Umeå universitets logga

Environmental changes trigger stress responses in living organisms. Although the underlying mechanisms are only partly understood, they involve intricate signaling pathways and transcription factors (TFs). Mediator is a conserved co-regulator complex required for transcriptional regulation of all eukaryotic protein-encoding genes. However, its function in abiotic stress responses is elusive. Here, we describe global gene expression changes induced by salt stress in Arabidopsis thaliana . To investigate the involvement of Mediator, we analyzed med9, med16, med18 , and cdk8 mutants, each representing one of the four Mediator modules. Our results demonstrate that promoters of differentially expressed genes (DEGs) for each mutant are enriched for binding sites of specific TFs. Phenotypic analyses further support the transcriptomic data: med16 and med18 , and to a lesser extent cdk8 , exhibit defects typical to mutations that affect abscisic acid and anthocyanin metabolism and we identify dysregulated signaling molecules, TFs, and target genes in these pathways. Our results reveal how signals from different stress response pathways are dependent on, and integrated by, Mediator subunits to coordinate a functional response to salt stress.

MED7, a middle-module subunit of the transcriptional co-regulator Mediator complex, plays a critical role in gene regulation in Arabidopsis thaliana, where it is encoded by two paralogs, MED7A and MED7B. We present phenotypic analyses of homozygous MED7-silenced transgenic lines with significantly reduced expression of both MED7 paralogs under autotrophic conditions. Our findings demonstrate that MED7 is essential for proper cotyledon opening during de-etiolation, as the silenced lines showed a marked delay in this process. Additionally, these lines displayed distinct morphological alterations, including hyponastic cotyledons, elongated hypocotyls, and modified root architecture, such as shorter primary roots and impaired root hair development in light-grown seedlings. MED7 silencing also significantly hindered light-induced adventitious root (AR) formation on the hypocotyls of etiolated seedlings, leading to a notable reduction in AR production. Moreover, MED7 silencing impacted the timing of floral transition and shoot branching, resulting in delayed flowering and an increased number of primary cauline branches on the inflorescence stem. Together, these results underscore a central role for MED7 in orchestrating key developmental processes in plants.

The components of the mediator kinase module are highly conserved across all eukaryotic lineages, and cyclin-dependent kinase 8 (CDK8) is essential for correct cell proliferation and differentiation in diverse eukaryotic systems. We show that CDK8 couples leaf development with the establishment of correct stomata patterning for prevailing CO₂ conditions. In Arabidopsis, the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS (SPCH) controls cellular entry into the stomatal cell lineage, and CDK8 interacts with and phosphorylates SPCH, controlling SPCH protein levels and thereby also expression of the SPCH target genes encoding key regulators of cell fate and asymmetric cell divisions. The lack of the CDK8-mediated control of SPCH results in an increased number of meristemoid and guard mother cells, and increased stomata index in the cdk8 mutants. Increasing atmospheric CO₂ concentrations trigger a developmental programme controlling cell entry into stomatal lineage by limiting the asymmetric divisions. In cdk8, the number of meristemoids and guard mother cells remains the same under ambient and high CO₂ concentrations, as the accumulated levels of SPCH caused by the lack of CDK8 appear to override the negative regulation of increased CO₂. Thus, our work provides novel mechanistic understanding of how plants alter critical leaf properties in response to increasing atmospheric CO₂.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2–8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3́-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3́-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron–exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections.

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.

MED7 is a subunit of the Mediator middle module and is encoded by two paralogs in Arabidopsis. We generated MED7 silenced lines using RNAi to study its impact on Arabidopsis growth and development. Compared with wild type, etiolated seedlings of the MED7 silenced lines exhibited reduced hypocotyl length caused by reduced cell elongation when grown in the dark. The hypocotyl length phenotype was rescued by exogenously supplied brassinosteroid. In addition, MED7 silenced seedlings exhibited defective hook opening in the dark as well as defective cotyledon expansion in the presence of the brassinosteroid inhibitor brassinazole. Whole transcriptome analysis on etiolated seedlings using RNA sequencing revealed several genes known to be regulated by auxin and brassinosteroids, and a broad range of cell wall-related genes that were differentially expressed in the MED7 silenced lines. This was especially evident for genes involved in cell wall extension and remodeling, such as EXPANSINs and XTHs. Conditional complementation with each MED7 paralog individually restored the hypocotyl phenotype as well as the gene expression defects. Additionally, conditional expression of MED7 had no effects that were independent of the Mediator complex on the observed phenotypes. We concluded that the MED7 paralogs function redundantly in regulating genes required for the normal development of etiolated Arabidopsis seedlings.

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.