Changes in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley

26 p.-5 fig.The microspore can be induced in vitro, under specific stress treatments, to deviate from its gametophytic development and to reprogram towards embryogenesis, becoming a totipotent cell and forming haploid embryos which 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 fibre which regulates gene expression. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation, however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood.In this work, the dynamics of global DNA methylation levels and distribution patterns have been analyzed during microspore reprogramming to embryogenesis and during pollen development in Hordeum vulgare. Quantification of global DNA methylation levels and 5-methyl-deoxy-cytidine (5mdC) immunofluorescence has been conducted at specific stages of pollen development and after reprogramming to embryogenesis, to analyze the epigenetic changes that accompany the change of developmental programme and cell fate. 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 lower DNA methylation signal. After the inductive stress treatment, low methylation levels and faint 5mdC signal were observed on nuclei of reprogrammed microspores and 2-4 cell proembryos. This data revealed a global DNA hypomethylation during the change of the developmental programme and first embryogenic divisions, in contrast with the hypermethylation of generative and sperm cells of the male germline accomplished during pollen maturation, suggesting an epigenetic regulation after microspore embryogenesis induction. At later embryogenesis stages global DNA methylation progressively increased, accompanying embryo development and differentiation events, like in zygotic embryos, supporting that DNA methylation is critical for the regulation of microspore embryogenesis gene expression.Work supported by Spanish Ministry of Economy and Competitivity (MINECO) project BFU2011-23752 and Spanish National Research Council (CSIC) project PIE 201020E038. AA-ET is recipient of a JAE predoctoral fellowship of CSIC (JAEPre2010-052).Peer reviewe