INVESTIGATING THE FUNCTIONAL ROLE OF MED5 AND CDK8 IN ARABIDOPSIS MEDIATOR COMPLEX
Creators: name: Mao, Xiangying
Publisher: name: Purdue University Graduate School
Year: 2019
Subjects: Bioinformatics and computational biology not elsewhere classified
Plant cell and molecular biology
Animal cell and molecular biology
Plant biology not elsewhere classified
Biochemistry and cell biology not elsewhere classified

description: The Mediator (Med) complex comprises about 30 subunits and is a transcriptional co-regulator in eukaryotic systems. The core Mediator complex, consisting of the head, middle and tail modules, functions as a bridge between transcription factors and basal transcription machinery, whereas the CDK8 kinase module can attenuate Mediator’s ability to function as either a co-activator or co-repressor. Many Arabidopsis Mediator subunit has been functionally characterized, which reveals critical roles of Mediator in many aspects of plant growth and development, responses to biotic and abiotic stimuli, and metabolic homeostasis. Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In Chapter 1, we highlight recent developments in the investigation of Mediator and plant metabolism, with emphasis on the large-scale biology studies of <i>med</i> mutants. We previously found that MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi-dominant mutation (<i>reduced epidermal fluorescence 4-3</i>, <i>ref4-3</i>) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. In contrast, loss of MED5a and MED5b (<i>med5</i>) results in increased levels of phenylpropanoids. In Chapter 2, we present our finding that <i>ref4-3</i> requires CDK8, a Mediator kinase module subunit, to repress plant growth even though the repression of phenylpropanoid metabolism in <i>ref4-3 </i>is CDK8-independent. Transcriptome profiling revealed that salicylic acid (SA) biosynthesis genes are up-regulated in a CDK8-dependent manner in <i>ref4-3,</i> resulting in hyper-accumulation of SA and up-regulation of SA response genes. Both growth repression and hyper-accumulation of SA in <i>ref4-3</i> require CDK8 with intact kinase activity, but these SA phenotypes are not connected with dwarfing. In contrast, mRNA-sequencing (RNA-seq) analysis revealed the up-regulation of a DNA J protein-encoding gene in <i>ref4-3</i>, the elimination of which partially suppresses dwarfing. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants. In Chapter 3, we characterize other phenotypes of <i>med5</i> and <i>ref4-3</i>, and find that in addition to the up-regulated phenylpropanoid metabolism, <i>med5</i> show other interesting phenotypes including hypocotyl and petiole elongation as well as accelerated flowering, all of which are known collectively as the shade avoidance syndrome (SAS), suggesting that MED5 antagonize shade avoidance in wild-type plants. In contrast, the constitutive <i>ref4-3 </i>mutant protein inhibits the process, and the stunted growth of <i>ref4-3 </i>mutants is substantially alleviated by the light treatment that triggers SAS. Moreover, <i>ref4-3</i> mimics the loss-of-function <i>med5</i> mutants in maintaining abscisic acid (ABA) levels under both normal and drought growth conditions. The phenotypic characterization of <i>med5</i> mutants extend our understanding of the role of Mediator in SAS and ABA signaling, providing further insight into the physiological and metabolic responses that require MED5. In Chapter 4, we explore the function of MED5 and CDK8 in gene expression regulation by investigating the effect of mutations in Mediator including <i>med5</i>, <i>ref4-3</i>, <i>cdk8-1</i> and <i>ref4-3 cdk8-1</i> on genome-wide Pol II distribution. We find that loss of MED5 results in loss of Pol II occupancy at many target genes. In contrast, many genes show enriched Pol II levels in <i>ref4-3</i>, some of which overlap with those showing reduced Pol II occupancy in <i>med5</i>. In addition, Pol II occupancy is significantly reduced when CDK8 is disrupted in <i>ref4-3</i>. Our results help to narrow down the direct gene targets of MED5 and identify genes that may be closely related to the growth deficiency observed in <i>ref4-3</i> plants, providing a critical foundation to elucidate the molecular function of Mediator in transcription regulation.