ABSTRACT: Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress
Project description:Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.
Project description:Plant responses to abiotic stresses are accompanied by massive changes in transcriptome composition. To provide a comprehensive view of stress-induced changes in the Arabidopsis thaliana transcriptome, we have used whole-genome tiling arrays to analyze the effects of salt, osmotic, cold and heat stress as well as application of the hormone abscisic acid (ABA), an important mediator of stress responses.
Project description:Identification of differentially expressed genes in Arabidopsis thaliana mutants in response to combined abiotic stress treatment through Microarray experiment.
Project description:Mutation of the lignin biosynthetic gene REF8, which encodes p-coumaroylshikimate 3'-hydroxylase, causes widespread changes in gene expression in Arabidopsis thaliana. Disruption of the genes encoding MED5a and MED5b, subunits of the transcriptional coregulatory complex Mediator, rescue the stunted growth of ref8-1 mutants. By comparing gene expression profiles of wild type, ref8-1, med5a/5b, and med5a/5b ref8-1 mutants, we found that the majority of gene expression changes in ref8-1 mutants depend directly or indirectly on MED5a/5b
Project description:Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.
Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. We isolated RNA from each strain as early as 45 minutes after changing from the permissive to the restrictive growth conditions to minimize possible secondary effects on gene expression that are not directly caused by the Degron construct(s).
Project description:Genetically engineering Arabidopsis thaliana to express Isoprene Synthase (ISPS) leads to changes in expression of genes assoiated with many growth regulator signaling pathways and signaling networks involved in abiotic and biotic stress responses.
Project description:Recent studies have shown that several plant species require microbial associations for stress tolerance and survival. In this work, we show that the desert endophytic bacterium Enterobacter sp. SA187 enhances yield and biomass of alfalfa in field trials, revealing a high potential for improving desert agriculture. To understand the underlying molecular mechanisms, we studied SA187 interaction with Arabidopsis thaliana. SA187 colonized surface and inner tissues of Arabidopsis roots and shoots and conferred tolerance to salt and osmotic stresses. Transcriptome, genetic and pharmacological studies revealed that the ethylene signaling pathway plays a key role in mediating SA187-triggered abiotic stress tolerance to plants. While plant ethylene production is not required, our data suggest that SA187 induces abiotic stress tolerance by bacterial production of 2-keto-4-methylthiobutyric acid (KMBA), known be converted into ethylene in planta. These results reveal a part of the complex molecular communication process during beneficial plant-microbe interactions and unravel an important role of ethylene in protecting plants under abiotic stress conditions.
Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together.
Project description:Plants acclimate to environmental fluctuations by transitory reconfigurations the homeostatic network. Primary studies suggested that transcriptome responses to deal with fluctuations in light intensity and temperature tend to reversibility after stress removal in the model plant Arabidopsis thaliana. To gain more insight into this pattern in the context of acclimation, RNA-Seq analysis were conducted in Arabidopsis thaliana after different abiotic stress treatments consisting in high light (HL), high humidity, drought, heat, cold and combinations among factors or after recovery periods. Our transcriptome study is in line of a general pattern wherby transcriptome changes in response to adverse environments are prone to return to the basal state during the de-acclimation phase.