Abstract
Summary Plant metabolism drives traits essential for productivity and resilience, yet understanding metabolic networks requires subcellular, cellular, and tissue-level spatial context that remains limited, particularly in crop species. Experimentally-derived subcellular localization data for enzymes are sparse, constraining analyses of metabolic organization in the cell. We developed a high-throughput protoplast transformation and fluorescent protein (FP) tagging system optimized for Sorghum bicolor , a climate-resilient C 4 crop. Using this platform, we experimentally determined the subcellular localization of 234 metabolic enzymes spanning 184 pathways. The sorghum enzymes we characterized localize to 12 subcellular compartments. Comparison with computational predictions highlights variable accuracy across compartments, and cross-species comparison with Arabidopsis thaliana shows partial agreement with available experimental data. All data are accessible through the Sorghum Metabolic Atlas ( www.sorghummetabolicatlas.org ) web platform, enabling search, visualization, and download. This study presents a large-scale experimental dataset of enzyme localization in sorghum, providing a resource for studies of plant metabolic organization and comparative analyses.
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Abstract
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 C4 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.
Competing Interest Statement
The authors have declared no competing interest.
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