Sorghum Metabolic Atlas: Large-Scale Mapping of Subcellular Enzyme Localization in Sorghum bicolor

Plant metabolism underpins growth and stress resilience, yet spatial information about metabolic networks and enzyme localization remains limited. We developed a high-throughput protoplast transformation and fluorescent-protein tagging system optimized for Sorghum bicolor , a climate-resilient C ₄ crop. Using this platform, we experimentally determined the subcellular localization of 234 enzymes spanning 184 pathways. These enzymes localized to 12 subcellular compartment classes, revealing conserved and species-specific patterns relative to Arabidopsis thaliana , including some unexpected. In the chorismate biosynthesis pathway, a key enzyme unexpectedly localized to the cytosol, suggesting functional divergence in pathway architecture. In the Calvin-Benson cycle, several enzymes formed puncta in the chloroplast, indicative of possible biocondensate formation. All data are available online via the Sorghum Metabolic Atlas ( sorghummetabolicatlas.org ). This dataset represents the first expansive experimental map of enzyme localization in sorghum, offering a foundational resource for investigating metabolic compartmentalization and advancing pathway engineering in bioenergy and food crops.