Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.
BackgroundMicrospore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.ResultsWe have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants.ConclusionThe present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.
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.
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.
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