Activity of Carbon Metabolism Enzymes in Wheat Plants Treated with Kartolin-4 and Exposed to Water Stress

Enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39), phospho(enol)pyruvate carboxylase (EC 4.1.1.31), NAD malate dehydrogenase (EC 1.1.1.37), and NADP glyceraldehyde phosphate dehydrogenase complex including phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) were comparatively assayed in wheat seedlings of the cultivar Lyutestsens 758 grown under normal conditions, water deficiency conditions, and subsequent rehydration. Water stress was found to decrease the activity of all enzymes tested, the effect being most pronounced in the case of Rubisco. The content of Rubisco in wheat plants exposed to water deficiency was reduced less significantly than the activity of the enzyme. Pretreatment of plant seeds with kartolin-4 (o-isopropyl-N-2-hydroxyethyl carbamate), a preparation with cytokinin activity, reduced the dehydration-induced inhibition of enzymatic activity. Upon a subsequent rehydration, kartolin-4 facilitated rapid recovery of the photosynthetic activity, the process being based on the kartolin-induced stimulation of reparation reactions. Under conditions of water stress, a partial decrease in the activity of carbon metabolism enzymes in vitrowas accompanied by complete inhibition of photosynthesis in vivo, perhaps, as a result of an abrupt increase in the stomatal resistance.     

Activity of NADP-dependent glyceraldehyde-phosphate dehydrogenase and phosphoenolpyruvate carboxylase in wheat leaves under water stress

The activities of NADP: glyceraldehyde-phosphate dehydrogenase (GAPDH), an enzyme complex comprising of phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.13), and phosphoenolpyruvate carboxylase (PEPK; EC 4.1.1.31) in seedlings and leaves of wheat (Triticum aestivum L.) plants of the cultivars Mironovskaya 808 and Lutescens 758 have been compared under conditions of normal water supply, water deficiency, and subsequent rehydration. GAPDH activity, which determines the carbohydrate route of photosynthetic metabolism at the initial stages, is decreased by water stress to a greater extent than that of PEPK, on the activity of which non-carbohydrate metabolic pathways depend. Pretreatment of seedlings and mature plants with natural (6-benzylaminopurine) and synthetic (tidiazuron, kartolin-2, and kartolin-4) cytokinins attenuates the loss of enzyme activities during drought and facilitates their recovery within the period of rehydration; both effects are underlain by augmentation of reparation processes. The relative intensification of non-carbohydrate pathways of photosynthetic metabolism, observed under conditions of water deficiency, is accompanied by an increase in the osmotic pressure of cell sap. Possible mechanisms of this protector effect of cytokinin preparations are discussed.     

Activity of NADP-dependent glyceraldehyde-phosphate dehydrogenase and phosphoenolpyruvate carboxylase in wheat leaves under water stress

The activities of NADP: glyceraldehyde-phosphate dehydrogenase (GAPDH), an enzyme complex comprising of phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.13), and phosphoenolpyruvate carboxylase (PEPK; EC 4.1.1.31) in seedlings and leaves of wheat (Triticum aestivum L.) plants of the cultivars Mironovskaya 808 and Lutescens 758 have been compared under conditions of normal water supply, water deficiency, and subsequent rehydration. GAPDH activity, which determines the carbohydrate route of photosynthetic metabolism at the initial stages, is decreased by water stress to a greater extent than that of PEPK, on the activity of which non-carbohydrate metabolic pathways depend. Pretreatment of seedlings and mature plants with natural (6-benzylaminopurine) and synthetic (tidiazuron, kartolin-2, and kartolin-4) cytokinins attenuates the loss of enzyme activities during drought and facilitates their recovery within the period of rehydration; both effects are underlain by augmentation of reparation processes. The relative intensification of non-carbohydrate pathways of photosynthetic metabolism, observed under conditions of water deficiency, is accompanied by an increase in the osmotic pressure of cell sap. Possible mechanisms of this protector effect of cytokinin preparations are discussed.     

