NADP-isocitrate Dehydrogenase Research Articles

Peroxisomal plant metabolism – an update on nitric oxide, Ca2+ and the NADPH recycling network

Plant peroxisomes are recognized organelles that - with their capacity to generate greater amounts of H2O2 than other subcellular compartments - have a remarkable oxidative metabolism. However, over the last 15 years, new information has shown that plant peroxisomes contain other important molecules and enzymes, including nitric oxide (NO), peroxynitrite, a NADPH-recycling system, Ca2+ and lipid-derived signals, such as jasmonic acid (JA) and nitro-fatty acid (NO2-FA). This highlights the potential for complex interactions within the peroxisomal nitro-oxidative metabolism, which also affects the status of the cell and consequently its physiological processes. In this review, we provide an update on the peroxisomal interactions between all these molecules. Particular emphasis will be placed on the generation of the free-radical NO, which requires the presence of Ca2+, calmodulin and NADPH redox power. Peroxisomes possess several NADPH regeneration mechanisms, such as those mediated by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) proteins, which are involved in the oxidative phase of the pentose phosphate pathway, as well as that mediated by NADP-isocitrate dehydrogenase (ICDH). The generated NADPH is also an essential cofactor across other peroxisomal pathways, including the antioxidant ascorbate-glutathione cycle and unsaturated fatty acid β-oxidation, the latter being a source of powerful signaling molecules such as JA and NO2-FA.

The effect of the biologically active additive epiphamine on antioxidant and NADPH-generating enzymes activity under experimental cerebral ischemia/reperfusion in rats

The effect of biologically active additive with immunomodulator properties epiphamine on the activity of antioxidant (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase) and NADPH-generating (glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) enzymes has been investigated at experimental cerebral ischemia/reperfusion in rats. The results obtained indicate epiphamine-induced changes of these enzymes activities towards control values. Changes in the content of lactate, a marker of the pathology development, have also been found in experimental animals under ischemia and epiphamine administration caused changes similar to those observed in the case of enzyme activities studied. In most cases, the changes were dose-dependent. Thus, epiphamine can be of considerable interest from the point of view of metabolic changes pharmacological correction at the development of the pathology accompanied by oxidative stress.

Primary carbon metabolism-related changes in cucumber exposed to single and sequential treatments with salt stress and bacterial infection

This study examines how salt stress interacts with bacterial infection at the metabolic level. We measured chlorophyll a fluorescence as well as profiles of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), NADP-isocitrate dehydrogenase (NADP-ICDH) and fumarase activities, malic and citric acids contents and the expression of NADP-ICDH and NADP-ME in the organ-dependent (root vs leaves) response of cucumber plants exposed to individual or sequential action of salt stress (50 mM or 100 mM NaCl) and Pseudomonas syringae pv lachrymans (Psl). NaCl treatment, Psl infection and the combination of these stresses caused disturbances in the activity of photosystem II which were suggested to specifically transmit the stress signals. PEPC and NADP-ME were induced in cucumber plants under stress, confirming that in C3 plants they function in defence responses. The profiles of malate and citrate contents, PEPC as well as NADP-ICDH and NADP-ME activities and gene expression in response to a combination of salt and pathogen stresses differed from those provoked by individual stress with respect to the direction, intensity and timing. The results indicated that the most pronounced defence response related to the readjustment of the carbon metabolism was observed in the leaves of plants exposed to combined stress. Intense activity changes of NADPH-generating enzymes, NADP-ICDH and NADP-ME, characterized the tailored response to combined stress and could be important for the integration of defence mechanisms between organs.

Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H2O2, ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H202, nitric oxide or peroxynitrite.

Nitrogen Addition Exacerbates the Negative Effects of Low Temperature Stress on Carbon and Nitrogen Metabolism in Moss.

