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2.
The activity of NADP-malate dehydrogenase (NADP-MDH) was determined in the developing first leaf of the C 3 plants wheat, barley and pea. Light dependent activation of the enzyme was observed in all three species following rapid extraction and immediate assay. Maximum activity was obtained following extraction from preilluminated leaves and incubation on ice for 45 min in the presence of dithiothreitol. In all three species, maximum activity was obtained in the young leaf 4 days after emergence of the seedling (about 2.5 to 3 moles per milligram chlorophyll per min in wheat and barley, and 6 moles per milligram chlorophyll per min in pea). On a chlorophyll basis there was an approximate five-fold decrease in NADP-MDH activity as the leaf matured. A similar pattern was found for phospho-enolpyruvate carboxylase and NADP-malic enzyme which had maximum activity in younger leaf tissue. Similarly, the activity of nitrate reductase in wheat and barley was high in the young leaf and it rapidly declined as the leaf matured. In contrast, the capacity for photosynthesis was relatively low in the young leaf, reaching a maximum 6 to 8 days after seedling emergence. The pattern of change in activity of phosphoribulokinase, an enzyme of the reductive pentose phosphate pathway, was similar to that of photosynthesis. The results suggest NADP-MDH and phospho-enolpyruvate carboxylase have important function(s) in the young leaf, which are not directly linked to C 3 photosynthesis, and which, in part, may be linked to nitrate assimilation and provision of malate to mitochondria.Abbreviations Chl
Chlorophyll
- DTT
Dithiothreitol
- NADP-MDH
NADP-malate Dehydrogenase
- NADP-ME
NADP-malic enzyme 相似文献
4.
The presence and properties of the enzyme xanthine oxidoreductase (XOR) in peroxisomes from pea (Pisum sativum L.) leaves were studied using biochemical and immunological methods. The activity analysis showed that, in leaf peroxisomes, the superoxide-generating XOR form, xanthine oxidase (XOD), was predominant over the xanthine dehydrogenase form (XDH), with a XDH/XOD ratio of 0.5. However, in crude extracts of pea leaves, the XDH form was more abundant, with a XDH/XOD ratio of 1.6. The native molecular mass of the peroxisomal XOR determined by polyacrylamide gel electrophoresis was 290kDa. Using western blot assays, we identified an immunoreactive band of 59kDa that was not affected by the reducing reagent DTT or endogenous proteases. The analysis of pea leaves by electron microscopy immunogold labeling with affinity-purified antibodies against rat XOD confirmed that this enzyme was localized in the matrix of peroxisomes, as well as in chloroplasts and cytosol. In pea plants subjected to abiotic stress by cadmium, the activity of the peroxisomal XOR was reduced, whereas its protein level expression increased. The results confirmed that leaf peroxisomes contain XOR, and suggest that this peroxisomal metalloflavoprotein enzyme is involved in the mechanism of response of pea plants to abiotic stress by heavy metals. 相似文献
5.
A difference was found between the temperature of control and heat-treated winter wheat and pea seedlings shoots during low temperature stress. Functioning of three thermogenic mitochondrial systems was established: (i) alternative cyanide-resistant oxidase, (ii) plant uncoupling mitochondrial protein and (iii) stress protein CSP 310 and these three caused the higher temperature of winter wheat control shoots. In peas only two thermogenic systems, the alternative cyanide-resistant oxidase and plant uncoupling mitochondrial proteins were found. 相似文献
6.
Investigations on the sites of ent-kaur-16-ene ( ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea ( Pisum sativum L.) seedlings. [ 14C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue. 相似文献
7.
Salicylhydroxamic acid (SHAM), an alternative oxidase inhibitor of plant mitochondria, enhances the NADH-oxidase activity in mitochondrial and chloroplast suspensions obtained from pea roots or leaves, respectively. This reaction is inhibited by the washing of mitochondria or chloroplasts and is observed in supernatants after the removal of the organelles by centrifugation. The reaction is sensitive to CN – and to antioxidant propyl gallate. The NADH oxidation is also enhanced by 2,4-dichlorophenol or phenol, but not salicylic acid. The acceleration of NADH oxidation by phenolic compounds is observed with presence of commercial horseradish peroxidase and is connected with the involvement of these compounds in NADH-dependent peroxidase reaction. SHAM and 2,4-dichlorophenol significantly enhance the destruction of nuclei in guard cells of pea leaf epidermis caused by the generation of reactive oxygen species during the oxidation of exogenous NADH by apoplastic peroxidase. 相似文献
8.
