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Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.