Genetic Interactions of the Arabidopsis Mediator Complex in the Regulation of Phenylpropanoid Metabolism and Global Gene Expression

The Mediator complex is a large, multi-subunit, transcription co-regulator that is conserved across eukaryotes. Studies of the Arabidopsis Mediator complex and its subunits have shown that it functions in nearly every aspect of plant development and fitness. In addition to revealing mechanisms of regulation of plant-specific pathways, studies of plant Mediator complexes have the potential to shed light on the conservation and divergence of Mediator structure and function across Kingdoms and plant lineages. So far, these studies indicate that, despite low sequence similarity between many orthologous subunits, the overall structure and function of Mediator is well conserved between Kingdoms. Several studies have also expanded our knowledge of Mediator to other plant species, opening avenues of investigation into the role of Mediator in plant adaptation and fitness. We summarize these insights to date in Chapter 1. The phenylpropanoid pathway is a major global carbon sink and its regulation is important not only for plant fitness but for the rational engineering of chemical and bioenergy feedstocks. The Arabidopsis Mediator complex subunits MED5a and MED5b are required for phenylpropanoid homeostasis and disruption of both paralogs results in an increase in phenylpropanoid accumulation. In contrast, the semi-dominant MED5b mutant reduced epidermal fluorescence4-3 (ref4-3) is dwarf and has constitutively repressed phenylpropanoid biosynthesis. In Chapter 2, we present the results of a forward genetic screen for suppressors of ref4-3. Whole-genome sequencing of the suppressors revealed that MED2, MED16, MED23, and particular residues in MED5b, are required for the phenotypes associated with ref4-3. Conversely, disruption of MED3 or MED25 has no discernable effect on ref4-3, indicating that the Mediator subunit interactions identified in our screen are specific. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression and that these changes are largely reversed by the suppressors. Our data also show that ref4-3 plants are upregulated in the expression of negative regulators of phenylpropanoid biosynthesis and identifies other pathways that may impinge on plant growth and phenylpropanoid metabolism. Together, our data highlight the functional interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. In Chapter 3, we explore the functions of MED2, MED5a/b, MED16, and MED23 beyond the phenylpropanoid pathway by comparing the impact of mutations in each on the Arabidopsis transcriptome. We find that these subunits have both overlapping and unique roles in gene expression, leading to the identification of several interesting functional relationships. We also show that, under our growth conditions, the mutants primarily affect the expression of genes in pathways related to biotic and abiotic stress. We also present evidence for a tissue specific role for MED23, as well as evidence for a role for MED23 in the production of alternative transcripts. Together, our data help disentangle the individual contributions of these MED subunits to global gene expression and suggest new avenues for future research into their functions