New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

Abstract

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

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. The diversity in the SE-associated DNA methylation levels suggests that various exo-and endogenous culture factors rather than the induction of embryonic development per se appear to contribute to the global DNA methylation level in cultured tissue (Huang et al 2012;Rival et al 2013;Li et al 2014;Rathore et al 2014).…”

“…Relevant to the BBM-mediated SE induction mechanism, we found the BBM expression to be distinctly repressed in the 5-AzaC-treated culture that was incapable of SE induction. 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.…”

“…5-AzaC treatment was indicated as distinctly changing the gene expression in the plants (Chang and Pikaard 2005;Song et al 2017) and in vitro cultured plant cells/tissue and both the stimulation and repression of the gene transcription has been reported (Berdasco et al 2008;Tokuji et al 2011;Nic-Can et al 2013). Consistent with the diverse effects of 5-AzaC on gene expression level, the hypo-and hypermethylation of DNA in response to this chemical were reported in an embryogenic culture of Acca sellowiana, which were dependent on the plant genotype and the supplementation of the culture medium with 2,4-D (Fraga et al 2012).…”

Identification of LEC1, L1L and Polycomb Repressive Complex 2 genes and their expression during the induction phase of Medicago truncatula Gaertn. somatic embryogenesis

“…Analysis of mutants showed that the lack of clf/swn activity resulted in an up-regulation of LEC1 and LEC2 genes in Arabidopsis thaliana . Regulation of LEC1 by epigenetic mechanisms was also proposed for Coffea camephora (Nic-Can et al 2013) where the presence of H3K27me3 marks within the LEC1 histoneassociated region was a possible cause of reduced transcriptional activity of the gene during early stages of induction somatic embryogenesis. In these experiments, LEC1 expression increased with progress of somatic embryogenesis induction, which was consistent with our results where LEC1 and L1L transcripts started to appear between days 7 and 21.…”

“…One of them, LEC1, is a CCAAT-binding (CBF) transcription factor with a HAP3 subunit . The LEC1 genes expression pattern was analyzed during somatic embryogenesis from different initial explants representing different genetic program such as immature embryos of Zea mays and Arabidopsis thaliana Gaj et al 2005), Coffea canephora seedling leaves (Nic-Can et al 2013), Daucus carota hypocotyl (Yazawa et al 2004), and Medicago sativa protoplasts (Domoki et al 2006). LEAFY COTYLEDON 1-LIKE B-domain of HAP3 shared 83% similarity to that of LEC1, which means that they define distinct class of HAP3 subunits (Kwong et al 2003).…”