Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions.

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

The role of promoter methylation of the genes encoding the enzymes metabolizing di- and tricarboxylic acids in the regulation of plant respiration by light.

We discuss the role of epigenetic changes at the level of promoter methylation of the key enzymes of carbon metabolism in the regulation of respiration by light. While the direct regulation of enzymes via modulation of their activity and post-translational modifications is fast and readily reversible, the role of cytosine methylation is important for providing a prolonged response to environmental changes. In addition, adenine methylation can play a role in the regulation of transcription of genes. The mitochondrial and extramitochondrial forms of several enzymes participating in the tricarboxylic acid cycle and associated reactions are regulated via promoter methylation in opposite ways. The mitochondrial forms of citrate synthase, aconitase, fumarase, NAD-malate dehydrogenase are inhibited while the cytosolic forms of aconitase, fumarase, NAD-malate dehydrogenase, and the peroxisomal form of citrate synthase are activated. It is concluded that promoter methylation represents a universal mechanism of the regulation of activity of respiratory enzymes in plant cells by light. The role of the regulation of the mitochondrial and cytosolic forms of respiratory enzymes in the operation of malate and citrate valves and in controlling the redox state and balancing the energy level of photosynthesizing plant cells is discussed.

Light-Dependent Expression and Promoter Methylation of the Genes Encoding Succinate Dehydrogenase, Fumarase, and NAD-Malate Dehydrogenase in Maize (Zea mays L.) Leaves.

The expression and methylation of promoters of the genes encoding succinate dehydrogenase, fumarase, and NAD-malate dehydrogenase in maize (Zea mays L.) leaves depending on the light regime were studied. The genes encoding the catalytic subunits of succinate dehydrogenase showed suppression of expression upon irradiation by red light, which was abolished by far-red light. This was accompanied by an increase in promoter methylation of the gene Sdh1-2 encoding the flavoprotein subunit A, while methylation was low for Sdh2-3 encoding the iron-sulfur subunit B under all conditions. The expression of Sdh3-1 and Sdh4 encoding the anchoring subunits C and D was not affected by red light. The expression of Fum1 encoding the mitochondrial form of fumarase was regulated by red and far-red light via methylation of its promoter. Only one gene encoding the mitochondrial NAD-malate dehydrogenase gene (mMdh1) was regulated by red and far-red light, while the second gene (mMdh2) did not respond to irradiation, and neither gene was controlled by promoter methylation. It is concluded that the dicarboxylic branch of the tricarboxylic acid cycle is regulated by light via the phytochrome mechanism, and promoter methylation is involved with the flavoprotein subunit of succinate dehydrogenase and the mitochondrial fumarase.

Light Dependent Changes in Adenylate Methylation of the Promoter of the Mitochondrial Citrate Synthase Gene in Maize (Zea mays L.) Leaves.

Limited methyl-specific restriction of genomic DNA by endonuclease MAL1 revealed the changes in its methyl status caused by adenine modification in maize (Zea mays L.) leaves under different light conditions (dark, light, irradiation by red and far-red light). Incubation in the light and irradiation by red light exhibited an activating effect on DNA adenine methylase activity, which was reflected in an increase in the number of methylated adenines in GATC sites. Far-red light and darkness exhibited an opposite effect. The use of nitrite conversion of DNA followed by methyladenine-dependent restriction by MboI nuclease revealed a phytochrome B-dependent mechanism of regulation of the methyl status of adenine in the GATC sites in the promoter of the gene encoding the mitochondrial isoform of citrate synthase. Irradiation of plants with red light caused changes in the adenine methyl status of the analyzed amplicon, as evidenced by the presence of restriction products of 290, 254, and 121 nucleotides. Adenine methylation occurred at all three GATC sites in the analyzed DNA sequence. It is concluded that adenylate methylation is controlled by phytochrome B via the transcription factor PIF4 and represents an important mechanism for the tricarboxylic acid cycle regulation by light.

Effect of Salt Stress on the Activity, Expression, and Promoter Methylation of Succinate Dehydrogenase and Succinic Semialdehyde Dehydrogenase in Maize (Zea mays L.) Leaves.

