Abstract
Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. 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. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for 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 formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.
Highlights
Adaptation to the changing environments is vitally important for long-lived plant species like forest trees
We show an extensive number of miRNAs that can target epigenetic regulators including those modifying DNA and histone methylation, and small RNA (sRNA) pathways genes, supporting the notion that these predicted miRNAs and their target genes could be among central players in epigenetic memory formation
Three clear read length peaks were found in the embryonic sRNA pool after the trimming—and these corresponded with the lengths of 24–23, 21–20, and 31–32 nt (Figure 1A)
Summary
Adaptation to the changing environments is vitally important for long-lived plant species like forest trees. Much remains to be known about the enigmatic repertoire of epigenetic mechanisms that operate in forest trees but earlier studies firmly confirmed the presence of epitype inducing (EpI) temperature-dependent plant phenotypes (Yakovlev et al, 2012; Liu et al, 2015) and significant transcriptomic changes in such epitypes (Yakovlev et al, 2016) Both long non-coding RNAs (lncRNAs) and small RNAs (sRNAs) such as short non-coding RNAs are known to be core components of signaling networks involved in epigenetic modification, transcription regulation and participate in transgenerational epigenetic inheritance in plants and animals (Hauser et al, 2011; Heard and Martienssen, 2014). Epigenetic regulation can be mediated through a dynamic interplay between sRNAs, DNA methylation, histone modifications, histone variants, and chromatin architecture, which together modulate transcriptional silencing, activation and the accessibility of DNA in variety of ways (Heo and Sung, 2011; Simon and Meyers, 2011; Lee, 2012; Mirouze, 2012; Bond and Baulcombe, 2014)
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