Final Report Summary - EPIPRIMING (Keeping ready for battle: understanding fundamental mechanisms of establishment and maintenance of epigenetic marks underlying abiotic stress memory in plants.) The overall aim of the EPIPRIMING project was to gain a better insight into chromatin-based mechanisms of plant responses to environmental stress to build on preliminary results by the host group.The specific objectives were:1. To test the requirement of specific demethylases and chromatin remodellers for H3K27me3 etching during the priming treatment.2. To investigate the mechanisms of H3K27me3 spreading after return of plants to control conditions.3. To assess the effect of repeated salt exposure on H3K27me3.4. To perform a detailed analysis of H3K27me3 changes in the HKT1 gene using cell-type specific ChIP.Experiments were carried out using several Arabidopsis thaliana chromatin mutants. ChIP-sequencing analysis was done and the results encouraged additional experiments exposing a selected mutant to different growth conditions and perform further analyses (ChIP-sequencing, qRT-PCR, phenotyping). We discovered that the mutants displayed a light-dependent phenotype. RNA-sequencing allowed us to pinpoint candidate genes that may mediate light-dependence. These findings have led to a new PhD project in the host group starting October 2016.The levels of H3K27me3 were fine-mapped by ChIP-qPCR along selected regions of the genome for a number of time points before and after the priming treatment, and additional experiments involving repeated priming were performed. Furthermore, transgenic plants were generated using the INTACT method to target specific root tissues by using tissue-specific promoters. Subsequent generations of plants were grown and selected to get stable homozygous plants, and plants were also screened for the correct location of the NTF fusion protein by confocal microscopy. A successful protocol for isolating and purifying the nuclei from the INTACT lines was established. A salt-priming experiment to resolve the profile of different histone marks and their relationship with gene expression at the cell-specific level was carried out. ChIP with antibodies against several histone modifications was carried out for 24 samples including tissue-specific and whole-root nuclei from replicated plant batches treated with or without salt. Illumina-sequencing and data analysis are still ongoing but first results are promising. The full dataset is expected to reveal for the first time the response of histone marks to salt treatment at single-tissue level, which will provide a better resolution of chromatin-based processes and thus make an important contribution to research on plant stress and epigenetics.
