Rice fructokinases orchestrate carbon partitioning to regulate photosynthesis and sugar metabolism

Fructokinases (FRKs) are key regulators in carbohydrate metabolism and plant development. However, their specific functions in crop photosynthesis and sugar metabolism remain unclear. In this study, we identified three rice FRKs (OsFRK1, OsFRK2, and OsFRK3), investigating their catalytic activities, expression pattern, and subcellular localization. OsFRK1 and OsFRK2 localized to the cytosol, whereas OsFRK3 localized to chloroplasts, indicating spatially distinct functions. Using CRISPR/Cas9, we generated single mutants (osfrk1, osfrk2, osfrk3) and a double mutant osfrk1×osfrk2 deficient in both cytosolic OsFRKs. All mutants showed significantly reduced plant height, biomass, and grain yield under field conditions. At the physiological level, OsFRK mutants displayed markedly reduced OsFRK enzyme activity, accompanied by impaired photosynthetic efficiency, excessive starch and fructose accumulation in leaves, and elevated sucrose and fructose levels in grains. Loss function of OsFRKs also triggered metabolic compensation in source leaves, including elevated activities of sucrose-degradation enzymes and hexokinase. Additionally, haplotype analysis identified elite alleles of OsFRKs: FRK1^C (higher panicle number), FRK2^C (greater 1000-grain weight), and FRK3^A (higher panicle number). Cultivars carrying the combined haplotypes (FRK1^C–FRK2^C–FRK3^A) exhibited higher grain yields than other genotype combinations. Together, these findings revealed cytosolic and chloroplastic OsFRKs in regulating rice photosynthesis, sugar allocation, and yield formation, and highlighted the elite OsFRK haplotypes as valuable genetic resources for future rice improvement.