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 <i>FRK</i> gene family in foxtail millet (<i>Setaria italica</i>), identifying 697 <i>SiFRKs</i> across 110 accessions and revealing extensive presence-absence variation shaped by evolution and domestication. Among nine characterized members in the reference genome, <i>SiFRK4</i> 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 <i>SiPhyC</i> mutants demonstrated that loss of <i>SiPhyC</i> 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.