The effect of salt stress on the expression of genes, the methylation of their promoters, and the enzymatic activity of succinate dehydrogenase (SDH) and succinic semialdehyde dehydrogenase (SSADH) was investigated in maize (Zea mays L.). The incubation of maize seedlings in a 150 mM NaCl solution for 24 h led to a several-fold increase in the activity of SSADH that peaked at 6 h of NaCl treatment, which was preceded by an increase in the Ssadh1 gene expression and a decrease in its promoter methylation observed at 3 h of salt stress. The increase in SDH activity and succinate oxidation by mitochondria was slower, developing by 24 h of NaCl treatment, which corresponded to the increase in expression of the genes Sdh1-2 and Sdh2-3 encoding SDH catalytic subunits and of the gene Sdh3-1 encoding the anchoring SDH subunit. The increase in the Sdh2-3 expression was accompanied by the decrease in promoter methylation. It is concluded that salt stress results in the rapid increase in succinate production via SSADH operating in the GABA shunt, which leads to the activation of SDH, the process partially regulated via epigenetic mechanisms. The role of succinate metabolism under the conditions of salt stress is discussed.

Effects of light, anoxia and salinity on the expression of dihydroxyacid dehydratase in maize.

Dihydroxyacid dehydratase (EC 4.2.1.9) participates in metabolism of branched chain amino acids, in CoA biosynthesis and in the conversion of hydroxycitric acid that accumulates in several plants. In maize (Zea mays L.), this enzyme is encoded by the two genes (Dhad1 and Dhad2), having different patterns of their expression during germination. We have demonstrated the inhibition of Dhad1 expression by light and the opposite effect of light on Dhad2. These effects were phytochrome-dependent and involved methylation/demethylation of promoters. Incubation of maize plants in a nitrogen atmosphere resulted in Dhad1 activation peaking at 12 h, which coincided with the decrease in promoter methylation. The gene Dhad2 was activated only during the first 6 h of anoxia, with no correlation with the level of promoter methylation. Salt stress (150 mM NaCl) caused the activation of expression of Dhad2 while the expression of Dhad1 was inhibited in the first hour and then after 12 h incubation with NaCl. We conclude that the expression of two genes encoding dihydroxyacid dehydratase reveals the opposite or different patterns of regulation by light, anoxia and salinity. The mechanisms underlying these modifications involve promoter methylation and result in corresponding changes in the enzymatic activity of the conversion of hydroxycitrate to 2-oxoglutarate.

Effect of Salt Stress on the Expression and Promoter Methylation of the Genes Encoding the Mitochondrial and Cytosolic Forms of Aconitase and Fumarase in Maize.

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted in a several-fold increase of the mitochondrial activities of aconitase and fumarase that peaked at 6 h of NaCl treatment, while the cytosolic activity of aconitase and fumarase decreased. This corresponded to the decrease in promoter methylation of the genes Aco1 and Fum1 encoding the mitochondrial forms of these enzymes and the increase in promoter methylation of the genes Aco2 and Fum2 encoding the cytosolic forms. The pattern of expression of the genes encoding the mitochondrial forms of aconitase and fumarase corresponded to the profile of the increase of the stress marker gene ZmCOI6.1. It is concluded that the mitochondrial and cytosolic forms of aconitase and fumarase are regulated via the epigenetic mechanism of promoter methylation of their genes in the opposite ways in response to salt stress. The role of the mitochondrial isoforms of aconitase and fumarase in the elevation of respiration under salt stress is discussed.

Aconitate isomerase from maize leaves: Light-dependent expression and kinetic properties.

Aconitate isomerase (EC 5.3.3.7) interconverts cis- and trans-isomers of aconitic acid. Expression of the gene encoding this enzyme was studied in maize (Zea mays L.) leaves depending on light regime. Aconitate isomerase was induced by white and by red light indicating the involvement of phytochrome in the regulation of gene expression. The enzyme was partially purified from maize leaves. The value of Km was 0.75 mM with cis-aconitate and 0.92 mM with trans-aconitate, pH optimum was 8.0-8.2 with both substrates, citrate and malate suppressed its activity. It is concluded that aconitate isomerase actively participates in the interconversion of cis- and trans-aconitate in the light providing a possibility of using the pool of trans-aconitate for the regulation of the tricarboxylic acid cycle activity and mediating citrate/isocitrate supply for the biosynthetic and signaling purposes in photosynthetic cells.