Global environmental changes are leading to an increase in localized abnormally low temperatures and increasing nitrogen (N) deposition is a phenomenon recognized worldwide. Both low temperature stress (LTS) and excess N induce oxidative stress in plants, and excess N also reduces their resistance to LTS. Mosses are primitive plants that are generally more sensitive to alterations in environmental factors than vascular species. To study the combined effects of N deposition and LTS on carbon (C) and N metabolism in moss, two moss species, Pogonatum cirratum subsp. fuscatum, and Hypnum plumaeforme, exposed to various concentrations of nitrate (KNO3) or ammonium (NH4Cl), were treated with or without LTS. C/N metabolism indices were then monitored, both immediately after the stress and after a short recovery period (10 days). LTS decreased the photosystem II (PSII) performance index and inhibited non-cyclic photophosphorylation, ribulose-1,5-bisphosphate carboxylase, and glutamine synthetase activities, indicating damage to PSII and reductions in C/N assimilation in these mosses. LTS did not affect cyclic photophosphorylation, sucrose synthase, sucrose-phosphate synthase, and NADP-isocitrate dehydrogenase activities, suggesting a certain level of energy and C skeleton generation were maintained in the mosses to combat LTS; however, LTS inhibited the activity of glycolate oxidase. As predicted, N supply increased the sensitivity of the mosses to LTS, resulting in greater damage to PSII and a sharper decrease in C/N assimilation. After the recovery period, the performance of PSII and C/N metabolism, which were inhibited by LTS increased significantly, and were generally higher than those of control samples not exposed to LTS, suggesting overcompensation effects; however, N application reduced the extent of compensation effects. Both C and N metabolism exhibited stronger compensation effects in H. plumaeforme than in P. cirratum subsp. fuscatum. The difference was especially pronounced after addition of N, indicating that H. plumaeforme may be more resilient to temperature and N variation, which could explain its wider distribution in the natural environment.

Mechanical wounding promotes local and long distance response in the halophyte Cakile maritima through the involvement of the ROS and RNS metabolism

Mechanical wounding in plants, which are capable of generating defense responses possibly associated with nitro-oxidative stress, can be caused by (a)biotic factors such as rain, wind, herbivores and insects. Sea rocket (Cakile maritima L.), a halophyte plant belonging to the mustard family Brassicaceae, is commonly found on sandy coasts throughout Europe. Using 7-day-old Cakile maritima L. seedlings, mechanical wounding was induced in hypocotyls by pinching with a striped-tip forceps; after 3 h, several biochemical parameters were analyzed in both the damaged and unwounded organs (green cotyledons and roots). We thus determined NO production, H2O2 content, lipid oxidation as well as protein nitration patterns; we also identified several antioxidant enzymes including catalase, superoxide dismutase (SOD) isozymes, peroxidases, ascorbate-glutathione cycle enzymes and NADP-dehydrogenases. All these parameters were differentially modulated in the damaged (hypocotyls) and unwounded organs, which clearly indicated an induction of CuZnSOD V in the three organs, an increase in protein nitration in green cotyledons and an induction of NADP-isocitrate dehydrogenase activity in roots. On the whole, our results indicate that the wounding of hypocotyls, which showed an active ROS metabolism and oxidative stress, causes long-distance signals that also trigger responses in unwounded tissues with a more active RNS metabolism. These data therefore confirm the existence of local and long-distance responses which counteract negative effects and provide appropriate responses, enabling the wounded seedlings to survive.

Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200μM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs.

NADP-dependent enzymes are involved in response to salt and hypoosmotic stress in cucumber plants.

