The expression of two genes, Fum1 and Fum2, encoding the mitochondrial and cytosolic forms of fumarase (EC 4.2.1.2); the methylation of individual CpGs of their promoters; and fumarase activity were studied in sunflower (Helianthus annuus L.) leaves depending on irradiation and salinity. Fumarase activity was twice as high in darkness compared to irradiation by white light and red light, while far-red light applied after darkness or after red light reverted the activity to the values in darkness, which indicates the involvement of phytochrome. Using qRT-PCR, it was demonstrated that this corresponded to the pattern of expression of the Fum1 gene, while the expression of the Fum2 gene was higher upon irradiation by white and red light, and lower in darkness and under far-red light. Under the application of 150 mM NaCl for 1, 3, 6, 12, and 24 h, fumarase activity increased fivefold from the start of incubation to 6 h, and then decreased after 12 h. These changes were associated with the transcriptional regulation of the Fum1 and Fum2 genes. Changes in the methylation status of the analyzed CpGs in their gene promoters, detected via semi-quantitative methylation-specific PCR, were associated with differences in their expression. The higher methylation levels of the analyzed CpGs in the Fum1 gene promoter under different light conditions and in the Fum2 gene promoter under salinity corresponded to low levels of their transcripts in sunflower leaves. It is suggested that the mitochondrial and cytosolic forms of fumarase are regulated by light and salinity at the gene expression level, presumably through changes in the methylation status of individual CpGs in their promoters.

Hypoxia-dependent microRNAs play an important role in orchestrating a plant's response to low-oxygen stress. To assess the regulatory mechanisms of the adaptive response of maize (Zea mays L.) to hypoxia, an antisense sequence was developed, and the short tandem target mimic (STTM) system was used to induce the loss of function of the mature microRNA775A (miR775a) in maize. A recombinant binary vector pBI121 cloned in E. coli cells containing the antisense sequence anti-miR775A to maize miR775A was acquired to create a line of modified A. tumefaciens EHA105. Using the puncturing method on soaked seeds, maize plants with an active anti-miR775A construct were obtained, as evidenced by a decrease of more than 10-fold in mature miR775A content and by developmental changes in the seedlings. The size of seedlings of the maize knockdown line was almost twice smaller than that of the wild-type (WT) plants. An assessment of the effects of hypoxic conditions induced by flooding of 14-day-old maize plants revealed differences in the expression and activity of several enzymes between WT and knockdown plants. The reduced miR775A levels led to a 2.1-fold drop in pyruvate levels, which resulted in decreased pyruvate kinase, pyruvate dehydrogenase, and lactate dehydrogenase activities as compared to WT plants. A decrease in miR775A content in the maize knockdown cell line also affected the function of mitochondrial and extramitochondrial isoenzymes of citrate synthase, aconitase, and fumarase under hypoxic conditions.

The cytosine methylation status of symmetric and asymmetric sites of promoters of the genes encoding the membrane-bound subunits C and D of succinate dehydrogenase (SDH) was assessed during the germination of maize (Zea mays L.) seeds, when the stored lipids were utilized and the glyoxylate cycle produced succinate. The results of bisulfite sequencing of the promoters of Sdh genes in maize scutella allowed us to determine the cytosine methylation status in the CG, CNG, and CNN sites. The observed site-specific changes in the cytosine methylation status of the Sdh3-1 and Sdh3-2 genes encoding the SDH subunit C indicate an important role in controlling their transcriptional activity. In contrast, no marked changes were observed in the methylation of promoters of the Sdh4 gene, encoding SDH subunit D. The analysis of changes in the activity of the CG, CNG, and CNN DNA methyltransferases revealed the redistribution of activity between CG, CNG, and CNN DNA methyltransferases toward an increase in the proportion of CG DNA methyltransferases. The locus-specific methylation dynamics of SDH membrane subunit promoters during maize germination have been demonstrated. It is concluded that the changes in the cytosine methylation status may play a role in the regulation of the expression of the Sdh genes in the course of the conversion of succinate formed in the glyoxylate cycle.

