A. T. Eprintsev

Succinate dehydrogenase in Arabidopsis thaliana is regulated by light via phytochrome A.

The effect of light on succinate dehydrogenase (SDH) activity and mRNA content was studied in Arabidopsis thaliana plants. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down‐regulating factors for the mRNA content of the sdh1‐2 and sdh2‐3 genes and SDH catalytic activity both in A. thaliana wild‐type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far‐red light of 730 nm reversed the red light effect. It is concluded that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression.

Phytochrome-mediated regulation of plant respiration and photorespiration

The expression of genes encoding various enzymes participating in photosynthetic and respiratory metabolism is regulated by light via the phytochrome system. While many photosynthetic, photorespiratory and some respiratory enzymes, such as the rotenone-insensitive NADH and NADPH dehydrogenases and the alternative oxidase, are stimulated by light, succinate dehydrogenase, subunits of the pyruvate dehydrogenase complex, cytochrome oxidase and fumarase are inhibited via the phytochrome mechanism. The effect of light, therefore, imposes limitations on the tricarboxylic acid cycle and on the mitochondrial electron transport coupled to ATP synthesis, while the non-coupled pathways become activated. Phytochrome-mediated regulation of gene expression also creates characteristic distribution patterns of photosynthetic, photorespiratory and respiratory enzymes across the leaf generating different populations of mitochondria, either enriched by glycine decarboxylase (in the upper part) or by succinate dehydrogenase (in the bottom part of the leaf).

Organic Acids: The Pools of Fixed Carbon Involved in Redox Regulation and Energy Balance in Higher Plants

Organic acids are synthesized in plants as a result of the incomplete oxidation of photosynthetic products and represent the stored pools of fixed carbon accumulated due to different transient times of conversion of carbon compounds in metabolic pathways. When redox level in the cell increases, e.g., in conditions of active photosynthesis, the tricarboxylic acid (TCA) cycle in mitochondria is transformed to a partial cycle supplying citrate for the synthesis of 2-oxoglutarate and glutamate (citrate valve), while malate is accumulated and participates in the redox balance in different cell compartments (via malate valve). This results in malate and citrate frequently being the most accumulated acids in plants. However, the intensity of reactions linked to the conversion of these compounds can cause preferential accumulation of other organic acids, e.g., fumarate or isocitrate, in higher concentrations than malate and citrate. The secondary reactions, associated with the central metabolic pathways, in particularly with the TCA cycle, result in accumulation of other organic acids that are derived from the intermediates of the cycle. They form the additional pools of fixed carbon and stabilize the TCA cycle. Trans-aconitate is formed from citrate or cis-aconitate, accumulation of hydroxycitrate can be linked to metabolism of 2-oxoglutarate, while 4-hydroxy-2-oxoglutarate can be formed from pyruvate and glyoxylate. Glyoxylate, a product of either glycolate oxidase or isocitrate lyase, can be converted to oxalate. Malonate is accumulated at high concentrations in legume plants. Organic acids play a role in plants in providing redox equilibrium, supporting ionic gradients on membranes, and acidification of the extracellular medium.

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves.

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.

Glyoxylate cycle enzymes are present in liver peroxisomes of alloxan-treated rats.

Key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan‐treated rats. The activity of ICL in rat liver was 0.040 μmol/min/mg protein and the activity of MS was 0.022 μmol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87–90).

Expression and properties of the mitochondrial and cytosolic forms of fumarase in germinating maize seeds.

Fumarase (EC 4.2.1.2) catalyzes reversible interconversion of malate and fumarate. It is usually associated with the tricarboxylic acid cycle in mitochondria, although the cytosolic form has also been detected. We investigated the expression of two fumarase genes and activities of the mitochondrial and cytosolic isoforms of fumarase in maize (Zea mays) scutellum during germination. Both isoforms were purified to electrophoretic homogeneity. The cytosolic form had low optimum pH (6.5) and high affinity to malate (Km 5 μM) when compared with the mitochondrial form (optimum pH 7.0, Km 50 μM). The cytosolic form was strongly activated by Mg(2+) and even more by Mn(2+) , whereas the mitochondrial form was moderately activated by Mg(2+) and Mn(2+) was less effective. The highest fumarase activity in scutellum and a high expression of the gene encoding the cytosolic form were observed during the maximal activity of the glyoxylate cycle. In leaves, the localization of fumarase is only mitochondrial and only one fumarase gene is expressed. It is concluded that the function of cytosolic fumarase in maize scutellum can be related to metabolism of succinate formed in the glyoxylate cycle.

Ca2+ is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.

Light regulation of succinate dehydrogenase expression in Arabidopsis thaliana leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.

Expression and properties of the glyoxysomal and cytosolic forms of isocitrate lyase in Amaranthus caudatus L.

Isocitrate lyase (EC 4.1.3.1) catalyzes the reversible conversion of d-isocitrate to succinate and glyoxylate. It is usually associated with the glyoxylate cycle in glyoxysomes, although the non-glyoxysomal form has been reported and its relation to interconversion of organic acids outside the glyoxylate cycle suggested. We investigated the expression of two isocitrate lyase genes and activities of the glyoxysomal (ICL1) and cytosolic (ICL2) forms of isocitrate lyase in amaranth (Amaranthus caudatus L.) seedlings. Both forms were separated and purified. The cytosolic form had a low optimum pH (6.5) and was activated by Mn(2+) ions, while Mg(2+) was ineffective, and had a lower affinity to d, l-isocitrate (Km 63 μM) as compared to the glyoxysomal form (optimum pH 7.5, K(m) 45 μM), which was activated by Mg(2+). The highest ICL1 activity was observed on the 3rd day of germination; then the activity and expression of the corresponding gene decreased, while the activity of ICL2 and gene expression increased to the 7th day of germination and then remained at the same level. It is concluded that the function of ICL1 is related to the glyoxylate cycle while ICL2 functions independently from the glyoxylate cycle and interconverts organic acids in the cytosol.

[Isolation and properties of malate dehydrogenase from meso- and thermophilic bacteria].

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 ± 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 ± 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.