Two forms of NAD-malic enzyme in maize leaves are regulated by light in opposite ways via promoter methylation.

NAD-malic enzyme (EC 1.1.1.39) activity, expression and methylation of promoters of its two genes was studied in maize (Zea mays L.) leaves depending on light regime. The total activity was high in darkness and upon irradiation by far-red light and suppressed by white light and by red light. The changes in the levels of transcripts of the genes Me1 and Me2 encoding NAD-malic enzyme revealed their dependence on irradiation in opposite ways. White and red light decreased the quantity of mRNA of the gene Me1, while far-red light led to the increase of its transcripts. The opposite pattern was observed for the transcripts of Me2, the level of which was low in darkness and upon irradiation by far-red light, and was higher in white light and after irradiation by red light. The study of methylation of the promoters of the genes encoding NAD-ME showed a strong dependence between the levels of transcripts and the state of methylation of CG dinucleotides. The two isoforms of NAD-malic enzyme were partially purified from maize leaves and characterized. The first isoform had a pH optimum of 6.4 while the second had a pH optimum of 6.9; in the reverse reaction, the pH optimum was ∼0.5 units higher. It is concluded that the two genes encode different isoforms of NAD-malic enzyme with different kinetic properties. The role of both isoforms in the operation of the tricarboxylic acid cycle in the open mode is discussed.

Regulation of expression of the mitochondrial and cytosolic forms of aconitase in maize leaves via phytochrome.

Regulation of expression and methylation of promoters of two aconitase (EC 4.2.1.3) genes by light have been investigated in maize (Zea mays L.) in relation to the involvement of phytochrome. Transferring of plants from light to darkness resulted in the stimulation of aconitase activity in mitochondria and in its suppression in the cytosol. Irradiation by red light reversed aconitase activity to the levels observed under white light while far red light reverted the effect of red light. Electrophoretic staining of aconitase activity revealed the preference of the cytosolic form in white and red light and of the mitochondrial form in darkness and in far red light. Both forms of aconitase were purified, the mitochondrial form revealed lower affinity to citrate and higher to isocitrate as compared to the cytosolic form. The study of the aconitase gene Aco1 encoding the mitochondrial form revealed its low expression and high promoter methylation in the light and upon irradiation by red light as compared to high expression and low promoter methylation in darkness and in far red light. The pattern of expression and promoter methylation of the gene Aco2 encoding the cytosolic form was opposite. It is concluded that expression of the mitochondrial and cytosolic forms of aconitase is under control of light via phytochrome in opposite ways at the level of promoter methylation. Light inhibits expression of the mitochondrial aconitase, while it stimulates expression of the cytosolic aconitase which is important for directing citrate exported from mitochondria to the synthesis of amino acids.

Enzymatic Conversions of Glutamate and γ-Aminobutyric Acid as Indicators of Plant Stress Response.

Glutamate plays a central role in amino acid metabolism, in particular, in aminotransferase reactions leading to formation of many other proteinogenic and nonproteinogenic amino acids. In stress conditions, glutamate can be either metabolized to γ-aminobutyric acid (GABA) by glutamate decarboxylase which initiates a GABA shunt bypassing several reactions of the tricarboxylic acid cycle or converted to 2-oxoglutarate by glutamate dehydrogenase. Both reactions direct protein carbon to respiration but also link glutamate metabolism to other cellular pathways, resulting in the regulation of redox level and pH balance. Assays for determination of activities of glutamate dehydrogenase and of the GABA shunt enzymes as the markers of stress response is described in this chapter. These assays are important in the studies of the strategy of biochemical adaptation of plants to changing environmental conditions including elevated CO2, temperature increase, flooding, and other stresses.

Expression and properties of the mitochondrial and cytosolic forms of fumarase in sunflower cotyledons.

