The project investigated plant responses to long-term cold exposure using temperature regimes that mimic natural autumn and early winter conditions. By integrating transcriptomic and chromatin accessibility data, the project provides new insights into the molecular mechanisms underlying cold acclimation in Arabidopsis thaliana. To achieve these objectives, COOLCASE was structured into four interconnected Work Packages (WP1–WP4) described below.
WP1 – Transcriptomic analysis under decreasing temperature regimes Genome-wide RNA sequencing was performed on Arabidopsis plants exposed to different temperature regimes, including constant warm conditions and two progressively decreasing-temperature treatments. This work enabled the identification of dynamic transcriptional responses to cold over time, revealing distinct expression patterns associated with either moderate cold or temperature below 10°C. Progressive cold induces a major reorganization of plant transcriptome, defining two sequential gene regulatory programs corresponding to genes modulated above or below 10°C.
WP2 – Chromatin accessibility dynamics during cold acclimation Chromatin accessibility was profiled using ATAC-seq across the same temperature regimes. This analysis generated genome-wide maps of accessible chromatin regions that change in response to decreasing temperatures. The results showed that the induction of cold-responsive genes is associated with increased promoter accessibility, while genes down-regulated during cold exposure retain promoter accessibility, suggesting a mechanism that allows rapid transcriptional reactivation upon return to warmer conditions.
WP3 – Integration of transcriptomic and chromatin accessibility data RNA-seq and ATAC-seq datasets were integrated to investigate the regulatory mechanisms underlying long-term cold responses. This integrative analysis enabled the identification of transcription factors and regulatory networks involved in cold acclimation. Notably, we found transcription factors acting at the interface between moderate cold and chilling conditions, as well as regulators required across different temperature ranges.
WP4 – Functional characterization of candidate genes Based on the transcriptomic analyses, a subset of cold-responsive coding genes was selected for functional investigation. Phenotypic analyses of mutant lines were initiated under different temperature regimes. While the functional characterization of candidate genes is ongoing, this work provided initial insights into the contribution of newly identified genes to plant cold responses.
Overall outcomes Overall, the project delivered high-quality datasets and a refined conceptual framework describing how plants respond to long-term, naturally relevant cold conditions. The results extend current models of cold acclimation by incorporating temporal and regulatory dimensions that were previously unexplored.
