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.
