Knockout of GmCKX3 Enhances Soybean Seed Yield via Cytokinin-Mediated Cell Expansion and Lipid Accumulation

Soybean is a dual-purpose crop for food and oil, playing a crucial role in China's grain production. Seed size and weight are key agronomic traits directly influencing the yield. Cytokinin oxidases/dehydrogenases (CKXs) specifically degrade certain isoforms of endogenous cytokinins (CKs), thereby modulating plant growth and seed development. However, their role in soybeans remains largely uncharacterized. In a previous genome-wide association study of 250 soybean core germplasms, we identified <i>GmCKX3</i> as a yield-related gene. To elucidate its function, we developed <i>GmCKX3</i>-deficient mutants using CRISPR/Cas9 gene editing in soybean Williams82 and conducted a three-year phenotypic analysis. Loss of <i>GmCKX3</i> function significantly enhanced the seed size and weight, which was attributed to an increased cell size and fat accumulation in the endosperm. This enhancement was driven by elevated endogenous CK levels resulting from suppressed <i>GmCKX3</i> expression. Subcellular localization revealed that <i>GmCKX3</i> resides in the endoplasmic reticulum and predominantly degrades the isopentenyladenine (iP)-type CK. Integrated transcriptomic and metabolomic analyses uncovered key genes and pathways involved in CK regulation, supporting <i>GmCKX3</i>'s central role in seed-trait modulation. These findings advance our understanding of cytokinin-mediated seed development and offer promising targets for molecular breeding aimed at improving the soybean yield.