Fully Tunable Phosphorylation of RPS6A Ensures the Successful Development of Arabidopsis Seedlings

Light enhances protein translation, enabling young seedlings to rapidly and timely acquire photosynthetic capacities. Sequential phosphorylation of ribosomal protein S6 (RPS6) was implicated in the light-enhanced translation; however, the exact phosphorylation sites and the biological relevance of RPS6 multi-phosphorylation in seedling development remain elusive. Here, we report the identification and quantification of RPS6A residues that exhibit dynamic, differential phosphorylation in seedlings grown in darkness or during the initial exposure to light. Among six C-terminal sites, four serine residues, serine-229 (S229), S231, S237 and S240, serve as seed sites for light-regulated sequential phosphorylation. Combinatorial mutations of the C-terminal serines/threonine (S/T) to aspartic acids (phospho-mimic) or alanines (phospho-null) partially rescued the reduced hypocotyl elongation in etiolated rps6a seedlings. De-etiolating rps6a seedlings expressing phospho-mimic or phospho-null RPS6A showed decreased photosynthetic protein accumulation and reduced translation capacity. These findings indicate that fully tunable phosphorylation of RPS6A is essential for its complete function in hypocotyl elongation, translation efficiency, and photosynthetic capacities in both etiolated and de-etiolating seedlings. Our results demonstrate that the structural integrity of the C-terminal S/T residues is vital for establishing precise phosphorylation codes of RPS6A in light or dark conditions. Even a single substitution at these conserved residues can disrupt the light-regulated phosphorylation-dephosphorylation dynamics of RPS6A, thereby impairing its functions. This also explains the evolutionary conservation and importance of these C-terminal S/T residues to warrant young seedlings' capacities to adapt effectively to changing light environments in their natural habitats.