Periodic Reporting for period 1 - CRoSh (“Chromatin Re-organization during Shade Avoidance”)
Période du rapport: 2019-09-01 au 2021-08-31
Light is vital for plants because it is their source of energy and provides important information about the environment. Quality and quantity of light change continuously and plants developed systems to sense these changes, which can be rapid (within minutes) or slow (seasonal changes) and adapt their growth to maximize photosynthesis and improve fitness. One example of growth response driven by changes in the light spectrum is the shade avoidance response. Shade avoidance happens when plants grow in dense vegetation and leaves reflect the Far-Red component of sunlight creating a shaded environment characterized by a low ratio between Red and Far Red light (LRFR). LRFR is not tolerated by plants loving full sunlight, where the Red/ Far Red ratio is high (HRFR), such as our model organism Arabidopsis thaliana. Arabidopsis deploys a series of growth responses to escape from this situation, which includes the elongation of stem-like organs to position the leaves towards the sun and outside the shade. The architectural changes are initiated by the deactivation of the photoreceptor phyB by LRFR, which triggers the accumulation/activation of a class of transcription factors called PIFs. PIFs start the remodelling of the expression of numerous genes, that includes genes involved in the biosynthesis of the hormone auxin. The increase of auxin levels is the limiting step for the elongation phenotype typical of the shade avoidance response. Until now, researchers have focused mostly on the characterization of the activity of PIFs and other TFs controlled by auxin levels, but a description of the chromatin landscape of the regulatory regions involved in this process was lacking and this is why we decided to initiate this project. Better understand the molecular mechanisms regulating gene expression underlying shade avoidance response is important for two main reasons: 1) shade avoidance is a beautiful example of how an external stimulus sensed by an organism starts a molecular cascade, which is translated into a physiological response, that facilitate the plants' adaptation to new environmental conditions. Given that the mechanisms of genic response are quite conserved, knowledge acquired from this response could be applied to other environmental conditions, species or organisms. 2) In monoculture, plants tend to be grown densely to improve food production, but this triggers shade avoidance responses that have an impact on traits that affect the final yield, such as the acceleration of flowering, decrease of biomass, and increase of sensitivity to the attack of pathogens and herbivores. Hence modern agriculture needs to solve the conflict between rearrangements of organ architecture to improve light harvesting and maintain yield stability, which can be achieved by deciphering the molecular mechanisms controlling these processes.
The fundamental element of chromatin is the nucleosome, which consists of 150pb of DNA wrapped around the histone core. Well-spaced nucleosomes are present in the body gene, while intergenic regions are characterized by fuzzy nucleosomes, with the presence of nucleosome-free DNA portions, which are also called accessible chromatin regions (ACRs). These short DNA sequences, placed in the non-coding genome, host regulatory elements for gene expression, and the lack of nucleosomes ensures access to the DNA of TFs and the transcription machinery. Their prediction can be challenging because they are small DNA sequences disperse in long intergenic -region, but the combination of information about chromatin accessibility, TFs binding profile and DNA sequence will facilitate their identification and it will help the scientific community to predict the expression pattern of specific genes and produce valuable tools. To characterize the chromatin architecture of the regulatory elements of genes responding to LRFR, we used ATAC-seq, which is a technique that uses the enzyme Tn5, a modified version of bacterial transposase, to fragment DNA and insert adaptors used by Illumina sequencing. Tn5 preferentially binds nucleosome-free DNA and hence it allows the isolation and sequencing of ACRs. The limiting step for this technique is the preparation of high-quality nuclei, which we achieved using a purification system based on biotin-streptavidin which is called INTACT. For INTACT, we needed Arabidopsis transgenic lines expressing a nuclear protein tagged with a peptide recognized by BirA, which is a bacterial protein driving the biotinylation of this peptide. Hence, during the first period of the project, I worked on the selection of these transgenic lines and the optimization of the INTACT protocol. The transgenic lines were in two genetic backgrounds: wild type and plants defective for LRFR-mediated auxin biosynthesis, this allowed to test whether the increase of auxin levels affects gene expression through the remodelling of chromatin accessibility. Both genetic backgrounds were analysed in HRFR (control condition), and after 1h or 25h of LRFR, to detect the short and long-term effects of LRFR. In this way, we produced a nice atlas of the ACRs for the LRFR-related genes, which can be used by other members of the host lab and, once the data will be published, by the scientific community interested in studying the expression pattern of specific genes in Arabidopsis at the early developmental stage of seedlings. Moreover, differential analyses revealed the regions showing an increase or decrease of accessibility after 25h of LRFR treatment, leading us to the discovery of new molecular mechanisms.
The results of this project were presented at seminars organized by the CIG and DBMV departments of our university (UNIL), at the Plant Biology Europe conference (June-July 2021) and the International Plant Photobiology Symposium (July 2021). Moreover, the writing of the paper describing the results is undergoing. Moreover, together with Martina Legris, another MSCA fellow of our lab, I participated in the following outreach activities for families and schools: “Science is Wonderful!” organized by the European Commission in Brussels, Belgium on 25-26 September 2019 and “Les Mystères de l'UNIL”, an event organized by the University of Lausanne in 2021, showing the amazing movements of plants mediated by light and experiments that boys and girls could easily reproduce at home to answer basic questions of plant biology.
The results of this project were presented at seminars organized by the CIG and DBMV departments of our university (UNIL), at the Plant Biology Europe conference (June-July 2021) and the International Plant Photobiology Symposium (July 2021). Moreover, the writing of the paper describing the results is undergoing. Moreover, together with Martina Legris, another MSCA fellow of our lab, I participated in the following outreach activities for families and schools: “Science is Wonderful!” organized by the European Commission in Brussels, Belgium on 25-26 September 2019 and “Les Mystères de l'UNIL”, an event organized by the University of Lausanne in 2021, showing the amazing movements of plants mediated by light and experiments that boys and girls could easily reproduce at home to answer basic questions of plant biology.
Among the regions showing changes in chromatin accessibility in response to LRFR we found the gene HFR1. HFR1 is a well-known factor involved in the LRFR response that counteracts the activity of PIFs establishing a gas-and-brake mechanism that prevents an excessive shade response. Moreover, HFR1 has a particular expression pattern, in fact, it is the only one among the early LRFR-induced genes whose expression is reinforced over the days, instead of being attenuated. The ATAC-seq allowed us to link the persistent LRFR-induction of HFR1 to the increase of chromatin accessibility of the HFR1 locus, suggesting that the remodelling of chromatin accessibility contributes to the creation of specific gene expression patterns.