Chun Pong Lee

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

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 NAD-linked 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 CO2 assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO2 assimilation/intercellular CO2 curves at low CO2, 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.

TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana.

The TCP family of transcription factors and site II promoter elements that they bind in Arabidopsis link the regulation of gene expression for mitochondrial proteins with a variety of circadian clock components to provide specific time-of-day expression for a variety of genes. 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 for Photosynthetic and Non-photosynthetic Arabidopsis Metabolism

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.

Mitochondrial Composition, Function and Stress Response in Plants†

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to ATP synthesis via oxidative phosphorylation. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to support photosynthetic processes in illuminated leaves. This review begins with outlining current models of mitochondrial composition in plant cells, with an emphasis upon the assembly of the complexes of the classical electron transport chain (ETC). Next, we focus upon the comparative analysis of mitochondrial function from different tissue types. A prominent theme in the plant mitochondrial literature involves linking mitochondrial composition to environmental stress responses, and this review then gives a detailed outline of how oxidative stress impacts upon the plant mitochondrial proteome with particular attention to the role of transition metals. This is followed by an analysis of the signaling capacity of mitochondrial reactive oxygen species, which studies the transcriptional changes of stress responsive genes as a framework to define specific signals emanating from the mitochondrion. Finally, specific mitochondrial roles during exposure to harsh environments are outlined, with attention paid to mitochondrial delivery of energy and intermediates, mitochondrial support for photosynthesis, and mitochondrial processes operating within root cells that mediate tolerance to anoxia and unfavorable soil chemistries. [Formula: see text] [ A. Harvey Millar (Corresponding author)].

Diurnal Changes in Mitochondrial Function Reveal Daily Optimization of Light and Dark Respiratory Metabolism in Arabidopsis

Biomass production by plants is often negatively correlated with respiratory rate, but the value of this rate changes dramatically during diurnal cycles, and hence, biomass is the cumulative result of complex environment-dependent metabolic processes. Mitochondria in photosynthetic plant tissues undertake substantially different metabolic roles during light and dark periods that are dictated by substrate availability and the functional capacity of mitochondria defined by their protein composition. We surveyed the heterogeneity of the mitochondrial proteome and its function during a typical night and day cycle in Arabidopsis shoots. This used a staged, quantitative analysis of the proteome across 10 time points covering 24 h of the life of 3-week-old Arabidopsis shoots grown under 12-h dark and 12-h light conditions. Detailed analysis of enzyme capacities and substrate-dependent respiratory processes of isolated mitochondria were also undertaken during the same time course. Together these data reveal a range of dynamic changes in mitochondrial capacity and uncover day- and night-enhanced protein components. Clear diurnal changes were evident in mitochondrial capacities to drive the TCA cycle and to undertake functions associated with nitrogen and sulfur metabolism, redox poise, and mitochondrial antioxidant defense. These data quantify the nature and nuances of a daily rhythm in Arabidopsis mitochondrial respiratory capacity.

Recent advances in the composition and heterogeneity of the Arabidopsis mitochondrial proteome

Mitochondria are important organelles for providing the ATP and carbon skeletons required to sustain cell growth. While these organelles also participate in other key metabolic functions across species, they have a specialized role in plants of optimizing photosynthesis through participating in photorespiration. It is therefore critical to map the protein composition of mitochondria in plants to gain a better understanding of their regulation and define the uniqueness of their metabolic networks. To date, <30% of the predicted number of mitochondrial proteins has been verified experimentally by proteomics and/or GFP localization studies. In this mini-review, we will provide an overview of the advances in mitochondrial proteomics in the model plant Arabidopsis thaliana over the past 5 years. The ultimate goal of mapping the mitochondrial proteome in Arabidopsis is to discover novel mitochondrial components that are critical during development in plants as well as genes involved in developmental abnormalities, such as those implicated in mitochondrial-linked cytoplasmic male sterility.

Combining proteomics of root and shoot mitochondria and transcript analysis to define constitutive and variable components in plant mitochondria.

