Exploring the Role and Mechanisms of Ubiquitin-Mediated Degradation of H1 during Reproduction in Arabidopsis

Abstract

Sexually reproducing organisms require the establishment of a reproductive lineage, but the mechanisms of germline establishment differ significantly between plants and mammals. In mammals, primordial germ cells (PGCs) are isolated early in development during embryogenesis. In contrast, flowering plants establish germ cells de novo in ovules and anthers during adult development. The somatic to reproductive cell fate transition is associated with epigenetic reprogramming in spore mother cells (SMCs), founder cells of the reproductive lineage, committed to meiosis. The process initiates the eviction of linker histone (H1), preceding additional changes in chromatin composition and organization. This study explored the mechanisms and role of H1 eviction during SMC differentiation in Arabidopsis thaliana, focusing on the ubiquitin-proteasome pathway that mediates protein degradation. The downregulation of CULLIN4 confirmed the involvement of the E3 ubiquitin ligase CULLIN4 in H1 eviction in SMCs. Yet a direct interaction between H1 and CULLIN4 could not be confirmed using immunoprecipitation followed by mass spectrometry (IP-MS), in seedlings and inflorescences. Unexpectedly, however, the analysis identified another E3 ligase, UPL2/3 from the HECT complex family as a potential H1 interaction. To determine which of the many lysine residues in H1 could be possible targets of ubiquitination, and participate in H1 degradation, six H1.1 mutant variants were created, with batches of Lysine-to-Arginine (K-to-R) substitutions. Among these, the H1.16xGC expressing line, which had one lysine mutated in the globular domain (K89) and five lysines mutated in the C-terminal tail (K204 K206 K211 K213 and K215), showed a strong persistence (95%) of the H1.1 mutant variant in the SMC. Further screening of three additional mutants (H1.15xC H1.1K89R and H1.1K204R) confirmed that H1.1 K89 residue is a critical target in the ubiquitination process, with 78% persistence of H1.1 in SMCs. This study also examined the functional implications of H1 eviction in SMCs for sporogenesis, meiosis, gametogenesis, and fertility using the H1.16xGC expressing line. While the persistence of H1.1 in SMCs did not affect SMC identity or meiosis, it impaired germline development, with the embryo sac arrested at the FG1/FG2 stage. The complete sterility of the line however suggested that this lysine residue plays a role in H1 function beyond embryo sac development. Another research question was whether these lysine residues would also affect the stability and function of H1.1 in somatic tissue. Since H1 is known to regulate heterochromatin, the study examined heterochromatin content and nuclear structure, including chromocenter number and nuclear area, in H1.16xGC and H1.1K89R mutants. The results indicated that these lysines are not essential for heterochromatin content nor nuclear structure. Given H1’s influence on lateral root development, the study also assessed lateral root number and root hair density in the H1.1 mutant variants, finding no differences among H1.16xGC, H1.1K89R and H1.1wt, suggesting that lysine mutations do not affect root development. The work involved extensive 3D image analysis, including reconstruction, segmentation and signal measurements, primarily using the Imaris software (Bitplane, CH). Imaris is predominantly used in animal cell and developmental biology, with limited representation in plant sciences. To address this gap, I conducted a review of 120 plant sciences publications that utilized Imaris for image analysis. The research fields, tools and objectives of these studies were summarized to promote advanced image processing techniques in plant sciences.