A single amino-acid switch reprograms ROP6 signaling to drive functional evolution in plants

Rho-related GTPases of plants (ROPs) are evolutionarily conserved molecular switches that typically maintain PIN-formed proteins at the plasma membrane (PM), yet their functional divergence remains poorly understood. Here, we reveal that a single amino-acid substitution at residue 194 acts as a core determinant, driving the evolutionary split between Brassicaceae and Malvaceae ROP6 orthologs: glycine enforces PM stabilization, whereas alanine licenses endocytic function. In cotton, the Malvaceae-predominant alanine mutated GhROP6 displays an auxin-independent manner. The distinct activity is attributed to the recruitment of three GhRIC effectors that directly bind the actin cytoskeleton and promote F-actin assembly and bundling. The resulting actin reorganization facilitates GhPIN3a internalization and proteolysis, consequently enhancing cotton fiber elongation. Our findings demonstrate that a single amino-acid change can reprogram ROP signaling and uncover a molecular switch underlying the functional evolution of ROP6 in regulating plant development.