Project description:To better understand the roles of SR (calculated from the nascent TR by dividing it by cell volume) (Pérez-Ortín, BioEssays 2013), and mRNA stability in stress adaptation, we investigated the yeast cell wall stress response and compared it with other stress responses using the GRO method. In this study, we analyzed genome-wide changes in RNA abundance to determine how synthesis rates and mRNA stabilization influence RA along the treatment with Congo Red. Our findings indicate that alterations in synthesis rates primarily drive changes in RA, whereas mRNA stability remains largely unaffected under our conditions, in contrast to other stress responses. Additionally, the RA of RP, RiBi, and ESRup genes is influenced by both mRNA stability and SR, albeit to a lesser extent than in other stress conditions. Moreover, we identified: a) a small subset of genes whose mRNA levels are co-regulated by both changes in synthesis rates and mRNA stability; b) previously unidentified genes whose mRNA levels increase in the presence of Congo Red; and c) RNA-binding proteins (RBPs) such as Nab2 and Hrp1 as potential regulators of genes induced in response to cell wall stress.
Project description:In response to stress, cells activate signaling pathways that coordinate broad changes in gene expression to enhance cell survival. Remarkably, complex variations in gene expression occur even in isogenic populations and in response to similar signaling inputs. However, the molecular mechanisms underlying this variability and their influence on adaptive cell fate decisions are not fully understood. Here, we use scRNA-seq to longitudinally assess transcriptional dynamics during osmoadaptation in yeast. Our findings reveal highly heterogeneous expression of the osmoresponsive program, which organizes into combinatorial patterns that generate distinct cellular programs. The induction of these programs is favored by global transcriptome repression upon stress. Cells displaying basal expression of the osmoresponsive program are hyper-responsive and resistant to stress. Through a transcription-focused analysis of more than 300 RNA-barcoded deletion mutants, we identify genetic factors that shape the heterogeneity of the osmostress-induced transcriptome, define regulators of stress-related subpopulations and find a link between transcriptional heterogeneity and increased cell fitness. Our findings provide a regulatory map of the complex transcriptional phenotypes underlying osmoadaptation in yeast and highlight the importance of transcriptional heterogeneity in generating distinct adaptive strategies.
Project description:The structure and composition of plant cell walls are modified to accommodate the needs of the cell and in response to environmental stimuli. Growth, development, and defense may demand potentially conflicting functional cell wall requirements, and thus modifications of the cell wall are tightly controlled in an adaptive manner. These modifications are mediated by a dedicated cell wall integrity (CWI) maintenance mechanism. We investigated the responses to cell wall damage (CWD) that compromise CWI and the underlying mechanisms in Arabidopsis thaliana. Inhibitor- and enzyme-triggered CWD induced similar, turgor-sensitive stress responses. Genetic analysis showed that the receptor-like kinase (RLK) FEI2 and the plasma membrane-localized mechano-sensitive Ca2+- channel MCA1 function downstream of the RLK THE1 in CWD perception. Phenotypic clustering with 27 genotypes identified a core group of RLKs and ion channels required for activation of CWD responses. In contrast, the responses were repressed by pattern-triggered immunity (PTI) signaling components including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2 (PEPR1/2). Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration dependent manner. CWD induced the expression of both PROPEP1 and PROPEP3 as well as the release of a PROPEP3 fusion protein into the growth medium. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses. If key PTI signaling elements acting downstream of PEPR1/2 are dysfunctional, suppression of CWD-induced responses is alleviated, thus compensating for the impairment.
Project description:Plants have evolved cell wall integrity signaling pathways to maintain cell wall homeostasis during rapid growth and in response to environmental stress. The cell wall leucine-rich repeat extensins LRX3/4/5, the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23, and FERONIA (FER) function as a module to regulate plant growth and salt stress responses via the sense of cell wall integrity. However, the intracellular signaling pathways that mediate the effects of the LRX3/4/5-RALF22/23-FER module are still largely unknown. Here, we report that jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) accumulate constitutively in lrx345 and fer mutants. Blocking JA pathway rescues the retarded growth phenotype of the lrx345 and fer-4 mutants, while disruption of ABA biosynthesis suppresses the salt-hypersensitivity of these mutants. Many salt stress-responsive genes display abnormal expression patterns in the lrx345 and fer-4 mutants, as well as in wild type plants treated with epigallocatechin gallate (EGCG), an inhibitor of pectin methylesterases, suggesting that the cell wall integrity is a critical factor that determines the expression of stress-responsive genes. Production of reactive oxygen species (ROS) is constitutively increased in the lrx345 and fer-4 mutants, and inhibition of ROS accumulation suppresses the salt-hypersensitivity of these mutants. Together, our results suggest that the LRX3/4/5-RALF22/23-FER module controls plant growth and salt stress responses by regulating hormonal homeostasis and ROS accumulation.
Project description:Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent of their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most ap-plied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed a considerable contamination with non-cell wall proteins, mainly comprising mitochondrial proteins. Here-in, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial proteins removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and enriching significant-ly their amounts. This promising method could be reliably implemented in lab-scale and industrial processes for “pure” cell wall isolation.
Project description:Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the GC/MS technology with in-vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up and downregulation relative to control, mid-temperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism. Reference: Strassburg K, Walther D, Takahashi H, Kanaya S, and Kopka J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. OMICS JIB, 2010, 14(3), in press.
Project description:Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the GC/MS technology with in-vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up and downregulation relative to control, mid-temperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism. Reference: Strassburg K, Walther D, Takahashi H, Kanaya S, and Kopka J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. OMICS JIB, 2010, 14(3), in press. Time-series with 8 time points for three different ambient temperatures. Single replicate per time point and temperature series.