Different types of microRNA participate in the post-transcriptional regulation of target genes. The content of several hypoxia-dependent miRNAs in plant cells, including miR775, increases in the conditions of oxygen deficiency. Electrophoretic studies of total RNA samples from the leaves of flooded seedlings of maize (Zea mays L.) revealed the presence of two interfering complexes with miR775 at 12 h of hypoxic incubation. A nucleotide sequence analysis of a sample containing the interfering complex of miR775 with mRNA from maize leaves showed a high degree of homology with the ICL/PEPM_KPHMT lyase family domain. It corresponded to a fragment of fructose-1,6-bisphosphate aldolase mRNA. By real-time PCR, we established the dynamics of the content of transcripts of aldolase isoenzyme genes under hypoxia in maize leaves. A decrease in the transcriptional activity of the aldolase 1 gene (Aldo1) correlated with a high content of miR775 in maize leaf cells. The fraction of extracellular vesicles sedimented at 100,000× g, was enriched with miR775. The accumulation of aldolase 2 (Aldo2) mRNA transcripts under hypoxic conditions indicates its participation in maintaining glycolysis when Aldo1 expression is inhibited. We conclude that an increase in the total content of free miR775 and its participation in the suppression of the Aldo1 gene represents an important mechanism in developing the adaptive reaction of cellular metabolism in response to hypoxia.

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.

The effect of salt stress (150 mM NaCl) on the expression of genes, methylation of their promoters, and enzymatic activity of glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), and the 2-oxoglutarate (2-OG)–dehydrogenase (2-OGDH) complex was studied in maize (Zea mays L.). GDH activity increased continuously under salt stress, being 3-fold higher after 24 h. This was accompanied by the appearance of a second isoform with lower electrophoretic mobility. The expression of the Gdh1 gene strongly increased after 6–12 h of incubation, which corresponded to the demethylation of its promoter, while Gdh2 gene expression slightly increased after 2–6 h and then decreased. GAD activity gradually increased in the first 12 h, and then returned to the control level. This corresponded to the increase of Gad expression and its demethylation. Salt stress led to a 2-fold increase in the activity of 2-OGDH during the first 6 h of NaCl treatment, then the activity returned to the control level. Expression of the genes Ogdh1 and Ogdh3 peaked after 1–2 h of incubation. After 6–8 h with NaCl, the expression of these genes declined below the control levels, which correlated with the higher methylation of their promoters. We conclude that salt stress causes a redirection of the 2-OG flux to the γ-aminobutyric acid shunt via its amination to glutamate, by altering the expression of the Gdh1 and Gdh2 genes, which likely promotes the assembly of the native GDH molecule having a different subunit composition and greater affinity for 2-OG.

An increase in GDH activity has been established in the first six hours after plants are exposed to hypoxic conditions, which ensures the formation of an adaptive response of cellular metabolism to the lack of oxygen in the cell. A difference in the transcriptional activity of homologous GDH-1 genes in wheat leaves under stress conditions was shown. An increase in the mRNA content of the GDH-1(5A) gene in wheat leaves after plants are exposed to hypoxic conditions is observed in the first hours of the experiment, which correlates with a change in the catalytic activity of glutamate dehydrogenase. Regulation of this gene is carried out by the transcription factor HIF, the specific landing site of which is found in the transcription initiation site of the promoter of this gene.

Using the real-time polymerase chain reaction method, a change in the level of relative transcription of the SSADH gene in wheat leaves under salt stress conditions was established, correlating with changes in activity. A study of the nucleotide composition of the wheat SSADH gene showed a certain distribution pattern of GATC sequences in the promoter region, which are methylation sites for adenine DNA methyltransferase. Their content is quite high, which may indicate regulation of the expression of this gene by changing the degree of their methylation. Based on the analysis of the nucleotide sequence of this promoter, primers were developed for its amplification and analysis of the adenylate methyl status. A change in the methylation status of adenines in the GATC sites of the succinic semialdehyde dehydrogenase SSADH gene promoter in wheat leaves under salinity was shown.

