An investigation into the molecular basis of secondary vascular tissue formation in poplar and Arabidopsis with an emphasis on the role of auxin and the auxin response factor MONOPTEROS

The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value