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...

We tested the hypothesis that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of triacylglycerol in Arabidopsis thaliana seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings, and seeds from plants with both CSY genes disrupted were dormant and did not metabolize triacylglycerol. Germination was achieved by removing the seed coat and supplying sucrose, but the seedlings still did not use triacylglycerol. The mutant seedlings were resistant to 2,4-dichlorophenoxybutyric acid, indicating a block in peroxisomal b-oxidation, and were unable to develop further after transfer to soil. The mutant phenotype was complemented with a cDNA encoding CSY with either its native peroxisomal targeting sequence (PTS2) or a heterologous PTS1 sequence from pumpkin (Cucurbita pepo) malate synthase. These results suggest that peroxisomal CSY in Arabidopsis is not only a key enzyme of the glyoxylate cycle but also catalyzes an essential step in the respiration of fatty acids. We conclude that citrate is exported from the peroxisome during fatty acid respiration, whereas in yeast, acetylcarnitine is exported.

The glyoxylate cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds. The conventional view that the glyoxylate cycle is contained within peroxisomes in fungi and plants is no longer valid. Glyoxylate cycle enzymes are located both inside and outside the peroxisome. Thus, the operation of the glyoxylate cycle requires transport of several intermediates across the peroxisomal membrane. Glyoxylate cycle progression is also dependent upon mitochondrial metabolism. An understanding of the operation and regulation of the glyoxylate cycle, and its integration with cellular metabolism, will require further investigation of the participating metabolite transporters in the peroxisomal membrane.

SUMMARYThe Arabidopsis genome has two fumarase genes, one of which encodes a protein with mitochondrial targeting information (FUM1) while the other (FUM2) does not. We show that a FUM1-green fluorescent protein fusion is directed to mitochondria while FUM2-red fluorescent protein remains in the cytosol. While mitochondrial FUM1 is an essential gene, cytosolic FUM2 is not required for plant growth. However FUM2 is required for the massive accumulation of carbon into fumarate that occurs in Arabidopsis leaves during the day. In fum2 knock-out mutants, fumarate levels remain low while malate increases, and these changes can be reversed with a FUM2 transgene. The fum2 mutant has lower levels of many amino acids in leaves during the day compared with the wild type, but higher levels at night, consistent with a link between fumarate and amino acid metabolism. To further test this relationship we grew plants in the absence or presence of nitrogen fertilizer. The amount of fumarate in leaves increased several fold in response to nitrogen in wild-type plants, but not in fum2. Malate increased to a small extent in the wild type but to a greater extent in fum2. Growth of fum2 plants was similar to that of the wild type in low nitrogen but much slower in the presence of high nitrogen. Activities of key enzymes of nitrogen assimilation were similar in both genotypes. We conclude that FUM2 is required for the accumulation of fumarate in leaves, which is in turn required for rapid nitrogen assimilation and growth on high nitrogen.

SummaryThe aim was to determine the function of peroxisomal NAD + -malate dehydrogenase (PMDH) in fatty acid boxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in b-oxidation, even though microarray analysis shows that b-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in b-oxidation in mutant seedlings, [14 C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid b-oxidation and does not participate directly in the glyoxylate cycle.

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenase (PMDH). PMDH, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana), reduces NAD + to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than in the wild type. This is in contrast to other photorespiratory mutants, which have severely reduced rates of photosynthesis and require high CO 2 to grow. The pmdh mutant had a higher O 2 -dependent CO 2 compensation point than the wild type, implying that either Rubisco specificity had changed or that the rate of CO 2 released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O 2 evolution and uptake were similar in the pmdh and wild-type plants, indicating that chloroplast linear electron transport and photorespiratory O 2 uptake were similar between genotypes. The CO 2 postillumination burst and the rate of CO 2 released during photorespiration were both greater in the pmdh mutant compared with the wild type, suggesting that the ratio of photorespiratory CO 2 release to Rubisco oxygenation was altered in the pmdh mutant. Without PMDH in the peroxisome, the CO 2 released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration in air; however, in its absence, significant rates of photorespiration are still possible, indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or that the HPR reaction is altogether circumvented.

Recycling of carbon by the photorespiratory pathway involves enzymatic steps in the chloroplast, mitochondria, and peroxisomes. Most of these reactions are essential for plants growing under ambient CO(2) concentrations. However, some disruptions of photorespiratory metabolism cause subtle phenotypes in plants grown in air. For example, Arabidopsis thaliana lacking both of the peroxisomal malate dehydrogenase genes (pmdh1pmdh2) or hydroxypyruvate reductase (hpr1) are viable in air and have rates of photosynthesis only slightly lower than wild-type plants. To investigate how disruption of the peroxisomal reduction of hydroxypyruvate to glycerate influences photorespiratory carbon metabolism we analyzed leaf gas exchange in A. thaliana plants lacking peroxisomal HPR1 expression. In addition, because the lack of HPR1 could be compensated for by other reactions within the peroxisomes using reductant supplied by PMDH a triple mutant lacking expression of both peroxisomal PMDH genes and HPR1 (pmdh1pmdh2hpr1) was analyzed. Rates of photosynthesis under photorespiratory conditions (ambient CO(2) and O(2) concentrations) were slightly reduced in the hpr1 and pmdh1pmdh2hpr1 plants indicating other reactions can help bypass this disruption in the photorespiratory pathway. However, the CO(2) compensation points (Γ) increased under photorespiratory conditions in both mutants indicating changes in photorespiratory carbon metabolism in these plants. Measurements of Γ*, the CO(2) compensation point in the absence of mitochondrial respiration, and the CO(2) released per Rubisco oxygenation reaction demonstrated that the increase in Γ in the hpr1 and pmdh1pmdh2hpr1 plants is not associated with changes in mitochondrial respiration but with an increase in the non-respiratory CO(2) released per Rubisco oxygenation reaction.

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