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

Hater F; Eirich J; Heinkow P; Joshi S; Ehlert Y; Palovaara J; Erbasol Serbes I; Brahmia A; Diederichs M; Jalili S; Mukherjee N; Ssemanda P; Peter A; Schweser O; Kubitschke M; Madduri MK; Kirstein J; Maedler K; Masseck OA; Finkemeier I; Groß-Hardt R

Abstract

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.