Abstract
<title>Abstract</title> <p>Cysteine-rich peptides (CRPs) are vital components in many prokaryotic and eukaryotic organisms. However, their remarkable sequence diversity has left them as a largely uncharted “dark proteome” within plant genomes. Here, we establish a structure-based phylogenomic approach to decode the evolutionary landscape of CRPs across 868 species. By integrating high-throughput structural clustering, we redefined the CRP superfamily into Major Superfamilies (MSs), Subfamilies (SFs), and Emerging Families (EFs), achieving a resolution that far surpasses traditional sequence-based methods. Our analysis reveals a massive expansion of CRPs in Poaceae, predominantly driven by the proliferation of EFs within transposable element-depleted, recombination-rich distal chromosomal regions. We propose that these EFs constitute an "evolutionary reservoir" characterized by structural and sequence plasticity. Crucially, we exemplified and functionally validated this model through the characterization of Reduced Male Fertility 1 (RMF1), a Poaceae-specific CRP-lineage that evolved from the evolutionary reservoir into a fixed functional state. The transition of RMF1 is marked by a structural reorganization from a beta-sheet to a helical fold, accompanied by a shift from neutral drift to intense purifying selection. CRISPR/Cas9-mediated knockout of RMF1 in rice and the identification of its TILLING mutants in wheat both lead to partly male sterility, confirming its essential role in cereal reproduction. Furthermore, as a proof-of-concept for breeding applications, we identified climate-adaptive CRP clusters on chromosome 6H and breeding-selected thionin-like clusters on 7H in European barley. Together, our study uncovers the evolutionary driving force underlying CRP expansion and provides a structure-based classification system which may facilitate harnessing the adaptive potential of the small-peptide dark proteome for future crop improvement</p>