In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses

Abstract

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Azacitidine (5-AzaC)-treatment and mutations in DNA methylase genes affect embryogenic response and expression of the genes that are involved in somatic embryogenesis in Arabidopsis

“…In line with this postulate, we observed a significantly decreased level of 5 mC in the embryogenic culture of Arabidopsis and the reduction in the DNA methylation level has also been associated with SE induction in other plants (Santos and Fevereiro 2002;Xu et al 2004;Noceda et al 2009;Rodríguez-Sanz et al 2014;Teyssier et al 2014). However, in some plants, an increased (Fraga et al 2012;Rival et al 2013), differentially modulated (Leljak-Levanić et al 2004;Nic-Can et al 2013) and steady (Parra et al 2001) level of DNA methylation during SE has been reported.…”

“…In the present study, we demonstrated that in Arabidopsis, similar to the embryogenic cultures of other plants (Santos and Fevereiro 2002;Yamamoto et al 2005;Teyssier et al 2014), treating the tissue with 5-AzaC severely and negatively affected the embryogenic response of the explants. We observed that the decreased embryogenic response of the 5-AzaC-treated explants is accompanied by a deregulated expression of LEC1, LEC2 and BBM that have a key regulatory role in SE induction.…”

“…Treatment with 5-AzaC has been shown to induce various plant phenotypes including dwarfism, early flowering and an inhibition of vegetative growth (Kondo et al 2006). Although 5-AzaC treatment has also been indicated to impact the morphogenic processes that are induced in vitro including the capacity of a tissue for SE (Santos and Fevereiro 2002;Yamamoto et al 2005;Tokuji et al 2011;Fraga et al 2012;Teyssier et al 2014;Solís et al 2015), our knowledge about the role of this modification in the epigenetic regulation of the genes that control the developmental plasticity of somatic plant cells is still limited.…”

Analysis of Two Clonal Lines (Embryogenic and Non-Embryogenic) of <i>Agave fourcroydes</i> Using AFLP and MSAP

“…This plasticity (methylation/demethylation) is related to the regulation of several genes involved in SE [2] [61] and to embryo development [11] [62].…”

Different environmental conditions at initiation of radiata pine somatic embryogenesis determine the protein profile of somatic embryos

“…In conifers, some efforts have recently been made to describe SE development at the molecular level (Jo et al 2014) and differences in proteins abundance between cell lines with contrasting embryogenic potential have been found (dos Santos et al 2016). Proteomic studies combining two-dimensional (2D) gel electrophoresis with a high degree of accuracy in protein identification by mass spectrometry (MS), are available for Cupressus sempervirens (Sallandrouze et al 1999), Picea glauca (Lippert et al 2005), Larix×eurolepis (Teyssier et al 2014) and other conifers as it was reviewed by Abril et al (2011).…”