The role of epigenetics in leaf cell differentiation during drought stress in Brachypodium distachyon

Epigenetic modifications play crucial roles in plant development and plant response to environmental stimuli. These modifications determine changes in plant phenotypes without affecting the DNA sequence. The availability of high-throughput DNA sequencing technologies provides powerful tools to characterize the epigenetic modifications at a genome-wide level. This PhD project aims to investigate the epigenetic network underlying leaf cell differentiation during drought stress in the model species Brachypodium distachyon (Bd), focusing on two developmental leaf zones: proliferating and expanding leaf cells. Drought stress is applied because it has been shown to perturb leaf development. The investigation of the molecular mechanism controlling leaf growth includes the analysis of long noncoding RNAs (lncRNAs), protein-coding genes and DNA methylation of the entire genome applying next-generation sequencing (NGS) approaches. First, we studied the phenotypic effect of drought stress on leaf growth of the B. distachyon inbred line 21, performing a kinematic growth analysis of the third and fifth leaf of plants grown in control and severe drought conditions. Leaf growth was also characterized when optimal growing conditions were restored after the drought stress. We showed that Brachypodium distachyon under drought stress reduced its final leaf size, but was still able to produce viable seeds even if the drought stress was maintained for the entire life cycle. Secondly, we started the analysis of the regulatory network of leaf growth by annotating Brachypodium lncRNAs. We exploited RNA-seq libraries previously produced in our laboratory from Bd21 young developing third leaf and also by thoroughly analyzing data from several RNA-seq experiments produce in Bd21 and other Bd lines, Bd21-3 and Bd1-1. All RNA-seq datasets analyzed are public available. By applying an in house developed bioinformatic pipeline, considering several filters based on the main lncRNA features, we produced a lncRNA atlas of Brachypodium distachyon including a total of 25,338 Bd lncRNAs. Those lncRNAs displayed organ, tissue and stress-specific expression profiles and target mimicry activity. Successively, the methylation level was assessed at a single base resolution performing a whole-genome bisulfite sequencing (BS-Seq) of proliferating and expanding leaf cells. After having applied the drought stress, we collected the third leaf of plants grown in both severe drought and in optimal conditions and proliferating, and expansion leaf zones were dissected. For each leaf tissue three biological replicates were considered. We generated 12 BS-Seq libraries and sequenced the DNA treated with sodium bisulfite using a 2 x 125 bp sequencing strategy with an expected 20X coverage. Cytosine methylation level of each cell type was analyzed at genome level in the three methylation contexts, i.e. CG, CHG and CHH, where H can be either A, C or T. DNA methylation was assessed within annotated protein-coding genes, lncRNAs, and transposable elements. Differentially methylated regions (DMRs) and differentially expressed (DE) genes and lncRNAs were identified between cell types and considering the treatment. We identified 7,867 DMRs, 15,689 DE protein-coding genes and 1,337 DE lncRNAs between proliferating and expanding leaf cells during leaf development. Fewer DMRs and DE genes and lncRNAs were identified in response to the drought stress in both leaf cell-types. This result suggests that during cell differentiation under control and severe drought conditions drastic changes in expression level of both coding and noncoding transcripts and changes in DNA methylation take place between proliferating and expanding leaf cells. Finally, the relationship between transcriptomic profiles of both coding and noncoding transcripts and the DNA methylation were investigated.