Carl Gunnar Fossdal

Main conclusion: Dynamic DNA methylation differences between epitypes throughout the annual cycle, circannual clocks affect methylation levels, stable methylation marks in the promoters of 30 candidate genes, embryo–adult transmission of methylation marks. DNA methylation can change DNA properties, affecting chromatin accessibility, gene expression, and phenotypic variation. In clonal Norway spruce, warmer (WE) versus cooler (CE) embryogenic conditions produce phenotypically different trees. This climatic memory, induced during embryogenesis, remains stable in the resulting epitype trees, and the epigenetically altered timing of bud phenology persists between WE and CE epitypes even after decades under common garden conditions. We examined DNA methylation patterns in 14-year-old epitypes throughout the annual developmental cycle. Using targeted bisulfite sequencing, we screened for differential DNA methylation over a 3000 bp region in 2744 genes related to the epigenetic machinery, circadian clock, and phenology. Clustering DNA methylation differences in the CG context clearly separated epitype trees, confirming epigenetic mark differences. Differences in methylation of cytosines in all contexts were highly dynamic and varied markedly among annual developmental stages, suggesting the existence of circannual clocks affecting methylation levels in the studied genomic regions. Most stable methylation marks were identified in CG contexts, fewer in CHG and none in CHH contexts, consistent with differences in inheritance among methylation contexts. We identified stable CG and CHG methylation marks in the promoter regions of 30 specific genes. Two ARGONAUTE genes and 4 other genes exhibited stable marks across all time points for CE or WE, and putative embryo–adult transmission for some genes. These findings indicate that DNA methylation marks maintained in genomic regions throughout the annual cycle may contribute to an induced epigenetic memory established in embryos and later manifested as phenologically different epitype trees.

Seed multiplication of perennial grasses outside their area of adaptation is common practice globally. This also applies to timothy ( Phleum pratense L.), the most important forage grass in Arctic Norway (67°–71° N). Early research suggests little risk for loss of winter hardiness if seed multiplication of Arctic timothy is limited to one or two generations in the south, but recent experiences by North Norwegian farmers suggest otherwise. A study was conducted in which original seed of two northerly adapted timothy varieties ‘Engmo’ (an old landrace originating at 69° N) and ‘Noreng’ (a synthetic variety consisting of 10 clones of ‘Engmo’ and four clones of the South‐Norwegian variety ‘Grindstad’) was multiplied for one, two or three generations at 70° N, 64° N and 58° N. Plants raised from the different generations at each site were subjected to freezing tests to determine LT ₅₀ and ice encasement (IE) tests to determine LD ₅₀ , both tests followed by the weighing of regrowth of surviving plants. On average for generations and compared with the original seed, we found no changes in freezing tolerance or IE tolerance after seed production at 70° N, but significant losses in freezing tolerance of both varieties after seed production at 64° N and in particular at 58° N. The fact that major shifts occurred already in the first generation and were later, at least to some extent, reversible and influenced by the winter before each seed harvest raises the possibility that epigenetic memory of environmental conditions was transferred to the seed progeny in addition to the classical genetic effects.

Gymnosperms are long-lived, cone-bearing seed plants that include some of the most ancient extant plant species. These relict land plants have evolved to survive in habitats marked by chronic or episodic stress. Their ability to thrive in these environments is partly due to their phenotypic flexibility, and epigenetic regulation likely plays a crucial part in this plasticity. We review the current knowledge on abiotic and biotic stress memory in gymnosperms and the possible epigenetic mechanisms underlying long-term phenotypic adaptations. We also discuss recent technological improvements and new experimental possibilities that likely will advance our understanding of epigenetic regulation in these ancient and hard-to-study plants.

