Mitochondrial Cell-Type-Specific Profiling: Differential Function of Mesophyll and Guard Cells is Reflected by Their...

Cell specification and ensuing division of labor enable the accomplishment of complex tasks in multicellular organisms. In plants, mesophyll and guard cells exhibit striking functional, metabolic, and mechanophysical differences: mesophyll cells are highly specialized for efficient light-energy conversion, whereas guard cells regulate gas exchange. Here, we address whether the cellular specialization is sustained by mitochondrial diversity between cells. We developed mRACE (RApid CEll-type-specific isolation of mitochondria), an innovative method for rapid cell-type-specific mitochondrial isolation. mRACE involves biotin labeling of mitochondria in target cells, followed by affinity-based organelle enrichment with streptavidin-coated microbeads or immobilization on functionalized glass slides. Using mitochondrial activity modulators, we confirmed that mRACE-isolated mitochondria remain physiologically active, validating their suitability for downstream analyses. Applying mRACE, we isolated mitochondria from mesophyll and guard cells and performed a comprehensive proteome analysis. Notably, guard cells are enriched in active branched-chain amino acid (BCAA) catabolism components, suggesting they exploit this unconventional carbon source for energy. Additionally, the two cell types differ in ribosomal protein composition and RNA-editing proteins, highlighting mitochondrial specialization at multiple regulatory levels. We investigated gene activity for seven proteins differentially enriched between mesophyll and guard cells. Five promoter-reporter constructs showed distinct activities between both cell types, validating our approach and indicating that distinct molecular make-ups of guard and mesophyll mitochondria are substantially already established at the transcriptional level. Our findings reveal a new layer of mitochondrial specialization between mesophyll and guard cells, and establish mRACE as a powerful tool for isolating and analyzing mitochondria with cell-type specificity.