Heterogeneity of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles in different tissues. We quantitatively compared the mitochondrial proteome isolated from a non-photosynthetic cell culture model with more specialized mitochondria isolated from photosynthetic shoots. Differences in intact mitochondrial respiratory rates with various substrates and activities of specific enzymes provided a backdrop of the functional variation between these mitochondrial populations. Proteomics comparisons provided a deep insight into the different steady-state abundances of specific mitochondrial proteins. Combined these data showed the elevated level of the photorespiratory apparatus and its complex interplay with glycolate, cysteine, formate, and one-carbon metabolism as well as the decrease of selected parts of the tricarboxylic acid cycle, alterations in amino acid metabolism focused on 2-oxoglutarate generation, and degradation of branched chain amino acids. Comparisons with microarray analysis of these tissue types showed a positive, mild correlation between mRNA and mitochondrial protein abundance, a tighter correlation for specific biochemical pathways, but over 78% concordance in direction between changes in protein and transcript abundance in the two tissues. Overall these results indicated that the majority of the variation in the plant mitochondrial proteome occurred in the matrix, highlighted the constitutive nature of the respiratory apparatus, and showed the differences in substrate choice and/or availability during photosynthetic and non-photosynthetic metabolism.