Pan-species genome-wide evolution of the RGF peptide gene family and its impact on adventitious root regeneration in apple.

Root meristem growth factors (RGFs) are pivotal regulators of root growth in plants and are critically involved in the regulation of root development in fruit trees. In this study, we constructed a comprehensive small secreted peptide library from 64 plant species. Using a structure-based screening strategy, we identified a total of 495 RGF genes. Phylogenetic analysis revealed that the modern RGF gene family originated in ferns and exhibited an evolutionary pattern of initial contraction and subsequent expansion, consistent with the evolutionary emergence and diversification of plant root systems. Analysis of the Malus pan-genome indicated that RGF genes within the genus were classified into 12 clades, with some clades present exclusively in wild Malus species. Notably, the repertoire and copy number of RGF peptides vary significantly among Malus species with different ploidy levels, suggesting a potential association between RGF gene expansion and polyploidization. Functional analysis of a gene encoding the canonical RGF mature peptide 'DYTPARKKPPIHN' in Malus demonstrated a dose-dependent effect on primary root elongation in apple seedlings following exogenous application. Subsequent experiments confirmed that overexpression of MdglRGF1 negatively regulates adventitious root growth during regeneration-associated and hormone-induced adventitious rooting, a phenotype sustained throughout adventitious root development in soil-grown seedlings. Collectively, our research provides novel insights into the identification and functional characterization of small peptides and establishes a foundation for their potential application in apple rootstock breeding and the development of ideal root system architecture.