The plant Arabidopsis thaliana faces the ABA-mediated processes through the DAG1, WRKY6 and WRKY18 proteins,with the control of the Polycomb Repressive Complex2.

Plants, as sessile organism, have to survive to changing environmental conditions. The phytohormone abscisic acid (ABA) has a pivotal role in the adaptation to environmental challenges as well as in the regulation of plant growth and development. During maturation, dormancy and germination of seed ABA action is crucial to induce dormancy -an adaptative trait necessary to prevent early germination- and repress germination. This activity is counteracted by gibberellins (GAs), which repress dormancy while inducing germination. Therefore, the seed-to-seedling transition is mainly controlled through the ratio between ABA and GA. In Arabidopsis thaliana, thetranscription factor Dof AFFECTING GERMINATION 1 (DAG1) controls the balance between ABA and GA to promote seed dormancy while repressing seed germination by the negative control of the ABA catabolic gene CYP707A2 and the GA biosynthetic gene GA3ox1. DAG1 not only functions during the seed-to-seedling transition, but also during seedling development, as it promotes hypocotyl elongation. RNA-seq analysis on dag1 hypocotyls proved that DAG1 is involved in the promotion of hypocotyl elongation through the control of ABA, ethylene and auxin signaling. This geneme-wide analysis revealed that seven factors belonging to the WRKY family of transcription factors, are deregulated in dag1 hypocotyls. In particular, WRKY6 and WRKY18 are involved in the ABA-mediated developmental processes and stress response. Given the close relationship between DAG1 and ABA, we have focused our study on the putative role of DAG1 in ABA-mediated stress response, and on the putative interaction between DAG1, WRKY6 and WRKY18. The results of this study revealed that DAG1 plays a role in abiotic stress response, and in particular in cold tolerance; indeed, dag1 mutant plants are significantly more tolerant to freezing temperatures respect to the wild type, suggesting that DAG1 may function as a repressor of this process. Consistently, DAG1 expression is induced by cold treatment, as well as the expression of WRKY18, which we proved is a direct target of DAG1. Interestingly, while wrky6 mutants show an increase tolerance to cold treatment respect to the wild type suggesting that the protein has a negative role in cold tolerance,wrky18 mutant plants do not show differences in the survival rate after the cold exposure respect to the wild type. Conversely the simultaneous inactivation of dag1 and wrky18 results in an increased cold tolerance, corroborating the role of DAG1 in this process. The response to abiotic stress is mediated by a wide epigenetic transcriptomic reprogramming, which involves the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). Indeed, PRC2 is known for its key role during plant development as well as in plant response to abiotic stress. The DAG1 locus is a target of PRC2, and is marked by H3K27me3 in seeds and seedlings.. Mutations inthe catalytic subunit of PRC2 results in a severe phenotype, like embyo-lethality in plants or cancer in animals. Although theeffects of several inhibitors of the PRC2 catalytic subunit (EZH2)have long been tested in animals as a possible anti-cancer therapy, no trial with any inhibitor has ever been reported. Taking advantage of the homology of EZH2 in animals and plants, we assessed the efficacy of a EZH2 inhibitor on Arabidopsis seeds, in order to provide a powerful tool in studying PRC2 action in plants. We performed treatments with a compound previously reported as an EZH2 inhibitor in human leukemia cells, and we proved that is active on the Arabidopsis catalytic subunit of PRC2. Indeed, treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, the expression level of two PRC2 targets (DAG1 and WRKY70) is significantly increased following treatment with this compound. ChIP analysis confirms that enrichment of H3K27me3 on these targets significantly decreases in the presence of the inhibitor. In addition, impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. The pharmacological approach to inhibit PRC2 is efficient in plants ; therefore, this inhibitor could represent a powerful tool to further investigate the effects of the transcriptional control mediated by PRC2 in plants, also in the response to abiotic stresses.