Foundational characterization of tomato fruit RALF peptides reveals structural and functional specialization within the SlRALF

Rapid Alkalinization Factor (RALF) peptides regulate plant growth and cell wall signaling, but their roles in fruit development remain unclear. Here, we characterized tomato (Solanum lycopersicum) fruit-associated RALF peptides and their interactions with leucine-rich repeat extensins (LRXs). Expression analyses identified SlRALF5, SlRALF7, and SlRALF10 as the main fruit-expressed RALFs. SlRALF10 was associated with early fruit development, whereas SlRALF5 and SlRALF7 remained expressed during ripening. Sequence analyses showed that SlRALF5/7 retain conserved motifs of canonical RALFs, while SlRALF10 displays divergent structural features and altered charge distribution. Synthetic SlRALF5 and SlRALF7 inhibited root growth and induced extracellular alkalinization, whereas SlRALF10 lacked both activities. Co-immunoprecipitation assays showed that all three peptides interact with the fruit-expressed proteins SlLRX2 and SlLRX5. Structural modeling predicted distinct electrostatic properties for the SlLRX5/SlRALF10 complex compared with SlRALF5. These results reveal structural and functional specialization among tomato fruit RALF peptides and suggest that distinct SlRALFs may differentially respond to cell wall remodeling during fruit development and ripening.