Salt stress is one of the most damaging plant stressors, whereas hypoosmotic stress is not considered to be a dangerous type of stress in plants and has been less extensively studied. This study was performed to compare the metabolism of cucumber plants grown in soil with plants transferred to distilled water and to a 100 mM NaCl solution. Even though hypoosmotic stress caused by distilled water did not cause such significant changes in the relative water content, Na+/K+ ratio and Rubisco content as those caused by salt stress, it was accompanied by more pronounced changes in the specific activities of NADP-dependent enzymes. After 3 days, the specific activities of NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves were highest under hypoosmotic stress, and lowest in plants grown in soil. In roots, salt stress caused a decrease in the specific activities of major NADP-enzymes. However, at the beginning of salt stress, NADP-galactose-1-dehydrogenase and ribose-1-dehydrogenase were involved in a plant defense response in both roots and leaves. Therefore, the enhanced demands of NADPH in stress can be replenished by a wide range of NADP-dependent enzymes.

English

Chloris virgata, an alkali-tolerant halophyte, was chosen as the test material for our research. The seedlings of C. virgata were treated with varying salt and alkali stress. First, the composition and content of organic acids in shoots were analyzed and the results indicated that there was not only a significant increase in total organic acids, but there were also obvious changes in different components of organic acids under alkali stress. The increments in citrate were the largest, followed by malate. However, none of the organic acids showed significant alterations in the content and components under salt stress. Also, activity of some enzymes (citrate synthase, malate synthase, NADP-isocitrate dehydrogenase, and isocitrate lyase) associated with such organic acids did not change significantly under alkali stress, but malate dehydrogenase activity markedly decreased under a stronger alkali stress (80 mM). Under salt stress as well as increased malate synthase (MS) activity, however, there was no significant change for other enzymes. These results strongly demonstrated that the enzymatic regulation of organic acid metabolism may be the biochemical basis of alkali tolerance for C. virgata. Citrate synthase (CS), MS and isocitrate lyase (ICL) might be the key enzymes that determine the alkali tolerance of C. virgata. Key words: Salinity, ion balance, enzyme activity, Chloris virgata.

The evolution of citrate metabolism in acidic and acidless citrus genotypes during fruit development and ripening

In the present work, citric acid accumulation and degradation was monitored during fruit development and ripening in two acidless varieties, Tarocco Ferreri (TF) and Vaniglia biondo (VB), in comparison with three acidic ones, namely Lanelate (L), Tarocco Meli (TM) and Mandalate [Fortune mandarin (2x) × Avana mandarin (4x)] (M), a tryploid hydrid. We evaluated titratable acidity (TA), pH and expression of genes involved in the critical steps of Krebs cycle, GABA shunt, acetyl-CoA metabolism and 'acid trap' from about 100 days post anthesis (DPA) until ripening. Based on the correlation between TA and pH, TF could not be considered a true ''acidless' as VB: even if the acidity levels of TF and VB at maturity reached lower values compared to acidic varieties (0.1 versus 1.6), the relation between TA and pH was negatively correlated in acidic varieties and TF, but weakly positively correlated in VB. The maximum of acidity in VB was identified at 160 DPA (0.28), while surprisingly TF anticipated the peak of acidity at 100 DPA, showing a higher value (2.35). Gene expression data showed marked differences between acidic and acidless varieties, as well as within acidic and within acidless ones. The main differences between acidic and acidless varieties were related to genes involved in Krebs cycle, as citrate synthase (CS), mitochondrial isoforms of aconitase (ACO) and NADP-isocitrate dehydrogenase (NADP-IDH), higher expressed in acidic varieties. We also observed that differences in gene expression within the acidic varieties are likely associated with differences in their citrate content. Specifically, CS, NADP-IDH and glutamate decarboxylase (GAD) are higher expressed in M, while ACO and ATP citrate lyase (ATP-CL) in TM, all compared to L. Differences between TF and VB were found in the expression of NADP-IDH, GAD, ATP-CL and H+ transporting ATPase isoform I, supporting the hypothesis of a different genetic basis of the acidless character in the two genotypes.

