Trees exhibit many unique aspects of plant biology, one of which is the formation of wood. Wood is one of the most important natural products with a multitude of applications. The formation of wood (xylogenesis) is a highly ordered developmental process involving the patterned division and differentiation of the vascular cambium into secondary xylem and phloem tissue types. The progression of xylogenesis developmental process requires differential gene expression across the different tissue types. The tight regulation of wood formation is mediated by genes that regulate cambial meristem differentiation and xylem cell fate. MicroRNAs (miRNAs) are a group of endogenous ~ 20 to 24 nt RNA molecules that down regulate gene expression at the post-transcriptional level. MicroRNAs have validated roles in developmental processes through the regulation of meristem cell differentiation and developmental patterning in plants. They have been shown to spatially regulate differential gene expression patterns at different developmental stages. Thus, the vascular cambium and its derivatives are excellent candidate tissues for miRNA discovery. The aim of this M.Sc. study was to isolate microRNAs from actively differentiating tissues of two tree species in order to determine possible gene regulatory networks involved in early meristem differentiation, tissue patterning and secondary vascular development. A small RNA library from two-month old in vitro Populus trichocarpa plantlets was constructed to identify putative miRNAs contributing to the early postembryonic development of trees. This library, in conjunction with computational prediction of poplar miRNA homologues and precursor secondary structures, was used to identify a total of 72 poplar miRNAs. Sixteen of these were putative novel miRNAs, belonging to nine new miRNA families. A genome-wide search identified 55 putative target genes for the newly identified miRNAs. The target genes had diverse biological roles in developmental events and maintenance of cellular homeostasis. A number of the predicted targets were involved in plant organ development such as leaf cell fate, floral organ development and meristem differentiation. Other targets were involved in response to hormones, such as growth regulating factors and signaling proteins. Additionally, several targets were related to cellular metabolic processes, such as protein modification and ubiquitination. By isolating miRNAs from developing poplar plantlets, we were able to suggest possible developmental programmes under the control of these molecules, possibly affecting early seedling development and growth. A similar approach was used to identify miRNAs from three differentiating vascular tissues of Eucalyptus grandis. Isolated small RNA sequences were used in a search against all available bacterial artificial chromosome (BAC) shotgun genomic sequences from an ongoing Eucalyptus camaldulensis genome sequencing initiative at the Kazusa DNA Research Institute in Japan. We were able to characterize the first Eucalyptus miRNAs, and identified 48 putative miRNAs grouping into thirteen gene families. Twenty of the miRNAs belong to five families previously identified in other plant species, whereas the remaining 28 miRNAs grouped into eight putative novel miRNA families. Searches of the Populus and Arabidopsis annotated genomes revealed 45 putative target genes for the new families. Targets of particular interest included transcription factors involved in cell fate determination, including a MADS-box transcription factor involved in xylem formation. Further targets included auxin signaling proteins and auxin response factors, which could play a significant role during auxin regulation of vascular development. Expression profiling of the putative miRNAs using quantitative RT-PCR revealed that a number of the miRNAs exhibited differential expression patterns across xylogenic and non-xylogenic tissues. One miRNA showed expression in a single vascular tissue, whereas others were expressed at varying levels across the vascular tissues. This observation indicates a possible role for these putative miRNAs during vascular development and differentiation in eucalypt trees. In this study we used a combination of small RNA library construction and computational prediction to identify microRNAs from two tree species. We identified a total of 120 putative miRNAs grouping into 31 families. Of these, 44 group into 17 putative novel tree-specific miRNAs. This study has allowed the identification of novel miRNAs from a unique set of tissues, and has contributed to the ever-growing number of plant-specific miRNAs. The results of this study further contribute to our expanding knowledge of the unique developmental process of vascular tissue differentiation of perennial woody plants such as Eucalyptus and Populus species.
Dissertation (MSc (Genetics))--University of Pretoria, 2008.