GmAGL6 Genes Regulate Floral Proportion and Seed Size Rather than Keel Petal Identity in Soybean (Glycine max)

AGL6 genes are critical floral regulators in diverse angiosperms, yet their roles in legumes remain poorly understood. This study aimed to characterize GmAGL6 genes in soybean (Glycine max [L.] Merr. cv. Williams 82). We identified four homologs (GmAGL6a-d) featuring conserved MADS-box and K-box domains that cluster within the AGL6 lineage. Tissue-specific expression profiling revealed significant transcript enrichment during flower bud differentiation and maturation. Using CRISPR/Cas9, we generated quadruple knockout lines to evaluate gene function. Phenotypic analysis showed that, unlike the homeotic transformations typical of AGL6 loss in monocots, Gmagl6 quadruple mutants retained a standard papilionaceous floral structure without keel petal aberrations. However, the mutants did not show significant changes in floral height or width, but exhibited a significantly increased floral height-to-width ratio and smaller mature seeds, while vegetative architecture and podding capacity remained unaffected. These results suggest that GmAGL6 genes in soybean may function primarily in the regulation of floral proportion and seed development rather than floral organ identity. This research provides insights into the evolution of specialized legume flowers and suggests candidate genes for seed size improvement.