Activation of NADPH-recycling systems in leaves and roots of Arabidopsis thaliana under arsenic-induced stress conditions is accelerated by knock-out of Nudix hydrolase 19 (AtNUDX19) gene

NADPH is an important cofactor in cell growth, proliferation and detoxification. Arabidopsis thaliana Nudix hydrolase 19 (AtNUDX19) belongs to a family of proteins defined by the conserved amino-acid sequence GX5-EX7REUXEEXGU which has the capacity to hydrolyze NADPH as a physiological substrate in vivo. Given the importance of NADPH in the cellular redox homeostasis of plants, the present study compares the responses of the main NADPH-recycling systems including NADP-isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and NADP-malic enzyme (ME) in the leaves and roots of Arabidopsis wild-type (Wt) and knock-out (KO) AtNUDX19 mutant (Atnudx19) plants under physiological and arsenic-induced stress conditions. Two major features were observed in the behavior of the main NADPH-recycling systems: (i) under optimal conditions in both organs, the levels of these activities were higher in nudx19 mutants than in Wt plants; and, (ii) under 500μM AsV conditions, these activities increase, especially in nudx19 mutant plants. Moreover, G6PDH activity in roots was the most affected enzyme in both Wt and nudx19 mutant plants, with a 4.6-fold and 5.0-fold increase, respectively. In summary, the data reveals a connection between the absence of chloroplastic AtNUDX19 and the rise in all NADP-dehydrogenase activities under physiological and arsenic-induced stress conditions, particularly in roots. This suggests that AtNUDX19 could be a key factor in modulating the NADPH pool in plants and consequently in redox homeostasis.

Effect of SkQ1 on Activity of the Glutathione System and NADPH-Generating Enzymes in an Experimental Model of Hyperglycemia.

We studied the effect of mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on the antioxidant activity of the glutathione system and NADPH-generating enzymes in liver and blood serum of rats with hyperglycemia induced by protamine sulfate. It was found that intraperitoneal injection of SkQ1 prevented both decrease in reduced glutathione level and increase in activity of glutathione system enzymes--glutathione peroxidase, glutathione reductase, and glutathione transferase. Activity of NADPH-generating enzymes--glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase--was also attenuated by SkQ1. Probably, in this model of hyperglycemia, decreased level of reactive oxygen species in mitochondria led to the decreased burden on the glutathione antioxidant system and NADPH-generating enzymes. Thus, SkQ1 appears to be a promising compound for the treatment and/or prevention of the adverse effects of hyperglycemia.

Effect of linseed dietary supplementation on adipose tissue development, fatty acid composition, and lipogenic gene expression in lambs

The objective of this study was to examine the effect of linseed supplementation on growth and carcass parameters, adipocyte cellularity, lipogenic enzyme activity, fatty acid (FA) composition and expression of lipogenic genes in subcutaneous (SC) adipose tissue and intramuscular (IM) adipose tissue of longissimus thoracis muscle of lambs. Thirty-six male Navarra breed lambs allocated into 3 experimental groups (15.2±0.18kg) were fed a control diet (containing barley and soya) and two experimental diets (5% and 10% linseed, L5 and L10 respectively) and slaughtered at 26.5±0.28kg. In the SC adipose tissue and muscle, the activity of fatty acid synthetase (FAS), glycerol 3-phosphate dehydrogenase (G3PDH), glucose 6-phosphate dehydrogenase (G6PDH) and NADP-Isocitrate dehydrogenase (ICDH) was studied and the expression of lipoprotein lipase (LPL), acetyl-CoA carboxylase 1or alpha (ACACA), stearoyl-CoA desaturase (SCD), fatty acid desaturase 1 (FADS1) and 2 (FADS2) and fatty acid elongase 5 (ELOVL5) was quantified. Addition of linseed, rich in α-linolenic acid (C18:3n−3), had no effect on growth and carcass parameters. Linseed supplementation increased C18:3n−3, C18:1t10 + C18:1t11 and total n−3 polyunsaturated fatty acid (PUFA) contents of SC and IM fat (P<0.001) and decreased C18:2n−6 and C18:3n−6 (P<0.05). The n−6/n−3 ratio was also decreased by linseed addition (P<0.05). There were no significant effects of linseed on C18:2c9,t11, eicosapentaenoic acid (C20:5n−3), docosapentaenoic acid (C22:5n−3) and docosahexaenoic acid (C22:6n−3) contents of SC and IM depots. In the SC fat, expression of ACACA and SCD was downregulated in L10 compared to C group (P<0.001) and LPL was upregulated in L5 group (P<0.001); however, there were no effects of diet on FADS1, FADS2 and ELOVL5 mRNA levels. In the IM fat, downregulation of ACACA (L5 and L10), SCD (L10), FADS1 (L5 and L10) and FADS2 (L5) was observed (P<0.001). Expression of LPL was upregulated in L5 compared to C group (P<0.01). It was concluded that dietary linseed raised C18:3n−3 and n−3 PUFA in SC and IM fat in Navarra breed lambs and modified the FA synthesis through the regulation of key-lipogenic gene expression. Dietary linseed regulated de novo FA synthesis and shifted monounsaturated FA formation at the transcriptional level by downregulating ACACA and SCD, respectively. Hence, linseed supplementation did not appear to enhance the synthesis of C18:2c9,t11. Moreover, dietary PUFA affected the expression of genes involved in long chain PUFA synthesis (FADS1, FADS2 and ELOVL5) in a tissue-specific manner.

Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement.

Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells.

Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings.

The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development. The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate-glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide ((·)NO), superoxide radical (O2 (·-)) and peroxynitrite (ONOO(-)) was investigated using confocal laser scanning microscopy. The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme. There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H2O2 and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.

Differential response of NADP-dehydrogenases and carbon metabolism in leaves and roots of two durum wheat (Triticum durum Desf.) cultivars (Karim and Azizi) with different sensitivities to salt stress

Wheat (Triticum durum Desf.) is a common Mediterranean species of considerable agronomic importance. Salinity is one of the major threats to sustainable agricultural production mainly because it limits plant productivity. After exposing the Karim and Azizi durum wheat cultivars, which are of agronomic significance in Tunisia, to 100mM NaCl salinity, growth parameters (dry weight and length), proline content and chlorophylls were evaluated in their leaves and roots. In addition, we analyzed glutathione content and key enzymatic activities, including phosphoenolpyruvate carboxylase (PEPC), NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), involved in the carbon metabolism and NADPH-generating system. The sensitivity index indicates that cv Karim was more tolerant to salinity than cv Azizi. This higher tolerance was corroborated at the biochemical level, as cv Karim showed a greater capacity to accumulate proline, especially in leaves, while the enzyme activities studied were differentially regulated in both organs, with NADP-ICDH being the only activity to be unaffected in all organs. In summary, the data indicate that higher levels of proline accumulation and the differential responses of some key enzymes involved in the carbon metabolism and NADPH regeneration contribute to the salinity tolerance mechanism and lead to increased biomass accumulation in cv Karim.

Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxidase activity in cucumber roots. Plants were treated with cadmium for 1, 3 or 6 days. Some of the plants after 3-day exposure to cadmium were transferred to a medium without the heavy metal for the next 3 days. Treatment of plants with cadmium for 6 days stimulated the activity of NADPH oxidase. The highest stimulation of O2•− production by NADPH oxidase was observed in post-stressed plants, which was correlated with the stimulation of activity of PM H+-ATPase in the same conditions. In order to examine the effects of cadmium stresses on the expression level of genes encoding NADPH oxidase, putative cucumber homologs encoding RBOH proteins were selected and a real-time PCR assay was performed. NADPH is a substrate for oxidase; thus alterations in the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and NADP-malic enzyme under cadmium stress were studied. The activity of NADPH dehydrogenases was increased under cadmium stress. The results indicate that PM NADPH oxidase could be involved in plants’ response to cadmium stress by affecting the activity of PM H+-ATPase, and NADPH-generating enzymes could play important roles in this process.

