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Protein coding genes have been extensively studied in both plant and animal genomes, while non-coding portions of the genomes were considered not relevant for a long time. This was due to the fact that non-coding led immediately to not functional, until the discovery of let-7, the first conserved miRNA, in Caenorhabditis elegans. From here on, several studies on small RNAs (sRNAs) were performed, while long non-coding RNAs (lncRNAs) have risen to attention in the last two decades, also because of their usage as diagnostic biomarkers in cancer. Studies to assign function to RNAs have progressed more slowly in plants compared to the animal kingdom and there is still a lot to explore even in the protein coding space, above all if we consider huge genomes like Norway spruce and Scots pine, so the non-coding part of the genome still represents an abyss to discover. In my PhD I mostly focused on a subclass of non-coding RNAs in Norway spruce and aspen. Long non-coding RNAs are considered arbitrarily longer than 200 nucleotides (nt) and can have one small open reading frame (sORF, length < 300 nt) coding for a short peptide (not a complete protein). lncRNAs tend to be expressed at lower levels than genes, but with precise spatio-temporal patterns. They are mostly expressed in particular tissues, stages of a biological process and/or particular conditions, that are often related to biotic or abiotic stresses. They have low levels of sequence homology conservation, even in close related species. In particular, I studied the class of lncRNAs located in the intergenic space, the long intergenic non-coding RNAs (lincRNAs). 

In the first part of this thesis, I developed a pipeline to identify lincRNAs. This pipeline allows to identify in silico bona fide lincRNAs starting from an RNA-Sequencing dataset. It is an ensemble method, considering different tools and the characteristics of lincRNAs. 

In the second part of this thesis, I focused on functionally annotating lincRNAs. To achieve this challenge, I decided to use the guilt-by-association strategy. This method relies on a co-expression network containing both lincRNAs and protein coding genes. Through a functional enrichment of the protein coding genes, it is possible to transfer the same annotation to a lincRNA co-expressed in the same module. I have also tried to relate lincRNAs to a possible function in the de novo methylation of DNA via the RdDM pathway in Norway spruce.

In the last part of this thesis, I identified lincRNAs expressed during leaf development in aspen and produced CRISPR-Cas9 mutants lacking the sequence of two lincRNAs in order to provide a functional validation. 

In general, RNA-Sequencing has enabled and advanced the identification of lincRNAs, and this thesis demonstrates an implemented strategy to identify and assign putative functional information to lincRNAs, deepening the knowledge in the non-coding abyss.

Trees have evolved an impressive array of strategies to cope with the challenges of having a long and sessile life. Not only must they withstand a fluctuating climate, but they also face instantaneous pressures from herbivores and other attackers. To protect themselves, plants can produce defence compounds, many of which are highly specialised and taxon specific. Within the Salicaceae family, a key group of such defence compounds are the salicinoid phenolic glycosides (SPGs). Many structural variants of SPGs have been identified, in which acyl groups (e.g., cinnamoyl, benzoyl, and acetyl) are common. Some of these SPGs can have toxic and deterrent effects against attackers, and a few are known for their medicinal properties in humans. However, the biological function of most SPGs in planta remains unclear, and the causal enzymes for the majority of SPGs are yet to be identified. 

The aim of this thesis was to uncover the genetic basis of SPG biosynthesis in European aspen (Populus tremula L.) and to determine the extent of ontogenic and organ-specific variation among individuals. To achieve this, SPG variation within a collection of natural aspen, the Swedish aspen (SwAsp) collection, was investigated using an integrative multi-omic approach. By analysing the metabolome and transcriptome of multiple leaf ages from aspen individuals with varying levels of cinnamoyl and acetylated SPGs, a set of candidate transferases and novel putative SPGs were identified. These analyses further suggested that young leaf tissue is a highly active site of SPG biosynthesis, compared with mature leaves.  

To extend this analysis, we performed genome-wide association studies on transcriptomic and metabolomic data from leaf buds to identify genomic regions associated with variation in SPG abundance and gene expression. These data were integrated into a systems genetics network, visualising the intricate relationship between candidate genes and the diversity of SPGs. Among the candidates, an acyltransferase was highly associated with both acetyl- and cinnamoyl-SPGs. Heterologous expression assays in Escherichia coli (E. coli) confirmed its acetylation activity. In line with these findings, overexpression of the gene in planta led to increased levels of acetyl-SPGs, suggesting acetylation activity of the enzyme.

In summary, these results have enhanced our understanding of SPG biosynthesis and provide a foundation for future studies aimed at elucidating the in planta function of the remaining candidate genes.