Abstract
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.
Highlights
Heterogeneity of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles in different tissues
Mitochondria were purified from Arabidopsis cell culture and a hydroponic shoot culture based on density purification techniques using Percoll
We have shown previously that respiration by cell culture mitochondria via ETFQO best utilizes Val and the Val-derived organic acid (␣-ketoisovaleric acid) over Leu or Ile derivatives [41], whereas plant leaf mitochondria are best known for catabolism of Leu [75], and knock-out of ETFQO in Arabidopsis leads to accumulation primarily of Leu-derived organic acids in leaves [53, 76]
Summary
Maintenance of Arabidopsis Cell Culture and Hydroponic Seedling Culture—Arabidopsis cell suspension was cultured in growth medium (1ϫ Murashige and Skoog medium without vitamins, 3% sucrose, 0.5 mg/liter naphthaleneacetic acid, 0.05 mg/liter kinetin, pH 5.8) at 22 °C under a 16/8-h day/night regime and light intensity of 90 mol mϪ2 sϪ1 with orbital shaking at 120 rpm. The transferred mitochondrial band was diluted ϳ5-fold with sucrose wash medium (0.3 M sucrose, 0.1% (w/v) BSA, 10 mM TES, pH 7.5) and centrifuged at 24,000 ϫ g for 10 min. When proteins of different families were identified in a gel spot, a reference map of the Arabidopsis mitochondrial proteome was used to identify the most probable match, taking into account the number of peptides with ion score Ͼ38 and the quality of delta mass for each peptide. Microarray Analysis—Microarray analysis of the changes in transcript abundance in cell culture and shoot was performed using Affymetrix GeneChipா Arabidopsis ATH1 Genome Arrays (catalog number 900386). Washing, staining, scanning, and data extraction were performed using Affymetrix GeneChip Operating Software (version 1.4). Shoot mitochondria NADH Glutamate ϩ malate Glycine Malate ϩ pyruvate Succinate Glutamate Formate Integrity (%)
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