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

Summary 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.