Small non-coding RNA (sRNA) and the various other RNA species have tissue specific expression profiles. However, some size fractions may be missed or left out in the sequencing library preparation process, sequence data processing and downstream bioinformatic analysis. Here, we performed sRNA profiling in embryonic and vegetative tissues to elucidate a more complete picture of the gymnosperm sRNA populations than previously reported. We detected a novel group of sRNA between 31–34 nt in our Norway spruce sequencing data, with a prevalence in the 32–33 nt fraction, defined as the long small RNAs (lsRNAs). These lsRNAs were predominantly present in embryonic samples of Norway spruce. An in-silico analysis indicated that these lsRNA could originate from transfer RNAs (tRNA) and other non-coding transcripts and thus may target other non-coding RNAs (including tRNAs) or target repetitive elements such as transposons. We identified 18 putative orthologs of genes involved in Piwi-interacting RNA (piRNA) biogenesis but no spruce proteins were true homologs to the animal Piwi-proteins, thus lsRNAs cannot be considered as plant analogs to the animal piRNAs. Among the lsRNAs, tRNA-derived sequences from Asp, Glu and His iso-acceptors were in a majority and these sequences showed 3’ or 5’- bias dependent on the iso-acceptor type putatively targeted. The lsRNA sized fraction was detected in seeds of all the gymnosperms examined and in Arabidopsis thaliana, suggesting that these comprise a conserved type of sRNAs between gymnosperms and angiosperms. lsRNA levels differed significantly among tissue types and developmental stages, and interestingly their expression was impacted by epitype-inducing temperature conditions. The lsRNAs add to the complexity of the small RNA world and may play a role in epigenetic regulation of gene expression in plants.

Plants must adapt with increasing speed to global warming to maintain their fitness. One rapid adaptation mechanism is epigenetic memory, which may provide organisms sufficient time to adapt to climate change. We studied how the perennial Fragaria vesca adapted to warmer temperatures (28°C vs. 18°C) over three asexual generations. Differences in flowering time, stolon number, and petiole length were induced by warmer temperature in one or more ecotypes after three asexual generations and persisted in a common garden environment. Induced methylome changes differed between the four ecotypes from Norway, Iceland, Italy, and Spain, but shared methylome responses were also identified. Most differentially methylated regions (DMRs) occurred in the CHG context, and most CHG and CHH DMRs were hypermethylated at the warmer temperature. In eight CHG DMR peaks, a highly similar methylation pattern could be observed between ecotypes. On average, 13% of the differentially methylated genes between ecotypes also showed a temperature-induced change in gene expression. We observed ecotype-specific methylation and expression patterns for genes related to gibberellin metabolism, flowering time, and epigenetic mechanisms. Furthermore, we observed a negative correlation with gene expression when repetitive elements were found near (±2 kb) or inside genes. In conclusion, lasting phenotypic changes indicative of an epigenetic memory were induced by warmer temperature and were accompanied by changes in DNA methylation patterns. Both shared methylation patterns and transcriptome differences between F. vesca accessions were observed, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic variation.

A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic memory that can be transmitted both meiotically and mitotically. Such epigenetic memories may increase phenotypic variation to global warming and provide time for adaptation to occur through classical genetic selection. The goal of this study was to understand how warmer temperature conditions experienced during sexual and asexual reproduction affect the transcriptomes of different strawberry (Fragaria vesca) ecotypes. We let four European F. vesca ecotypes reproduce at two contrasting temperatures (18 and 28°C), either asexually through stolon formation for several generations, or sexually by seeds (achenes). We then analyzed the transcriptome of unfolding leaves, with emphasis on differential expression of genes belonging to the epigenetic machinery. For asexually reproduced plants we found a general transcriptomic response to temperature conditions but for sexually reproduced plants we found less significant responses. We predicted several splicing isoforms for important genes (e.g. a SOC1, LHY, and SVP homolog), and found significantly more differentially presented splicing event variants following asexual vs. sexual reproduction. This difference could be due to the stochastic character of recombination during meiosis or to differential creation or erasure of epigenetic marks during embryogenesis and seed development. Strikingly, very few differentially expressed genes were shared between ecotypes, perhaps because ecotypes differ greatly both genetically and epigenetically. Genes related to the epigenetic machinery were predominantly upregulated at 28°C during asexual reproduction but downregulated after sexual reproduction, indicating that temperature-induced change affects the epigenetic machinery differently during the two types of reproduction.