Under the influence of hypoxia in plants, the transcription of genes responsible for adaptation to stress caused by low oxygen levels changes. Changes in metabolic pathways under stress conditions may be regulated by microRNAs. It was found that the content of microRNA775A increases in corn leaves under the influence of hypoxia. The use of the fluorescent probe miR775A-ROX made it possible to establish an increase in the number of RNA-inducing silencing complexes (RISC), formed on the basis of microRNA775A, in maize leaves during the development of hypoxic stress. The results we obtained indicate that microRNA775A is involved in the processes of adaptation of the body to hypoxic conditions by regulating the expression of target genes at the post-transcriptional level using the RNA interference mechanism.

Glutamate is an essential amino acid in both the energy and biosynthetic processes in plant cells. The aim of this work was to study changes in glutamate metabolism upon irradiation of maize (Zea mays L.) leaves with light of different spectral compositions, as well as to identify mechanisms regulating the work of enzymes involved in the studied process. A study was conducted of light-induced changes in glutamate metabolism in maize leaves, mediated by redirecting the glutamate flow to the γ-aminobutyric acid (GABA) shunt. Glutamate dehydrogenase (GDH) was more active in darkness, and the irradiation by red light inhibited the expression of both the Gdh1 and Gdh2 genes. EGTA and ruthenium red abolished the effects of light, indicating the participation of Ca2+ ions in phytochrome signal transduction. Contrary to GDH, glutamate decarboxylase (GAD) activity was moderately higher in the light, stimulated by red light, while far-red light reversed the effect. The effect of light on Gad expression was more pronounced than on GAD activity. Irradiation by red light also resulted in the increase in activity of GABA transaminase (GTA), which was abolished by far-red light. The third enzyme of the GABA shunt, succinic semialdehyde dehydrogenase (SSADH), was also activated by light. The effect of light on the expression of Ssadh1, but not on Ssadh2, was phytochrome-dependent. It is concluded that irradiation by light shifts glutamate metabolism from GDH to GAD with the activation of GABA transaminase and SSADH. This suggests that the GABA pathway plays a role in the maintenance of the tricarboxylic acid cycle in the light via bypassing its reactions when the 2-oxoglutarate dehydrogenase complex is inhibited and the cycle switches to the open mode.

MicroRNAs are a class of small noncoding RNAs that are 18 to 25 nucleotides in length and are found in most eukaryotic organisms. MicroRNAs can play an important role in epigenetic mechanisms of genome regulation, including DNA methylation and RNA and histone modification. Current methods for detecting and quantifying miRNAs rely heavily on cloning, Northern blotting, or primer extension, but each requires a pure preparation of the RNA type being analyzed. The standard method of RNA isolation, based on phenol−chloroform extraction with specific coprecipitants of nucleic acids, allows one to obtain preparations of total cellular RNA with a predominance of high-molecular types of ribonucleic acids. This greatly complicates the identification and quantification of microRNAs in sample preparations. Modification of the method of phenol−chloroform extraction of RNA, based on its precipitation of DNA with a specific precipitant, such as lithium chloride, showed that the use of polyethylene glycol 1500 using 2.5 M LiCl as a precipitant in the presence of 96

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.

Elevation of the enzyme activity in the leaves of soft wheat (Triticum aestivum L.) observed under salt stress was related to the maintenance of the rate of tricarboxylic acid cycle at the expense of arrival of extra substrates. Activation of succinic semialdehyde dehydrogenase (SSADH) in the leaves of wheat exposed to salt stress induced by sodium chloride (NaCl) reaches its peak in 6 h and amounts to 12.2 E/g fr wt. Activation of the examined enzyme ensures maintenance of necessary ATP level owing to arrival of additional respiratory substrate in the TCA cycle under the effect of stress agent. It was shown that SSADH is genetically predetermined. On the basis of mRNA of homoeologous SSADH genes, specific primers were designed for quantification of their transcripts. Under the effect of salt stress, the content of transcripts of the genes encoding SSADH in the leaves of wheat changes. Comparison of changes in SSADH activity and expression of the examined genes in the leaves of wheat exposed to salt stress has shown that this enzyme is regulated by changes in their transcriptional activity. The main contribution to alteration of the content of SSADH transcripts is made by the gene SSADH belonging to subgenome A. A specific binding site for salt-dependent transcription factor WRKY was detected within promoter of gene SSADHA. Elevation of the content of WRKY transcripts may regulate expression of the gene SSADHA upon plant adaptation to stress impact via interaction with a specific binding site located in the transcription initiation region of its promoter.

