario de Biotecnología de Asturias (IUBA asociado con CSIC), Oviedo, Spain

    Marta Pérez & Maria Jesús Cañal

  3. Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex, RH17 6TN, UK

    Peter E. Toorop

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  1. Marta Pérez
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  2. Maria Jesús Cañal
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  3. Peter E. Toorop
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Correspondence to Marta Pérez.

Electronic supplementary material

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11240_2014_706_MOESM1_ESM.pdf

Online resource 1 Developmental stages defined during somatic embryogenesis procedure. Proliferative stages composed by white callogenic structures with globular embryos (PS), immature translucent cotyledonary embryos (E1); white opaque cotyledonary embryos (E2), cotyledonary embryos after one month onto maturation medium in darkness at 25 °C (E3), cotyledonary E3 embryos after cold stratification for 2 months in darkness (E4). (bars = 1 mm) (PDF 2536 kb)

Online resource 2 Sequences used for degenerated primer design and phylogenetic analysis (PDF 250 kb)

11240_2014_706_MOESM3_ESM.pdf

Online resource 3 List of EST sequences obtained after amplification with degenerated primers. The similarity (BLAST score, query coverage) is provided to the Arabidopsis orthologue and to the best hit for each gene in an NCBI BLAST search (PDF 105 kb)

Online resource 4 PCR amplification efficiency of each primer pair (± standard error) (PDF 19 kb)

11240_2014_706_MOESM5_ESM.pdf

Online resource 5 Arabidopsis thaliana embryo development gene expression levels. Taken from the Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) (Winter et al. 2007; Bassel et al. 2008). Embryo stages according to Le et al. (2010). Pre-globular-stage embryo (PGLOB), globular stage embryo (GLOB), heart-stage embryo (HRT), linear cotyledon-stage seed (LCOT), mature-green-stage seed (MG), Spearman correlation coefficient (rs)Correlations between Q. suber gene expression levels at stages E1–E2 were compared with A. thaliana embryos expression patterns LCOT and MG. A negative coefficient indicates an inverse proportion correlation. *indicates significant differences (p ≤ 0.01). (PDF 1395 kb)

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Pérez, M., Cañal, M.J. & Toorop, P.E. Expression analysis of epigenetic and abscisic acid-related genes during maturation of Quercus suber somatic embryos. Plant Cell Tiss Organ Cult 121, 353–366 (2015). https://doi.org/10.1007/s11240-014-0706-y

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