Fumarase (EC 4.2.1.2) is encoded in sunflower (Helianthus annuus L.) by two genes (FUM1 and FUM2) expressing correspondingly the mitochondrial and the cytosolic form. Both forms have been purified from sunflower cotyledons and characterized. Three quarters of fumarase activity is located in the mitochondrial and one quarter in the cytosolic fraction. The cytosolic form has lower pH optimum than the mitochondrial form, it possesses higher affinity to malate, activated by Mn2+ and less efficiently by Mg2+ while the mitochondrial form is activated only by Mg2+. It is proposed that the mitochondrial form is involved in the respiratory processes linked to the tricarboxylic acid cycle and the cytosolic form participates in the utilization of succinate produced in the glyoxylate cycle providing the flux to gluconeogenesis in germinating sunflower seeds.

Regulation of expression of the mitochondrial and peroxisomal forms of citrate synthase in maize during germination and in response to light.

Expression of genes encoding the mitochondrial and peroxisomal forms of citrate synthase (EC 2.3.3.1) was studied in maize (Zea mays L.) in scutella during germination and in leaves depending on light regime. During germination, citrate synthase activity increased in scutella both in mitochondria and in fatty-acid metabolizing peroxisomes (glyoxysomes) by day 6 and then declined. This was preceded by the peak of expression of the genes encoding the mitochondrial (Csy1) and peroxisomal (Csy2) forms of citrate synthase occurring on the day 3 of germination, after which the expression of Csy1 gradually and of Csy2 sharply declined. The decrease of expression of both genes was followed by the increase of promoter methylation which was more intensive for the gene encoding the mitochondrial form. In leaves, the activity of the mitochondrial form was much higher than that of the peroxisomal form and increased in darkness, while the peroxisomal form was almost undetectable in darkness and increased in the light. The mitochondrial form was inhibited by white and red light while the peroxisomal form was induced by white, red and blue light indicating the involvement of phytochrome and cryptochrome. The mechanism of light regulation of citrate synthase involved promoter methylation leading to the inhibition of corresponding genes and exhibiting opposite patterns for Csy1 and Csy2. Citrate synthase was purified from mitochondria and glyoxysomes of maize scutellum. The mitochondrial form had higher optimum pH as compared to the glyoxysomal form and possessed higher affinity to oxaloacetate and acetyl-CoA. It is concluded that expression of citrate synthase during germination and in response to light is regulated by methylation of promoters of corresponding genes.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

Expression of succinate dehydrogenase and fumarase genes in maize leaves is mediated by cryptochrome.

Blue light inhibits succinate dehydrogenase and fumarase enzyme activity and gene expression in green leaves of maize (Zea mays L.). Irradiation of maize plants by blue light resulted in the transient decrease of transcripts of genes Sdh1-2 and Sdh2-3 encoding correspondingly the flavoprotein and iron-sulfur protein subunits of succinate dehydrogenase, and of Fum1 encoding the mitochondrial form of fumarase. The blue light effect was probably mediated by transcription factors COP1 and HY5, with the expression of the latter increased upon blue light treatment. This was accompanied by a decrease in the expression of COP1, presumably involved in proteasome degradation of HY5. It was also demonstrated that calcium ions do not participate in this process.

Expression and promoter methylation of succinate dehydrogenase and fumarase genes in maize under anoxic conditions.

Succinate dehydrogenase (SDH) and fumarase enzyme activity and expression of genes encoding the SDH subunits A (Sdh1-2), B (Sdh2-3), C (Sdh3), D (Sdh4) and the mitochondrial (Fum1) and cytosolic (Fum2) isoforms of fumarase were quantified in maize (Zea mays L.) seedlings exposed to atmospheres of air (control), N2, and CO2. The catalytic activity of SDH gradually declined in plants exposed to N2 atmospheres, with ∼40% activity remaining after 24h. In seedlings incubated in CO2, the suppression was even more pronounced. Fumarase activity was more stable, decreasing by one third after 24h of anoxia. The level of Sdh1-2 transcripts in seedlings declined significantly under N2 and even more rapidly upon exposure to CO2, with a concomitant increase in methylation of the corresponding promoters. The level of Sdh2-3 and Sdh3 transcripts also decreased under N2 and CO2, but the changes in promoter methylation were less pronounced, whereas the changes in the level of Sdh4 expression and promoter methylation were minor. Expression of Fum1 and Fum2 was affected by N2 and CO2 atmospheres, however without changes in corresponding promoter methylation. It is concluded that, under conditions of oxygen deficiency, succinate accumulates mainly due to downregulation of SDH gene expression and reduction of enzyme activity, and to a lesser extent due to the decrease of fumarase gene expression.

