A RAF-SnRK2-PP2C functional module regulates cell survival in the chlorophyte Chlamydomonas reinhardtii

The SNF1-related protein kinase 2 (SnRK2) pathway is a central regulator of abiotic stress signaling in land plants, yet its evolutionary origins and functional conservation across the green lineage remain poorly understood. To determine whether this signaling module predates the streptophyte-chlorophyte divergence, we investigated the chlorophyte alga Chlamydomonas reinhardtii, which diverged from the streptophyte lineage over 1 billion years ago. Using a combination of reverse genetics and protein-protein interaction assays, we identified and characterized a bona fide SnRK2 signaling module in C. reinhardtii. We demonstrate that the isoform SnRK2.7 is essential for osmotic stress tolerance and general cellular viability. SnRK2.7 localizes to the contractile vacuole, an osmoregulatory organelle lost during streptophyte evolution, revealing a lineage-specific functional adaptation. Together with MAPKKK3, a B1/B3-RAF kinase, and the clade A protein phosphatase PP2C3, these components constitute a MAPKKK3-SnRK2.7-PP2C3 regulatory module in C. reinhardtii. Our findings demonstrate the presence of a functional SnRK2 pathway in a chlorophyte alga, suggesting that core components were established early in the green lineage. This work provides a foundation for comparative studies across green plants and underscores the need for broader taxonomic sampling to reconstruct the ancestral signaling networks underlying stress adaptation.