Target of Rapamycin (TOR) regulates translation via phosphorylation of two main substrate classes: 4EBPs and S6Ks. Although TOR inactivation decreases overall translation in all eukaryotic systems, 4EBP-like factors are considered as metazoan inventions and remain obscure in plants. In this project, we discovered and characterized Arabidopsis 4EBPs whose primary structure differs from mammalian 4EBPs. Yeast-two hybrid was used to dissect eIF4E-binding sites of 4EBP1/2 and reveal amino acids critical for binding (WP1.1). Monoclonal antibodies and phosphoantibodies were produced against two conserved phosphorylation sites on 4EBP1/2 and used to detect total and phosphorylation levels of 4EBP1/2 under TOR contrasting conditions (WP1.2). Furthermore, tagged 4EBP1/2 overexpression line, 4EBP1 knock-out by Crispr-CAS9 and 4EBP1/2 RNAi lines were generated. These lines were used to analyse the impact of 4EBP1/2 on plant growth and development (WP1.3 and 1.4). Tagged 4EBP1/2 overexpression line was used to identify phospho-peptides and interaction partners. However, due to the technical issues of 4EBP1/2 digestion, other predicted phosphorylation sites were not yet identified. The method is currently optimized to repeat the experiment (WP1.5). additional methods were applied to study possible 4EBP interaction partners, e.g. immunoprecipitation, glycerol gradient co-sedimentation (WP1.5). 4EBP1/2 knock-out lines were obtained and characterized. Global translatome profiling by Ribo-seq is on-going experiment (WP2.1). Polysome profiling and mesophyll protoplast approaches allowed us to identify a subset of mRNAs regulated by 4EBP1/2 and TOR (WP2.2). From WP2, we found more promising targets involved in cytokinin signalling pathway by polysome profiling. So we further analysed how TOR regulates meristem via CK signalling. It is still on-going work whether 4EBP regulates the translation of components in CK signalling (WP3.1). Different reporters for expression in 4EBP knock-out lines are under construction and will be used to study the role of 4EBP in shoot apical meristem (WP3.2). 4EBP phosphorylation by TOR responded to various abiotic stresses, e.g. salt, dark, submergence (WP3.3). It is still on-going work to construct 5’UTR-GFP reporter construct to transform Arabidopsis. However, as a nitrogen-rich compound upstream of TOR we identified spermidine and concentrated on studies of the Spd-TOR signaling axis role in regulation of TOR activity. We have identified subset of mRNAs regulated by the Spd-TOR relay. Whether the Spermidine-TOR relay is controlling the cap-dependent translation initiation via 4EBPs is still under investigation.
In summary, we studied 4EBP interaction with the cap-binding complex, phosphorylation by TOR and biological significance for plant development. Client mRNAs regulated by the TOR-4EBP pathway have been identified. Several over-expression and knock-out/down lines of 4EBPs have been generated, which will be further used for polysomal RNA-seq and ribosome profiling (ribo-seq). Dr. Dong has finished most of the proposed work on the project and the main results are in preparation for publication.
Some of the results from Dr Dong project have already been published in Salazar-Diaz and Dong et al., iScience, 2021 (results are related to Task 4—identification a nitrogen-rich compound as a new upstream TOR signal). Despite COVID lockdown, Dr. Dong has actively participated in three international conferences to present the new results: EMBO workshop: Protein synthesis and translational control 2020 online (poster presentation), TOR de France 2021 in Nice, France (oral presentation) and EMBO workshop: TOR signaling in photosynthetic organisms 2021 online (oral presentation). Dr. Dong also gave two institute seminars in IBMP.
