Pan-Genome Analysis of the Fructokinase Gene Family Reveals a Light-Regulated SiPhyC–SiFRK4 Module Controlling Carbon Partitioning in Foxtail Millet

Fructokinase (FRK) initiates fructose phosphorylation, channeling carbon into central metabolic pathways, yet its functional diversity and regulatory networks in C4 cereals remain poorly understood. Here, we performed a comprehensive pan-genome analysis of the FRK gene family in foxtail millet (Setaria italica), identifying 697 SiFRKs across 110 accessions and revealing extensive presence–absence variation shaped by evolution and domestication. Among nine characterized members in the reference genome, SiFRK4 exhibited broad and high expression, a diurnal rhythm, and substantial natural variation. Biochemical assays confirmed its fructokinase activity in vitro. We discovered a novel physical interaction between SiFRK4 and the key photoreceptor Phytochrome C (SiPhyC), which co-localized in the cytoplasm. Functional analysis of SiPhyC mutants demonstrated that loss of SiPhyC disrupted carbohydrate homeostasis, elevating fructose while depleting sucrose and starch. Our findings reveal a physical and genetic link between the light-signaling component SiPhyC and the metabolic enzyme SiFRK4, suggesting their interaction influences carbon partitioning. This study provides foundational insights into the sugar metabolism network of a resilient C4 model crop and identifies potential targets for metabolic engineering and breeding.