Thèse Etude des Interactions Entre les Voies de Régulation Lec2 et Prcs Durant le Développement de la Graine d'Arabidopsis. H/F

Doctorat.Gouv.Fr

De l'embryogenèse à la maturation puis à la germination, le développement de la graine est contrôlé par des régulations basées sur la chromatine (par exemple, les modifications des histones par PRC1/2) ainsi que par des régulations transcriptionnelles exercées par les facteurs de transcription LAFL (à savoir LEC1, ABI3, FUS3 et LEC2), largement conservés chez les angiospermes (Lepiniec et al. 2018). Malgré leurs rôles centraux et les preuves génétiques existantes, nous ignorons encore comment ces acteurs interagissent pour contrôler les différentes phases du développement de la graine. Notre équipe met en oeuvre des approches complémentaires afin d'élucider l'imbrication des régulations LAFL et celles basées sur la chromatine dans le contrôle du développement de la graine.

Ce projet de thèse se concentrera sur l'élucidation des interactions moléculaires entre LEC2 et PRC1/2. L'étudiant caractérisera la dynamique de la chromatine et du transcriptome des cibles connues de LEC2 au cours du développement de la graine en utilisant des approches de ChIP-qPCR et de qRT-PCR. Afin de décrypter les régulations croisées transcriptionnelles et chromatiniennes, ces analyses seront menées sur des fonds génétiques de type sauvage et sur des mutants spécifiques, incluant des pertes de fonction de lec2 et de sous-unités prc (fie et val1), ainsi que des allèles modifiés inductibles ou à effet dominant négatif.

En plus de constituer un modèle fascinant pour la compréhension de nombreux mécanismes biologiques, les graines jouent un rôle clé dans l'évolution des plantes supérieures, en agriculture et dans la production alimentaire. Ainsi, comprendre les mécanismes contrôlant le développement des graines représente un enjeu majeur en biologie végétale et pourrait apporter des bénéfices scientifiques, sociétaux et environnementaux.

The LAFL transcriptional regulators interact to form different regulatory complexes and have partially overlapping and redundant functions. They control the expression of both structural and regulatory genes and are crucial to accumulate storage compounds (e.g. proteins, oil), acquire desiccation tolerance and repress temporarily germination and, thus to determine seed quality and yield (Lepiniec et al. 2018). Moreover, the ectopic expression of LEC1 or LEC2 in vegetative tissues can trigger the formation of somatic embryos (SEM) (Salaun et al. 2025). However, we ignore how the LAFL control the different developmental transitions and programs (i.e. embryogenesis, maturation and germination).

Consistent with their key function, the expression of the LAFL genes is tightly controlled during seed development and strongly repressed in vegetative tissues through multiple pathways involving chromatin-based mechanisms such as, POLYCOMB REPRESSIVE COMPLEXES 1 and 2 (PRC1 and PRC2) (Lepiniec et al. 2018). On this line, we have discovered the first plant PRE (Polycomb Recruiting Element) in the promoter of LEC2, named RLE (for Repression of LEC2 Expression), that is involved in PRC2-mediated LEC2 silencing and that a similar negative regulatory sequence (NRS) is found in the FUS3 promoter (Berger et al. 2011, Roscoe et al. 2019). Consistent with these observations, mutations of a PRC2 subunit (i.e. clf) led to the ectopic expression of FUS3 and ABI3, associated with the enhanced accumulation of oil in seed (Liu et al. 2016).

Nevertheless, these chromatin-based regulations of the LAFL genes expression hid that the LAFL act as pioneer factors' and be themselves involved in reprogramming chromatin states (Lepiniec et al. 2018). This hypothesis emerged from discoveries demonstrating that (i) many LAFL target genes are regulated by chromatin-based mechanisms (Merini et al. 2017, Xiao et al. 2017, Wang et al. 2016) and (ii) the LAFL are directly involved in re-setting the chromatin of the major flowering time regulator FLC, from a PRC2-mediated repressive state during seed development (Questa et al. 2016). On this line, it has been shown recently that, LEC2 is involved in cell fate reprogramming of specific vegetative cells through chromatin-based regulations (Peng et al. 2025, Tang et al. 2025). Adding to the entanglement of these interactions, the LAFL could also induce their own chromatin-mediated silencing, at the end of the maturation phase (Wang al. 2013, Mu et al. 2008).

