INVESTIGATING THE FUNCTIONAL ROLE OF MED5 AND CDK8 IN ARABIDOPSIS MEDIATOR COMPLEX

Abstract

The Mediator (Med) complex\ncomprises about 30 subunits and is a transcriptional co-regulator in eukaryotic\nsystems. The core Mediator complex, consisting of the head, middle and tail\nmodules, functions as a bridge between transcription factors and basal transcription\nmachinery, whereas the CDK8 kinase module can attenuate Mediator’s ability to function\nas either a co-activator or co-repressor. Many Arabidopsis Mediator subunit has\nbeen functionally characterized, which reveals critical roles of Mediator in many\naspects of plant growth and development, responses to biotic and abiotic\nstimuli, and metabolic homeostasis. Traditional genetic and biochemical\napproaches laid the foundation for our understanding of Mediator function, but\nrecent transcriptomic and metabolomic studies have provided deeper insights\ninto how specific subunits cooperate in the regulation of plant metabolism. In Chapter\n1, we highlight recent developments in the investigation of Mediator and plant\nmetabolism, with emphasis on the large-scale biology studies of med mutants.\n\nWe previously found that MED5, an\nArabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid\nhomeostasis. A semi-dominant mutation (reduced\nepidermal fluorescence 4-3, ref4-3)\nthat causes a single amino acid substitution in MED5b functions as a strong\nsuppressor of the pathway, leading to decreased soluble\nphenylpropanoid accumulation, reduced lignin content and dwarfism. In\ncontrast, loss of MED5a and MED5b (med5)\nresults in increased levels of phenylpropanoids. In Chapter 2, we present our\nfinding that ref4-3 requires CDK8, a\nMediator kinase module subunit, to repress plant growth even though the\nrepression of phenylpropanoid metabolism in ref4-3\nis CDK8-independent. Transcriptome profiling revealed that salicylic acid\n(SA) biosynthesis genes are up-regulated in a CDK8-dependent manner in ref4-3, resulting in hyper-accumulation\nof SA and up-regulation of SA response genes. Both growth repression and\nhyper-accumulation of SA in ref4-3\nrequire CDK8 with intact kinase activity, but these SA phenotypes are not\nconnected with dwarfing. In contrast, mRNA-sequencing (RNA-seq) analysis\nrevealed the up-regulation of a DNA J protein-encoding gene in ref4-3, the elimination of which partially\nsuppresses dwarfing. Together, our study reveals genetic interactions between\nMediator tail and kinase module subunits and enhances our understanding of\ndwarfing in phenylpropanoid pathway mutants.\n\nIn Chapter 3, we characterize\nother phenotypes of med5 and ref4-3, and find that in addition to the\nup-regulated phenylpropanoid metabolism, med5\nshow other interesting phenotypes including hypocotyl and petiole elongation as\nwell as accelerated flowering, all of which are known collectively as the shade\navoidance syndrome (SAS), suggesting that MED5 antagonize shade avoidance in\nwild-type plants. In contrast, the constitutive ref4-3 mutant protein inhibits the process, and the stunted growth\nof ref4-3 mutants is substantially\nalleviated by the light treatment that triggers SAS. Moreover, ref4-3 mimics the loss-of-function med5 mutants in maintaining abscisic\nacid (ABA) levels under both normal and drought growth conditions. The\nphenotypic characterization of med5\nmutants extend our understanding of the role of Mediator in SAS and ABA\nsignaling, providing further insight into the physiological and metabolic\nresponses that require MED5.\n\nIn Chapter 4, we explore\nthe function of MED5 and CDK8 in gene expression regulation by investigating\nthe effect of mutations in Mediator including med5, ref4-3, cdk8-1 and ref4-3 cdk8-1 on genome-wide Pol II distribution. We find that loss\nof MED5 results in loss of Pol II occupancy at many target genes. In contrast, many\ngenes show enriched Pol II levels in ref4-3,\nsome of which overlap with those showing reduced Pol II occupancy in med5. In addition, Pol II occupancy is\nsignificantly reduced when CDK8 is disrupted in ref4-3. Our results help to narrow down the direct gene targets of\nMED5 and identify genes that may be closely related to the growth deficiency observed\nin ref4-3 plants, providing a\ncritical foundation to elucidate the molecular function of Mediator in\ntranscription regulation.