PhysiologyEpigenetic and hormonal profile during maturation of Quercus Suber L. somatic embryos
Introduction
Quercus suber L. (cork oak) has high social, ecological and economic value in the western Mediterranean region. Unfortunately, conventional tree breeding methods are not suitable for commercial propagation and tree improvement programs due to its long reproductive cycle and irregular seed yield (Vieitez et al., 2012). Moreover, cork oak seeds are recalcitrant (Royal Botanic Gardens Kew Seed Information Database (SID), 2008) and, as a result, seeds cannot be stored long-term without losing viability (Bewley and Black, 1994). Vegetative propagation is an appropriate alternative to conventional storage methods; however, this is not feasible for oak mature trees because of the low rooting percentage of cuttings (Wilhem, 2000). Somatic embryogenesis is a powerful alternative for the production of Quercus trees and has been widely studied in this genus (reviewed by Wilhem, 2000 and Vieitez et al., 2012). Nevertheless, proper maturation and germination of somatic embryos seem to be the most important factors that limit the correct conversion of embryos into plants (Rai et al., 2011) and are the main weaknesses of this technique for large scale propagation (reviewed by Vieitez et al., 2012). Therefore, a deeper study into the molecular mechanisms that regulate these two main steps is necessary.
The regulation of embryo development, maturation and subsequent germination requires the concerted action of several signaling pathways that integrate genetic and epigenetic programs as well as hormonal and metabolic signals (Gutierrez et al., 2007, Feng et al., 2010, Gao et al., 2012). Moreover, during Q. suber somatic embryogenesis, a relationship between abscisic acid (ABA) and indole-3-acetic acid (IAA) has been described previously (García-Martín et al., 2005). Abscisic acid (ABA) plays an important role during seed development, maintaining embryogenesis and delaying germination until they are fully formed and have accumulated sufficient reserves to permit successful germination and subsequent seedling establishment (Kermode, 2005). During seed development and maturation, ABA signaling controls the expression of transcription factors essential for development-specific gene expression as well as the promotion of the synthesis of seed proteins and inhibits precocious germination in mature seeds (Gutierrez et al., 2007, Nakashima and Yamaguchi-Shinozaki, 2013). During somatic embryogenesis, ABA also promotes the transition from the proliferation phase to the maturation phase (Rai et al., 2011). Furthermore, changes in seed moisture content as well as fresh and dry weight are efficient parameters for the characterization of seed development and for the determination of seed maturity (Bewley and Black, 1994, Prewein et al., 2006).
Cell differentiation and plant development are controlled through temporal and spatial activation and silencing of specific genes. During somatic embryogenesis, multiple steps including dedifferentiation and reprograming of somatic cells are often required in addition to initiation and progression of the developmental program (Zhang and Ogas, 2009). A specific interaction between the signaling pathways and developmental programs is required, and this gene regulation is controlled in part by epigenetic mechanisms (Feng et al., 2010). Such epigenetic control has an essential role during cell differentiation allowing cells to be reprogrammed in order to generate a new differentiation pathway (Meijón et al., 2009, Santamaría et al., 2009). DNA methylation is an epigenetic regulatory mechanism considered a determining factor in the transcriptional control of gene expression, being essential for correct plant development. Disruptions in the cytosine methylation pattern often produce abnormalities in the developmental program (Finnegan et al., 1998, Zluvova et al., 2001). DNA methylation profiles through development can also be used as molecular indicators of processes related to aging, reinvigoration and maturity both in angiosperms and gymnosperms (Valledor et al., 2007, Viejo et al., 2012).
During somatic embryogenesis, DNA methylation levels are critical for both morphogenesis and cell proliferation (Chakrabarty et al., 2003). For example, in Medicago truncatula embryogenic lines it was shown that the production of somatic embryos depends on a certain level of DNA methylation (Santos and Fevereiro, 2002) and a decrease of methylation levels was associated with the embryogenic capacity of Pinus nigra Arn. cell cultures (Noceda et al., 2009). Similarly to zygotic seed development, environmental signals and stresses can induce epigenetic modifications in somatic embryos during maturation and germination steps. It has been noted that a large and global demethylation event occurs in Silene latifolia seeds during germination and post-germination periods, reflecting the transition from a quiescent seed to active growing and plant development (Zluvova et al., 2001). Furthermore, demethylation events have been reported to be necessary for germination of wheat seeds (Meng et al., 2012). In Arabidopsis, more in-depth studies showed that, during germination, DNA is demethylated except in centromeric regions and some clusters of silenced genes (Nakabayashi et al., 2005). In general, reductions in a given methylation level in a specific cell population or organ usually occur prior to the beginning of any differentiation program while de-differentiation concurs with an increase in DNA methylation (Valledor et al., 2007). During maturation of Quercus somatic embryos, cultures must be exposed to cold in order to successfully germinate (Manzanera et al., 1993, Mauri and Manzanera, 2004). Moreover, prolonged exposure to low temperature has been demonstrated to produce demethylation events in Arabidopsis (Finnegan et al., 1998).
Although the developmental process of cork oak seeds has been described (Merouani et al., 2003), little is known about the maturation of somatic embryos. During the course of Q. suber somatic embryo development, physiological and morphological changes must be accomplished in order to produce mature embryos with an effective ability to germinate. Culture media and conditions aim at resembling the natural conditions for the correct development of embryos. However, a more detailed study of the characteristics that define the maturity of embryos is required. Additionally, many studies have been carried out on the epigenetic regulation of somatic embryogenesis in different species, but the role of DNA methylation during maturation and germination of somatic embryos is still unclear. Therefore, the aims of this work were to analyze ABA and methylation levels during Q. suber somatic embryo development as well as to determine the temporal and spatial distribution of ABA and 5-methyl-deoxycytidine (5-mdC) in embryos at a range of developmental stages to determine which domains of cells within the embryo showed different expression patterns.
Section snippets
Plant material
Embryogenic cultures were initiated from immature embryos of Quercus suber L. according to Bueno et al. (2000). Immature acorns were collected every week from late July to the beginning of September during the period of fruit development. Acorns were surface-sterilized for 20 min by immersion in 2% (v/v) sodium hypochlorite (35 g L−1 active chlorine) plus a few drops of Tween 20, followed by three rinses in sterile distilled water of 10 min each. Immature embryos where then isolated, dissected and
Determination of embryo moisture content
During the initial developmental stages, E1 and E2 embryos showed the lowest fresh and dry weights (Fig. 2). In later stages, a significant increase in the fresh weight was recorded and was associated with a size increase (1–2 cm) of the embryos, mainly in the cotyledonary area, reaching the highest fresh weight (0.5 g) in E4 embryos. Dry weight also continued to increase, reaching 0.07 g in E4 embryos. Although the initial developmental stages E1 and E2 showed no differences in fresh or dry
Discussion
Seed development is characterized by two developmental phases: a histodifferentiation phase where embryos develop through rapid cell divisions, and a maturation phase characterized by the accumulation of storage reserves (Bewley and Black, 1994). Seed formation and development form an intricate genetically programmed process that is associated with changes in metabolite levels and is regulated by a complex signaling network mediated by sugar, hormone levels and epigenetic mechanisms (Gao et
Acknowledgements
We acknowledge Dr. Sergio J. Ochatt from the Institut National de la Recherche Agronomique (INRA) (Dijon) for his useful comments and help. We also thank Victor Granda from University of Oviedo for technical support and recommendations. This work was supported by Spanish national projects AGL2007-62907/FOR and AGL2010-22351-C03-01. FICYT foundation supported the fellowship of M. Pérez and M. Viejo.
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