In the present work, we studied the influence of water (osmotic) stress on mitochondrial metabolic activity in etiolated pea ( Pisum sativum L.) seedlings. Three-day-old pea seedlings were subjected to stress by placing their roots in 0.6 M mannitol for 48 h. Epicotyl growth was severely suppressed, and tissue water content was decreased. We revealed the negative influence of the water stress on mitochondrial metabolic activity of seedlings, which effect was retained also after organelle isolation. In particular, in the mitochondria of stressed seedlings, the rate of oxidation of malate and other respiratory substrates (in state 3) was severely decreased, as well as respiratory control ratio. The rate of proline oxidation was reduced most seriously (by 70%). The efficiency of oxidative phosphorylation, according to the ADP/O ratio was not changed or was increased as compared to mitochondria in control plants. Activation of CN-resistant oxidase and other alternative pathways of electron transport in the mitochondrial electron-transport chain in stressed plants were not observed. In the epicotyl tissues under water stress, no MDA was accumulated and proline accumulation was insignificant. The role of mitochondria in adaptation responses of young seedlings is discussed. 相似文献
9.
Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed. 相似文献
10.
Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose.In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial.Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration.Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation. 相似文献
11.
Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins. 相似文献
12.
The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea ( Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism. 相似文献
13.
The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species. 相似文献
14.
Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis. 相似文献
15.
Concentrations of fluazifop-butyl sprayed on intact plants caused large decreases in the incorporation of radioactivity from [1-14C]acetate into lipids of barley (Hordeum vulgare) leaves and stems, but did not affect leaves or stems of pea (Pisum sativum). Labelling of all acyl lipids, but not pigments, was reduced. The effects of the active acid form, fluazifop, were also determined in leaf pieces and chloroplasts. Concentrations of (R,S)-fluazifop up to 100 microM had no affect upon quality or quantity of fatty acids produced from [1-14C]acetate in pea. In barley, however, 100 microM-(R,S)-fluazifop caused 89% (leaf) or 100% (chloroplasts) inhibition in labelling of fatty acids from [1-14C]acetate. Lower concentrations of fluazifop (less than 25 microM) caused incomplete inhibition and significant decreases in the proportion of C18 fatty acids synthesized, particularly by isolated chloroplasts. Synthesis of fatty acids from [2-14C]malonate was also inhibited (59%) in barley leaf tissue by 100 microM-(R,S)-fluazifop. The labelling pattern of products showed that elongation reactions were unaffected by the herbicide, but synthesis de novo was specifically diminished. By using resolved stereoisomers, it was found that the (R) isomer was the form which inhibited fatty acid synthesis, a finding that is in agreement with its herbicidal activity. These results suggest that inhibition of fatty acid synthesis de novo forms the basis for the selective mode of action of fluazifop. 相似文献
16.
Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic processes. S-deficient bean plants ( Phaseolus vulgaris L. cv. Złota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower dry matter production. Phenotypic effects of S deficiency appeared as depressed shoot growth, paling and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show that S deficiency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex I. Increased activities of internal NADH dehydrogenases (ND in) may at least partially compensate for Complex I impairment. External NADH dehydrogenases (ND ex) activities, as well as protein level and capacity of alternative oxidase (AOX), did not change in S-deficient bean plants. Total ATP concentration severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-deficient bean plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our findings indicate that flexible function of plant mitochondrial respiratory chain could be an important target during adaptations to S deficiency. 相似文献
17.
Gas exchange and protein metabolism were studied in expanding, mature, and near-senescent leaves of young clonal Populus × euramericana cv. Wisconsin-5 plants. Dark respiration, CO 2 evolution in the light, and CO 2 compensation concentrations were highest in unexpanded leaves but declined markedly as leaves matured and aged. Net photosynthesis was highest in nearly mature leaves. Fresh weight continued to increase after leaf expansion was complete, whereas soluble protein levels declined. Changes in the distribution of photosynthetically incorporated 14C indicated that a high level of protein synthesis and rapid formation of structural components occurred only in expanding leaves. Protein turnover was slight in expanding leaves but was substantial after leaves were mature. Expanding leaves synthesized predominantly fraction I protein (ribulose diphosphate carboxylase). However, formation of this protein from photosynthate was slight once leaves matured. 相似文献
18.
Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration. 相似文献
19.
Asparagine is present in the mature leaves of young pea ( Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [ 14C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [ 14C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine. Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue. 相似文献
20.
The addition of cold shock CSP310 protein to mitochondria isolated from both monocotyledonous (rye, wheat, and maize) and dicotyledonous (pea) plants uncoupled oxidation from phosphorylation. This uncoupling was caused neither by the damage to mitochondrial membranes nor by the activation of alternative cyanide-resistant oxidase. As distinct from the classical plant uncoupling mitochondrial protein (PUMP), CSP310 uncoupling effect was insensitive to BSA. Therefore, we believe that the mechanism of CSP310 action differs from that of known plant uncoupling proteins. 相似文献
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