Protective role of kartolin-4 in soil drought in wheat plants

Parameters of photosynthesis, heat-resistance, and osmotic pressure of cell exudate of leaves of the drought-sensitive cultivar Lyutestsens 758 of wheat, Triticum aestivum, were studied under conditions of normal water supply, soil drought, and subsequent rehydration. The plants preliminarily treated with kartolin-4 were compared to untreated plants. Kartolin-4, a preparation with cytokininlike activity, partially prevented the drought-induced inhibition of the photosynthetic assimilation of carbon dioxide and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), the key enzyme of carbon metabolism in plants. Upon a subsequent rehydration, kartolin-4 stimulated the reparation reactions and facilitated rapid recovery of normal photosynthetic activity. Kartolin-4 also increased plant resistance to overheating and water deficiency.     

Protective Role of Kartolin-4 in Wheat Plants Exposed to Soil Draught

Parameters of photosynthesis, heat-resistance, and osmotic pressure of cell exudate of leaves of the drought-sensitive cultivarLyutestsens 758 of wheat, Triticum aestivum, were studied under conditions of normal water supply, soil drought, and subsequent rehydration. The plants preliminarily treated with kartolin-4 were compared to untreated plants. Kartolin-4, a preparation with cytokinin-like activity, partially prevented the drought-induced inhibition of the photosynthetic assimilation of carbon dioxide and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), the key enzyme of carbon metabolism in plants. Upon subsequent rehydration, kartolin-4 stimulated the reparation reactions and facilitated the rapid recovery of normal photosynthetic activity. Kartolin-4 also increased the plant resistance to overheating and water deficiency.     

The Activity and Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Wheat Plants as Affected by Water Stress and Kartolin-4

The carboxylating activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39), and other soluble proteins in young seedlings and mature leaves of Lutescens-758, a drought-sensitive cultivar of soft spring wheat Triticum aestivum L., were studied under the conditions of drought and subsequent rehydration. Seedlings and mature plants preliminarily treated with the cytokinin-like compound kartolin-4 were compared to untreated plants. Drought-induced decrease in RuBPCO activity should be attributed not only to proteolytic decomposition of the enzyme protein itself but also to a partial inhibition of its catalytic activity. The decrease in RuBPCO activity was larger than that in RuBPCO content. Water stress induced a marked decrease in the soluble protein content. Kartolin-4 increased the resistance to drought.     

Effects of Cytokinin Preparations on the Pools of Pigments and Proteins of Wheat Cultivars Differing in Their Tolerance to Water Stress

Contents of chlorophylls, carotenoids, soluble leaf proteins, and the key enzyme of carbon metabolism—ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39)—in young seedlings and adult leaves of the wheat Triticum aestivum L. cultivars Mironovskaya 808 and Lyutescens 758, contrasting in their water stress tolerances, were compared under conditions of normal available water supply, water deficiency, and subsequent rehydration. It was discovered that compounds displaying a cytokinin activity (6-benzylaminopurine, thidiazuron, kartolin-2, and kartolin-4) reduced the decreases in contents of chlorophylls, carotenoids, soluble leaf proteins, and RuBisCO, progressing with development of water stress, as well as contributed to their more rapid recovery. These compounds with cytokinin activity also accelerated restoration of the compounds studied to their initial concentrations during rehydration. The kartolin preparations caused a maximal protective effect. Water stress had a more pronounced negative effect on the cultivar Lyutescens 758. Dehydration resulted in a more extensive destruction of seedlings compared to leaves of adult plants.     

A protector effect of cytokinin preparations on the photosynthetic apparatus of wheat plants under water deficiency conditions

The protective effects of the cytokinin 6-benzylaminopurine and the compounds thidiazuron and kartolin, displaying a cytokinin activity, on the photosynthetic apparatus of young seedlings and leaves of adult plants of two wheat (Triticum aestivum L.) cultivars, Mironovskaya 808 (more drought tolerant) and Lutescens 758 (less tolerant to water stress), were compared on the background of an increasing water deficiency. At the stages of drought and subsequent rehydration, cartolin preparations were the most efficient protectors, enhancing a less pronounced decrease in the intensity of photosynthesis, carboxylating activity of the key enzyme of carbon metabolism--ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39)-and the activity of NADP-glyceraldehyde phosphate dehydrogenase--the enzyme complex comprising phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13). This effect also included an increase in the leaf specific density and plant productivity. The negative influence of water stress on the photosynthetic apparatus was more pronounced in a less tolerant cultivar Lutescens 758 and in the seedlings as compared with the adult plants.     

