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 non-photosynthetic cell culture model to 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. Proteomic 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 TCA cycle, alterations in amino acid metabolism focused on 2-oxoglutarate generation, and degradation of branched chain amino acids. Comparisons to 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 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.