DIFFERENTIAL EXPRESSION OF PROTEINS RELATED TO PRIMARY METABOLISM IN 'MONCADA' MANDARIN LEAVES WITH CONTRASTING FRUIT LOAD

A proteomic study was used to know how the primary metabolism in Citrus leaves is affected by the fruit load. To this end, we researched the differential expression of proteins related to these processes between on-crop and off-crop ‘Moncada’ mandarin leaves. Samples were collected in November and proteins were extracted. From 2D DIGE gel of these extracts, 33 spots related to primary metabolism were isolated: 26 spots being associated with photosynthesis and carbohydrate metabolism, 4 spots related to Krebs cycle, 1 spot related to pentose phosphate pathway, and 2 spots related to nutrient reservoir activity. These spots were identified by MALDI-MS or LC-MS-MS. Between the proteins related to photosynthesis and carbohydrate metabolism, 16 were up-expressed in off-crop leaves, such as NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, RuBisCO large subunit, ADP-glucose pyrophosphorylase, cinnamoyl-CoA reductase, and carbonic anhydrase; while 10 proteins were up-expressed in on-crop leaves such as NADP-dependent malic enzyme, and 3,4-dihydroxy-2-butanone kinase. The isolated proteins related to pentose phosphate pathway (6-phosphogluconate dehydrogenase) and to nutrient reservoir activity (Granule-bound starch synthase Ib precursor) were up-expressed in off-crop leaves. Regarding the proteins related to Krebs cycle, only malate dehydrogenase was up-expressed in off-crop leaves, while the other proteins were up-expressed in on-crop leaves (citrate synthase and NADP-isocitrate dehydrogenase). According to these results, the primary metabolism seems to be more active in off-crop leaves, suggesting that fruit load inhibits the primary metabolism in ‘Moncada’ mandarins.

Peroxisomal enzymes in the liver of rats with experimental diabetes mellitus type 2.

Diabetes mellitus is relatively frequently associated with fatty liver disease. Increased oxidative stress probably plays an important role in the development of this hepatopathy. One of possible sources of reactive oxygen species in liver is peroxisomal system. There are several reports about changes of peroxisomal enzymes in experimental diabetes, mainly enzymes of fatty acid oxidation. The aim of our study was to investigate the possible changes of activities of liver peroxisomal enzymes, other than enzymes of beta-oxidation, in experimental diabetes mellitus type 2. Biochemical changes in liver of experimental animals suggest the presence of liver steatosis. The changes of serum parameters in experimental group are similar to changes in serum of patients with non-alcoholic fatty liver disease. We have shown that diabetes mellitus influenced peroxisomal enzymes by the different way. Despite of well-known induction of peroxisomal beta-oxidation, the activities of catalase, aminoacid oxidase and NADH-cytochrome b(5) reductase were not significantly changed and the activities of glycolate oxidase and NADP-isocitrate dehydrogenase were significantly decreased. The effect of diabetes on liver peroxisomes is probably due to the increased supply of fatty acids to liver in diabetic state and also due to increased oxidative stress. The changes of metabolic activity of peroxisomal compartment may participate on the development of diabetic hepatopathy.

NADPH-generating dehydrogenases: their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditions

NADPH is an essential reductive coenzyme in biosynthetic processes such as cell growth, proliferation and detoxification in eukaryotic cells. It is required by antioxidative systems such as the ascorbate-glutathione cycle and is also necessary for the generation of superoxide radicals by plant NADPH oxidases and for the generation of nitric oxide (NO) by L-arginine-dependent nitric oxide syntase. This coenzyme is principally re-generated by a group of NADP-dehydrogenases enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), both belonging to the pentose phosphate pathway, the NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). In this study, current perspectives on these enzymes in higher plants under different stress situations are reviewed and it is also pointed out that this group of NADPH-generating dehydrogenases is a key element in supporting the mechanism of response to nitro-oxidative stress situations.