<p>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.</p>
<p>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 <a>decreased soluble
phenylpropanoid accumulation, reduced lignin content and dwarfism</a>. 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.</p>
<p>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.</p>
<p>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.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8155952 |
| Date | 02 August 2019 |
| Creators | Xiangying Mao (6714896) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/INVESTIGATING_THE_FUNCTIONAL_ROLE_OF_MED5_AND_CDK8_IN_ARABIDOPSIS_MEDIATOR_COMPLEX/8155952 |
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