Expression of genes encoding subunits A and B of succinate dehydrogenase in germinating maize seeds is regulated by methylation of their promoters.

Succinate dehydrogenase (SDH) activity, isoenzyme pattern, and expression of two genes encoding subunit A and of three genes encoding subunit B have been investigated in the scutellum of germinating maize (Zea mays L.) seeds. Four SDH isoforms were detected electrophoretically and by ion-exchange chromatography at the peak of activity of the glyoxylate cycle (on the 4th and 5th day of germination), while in dry seeds and on the 8th and 9th day of germination only two isoforms were present, which can be related to differential expression of the genes encoding SDH subunits. The levels of transcription of Sdh1-1, Sdh1-2, Sdh2-1, Sdh2-2 and Sdh2-3 and the intensity of methylation of their promoters have been determined. In the course of seed germination, the level of methylation of the promoters of one gene encoding subunit A (Sdh1-1) and of two genes encoding subunit B (Sdh2-1 and Sdh2-2) changed from low to the highest, which resulted in suppression of their transcription during the period when the intensity of the glyoxylate cycle was decreasing, while methylation of the promoter of Sdh2-3 did not change and expression of this gene was constitutive during germination. Methylation of the promoter of Sdh1-2 increased but less sharply as compared to Sdh1-1. It is suggested that epigenetic mechanisms of SDH expression via methylation of promoters play an important role in the regulation of transcription of Sdh1-1, Sdh2-1 and Sdh2-2 in maize seeds during germination. These genes may play a role in the provision of operation of the glyoxylate cycle, while Sdh1-2 and Sdh2-3 are involved mainly in the respiratory processes that are not connected with utilization of succinate formed in the glyoxylate cycle.

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.

Light inhibition of fumarase in Arabidopsis leaves is phytochrome A-dependent and mediated by calcium.

Inhibition of fumarase activity in the light has been studied in Arabidopsis in relation to the involvement of phytochrome. Using knockout phytochrome mutants, we observed that the main regulator of FUM1 gene transcription, encoding the mitochondrial form of fumarase, is phytochrome A. The active form of phytochrome A suppressed FUM1 expression, while the expression of the FUM2 gene encoding the cytosolic form of fumarase was unaffected both in darkness and in light. The nuclear concentration of Ca(2+) was modulated by red and far-red light. We suggest that the signal transduction mechanism operates via Ca(2+) activation of expression of the gene encoding the transcription factor PIF3, which binds to promoters of phytochrome-regulated genes and inhibits FUM1 expression.

Expression and properties of the mitochondrial and cytosolic forms of aconitase in maize scutellum.

Aconitase (EC 4.2.1.3) catalyzes the reversible interconversion of citrate, cis-aconitate, and D-isocitrate. It operates in mitochondria and cytosol. We investigated the expression of two aconitase genes (Aco1 and Aco4) and activities of the mitochondrial and cytosolic forms in maize (Zea mays L.) scutellum during germination. Both forms were isolated and purified. The cytosolic form had a higher pH optimum (8.0), twice higher affinity to citrate (K(m) 9.5 mM), and slightly lower affinity to D,L-isocitrate (K(m) 1.7 mM) as compared to the mitochondrial form (optimum pH 7.5, K(m) with citrate 21 mM, and K(m) with isocitrate 1.5 mM). The highest activity of both forms of aconitase was observed on the 4th day of germination; then the activity and expression of the cytosolic form sharply decreased, while the mitochondrial form decreased more slowly. The mitochondrial aconitase was more strongly inhibited by H2O2 (half-inhibition at 35 µM) than the cytosolic form (60 µM). Aconitase activity was not detected in the glyoxysomal fraction beyond the cross-contamination level. It is suggested that the mitochondrial form operates in the tricarboxylic acid cycle, whereas the cytosolic form participates in the reactions of the glyoxylate cycle taking place outside the glyoxysome.