Mitochondria undertake respiration in plant cells, but through metabolic plasticity utilize differ proportions of substrates and deliver different proportions of products to cellular metabolic and biosynthetic pathways. In Arabidopsis the mitochondrial proteome from shoots and cell culture have been reported, but there has been little information on mitochondria in roots. We compare the root mitochondrial proteome with mitochondria isolated from photosynthetic shoots to define the role of protein abundance in these differences. The major differences observed were in the abundance and/or activities of enzymes in the TCA cycle and the mitochondrial enzymes involved in photorespiration. Metabolic pathways linked to TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation. Comparisons to microarray analysis of these same tissues showed a positive correlation between mRNA and mitochondrial protein abundance, but still ample evidence for the role of post-transcriptional processes in defining mitochondrial composition. Broader comparisons of transcript abundances for mitochondrial components across Arabidopsis tissues provided additional evidence for specialization of plant mitochondria, and clustering of these data in functional groups showed the constitutive vs variably expressed components of plant mitochondria.

Mitochondrial Proteome Heterogeneity between Tissues from the Vegetative and Reproductive Stages of Arabidopsis thaliana Development

Specialization of the mitochondrial proteome in Arabidopsis has the potential to underlie the roles of these organelles at different developmental time points and in specific organs; however, most research to date has been limited to studies of mitochondrial composition from a few vegetative tissue types. To provide further insight into the extent of mitochondrial heterogeneity in Arabidopsis, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, bolt stem, and silique, were analyzed. Of the 286 protein spots on a 2-D gel of the mitochondrial proteome, the abundance of 237 spots was significantly varied between different samples. Identification of these spots revealed a nonredundant set of 83 proteins which were differentially expressed between organ/cell types, and the protein identification information can be analyzed in an integrated manner in an interactive fashion online. A number of mitochondrial protein spots were identified as being derived from the same genes in Arabidopsis but differed in their pI, indicating organ-specific variation in the post-translational modifications, or in their MW, suggesting differences in truncated mitochondrial products accumulating in different tissues. Comparisons of the proteomic data for the major isoforms with microarray analysis showed a positive correlation between mRNA and mitochondrial protein abundance and 60-90% concordance between changes in protein and transcript abundance. These analyses demonstrate that, while mitochondrial proteins are controlled transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the state or regulation of proteins in key biochemical pathways in plant mitochondria for specific functions. The integration of protein abundance and protein modification data with respiratory measurements, enzyme assays, and transcript data sets has allowed the identification of organ-enhanced differences in central carbon and amino acid metabolism pathways and provides ranked lists of mitochondrial proteins that are strongly transcriptionally regulated vs those whose abundance or activity is strongly influenced by a variety of post-transcriptional processes.

An Arabidopsis Stomatin-Like Protein Affects Mitochondrial Respiratory Supercomplex Organization

A mitochondrial membrane protein forms a large oligomeric complex that affects the abundance of complex I and respiratory supercomplexes. Stomatins belong to the band-7 protein family, a diverse group of conserved eukaryotic and prokaryotic membrane proteins involved in the formation of large protein complexes as protein-lipid scaffolds. The Arabidopsis (Arabidopsis thaliana) genome contains two paralogous genes encoding stomatin-like proteins (SLPs; AtSLP1 and AtSLP2) that are phylogenetically related to human SLP2, a protein involved in mitochondrial fusion and protein complex formation in the mitochondrial inner membrane. We used reverse genetics in combination with biochemical methods to investigate the function of AtSLPs. We demonstrate that both SLPs localize to mitochondrial membranes. SLP1 migrates as a large (approximately 3 MDa) complex in blue-native gel electrophoresis. Remarkably, slp1 knockout mutants have reduced protein and activity levels of complex I and supercomplexes, indicating that SLP affects the assembly and/or stability of these complexes. These findings point to a role for SLP1 in the organization of respiratory supercomplexes in Arabidopsis.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.