Changes in DNA Methylation Levels and Nuclear Distribution Patterns after Microspore Reprogramming to Embryogenesis in Barley

Abstract

Under specific stress treatments, the microspore can be induced in vitro to deviate from its gametophytic development and to reprogram towards embryogenesis, becoming a totipotent cell and forming haploid embryos. These can further regenerate homozygous plants for production of new isogenic lines, an important biotechnological tool for crop breeding. DNA methylation constitutes a prominent epigenetic modification of the chromatin fiber which regulates gene expression. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation; however, the relationship between global DNA methylation and genome-wide expression patterns is still poorly understood. In this work, the dynamics of global DNA methylation levels and distribution patterns were analyzed during microspore reprogramming to embryogenesis and during pollen development in Hordeum vulgare. Quantification of global DNA methylation levels and 5-methyl-deoxycytidine (5mdC) immunofluorescence were conducted at specific stages of pollen development and after reprogramming to embryogenesis to analyze the epigenetic changes that accompany the change of developmental program and cell fate. The results showed low DNA methylation levels in microspores and a high increase along pollen development and maturation; an intense 5mdC signal was concentrated in the generative and sperm nuclei whereas the vegetative nucleus exhibited a weaker DNA methylation signal. After inductive stress treatment, low methylation levels and faint 5mdC signals were observed in nuclei of reprogrammed microspores and 2-4-cell proembryos. This data revealed a global DNA hypomethylation during the change of the developmental program and first embryogenic divisions. This is in contrast with the hypermethylation of generative and sperm cells of the male germline during pollen maturation, suggesting an epigenetic regulation after induction of microspore embryogenesis. At later embryogenesis stages, global DNA methylation progressively increased, accompanying embryo development and differentiation events like in zygotic embryos, corroborating that DNA methylation is critical for the regulation of gene expression in microspore embryogenesis.

Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suberL

“…This 5mdC distribution pattern over small heterochromatin masses correlates with the pattern found in active proliferating cells of root meristems [34], and early microspore-derived embryos of rapeseed, Brassica napus , with high transcriptional activity and decondensed chromatin patterns [11,14]. In contrast, 5mdC distribution over large heterochromatin masses has been associated with decreased cellular activity stages and cell differentiation, as in the generative/sperm cells of pollen grains, in the differentiating cells of advanced embryos [14,15] or in the tapetal cells during the high chromatin condensation occurring during their developmental programmed cell death [36]. …”

“…DNA methylation plays an essential role in plant growth as a mechanism to epigenetically maintain developmental fates in proliferating and differentiating cells [14,15,32-34]. In the present work, DNA methylation level and distribution pattern, in relation to chromatin changes, have been analyzed during early embryogenesis of the two pathways, of microspores and zygotic embryos, results suggesting that the initiation of both embryogenic processes is epigenetically regulated.…”

“…Changes in various cell activities and in the structural organization of subcellular compartments have been reported as accompanying the microspore reprogramming process in some herbaceous and woody species [4-11]. Increasing evidences have indicated the relevance of some cell features like epigenetic marks [12-15], cell wall components [10,16-19] and hormones [9] in the progression of in vitro organogenesis and embryogenesis in other systems, but no reports about the dynamics of these three cellular markers during in vitro early embryogenesis are available in two somatic embryogenesis pathways of the same species.…”

“…Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors of genome flexibility [13]. Previous studies have shown changes in DNA methylation levels and distribution patterns during microspore embryogenesis of Brassica napus and Hordeum vulgare [14,15], suggesting the existence of an epigenetic reprogramming after microspore induction to embryogenesis, but no information is available on DNA methylation dynamics during in vitro embryogenesis in trees.…”

Cork Oak Young and Traumatic Periderms Show PCD Typical Chromatin Patterns but Different Chromatin-Modifying Genes Expression

“…To achieve precise and reliable comparisons between signals observed at distinct cell differentiation stages, confocal analysis was performed using the same laser excitation and sample emission capture settings (El-Tantawy et al, 2014). The same procedure was applied to epifluorescence analysis on herbaceous sprigs and traumatic periderms preparations.…”