Phenotypic and molecular characterization of Ribosomal protein of the small subunit 6 phosphorylation in Arabidopsis thaliana.

The TOR kinase pathway is responsible for mediating the phosphorylation of eS6, a ribosomal protein unique to eukaryotes. The implications of this dynamic phosphorylation event in response to environmental stimuli are still a matter of debate and not fully comprehended despite extensive research conducted over more than five decades. Studies have revealed that eS6-P is implicated in the regulation of cell size, translation, and ribosome biogenesis in mammals and yeast. However, the extent to which it plays a similar role in plants is yet to be determined.

To address the role of eS6 phosphorylation in plants, the experimental approach was to generate multiple combinations of phospho-mutant versions of eS6 with the motivation of generating transgenic lines in a genetic background mutated for both paralogs of the eS6 genes (RPS6) in Arabidopsis, to have a system to investigate the function of eS6 phosphorylation. I analyzed these lines for several generations and identified the ones that were completely devoid of endogenous eS6, harboring either phospho-enabled eS6 or phospho-deficient eS6. Their phenotypes were characterized at the level of translation, photosynthesis, plant growth and development. Our data showed that eS6-P is not essential for the basic functions of eS6 as a ribosomal protein or for bulk translation but has subtle functional consequences at the level of cell-cycle and ribosome biogenesis. However, its primary biochemical readout remains a subject for future work.

eS6 phosphorylation is dynamically regulated in response to various external stimuli including abiotic stresses. Abiotic stress also triggers massive translational inhibition. I studied autophagic turnover in rps6a single mutant, phospho-deficient eS6A, and phospho-enabled eS6Acomplemented double mutants, after hypoxia treatment. The MDC vesicle staining pattern varied between seedlings that were phospho-defective and those that were phospho-enabled, suggesting a potential role of eS6-P in the response. Nonetheless, the results were complicated by factors such as high background staining, a distinct phenotype in rps6a, and the confounding effects of sucrose present in the medium during hypoxia.

The work presented here advances our understanding of the physiological and molecular implications of eS6 protein and its phosphorylation, with its modest but clear effect on early plant development.