A protector effect of cytokinin preparations on the photosynthetic apparatus of wheat plants under water deficiency conditions

The protective effects of the cytokinin 6-benzylaminopurine and the compounds thidiazuron and kartolin, displaying a cytokinin activity, on the photosynthetic apparatus of young seedlings and leaves of adult plants of two wheat (Triticum aestivum L.) cultivars, Mironovskaya 808 (more drought tolerant) and Lutescens 758 (less tolerant to water stress), were compared on the background of an increasing water deficiency. At the stages of drought and subsequent rehydration, kartolin preparations were the most efficient protectors, enhancing a less pronounced decrease in the intensity of photosynthesis, carboxylating activity of the key enzyme of carbon metabolism—ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39)—and the activity of NADP—glyceraldehyde phosphate dehydrogenase—the enzyme complex comprising phosphoglycerate kinase (EC 2.7.2.3.) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13). This effect also included an increase in the leaf specific density and plant productivity. The negative influence of water stress on the photosynthetic apparatus was more pronounced in a less tolerant cultivar Lutescens 758 and in the seedlings as compared with the adult plants.     

Effect of preparations exhibiting cytokinin-like activity on the specific density of leaf in grasses

The effects of synthetic preparations exhibiting cytokinin-like activity (6-benzylaminopurine, Thidiazuron, and kartolin-2) on the specific leaf area (SLA) were studied in plants of the family Gramineae (wheat, Triticum aestivum L.; meadow fescue, Festuca pratensis Huds.; and reed fescue, F. arindinacea Schreb.). At the early stages of ontogeny (until the leaf area reached 50-60% of the maximum value), treatment of plants of the three species with cytokinin-like preparations caused an increase in SLA. The SLA value in these plants was correlated with the rate of photosynthetic assimilation of carbon dioxide and activities of carbon metabolism enzymes: ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), NAD-malate dehydrogenase (EC 1.1.1.37), and NADP-glyceraldehydrophosphate dehydrogenase complex, which includes phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydrophosphate dehydrogenase (EC 1.2.1.13). However, there was no correlation of SLA with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31), an anaplerotic carboxylation enzyme of grasses. SLA is suggested to reflect the state and activity of the photosynthetic apparatus and can be recommended as a characteristic of photosynthesis variability (e.g., caused by cytokinin-like preparations).     

Effect of cytokinin preparations on the pools of pigments and proteins of wheat cultivars differing in their tolerance to water stress

Contents of chlorophylls, carotenoids, soluble leaf proteins, and the key enzyme of carbon metabolism--ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39)--in young seedlings and adult leaves of the wheat Triticum aestivum L. cultivars Mironovskaya 808 and Lyutestsens 758, contrasting in their water stress tolerances, were compared under conditions of normal available water supply, water deficiency, and subsequent rehydration. It was discovered that compounds displaying a cytokinin activity (6-benzylaminopurine, thidiazuron, cartolin-2, and cartolin-4) reduced the decreases in contents of chlorophylls, carotenoids, soluble leaf proteins, and RuBisCO progressing with development of water stress. These compounds with cytokinin activity also accelerated restoration of the compounds studied to their initial concentrations during rehydration. The cartolin preparations caused a maximal protective effect. Water stress had a more pronounced negative effect on cultivar Lyutestsens 758. Dehydration resulted in a more extensive destruction of seedlings compared to leaves of adult plants.     

Effect of Preparations Exhibiting Cytokinin-like Activity on the Specific Density of Leaf in Grasses

The effects of synthetic preparations exhibiting cytokinin-like activity (6-benzylaminopurine, Thidiazuron, and kartolin-2) on the specific leaf area (SLA) were studied in plants of the family Gramineae (wheat, Triticum aestivum L.; meadow fescue, Festuca pratensis Huds.; and reed fescue, F. arindinacea Schreb.). At the early stages of ontogeny (until the leaf area reached 50–60% of the maximum value), treatment of plants of the three species with cytokinin-like preparations caused an increase in SLA. The SLA value in these plants was correlated with the rate of photosynthetic assimilation of carbon dioxide and activities of carbon metabolism enzymes: ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), NAD-malate dehydrogenase (EC 1.1.1.37), and NADP-glyceraldehydephosphate dehydrogenase complex, which includes phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydephosphate dehydrogenase (EC 1.2.1.13). However, there was no correlation of SLA with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31), an anaplerotic carboxylation enzyme of grasses. SLA is suggested to reflect the state and activity of the photosynthetic apparatus and can be recommended as a characteristic of photosynthesis variability (e.g., caused by cytokinin-like preparations).     

Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase

The effects of ozone exposure on carbon-fixation-related processes in Pinus halepensis Mill. needles were assessed over 3 months under controlled conditions. Ozone fumigation (200 ppb) did not induce a modification of either net CO₂ assimilation or stomatal conductance in 1-year-old needles, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity was shown to be reduced by a half. Moreover, this ozone-induced reduction in Rubisco activity was associated with a decrease in the quantity of Rubisco, as determined by the decrease in the large subunit (LSU). On the other hand, 200-ppb ozone fumigation induced a strong increase in both activity and quantity of another carboxylating enzyme, phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), generally considered in C₃ plants to participate in carbon catabolism processes. Ozone induced a significant decrease in the Rubisco/PEPC activity ratio which promotes the role of PEPC in trees under ozone stress. The role of this carboxylase will be discussed.     

Effects of cytokinin preparations on the stability of the photosynthetic apparatus of two wheat cultivars under water deficiency

Carboxylase activities of the key enzyme of carbon metabolism, ribulose-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), as well as intensities of carbon dioxide photosynthetic assimilation in young seedlings and adult leaves of the wheat Triticum aestivum L. cultivars Mironovskaya 808 (a more tolerant) and Lyutestsens 758 (a less tolerant), were compared under conditions of progressive water deficiency. The water stress had more pronounced negative effects on all the studied characteristics of photosynthetic apparatus of cultivar Lyutestsens 758 photosynthetic machinery of the cultivar Lyutestsens 758. Its seedlings were more sensitive to water stress. Compounds with a cytokinin activity (6-benzylaminopurine, thidiazuron, cartolin 2, and cartolin 4) played a protective role, increasing the stability of the photosynthetic machinery under conditions of water deficiency. Preparations of cartolins displayed the maximum protective effect.     

Effects of Cytokinin Preparations on the Stability of the Photosynthetic Apparatus of Two Wheat Cultivars Experiencing Water Deficiency

Carboxylase activity of the key enzyme of carbon metabolism, ribulose-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), as well as the intensity of carbon dioxide photosynthetic assimilation in young seedlings and adult leaves of the wheat Triticum aestivum L. cultivars Mironovskaya 808 (a more tolerant) and Lyutestsens758 (a less tolerant), were compared under conditions of progressive water deficiency. The water stress had more pronounced negative effects on all the studied characteristics of the photosynthetic apparatus of the cultivar Lyutestsens758. Its seedlings were more sensitive to water stress. Compounds with a cytokinin activity (6-benzylaminopurine, thidiazuron, kartolin 2, and kartolin 4) played a protective role, increasing the stability of the photosynthetic apparatus under conditions of water deficiency. Preparations of kartolins displayed the maximum protective effect.     

Nickel-induced changes in carbon metabolism in wheat shoots

In this study, we analyzed the toxic effect of Ni during the development of wheat shoots. Typical developmental alterations in carbon metabolism-related parameters reflecting changes associated with the transition of the seedlings from heterotrophic to autotrophic metabolism were observed in the control shoots between the 1st and the 4th days. Adverse effects of 50 and 100 μM Ni became evident starting from the 4th day of growth on the metal-containing media. We found that Ni-induced stimulation of phosphoenolpyruvate carboxylase (PEPC) activity coincided with decrease in the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) level and with declines in net photosynthetic rate (P_N) and stomatal conductance (g_s). Application of Ni resulted in increased activities of several dehydrogenases: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (NADP-ICDH) and malate dehydrogenase (NADH-MDH). In contrast, the activities of malic enzymes (NADP-ME and NAD-ME) decreased due to Ni stress. Treatment with Ni led to accumulation of glucose and declined concentration of sucrose as well as considerable increases in concentrations of malic and citric acids. Our results indicate that Ni stress redirects the carbon metabolism of developing wheat shoots to provide carbon skeletons for synthesis of amino acids and organic acids as well as to supply reducing power to sustain normal metabolic processes and to support defense mechanisms against oxidative stress.     

