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MicroRNAs (miRNA, miR) are short non-protein coding RNA molecules that are involved in both the nuclear and the posttranscriptional regulation of gene expression. miRNAs are endogenous mediators of RNA-interference forming part of the epigenetic machinery and influence gene expression post-transcriptionally without affecting the DNA sequence. In Norway spruce, epigenetic memory is established in response to the temperature conditions during embryogenesis and it affects the timing of bud burst and bud set, vitally important adaptive traits for long-lived forest species. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. Developing Norway spruce embryos possesses a more complex small non-coding RNA (sRNA) structure than that reported for other somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature-dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the placement of epigenetic marks. Major features of miRNAs are related to their pleiotropic and synergistic actions, whereby a single miRNA can have several potential mRNA targets, and a single mRNA usually also has multiple miRNA binding sites. Moreover, miRNAs binding to a single mRNA often acts in a synergistic fashion. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation. Further functional and expression studies are necessary in order to elucidate the common miRNA-mediated regulatory mechanisms that underlie memory formation in plants. The use of artificial miRNAs, as well as overexpression and knockout/down of both miRNAs and their targets, will be the best techniques for determining the specific roles of individual miRNAs in memorizing the response to environmental stresses. In the present minireview, we illustrate the current knowledge regarding the epigenetics–miRNA regulatory networks aiming to provide biological insights into epigenetic memory formation in plants with a particular emphasis in spruce.

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5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous. Here, employing reversed-phase HPLC coupled with sensitive mass spectrometry, we demonstrated that, unlike 5caC, both 5hmC and 5fC are detectable in non-negligible quantities in the DNA of a conifer, Norway spruce. Remarkably, whereas 5hmC content of spruce DNA is approximately 100-fold lower relative to human colorectal carcinoma cells, the levels of both - 5fC and a thymine base modification, 5-hydroxymethyluracil, are comparable in these systems. We confirmed the presence of modified DNA bases by immunohistochemistry in Norway spruce buds based on peroxidase-conjugated antibodies and tyramide signal amplification. Our results reveal the presence of specific range of noncanonical DNA bases in conifer genomes implying potential roles for these modifications in plant development and homeostasis.

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Main conclusion: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burstrelated genes in genetically identical Norway spruce epitypes in a manner usually associated with ecotypes. In Norway spruce, a temperature-dependent epigenetic memory established during embryogenesis affects the timing of bud burst and bud set in a reproducible and predictable manner. We hypothesize that the clinal variation in these phenological traits, which is associated with adaptation to growth under frost-free conditions, has an epigenetic component. In Norway spruce, dehydrins (DHNs) have been associated with extreme frost tolerance. DHN transcript levels decrease gradually prior to flushing, a time when trees are highly sensitive to frost. Furthermore, EARLY BUD BREAK 1 genes (EBB1) and the FT-TFL1- LIKE 2-gene (PaFTL2) were previously suggested to be implied in control of bud phenology. Here we report an analysis of transcript levels of 12 DHNs, 3 EBB1 genes and FTL2 in epitypes of the same genotype generated at different epitype-inducing temperatures, before and during spring bud burst. Earlier flushing of epitypes originating from embryos developed at 18 C as compared to 28 C, was associated with differential expression of these genes between epitypes and between buds and last year’s needles. The majority of these genes showed significantly different expressions between epitypes in at least one time point. The general trend in DHN expression pattern in buds showed the expected reduction in transcript levels when approaching flushing, whereas, surprisingly, transcript levels peaked later in needles, mainly at the moment of bud burst. Collectively, our results demonstrate that the epigenetic memory of temperature during embryogenesis affects bud burst phenology and expression of the bud burst-related DHN, EBB1 and FTL2 genes in genetically identical Norway spruce epitypes.