Mitochondrial Β-Carbonic Anhydrase is a Conserved Metabolic Rheostat for Branched-Chain Amino Acid Catabolism and Metabolic Flexibility

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of CO2, enabling fundamental processes in organisms across all domains of life. Among all CAs, the role of mitochondrial βCA remains poorly understood. Here, we identify a mitochondrial βCA, βCA6, as a key regulator of branched-chain amino acid (BCAA) catabolism and metabolic flexibility during carbon starvation in Arabidopsis thaliana. Loss of βCA6 triggers hypersensitivity to prolonged darkness, marked by accelerated Chl degradation, early senescence, impaired BCAA degradation, and disrupted carbon-nitrogen remobilization. Transcriptomic and metabolic profiling revealed elevated expression of BCAA catabolic enzymes, as well as BCAA accumulation and reduced glutamate levels, indicating defective carbon-nitrogen remobilization. βca6 loss-of-function mutants exhibited a striking hypersensitivity to exogenous BCAAs, supporting a central role of βCA6 in BCAA homeostasis. These findings uncover a previously unrecognized function for mitochondrial CA in maintaining energy balance under dark stress. Given the evolutionary conservation of mitochondria and BCAA metabolism, our work highlights a broadly relevant mechanism by which eukaryotes integrate core metabolic pathways with environmental adaptation.