The project was structured around two research Work Packages (WPs), combining advanced molecular biology, omics technologies, and functional genetics in Arabidopsis thaliana.
WP1 – Selection of candidates focused on identifying genes regulated by THE1 during stress. Transcriptomic and phosphoproteomic datasets generated under cell-wall and osmotic stress were analyzed to identify candidate genes acting downstream of THE1. RNA-seq comparisons between the1 mutant alleles and wild-type plants treated with the cellulose biosynthesis inhibitor isoxaben or subjected to hyperosmotic stress identified 15 transcription factors (TFs) whose expression or phosphorylation was THE1-dependent.
The phosphoproteomics approach, initially planned as a complementary method, was expanded to provide post-translational insights beyond the original Description of Action (DoA). This effort generated a valuable dataset revealing phosphorylation-based regulation of signaling elements, substantially strengthening the project’s mechanistic framework.
WP2 – Functional characterization of candidates validated these findings experimentally. Fifteen T-DNA insertional mutant lines (12 knockouts, 2 knockdowns, and 1 over-expression line) were obtained and verified.
The following functional assays were performed to understand the functions of the TFs in plant growth, stress responses and cell wall metabolism:
Root growth phenotyping in medium containing isoxaben or sorbitol, identified altered sensitivity in multiple mutants; Cell-wall monosaccharide profiling revealed changes in glucose, rhamnose, galacturonic acid, and fucose content in particular TF mutants; qRT-PCR based expression analysis of genes required for cellulose biosynthesis (CesA1, CesA3, CesA6) and candidate TFs showed how certain TFs are required for moulating CESA gene expression; Phytohormone quantification (SA, JA, ABA) and lignin deposition assays identified TFs required for deposition of lignin and production of JA, thus connecting TFs required for transcriptional regulation with stress response and cell wall metabolism.
These studies demonstrated that several transcription factors act as stress-specific regulators of cell-wall metabolism and growth adaptation. Constructs for four key TFs were successfully generated to allow mechanistic experiments such as co-immunoprecipitation (co-IP) and chromatin-immunoprecipitation (ChIP). Unfortunately they could not be used for analysis in planta within the fellowship period due to the generation time of Arabidopsis.
Together, the integration of transcriptomics, phosphoproteomics, and mutant phenotyping provided a comprehensive understanding of THE1-dependent transcriptional regulation and established a strong foundation for future mechanistic and translational studies.