Comparative effect of water, heat and light stresses on photosynthetic reactions in Sorghum bicolor(L.) Moench

Five varieties of Sorghum bicolor (L.) Moench., differing in their drought tolerance under field conditions have been used to study the effect of individual components of drought stress, namely high light intensity stress, heat stress and water stress, on their photosynthetic performance. Chlorophyll content, chlorophyll fluorescence, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) content, phosphoenolpyruvate carboxylase (PEPcase, EC 4.1.1.31) activity and photo-synthetic oxygen evolution were used as key parameters to assess photosynthetic performance. The results indicated that photochemical efficiency of photosystem II (PSII) was severely reduced by all three stress components, whereas PEPcase activity was more specifically reduced by water stress. Degradation of Rubisco and chlorophyll loss occurred under high light and water stress conditions. Of the four drought-tolerant varieties, E 36-1 showed higher PEPcase activity, Rubisco content and photochemical efficiency of PSII, and was able to sustain a higher maximal rate of photosynthetic oxygen evolution under each stress condition as compared to the other varieties. A high stability to stress-induced damage, or acclimation of photosynthesis to the individual components of drought stress may contribute to the high yields of E 36-1 under drought conditions. In the E 36-1 variety markedly higher levels of the chloroplastic chaperonin 60 (cpn 60) were observed under all stress conditions than in the susceptible variety CSV 5.Key words: Chlorophyll fluorescence, drought stress, oxygen evolution, phosphoenopyruvate carboxylase, Sorghum.      

Activity of biochemical pH-stat enzymes in cereal root tips under oxygen deficiency

The activity of the enzymes of alcoholic and lactic-acid fermentation: pyruvate decarboxylase (PDC, EC 4.1.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and the enzymes of malic acid metabolism: phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.23), NAD-dependent malate dehydrogenase (NAD-MDH, EC 1.1.1.37), and NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) involved in the operation of biochemical pH-stat was investigated in the root tips of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under hypoxia and anoxia. Exposures lasted for 6, 12, and 18 h. The most pronounced response was detected for the enzymes of alcoholic fermentation. The activation of ADH and PDC in wheat occurred only under hypoxia, whereas in rice it was detected both under hypoxia and anoxia. The activation of LDH in wheat occurred under hypoxia, and in rice, the activity of this enzyme was slightly enhanced. The activity of the enzymes of malic acid metabolism did not change except in wheat root tips under hypoxia when PEPC activity decreased and NADP-ME activity simultaneously rose. The role of biochemical pH-stat in the regulation of cytoplasmic pH in plant cells under oxygen deficit and the mechanisms for regulating the activities of enzymes involved in biochemical pH-stat are discussed as well as the interaction between biochemical pH-stat and other mechanisms maintaining pH of plant cells. The results are analyzed within a context of intracellular pH regulation.     

Cold acclimation of wheat (Triticum aestivum): Properties of enzymes involved in proline metabolism

Two cultivars of wheat ( Triticum aestivum L.), a winter wheat, Kharkov, and a spring wheat, Glenlea, were acclimated under controlled conditions at 2 temperatures, 5°C and 25°C with a 12-h photoperiod. Water content, protein and proline concentrations were determined. Enzymatic properties (activity and apparent energy of activation) were investigated for enzymatic systems involved in 2 pathways of proline metabolism, the glutamic acid and ornithine pathways. Four enzymes were studied, proline dehydrogenase (PDH, EC 1.5.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), glutamine synthetase (GS, EC 6.3.1.2) and ornithine transaminase (OT, EC 2.6.1.13). Cold acclimation led to an accumulation of proline, a decrease in water content and an increase in soluble protein, especially in winter wheat. For both cultivars, cold acclimation modulated enzyme properties of PDH and GDH. Increased activities of GS and OT were observed as a result of cold acclimation in both cultivars, with the greatest increase in Kharkov. The apparent energy of activation of these 2 enzymes decreased, particularly for Kharkov, which accumulated proline in cold conditions.