La caractérisation moléculaire de la réponse des plantes aux contraintes environnementales permet de mieux comprendre les bases de l’adaptation des plantes à leur milieu, et pourrait aider à l’amélioration des plantes cultivées. L’épigénome est constitué de l’ensemble des marques présentes sur la chromatine et participe à la régulation de l’expression du génome, notamment au cours du développement. Il contribue aussi à la stabilité des génomes, notamment en empêchant la transposition des éléments transposables (ET). L’épigénome varie en fonction des contraintes environnementales et une meilleure compréhension de ces variations pourrait permettre d’apporter une vision nouvelle de l’interaction entre la plante et son environnement. Cependant, l’étendue des modifications de l’épigénome, le type de séquences affectées et les mécanismes impliqués restent à déterminer. Dans ce cadre, j’ai analysé l’impact du froid sur le méthylome du maïs, une plante à génome complexe riche en ET. Dans un premier axe, j’ai analysé le méthylome d’un génotype sensible au froid, B73, par séquençage haut-débit d’ADN traité au bisulfite de sodium (BS-seq). Cette analyse comparative entre plantes « stressées » et « non stressées » a été menée (i) à l’échelle chromosomique, sans a priori sur le niveau de variation de méthylation de l’ADN et (ii) à l’échelle locale (régions différentiellements méthylées, ou « DMR ») en fixant des niveaux de variations forts (>10%). Ces deux types d’analyses ont permis de montrer que le froid déclenche une hyperméthylation à l’échelle du génome, à laquelle se superposent des hyper- et hypométhylations à l’échelle locale. Ces variations sont observées pour les trois contextes de cytosine et dans différentes régions génomiques associées aux gènes et aux ET. Ceci suggère l’activation parallèle de plusieurs mécanismes de régulation de la méthylation de l’ADN en réponse au froid. Dans un second axe, j’ai suivi ces DMR au cours du développement et dans la descendance afin d’étudier leur transmission, en lien avec leur localisation génomique et les contextes de cytosine affectés. Dans un troisième axe, j’ai étudié le lien entre variations de méthylation et sensibilité au froid en comparant la réponse du méthylome chez trois génotypes de maïs (B73, F2 et F331) présentant une réponse phénotypique contrastée pour ce caractère. / Molecular characterization of plant response to environnemental constraints allows to both better understand plant adaptation and help crop improvement. The epigenome is composed of chromatin marks and participates to the regulation of genome expression, notably through development. It is also involved in genome stability, essentially by preventing the transposition of transposable elements (TE). The epigenome can be modified by environmental cues and better understanding this variation could give new insights on the interaction between the plant and its environnement. However, the extent of this modification, targeted sequences and underlying mecanisms remain to be elucidated. In this context, I analyzed the impact of cold on the methylome of maize, a plant with a complex genome with high proportion of TEs. In a first part, I analyzed the methylome of a cold-sensitive genotype, B73, using whole genome bisulfite sequecing (BS-seq). This comparative analysis between “stressed” and “unstressed” plants was carried out (i) at the chromosome scale, without a priori definition of a DNA methylation difference and (ii) at a localized scale (Differentially Methylated regions, « DMRs ») using high minimum methylation difference rate (10%). These two types of analysis revealed that cold triggers hypermethylation at the genome scale, as well as. hyper-and-hypo-methylation at the local scale. These variations were observed in the 3 contexts of cytosine and occur in different genomic regions associated with genes and TEs. This suggests the parallel activation of different regulatory pathways in response to cold. In a second part, I focused on following-up methylation changes through development and in the progeny in conjunction with the genomic sequences and the cytosine context involved. In a third part, I studied the relationship between methylome variations and cold sensitivity by comparing the methylomes of three maize genotypes (B73, F2 and F331) with a contrasted phenotypic response to cold.

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.