No abstract

Mediator is a multisubunit transcriptional co-regulator that is involved in the regulation of an array of processes including plant metabolism. The pathways regulated by Mediator-dependent processes include those for the synthesis of phenylpropanoids (MED5), cellulose (MED16), lipids (MED15 and CDK8), and the regulation of iron homeostasis (MED16 and MED25). 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 this review, we highlight recent developments in the investigation of Mediator and plant metabolism, with particular emphasis on the large-scale biology studies of med mutants.

Summary The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semidominant mutation (ref4‐3) 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. By contrast, loss of MED5 results in increased concentrations of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4‐3 and found that ref4‐3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA‐sequencing (RNA‐seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4‐3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4‐3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, misregulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.

Plant metabolic networks are precisely regulated by the spatial and temporal expression of suites of genes. Among the various transcription (co)factors, a multi‐protein complex, Mediator has been identified as a hub for transcription regulation. The core Mediator complex, comprising the head, middle and tail domains, functions as a bridge between transcription factors and basal transcription machinery, whereas the CDK8 kinase module plays a repressive regulatory role. It is still unclear, however, how the kinase module represses target genes especially in planta. Using a forward genetic screen, our lab determined that MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi‐dominant mutant (ref4‐3) characterized by a single amino acid substitution in MED5a (G383S) was isolated as a strong suppressor of phenylpropanoid pathway, indicated by decreased soluble phenylpropanoid metabolite accumulation, reduced lignin content and dwarfism. In contrast, knocking out MED5a and MED5b (med5a/5b) results in the accumulation of increased levels of phenylpropanoid pathway derivatives. Considering that the CDK8 kinase module is a repressive module in Mediator, we tested the hypothesis that Arabidopsis MED5 represses phenylpropanoid pathway by interacting with CDK8.To test this hypothesis, CDK8 knockout lines (cdk8‐1) were crossed with ref4‐3, and the phenylpropanoid content of the resulting double mutants was evaluated. In ref4‐3 cdk8‐1 plants, the concentration of sinapate esters and total lignin content are as low as they are in ref4‐3, yet the growth defect in ref4‐3 is largely rescued. To further determine the genes targeted by MED5 and CDK8 in maintaining proper plant growth, we performed an RNA‐seq analysis which showed that a majority of the genes involved in salicylic acid (SA) biosynthesis and signaling are up‐regulated in ref4‐3 compared to wild type and ref4‐3 cdk8‐1. Consistent with this observation, both free and total SA, both of which have been previously implicated in dwarfing in lignin‐modified plants, are accumulated to elevated levels in ref4‐3 but not in wild type and ref4‐3 cdk8‐1. Nevertheless, blocking SA biosynthesis is not sufficient to restore the growth deficiency of ref4‐3, suggesting that the hyperaccumulation of SA is more likely to be an effect rather than a cause for its dwarf phenotype.At the molecular level, to elucidate how ref4‐3 regulates downstream gene targets in either a CDK8‐dependent manner, we performed RNA polymerase II (Pol II) ChIP‐seq analysis in wild type, ref4‐3, cdk8‐1 and ref4‐3 cdk8‐1. The Pol II ChIP‐seq data may provide additional information for us to identify the genes that are causative for the dwarfism of ref4‐3.Taken together, this study identifies the genetic interaction between MED5 and CDK8 in Arabidopsis, which enhances our understanding in the function of Mediator in plant metabolism and its role in lignin‐modification‐induced dwarfism.Support or Funding InformationDepartment of Energy; Global Climate and Energy ProjectThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.