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Recent discoveries have highlighted multiple mitotically and meiotically inherited alterations in gene expression that could not be explained solely by changes in the DNA sequence but were acknowledged as epigenetic. The modern view on epigenetics considers it as an integral part of genetics. Epigenetic mechanisms are encoded by genes in the genome and contribute to an essential part of genomic diversity, significantly extending its regulatory abilities. Epigenetic mechanisms involve molecular chromatin alterations through DNA methylation and histone modifications, as well as, complex non-coding RNAs and related enzyme machinery leading to changes in gene expression and resulting in changing phenotypes. In plants, epigenetic mechanisms may occur over their lifetime and across multiple generations, and can contribute substantially to phenotypic plasticity, stress responses, disease resistance, acclimation and adaptation to habitat conditions. In this review, we summarize recent advances with regards to Norway spruce epigenomics. We first consider the large size of the spruce genome that is linked to epigenetic mechanisms and why epigenomics is vitally important for spruce. Then, we discuss the molecular machinery supporting epigenetic mechanisms in Norway spruce and putative gene models involved. We presume substantial extension of gene families of epigenetic regulators and non-coding RNAs, especially in reproductive tissues. Norway spruce was the first species among forest trees in which epigenetic memory and epigenetic mechanisms were studied. The induction of an epigenetic memory during sexual reproduction and somatic embryogenesis has been described in Norway spruce. We discuss the latest results of epigenomic variation and epigenetic memory studies in Norway spruce and define the future perspectives for epigenetic studies. However, there is still a long way to decipher how the epigenetic mechanisms are involved in maintaining the stability of the spruce epigenome, how the epigenome is set to produce the epigenetic memory phenomenon and how these may result in an increased rate of adaptation to a changing environment.

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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.