Cytosolic MAPK signaling gates chloroplast protein import and photosynthetic capacity

Chloroplast protein import is essential for photosynthesis, yet whether and how cytosolic signaling pathways dynamically regulate this process remains largely unknown. Here we uncover a signaling module that links mitogen activated protein kinase (MAPK) activity to fine tune the import of small subunit of Rubisco (RbcS) into the chloroplast. We show that MPK3 negatively regulates the cytosolic Raf-like kinase ACTPK1, which directly phosphorylates the transit peptide of RbcS precursor at Thr12. MPK3 directly phosphorylates ACTPK1, attenuating its kinase activity and thereby limiting RbcS transit peptide phosphorylation. Genetic and physiological analyses demonstrate that loss of MPK3 elevates ACTPK1 activity, increases RbcS phosphorylation and enhances Rubisco accumulation and CO₂ assimilation. On the other hand, ACTPK1 deficiency compromises these processes and photosynthetic performance. Phospho-mutant analyses further reveal that reversible phosphorylation of the RbcS transit peptide is required for efficient chloroplast import. Together, our findings establish chloroplast protein import as a signaling-regulated process and identify transit peptide phosphorylation a key check point integrating cytosolic MAPK signaling with photosynthetic capacity.