Malate dehydrogenase (MDH) catalyzes a reversible NAD + -dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NADlinked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO 2 assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO 2 assimilation/intercellular CO 2 curves at low CO 2 , and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.Plant tissues contain multiple isoforms of malate dehydrogenase (L-malate-NAD-oxidoreductase [MDH]; EC 1.1.1.37) that catalyze the interconversion of malate and oxaloacetate (OAA) coupled to reduction or oxidation of the NAD pool. These isoforms are encoded by separate genes in plants and have been shown to possess distinct kinetic properties as well as subcellular targeting and physiological functions (Gietl, 1992). While the MDH reaction is reversible, it strongly favors the reduction of OAA. The direction of the reaction in vivo depends on substrate/product ratios and the NAD redox state, and it can vary even in the same tissue due to prevailing physiological conditions. Isoforms operate in mitochondria, chloroplasts, peroxisomes, and the cytosol, but due to the ready transport and utilization of malate and OAA and the availability of NAD, this reaction can cooperate across compartments and is the basis for malate/OAA shuttling of reducing equivalents in many different metabolic schemes of plant cellular f...

Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested. These elements bind to the TCP family of transcriptions factors in Arabidopsis thaliana, which nearly all display distinct diurnal patterns of cycling transcript abundance. TCP2, TCP3, TCP11, and TCP15 were found to interact with different components of the core circadian clock in both yeast two-hybrid and direct protein-protein interaction assays, and tcp11 and tcp15 mutant plants showed altered transcript profiles for a number of core clock components, including LATE ELONGATED HYPOCOTYL1 and PSEUDO RESPONSE REGULATOR5. Thus, site II elements in the promoter regions of genes encoding mitochondrial, plastid, and peroxisomal proteins provide a direct mechanism for the coordination of expression for genes involved in a variety of organellar functions, including energy metabolism, with the time-of-day specific needs of the organism.

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 nonphotosynthetic 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 steadystate 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. Molecular & Cellular Proteomics 7:1297-1316, 2008.Plant mitochondria have been traditionally purified from total cell extracts of storage organs, etiolated tissues, or cell cultures by differential centrifugation and density gradient centrifugation using sucrose or Percoll (1-4). These organelles are derived from tissues where sugars or fats are used as energy sources and mitochondria have a primary role in ATP production for cellular activity. The combination of Percoll and PVP 1 gradients has also enabled purification of mitochondria away from chloroplasts in extracts of photosynthetic shoot or leaf samples from plants (3). These organelles are known to have a range of additional roles including carbon skeleton provision for nitrogen assimilation (5) and the recycling of carbon in the photorespiratory cycle (6, 7).Early reports using 1D SDS-PAGE documented apparent differences between mitochondria isolated from leaves, petioles, and roots from spinach (8); roots, leaves, and flowers from sugar beet (9); and shoot, scutella, endosperm, and cob from developing maize (10). Remy et al. (11) performed the first comparative 2D IEF/SDS-PAGE study of pea mitochondrial protein profiles isolated from different organs and quantified differences in the abundance of glycine decarboxylase complex subunits using protein spot intensities. Similar studies to compare mitochondrial proteomes were also performed on pot...