The purpose of this study was to obtain a purified preparation of the studied enzyme from corn leaves and to study its characteristics. In this work, kinetic and regulatory parameters of glyoxylate reductase were calculated for leaves of 14-day-old corn (Zea mays) seedlings grown hydroponically at 25°C. The following methods were used during the study: sample homogenisation, four-step purification including ammonium sulphate for desalting, gel filtration on G-25 columns, and ion exchange chromatography using DEAE-sephacel, as well as electrophoresis on polyacrylamide gels and quantitative analysis of protein. To study the properties of the enzyme, we used electrophoretically homogenous preparations. The influence of pH, substrate concentration, and cofactor on the rate of the enzymatic reaction was determined by a series of measurements with different values of the enzymatic reaction rate. As a result of four-stage purification, we obtained a homogeneous preparation with a specific activity of167 E/mg of protein. Ion exchange chromatography was important for purification; we obtained 1 peak of enzyme activity upon desorption in 104 mM sodium chloride. Studying the properties of glyoxylate reductase showed a significant dependence of activity on pH. The optimal pH value was 6.5 units.

Salt stress has a significant effect on plants, causing a whole range of changes in metabolism. Unfortunately, the mechanisms that ensure the adaptive response of cells in response to salinity have not been sufficiently studied. The study examined changes in the functioning of one of the enzymes of the anaplerotic pathway of the Krebs cycle - GABA shunt - GABA transaminase (GABA-T, EC 2.6.1.19). It has been shown that salt stress caused by incubation of corn seedlings in a 150 mM sodium chloride solution causes activation of GABA transaminase (GABA-T, EC 2.6.1.19). The established increase in GABA-T enzymatic activity in the first hours of incubation in saline solution reaches a maximum at 3 hours of incubation. At the same time, differences are observed in the expression profilees of the GTA-1 and GTA-2 genes, which encode this enzyme in the maize genome. Salinity in the first three hours induces an increase in the expression of the GTA-1 GABA-T gene, while the GTA-2 gene demonstrates an increase in transcriptional activity from 6 to 12 hours of the experiment. An increase in GABA-T enzymatic activity under salinity indicates activation of the GABA shunt to maintain the energy metabolism of the plant cell under stress conditions.

Aim. To study the features of transcriptional regulation of the activity and isoenzyme composition of lactate dehydrogenase in the kidneys of Rattus norvegicus L. in diabetic nephropathy.Materials and methods. The study included 20 male laboratory rats (Rattus norvegicus L.) divided into two equal groups: “Norm” – intact rats injected with 0.9% NaCl intraperitoneally and “Diabetes” – animals with alloxaninduced diabetes (DM1 model). The activity, subcellular localization, and mobility of lactate dehydrogenase (LDH, EC 1.1.1.27) isoenzymes were studied using spectrophotometry and electrophoresis. LDHA and LDHB gene transcripts were analyzed by the polymerase chain reaction.Results. Analysis of the LDH activity showed that this parameter increased by more than 6 times in the animals with diabetic nephropathy compared to the control group. Moreover, the increase in the rate of the LDH activity was a consequence of the enzyme activation in all the studied compartments of the cell and is consistent with the parameter in the homogenate. The increase in the LDH activity in diabetic nephropathy may result from redistribution of the activity rate between the available isoforms and may be associated with an increase in the transcription rate of genes encoding subunits A and B of this enzyme.Conclusion. The increase in the LDH activity is likely associated with the activation of renal gluconeogenesis, the main substrate for which is lactic acid reabsorbed in the renal glomeruli. The revealed increase in the LDH activity in the kidneys of rats with diabetic nephropathy may be associated with adaptation of their metabolism to the pathological state.

