A homolog of Growth Regulating Factor Targets Protein Synthesis Genes to Regulate Seed Protein Accumulation in Soybean

Abstract Growth Regulating Factor (GRF) genes encode a family of highly conserved plant transcription factors that control diverse developmental processes. However, no GRF5 orthologs have been directly linked to seed protein accumulation. Here we demonstrate that GmGRF5a, a soybean homolog of GRF5, functions as a key positive regulator of seed protein accumulation. GmGRF5a is preferentially expressed in reproductive tissues, with transcript levels gradually increasing throughout seed development. Overexpression of GmGRF5a driven by its native promoter or the CAB3 promoter increased seed protein content by approximately one percentage point—comparable to the effect of known protein QTLs in soybean—without yield penalty across three independent field seasons, whereas CRISPR/Cas9-generated mutants showed reduced protein and oil contents. Integrative analysis of genome-wide DAP-seq and transcriptome profiling identified hundreds of direct GmGRF5a target genes significantly enriched in pathways related to protein synthesis, ribosomal biogenesis, and energy metabolism. Multiple independent molecular assays, including ChIP-qPCR, EMSA, and dual-luciferase reporter assays, confirmed that GmGRF5a directly activates genes encoding seed storage proteins (e.g., GmCupin1-1) and translational components (e.g., GmRPS4 and GmGOGAT2). Furthermore, natural variation at amino acid position 124 near the WRC DNA-binding domain is associated with seed protein content and exhibits a latitude-dependent distribution pattern consistent with regional breeding priorities. Our findings demonstrate that GmGRF5a coordinates a transcriptional network governing seed protein synthesis and highlight its potential as a promising target for breeding high-protein soybean cultivars without compromising agronomic performance.