5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

Abstract

Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs.

Somatic Embryogenesis. An Overview

“…8). There is an epigenetic reprogramming during the SE and the presence of somaclonal variation among the regenerated plants ( De-la-Peña et al 2015;Mahdavi-Darvari et al 2015;Nic-Can et al 2015;Solís et al 2015). This variation can be the result of chromosomal aberrations, genetic alterations, epigenetic regulations, and transposable elements.…”

Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen

“…This phenomenon has been widely utilized to obtain homozygous parental materials in crops (Ferrie and Caswell, 2011; Corral-Martínez and Seguí-Simarro, 2012; Yuan et al, 2015). Several investigations have explored the natural basis of microspore embryogenesis (Belmonte et al, 2010; Rodríguez-Serrano et al, 2012; Solís et al, 2012, 2015; Ahmadi et al, 2016). Microspores or early binucleate pollen grains were demonstrated to more easily undergo embryogenesis in vitro (Keller et al, 1987; Ferrie, 2003).…”

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

“…5-AzaC was also found to strongly inhibit the embryogenic response in coffee by decreasing the expression of LEC1 and BBM1 (Nic-Can et al 2013). Given that 5-AzaC-treatment has been reported to distinctly reduce the global methylation level in the in vitro cultured tissue of different plants that have been cultured in vitro, including Arabidopsis (Tokuji et al 2011), rape and barley (Solís et al 2015) and coffee (Nic-Can et al 2013), we assumed that the repression of the SE-regulatory genes that are associated with a decrease in the SE response is related to the demethylation of DNA.…”

“…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.…”