Identification and Characterization of a Novel Antisense Non-Coding RNA from the T-DNA Locus of Rosewood, an Activation-Tagged Poplar Tree

Abstract

Activation-tagging is a functional genomics technique where strong enhancers are inserted randomly into target genomes and can over-activate endogenous genes. When interesting phenotypes occur as a result, it can lead to the discovery of new genes or the characterization of known genes. From a population of ~2000 activation-tagged poplar trees created in our lab, a mutant with red xylem was identified and named rosewood. Based on previously identified poplar mutants, the red-wood phenotype suggested a change in lignin biosynthesis, but work done during this thesis revealed that the red wood is actually a result of increased anthocyanin content. Efforts to engineer edible plants for increased anthocyanin content are on-going, due to their antioxidant properties and wide-ranging health benefits to humans. Although poplar trees are not eaten by humans, discovery of a novel aspect of anthocyanin regulation could have implications beyond the Populus genus. In this thesis, rosewood’s single T-DNA insertion was identified and characterized. Rosewood’s case turned out to be atypical in the analysis of activation-tagged mutants: no surrounding protein-coding genes could be implicated in rosewood’s phenotype, and instead several novel, non-coding, upregulated RNAs were discovered next to the T-DNA enhancers. Only four cases of activation-tagged non-coding RNAs appear in the literature, only one of which involved a novel gene. To date, there is no clear methodology on how to annotate plant non-coding RNAs discovered in this fashion. A major part of this thesis consisted of setting up a workflow for this purpose, which accounts especially for the current computational tools that can be used for microRNA prediction in plants. Based on molecular data and bioinformatics analyses, it is proposed that the locus at the T-DNA insertion contains one or more natural-antisense microRNAs and that these are somehow responsible for rosewood’s increased anthocyanin content. The results of a phenotype recapitulation attempt in stem tissue using one of the novel transcripts are discussed.