S²-PepAnalyst: A Web Tool for Predicting Plant Small Signalling Peptides

Small signalling peptides (SSPs) serve as crucial mediators of cell-to-cell communication in plants, orchestrating diverse physiological processes from development to stress responses. While recent advances in sequencing technologies have improved genome annotation, the identification of novel SSPs remains challenging due to their small size, sequence diversity, and often transient expression patterns. To address this bottleneck, we developed S²-PepAnalyst, a machine learning-powered web tool that integrates plant-specific datasets with advanced computational approaches for SSP prediction and classification. Our platform combines protein language models with geometric-topological feature analysis to capture both sequence and structural characteristics of known SSP families. When validated against experimentally confirmed peptides, S²-PepAnalyst achieved high predictive accuracy (99.5%) while maintaining low false-negative rates. The tool successfully classified peptides into functionally distinct families (e.g., CLE, RALF) and identified non-canonical SSPs that lack traditional signal peptides. Importantly, S²-PepAnalyst demonstrated robust performance across both model plants and agriculturally important species. As a freely available resource (https://www.s2-pepanalyst.uma.es), this tool will empower plant biologists to systematically explore the largely untapped repertoire of plant SSPs, facilitating discoveries in plant cell signalling and potential applications in crop improvement.