Objective: Malate dehydrogenase (MDH, ЕC 1.1.1.37) is a multifunctional enzyme complex that catalyzes the reversible conversion of malate to oxaloacetate using NADH as a coenzyme. Currently, the regulation of MDH expression is poorly understood and needs further study. The aim of the study was to study the role of transcription factor CREB1 in regulating the expression of the gene encoding the mitochondrial form of malate dehydrogenase in the liver of rats Rattus norvegicus L. with experimental diabetes. Methods: The work investigated the rate of malate dehydrogenase functioning and the features of its regulation by the transcription factor CREB. Results and Discussion: An increase in the rate of work of NAD-dependent malate dehydrogenase in rat liver cells during the development of experimental diabetes was shown, associated with the activation of the Mdh1 and Mdh2 genes encoding this enzyme. The analysis of the promoters of these genes showed that only in the Mdh2 gene there is a specific binding site with the transcription factor CREB1. It was found that in the liver of rats with pathology, there is an increase in the rate of expression of the gene encoding this transcription factor, which correlates with the expression of the Mdh2 gene. Conclusions: Thus, the data obtained by us confirm the possibility of positive regulation of the rate of the Mdh2 gene by the transcription factor CREB1.

Oxygen deficiency causes significant changes in plant metabolism related to the coordination of metabolic processes for stress adaptation. MicroRNAs provide regulation of target genes at the post-transcriptional level. The plant cell presents hypoxia-dependent microRNAs, including miR775A, whose quantity increases under these stress conditions. An electrophoretic study of total RNA samples from corn leaves under hypoxia has revealed the presence of two interfering complexes with miR775A at the 12th hour of the experiment. Sequencing and subsequent analysis of the nucleotide sequence of image 2 of the miR775A interfering complex with mRNA from corn leaves have showed a high degree of homology with the N-acyltransferase domain, and corresponded to a fragment of glycerol-3-phosphate acyltransferase mRNA. Inhibition of glycerol-3-phosphate acyltransferase in maize leaf cells under hypoxia can probably provide regulation of cellular fatty acid metabolism under the influence of a stress factor.

Установлено повышение активности фермента в листьях мягкой пшеницы (Triticum aestivum L.) при солевом стрессе, что связано с поддержанием скорости функционирования цикла трикарбоновых кислот за счет притока дополнительных субстратов. Увеличение активности дегидрогеназы полуальдегида янтарной кислоты (ПЯКДГ) в листьях пшеницы при солевом стрессе, вызванном хлоридом натрия (NaCl), достигает максимального значения на 6 ч и составляет 12.2 E/г сырой массы. Активация исследуемого фермента обеспечивает поддержание необходимого уровня АТФ за счет дополнительного поступления дыхательного субстрата в ЦТК при действии стрессового фактора. Установлена генетическая детерминация ПЯКДГ. На основании мРНК гомеологичных генов SSADH разработаны специфические праймеры для оценки уровня их транскриптов. Показано изменение содержания транскриптов генов фермента ПЯКДГ в листьях пшеницы при действии солевого стресса. Сравнительный анализ изменения активности ПЯКДГ и экспрессии исследуемых генов в листьях пшеницы в условиях солевого стресса свидетельствует о регуляции данного энзима за счет изменения их транскрипционной активности. Базовый вклад в изменение содержания транскриптов ПЯКДГ вносит ген SSADH А-субгенома. Выявлено наличие специфического сайта связывания солезависимого транскрипционного фактора WRKY в составе промотора гена SSADHA. Увеличение содержания транскриптов WRKY может обеспечивать регуляцию экспрессии гена SSADHA при адаптации растений к стрессовому воздействию при взаимодействии со специфическим сайтом связывания, расположенном в области инициации транскрипции его промотора.