The objective is to unravel the complex interactions between LEC2 and PRCs to control Arabidopsis seed development. To elucidate the mechanisms involved, the student will characterize chromatin and transcriptomic dynamics of LEC2 targets during seed development, using wild-type and specific mutant backgrounds, including mutants carrying inducible alleles of LEC2 and PRC subunits (e.g. FIE and VAL1).

In order to elucidate the multi-layered interactions between LEC2 and PRC, the student will use cutting-edge techniques recently adapted in our labs. These techniques are notably based on the extraction of low amounts of seed material, such as manual or laser-assisted micro-dissection of embryos.

The isolated material will be used to establish the dynamics of transcription and accumulation of the chromatin markers during seed development (in both wild-type and various mutant background) using targeted RT-qPCR and ChIP-qPCR, respectively. Genome-wide analyses (RNA-Seq, ChIP-seq or ATAC-seq) are not necessary in the frame of the PhD, but may be conducted depending on time available, paving the way to the characterization of new actors in the control of seed development and maturation.
The epistatic relationships between the LEC2 and PRC will be investigated by combining specific loss- and gain-of-function alleles. The student will generate and use original inducible, dominant negative and/or tissue-specific alleles of LEC2 and PRC1/2 subunits (FIE for PRC2 and VAL1 for PRC1).

1) Biological materials. Seed will be produced in a controlled environment. At least three independent biological replicates will be generated for transcriptomic, cytological and chromatin analyses. For dynamic analyses, five stages of seed development (S1-5), known for displaying highly contrasted transcriptional programs will be used: the globular stage of embryo development (S1, 3-4 days after pollination (dap)), at the peak of LEC2 expression (S2, hearth stage of embryo development, about 7-8 dap, corresponding to the induction of maturation), maturing seeds (S3, 13-15 dap), mature dry seeds (S4, 21-24 dap), and germination (S5, 24h of imbibition, based on our previous observations that chromatin changes occur already within a few hours).

2) Epistatic analyses using targeted transcriptomic and chromatin analyses. The epistatic relationships between LEC2 and PRC complexes will be investigated by mutant crosses. The impacts of the mutations on chromatin and gene expression will be analysed by targeted analyses (i.e. ChIP-qPCR and qRT-PCR) at the loci of interest (i.e. chromatin regulators and LAFL) and some of their well-characterized target loci involved in different pathways during seed development, including oil (i.e. OLE1, WRI1), storage proteins (At2S3) or hormones (GA3ox2 and YUCCA4) biosynthesis (Lepiniec et al. 2018, Stone et al. 2008).

3) Uncoupling LEC2 gene expression and PRC-based regulations. The PhD student will generate transgenic LEC2 and PRC inducible or dominant negative alleles to precisely control their spatio-temporal activity and decouple their expression from cross-regulations. He will fuse LEC2 and two PRC subunits (e.g. VAL1/2 or FIE) to a Glucocorticoid Receptor (GR) domain allowing to control their transport and hence activity into the nucleus upon addition of a synthetic hormone. We have already established inducible LEC2:GR lines allowing to ectopically activate the seed maturation program in leaves (Santos-Mendoza et al. 2005, 2008), underlining the relevance of this approach. In addition, LEC2-inducible constructs will be fused with a modified EAR repression domain used in the CRES-technology (Oshima et al., 2011), hence conferring LEC2 a dominant repressor activity. For these constructs, the student will use the well-characterized constitutive promoter pUBQ10 to uncouple the expression from epigenomic regulation.

Contingency and/or additional possible experiments. Depending on results and time available, the PhD student will be associated to genetic and molecular approaches developed in the lab to investigate the interaction of other LAFLs (LEC1 or FUS3) with chromatin-based regulations. He could also be involved in genome-wide analyses, such as RNA-seq or ChIP-seq/CUT&Tag, at specific key developmental stages on WT and mutant lines.