Temperature conditions experienced during embryogenesis and seed development may induce epigenetic changes that increase phenotypic variation in plants. Here we investigate if embryogenesis and seed development at two different temperatures (28 vs. 18°C) result in lasting phenotypic effects and DNA methylation changes in woodland strawberry (Fragaria vesca). Using five European ecotypes from Spain (ES12), Iceland (ICE2), Italy (IT4), and Norway (NOR2 and NOR29), we found statistically significant differences between plants from seeds produced at 18 or 28°C in three of four phenotypic features investigated under common garden conditions. This indicates the establishment of a temperature‐induced epigenetic memory‐like response during embryogenesis and seed development. The memory effect was significant in two ecotypes: in NOR2 flowering time, number of growth points and petiole length were affected, and in ES12 number of growth points was affected. This indicates that genetic differences between ecotypes in their epigenetic machinery, or other allelic differences, impact this type of plasticity. We observed statistically significant differences between ecotypes in DNA methylation marks in repetitive elements, pseudogenes, and genic elements. Leaf transcriptomes were also affected by embryonic temperature in an ecotype‐specific manner. Although we observed significant and lasting phenotypic change in at least some ecotypes, there was considerable variation in DNA methylation between individual plants within each temperature treatment. This within‐treatment variability in DNA methylation marks in F. vesca progeny may partly be a result of allelic redistribution from recombination during meiosis and subsequent epigenetic reprogramming during embryogenesis.

Plants must quickly adapt to a changing environment in order to maintain their fitness. One rapid adaptation mechanism that promotes plasticity is epigenetic memory, which may provide long-lived organisms the precious time needed to adapt to climate change. In this study, we used the perennial plant Fragaria vesca as a model to determine how the methylome and transcriptome adapt to elevated temperatures (28 vs. 18 °C) over three asexual generations. Changes in flowering time, stolon number, and petiole length were induced in responses to temperature treatment in one or more ecotypes after three asexual generations in a manner indicative of an epigenetic memory. Induced methylome changes differed between four different ecotypes from Norway, Iceland, Italy, and Spain, but there were also some shared responses. Elevated temperature conditions induced significant phenotypic and methylation changes, particularly in the Norwegian ecotype. Most of the differentially methylated regions (DMRs) were in the CHG context, and most CHG and CHH DMRs were hypermethylated. Notably, the four ecotypes shared only eight CHG DMR peaks. Several differentially methylated genes (DMGs) also showed a change in gene expression. Ecotype-specific methylation and expression patterns were observed for genes related to gibberellin metabolism, flowering time, epigenetics. Furthermore, when repetitive elements (REs) were found near (±2 kb) or inside a gene, they showed a negative correlation with gene expression. In conclusion, phenotypic changes induced by elevated temperatures during asexual reproduction were accompanied by changes in DNA methylation patterns. Also, positional influences of REs impacted gene expression, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic plasticity in F. vesca .

MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim of this study was to identify and characterize novel and conserved microRNAs expressed in methyl jasmonate-treated Scots pine needles. In addition, potential precursor sequences and target genes of the identified miRNAs were determined by alignment to the Pinus unigene set. Potential precursor sequences were identified using the miRAtool, conserved miRNA precursors were also tested for the ability to form the required stem-loop structure, and the minimal folding free energy indexes were calculated. By comparison with miRBase, 4975 annotated sequences were identified and assigned to 173 miRNA groups, belonging to a total of 60 conserved miRNA families. A total of 1029 potential novel miRNAs, grouped into 34 families were found, and 46 predicted precursor sequences were identified. A total of 136 potential target genes targeted by 28 families were identified. The majority of previously reported highly conserved plant miRNAs were identified in this study, as well as some conserved miRNAs previously reported to be monocot specific. No conserved dicot-specific miRNAs were identified. A number of potential gymnosperm or conifer specific miRNAs were found, shared among a range of conifer species.

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.