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1.
In the succulent leaves of Aloe arborescens Mill diurnal oscillations of the malic acid content, being indicative of Crassulacean Acid Metabolism (CAM), were exhibited only by the green mesophyll. In contrast, the malic acid level of the central chloroplast-free water-storing tissue remained constant throughout the day-night cycle. Apart from malate, the green tissue contained high amounts of isocitrat which was lacking in the water tissue. There was no significant transfer from the green mesophyll to the water tissue of 14C fixed originally via dark 14CO 2 fixation in the mesophyll. Both isolated mesophyll and water tissue were capable of dark CO 2 fixation yielding mainly malate as the first stable product. Both tissues have phosphoenolpyruvate carboxylase. However, the enzymes derived from the both sources could be distinguished by their molecular weights and by their kinetic properties, suggesting different phosphoenolpyruvate carboxylase proteins. The conclusion drawn from the experiments is that in a. arborescens the CAM cycle proceeds exclusively in the green mesophyll and that the water tissue, though capable of malate synthesis via -carboxylation of phosphoenolpyruvate, behaves as an independent metabolic system where CAM is lacking. This view is supported by the finding that the cell walls bordering the green mesophyll from the water tissue lack plasmodesmata, hence conveniant pathways of metabolite transport.Abbreviations CAM
Crassulacean acid metabolism
- PEP
phosphoenolpyruvate
- PEP-C
phosphoenolpyruvate carboxylase 相似文献
2.
Leaves of Peperomia camptotricha contain three distinct upper tissue layers and a one-cell thick lower epidermis. Light and dark CO 2 fixation rates and the activity of ribulose bisphosphate carboxylase/oxygenase and several C 4 enzymes were determined in the three distinct tissue layers. The majority of the C 4 enzyme activity and dark CO 2 fixation was associated with the spongy mesophyll, including the lower epidermis; and the least activity was found in the median palisade mesophyll. In contrast, the majority of the C 3 activity, that is ribulose bisphosphate carboxylase/oxygenase and light CO 2 fixation, was located in the palisade mesophyll. In addition, the diurnal flux in titratable acidity was greatest in the spongy mesophyll and lowest in the palisade mesophyll. The spatial separation of the C 3 and C 4 phases of carbon fixation in P. camptotricha suggests that this Crassulacean acid metabolism plant may have low photorespiratory rates when it exhibits daytime gas exchange (that is, when it is well watered). The results also indicate that this plant may be on an evolutionary path between a true Crassulacean acid metabolism plant and a true C 4 plant. 相似文献
3.
The metabolism of [ 13C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to 13CO 2 for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [ 13C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4- 13C]malate to [1- 13C]malate by fumarase, varied substantially during the dark period. At both 15 and 25°C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of 13CO 2 uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired 12CO 2 also served as a source of carbon for malate formation. At 15°C, 15% of the total malate was formed from respired 12CO 2, while at 25°C, 49% of the accumulated malate was derived from respired 12CO 2. Some of the malate synthesized from external 13CO 2 was also respired during the night. The proportion of the total [ 13C]malate respired during the dark period was similar at 15 and 25°C, and respiration of newly formed [ 13C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO 2 fixation. 相似文献
4.
A mechanical isolation procedure was developed to study the respiratory properties of mitochondria from the mesophyll and bundle sheath tissue of Panicum miliaceum, a NAD-malic enzyme C 4 plant. A mesophyll fraction and a bundle sheath fraction were obtained from young leaves by differential mechanical treatment. The purity of both fractions was about 80%, based on analysis of the cross-contamination of ribulose bisphosphate carboxylase activity and phosphoenolpyruvate carboxylase activity. Mitochondria were isolated from the two fractions by differential centrifugation and Percoll density gradient centrifugation. The enrichment of mitochondria relative to chloroplast material was about 75-fold in both preparations. Both types of mitochondria oxidized NADH and succinate with respiratory control. Malate oxidation in mesophyll mitochondria was sensitive to KCN and showed good respiratory control. In bundle sheath mitochondria, malate oxidation was largely insensitive to KCN and showed no respiratory control. The oxidation was strongly inhibited by salicylhydroxamic acid, showing that the alternative oxidase was involved. The bundle sheath mitochondria of this type of C4 species contribute to C4 photosynthesis through decarboxylation of malate. Malate oxidation linked to an uncoupled, alternative pathway may allow decarboxylation to proceed without the restraints which might occur via coupled electron flow through the cytochrome chain. 相似文献
5.
Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to 14CO 2 in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO 2 photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of 14CO 2 both in the light and in the dark. Presumably, the 14CO 2 is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with 14CO 2 showed high amounts of labeled starch after the incubation, whereas cells labeled with [4- 14C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant 14CO 2 fixation. However, adding PEP to an homogenate stimulated 14CO 2 uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO 2 and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs
guard-cell protoplasts
- MCPs
mesophyll cell protoplasts
- PEP
phosphoenolpyruvate
- DTT
dithiothreitol
- 3-PGA
3-phosphoglyceric acid
- RiBP
ribulose 1,5 bisphosphate
- MDH
malate dehydrogenase
- MES
2-(N-morpholino)ethane sulfonic acid
- CAM
crassulacean acid metabolism 相似文献
6.
The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O 2 evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C 4-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity. 相似文献
7.
Summary CO 2 fixation characteristics of a number of mature (but not senescing) tissues and organs (the outer layers of green pod and the seed testa of Vicia faba L.; the outer layers of green pod and seeds of Trigonella foenum-graecum L.; the outer layers of the green fruit of Lycopersicon esculentum Mill.) were studied and compared with their respective C 3 leaf characteristics. On a chlorophyll basis phosphoenolpyruvate carboxylase, malic enzyme (NADP) and malate dehydrogenase (NAD and NADP) acitivites were much higher in the non-leaf tissues (except for V. faba seed testa) than the leaf tissues. Generally, on a protein basis the differences were less significant. All tissues possessed ribulose-1.5-diphosphate carboxylase activity though there was great variation in activities both on a protein and chlorophyll basis. Protein: chlorophyll ratios varied greatly from tissue to tissue being lowest in the leaf tissue (11.5–14.0) and highest in V. faba seed testa (805.5). Chlorophyll a:b ratios were all between 2 and 3. 14CO 2 uptake in the dark by L. esculentum fruit slices was about 1/3 that in the light and the major, initially labelled product was malate both in the light and dark. Neither typical C 4-photosynthesis or crassulacean acid metabolism were exhibited by the non-leaf tissues and it was considered that the increased levels of certain enzyme activities were present to refix and recycle respired CO 2.Abbreviations PEP
phosphoenolpyruvate
- RuDP
ribulose -1,5-, diphosphate
- MDH
malate dehydrogenase
- CAM
Crassulacean acid metabolism
- OAA
oxaloacetic acid 相似文献
8.
The influence of indoleacetic acid, 0.03% CO 2, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO 2 stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO 2. Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO 2, and that indoleacetic acid increased the incorporation of 14C-bicarbonate into protein. These data are discussed in the context of CO 2-stimulated growth of etiolated tissue, and proposals that CO 2-stimulated growth involves dark CO 2 fixation. 相似文献
10.
Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH 4+ in the dark under anaerobic conditions. Addition of NH 4+ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO 2 efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phospho enolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H 14CO 3− to anaerobic cells assimilating NH 4+ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH 4+ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH 4+ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH 4+ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH 4+ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity. 相似文献
11.
The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O 2 evolution and O 2 uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m ?2 s ?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO 3‐dependent O 2 evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O 2 uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO 3‐dependent O 2 evolution or p‐benzoquinone (BQ)‐dependent O 2 evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model. 相似文献
12.
Abstract Crassulacean acid metabolism (CAM) was studied in mixotrophic callus tissue cultures of Kalanchoë blossfeldiana hybr. Montezuma and compared with plants propagated from the calli. The ultrastructural properties of the green callus cells are similar to mesophyll cells of CAM plants except that occasionally abnormal mitochondria were observed. There was permanent net CO 2 output by the calli in light and darkness, which was lower in darkness than in light. The calli exhibited a diurnal rhythm of malic acid, with accumulation during the night and depletion during the day. 14C previously incorporated by dark CO 2 fixation into malate was transferred upon subsequent illumination into end products of photosynthesis. All these data indicate that CAM operates in the calli tissue. The results revealed that the capacity for CAM is obviously lower in the calli compared with plantlets developing from the calli, or with ‘adult’ plants. The data suggest also that CAM in the calli was not limited by the activities of CAM enzymes. 相似文献
14.
Malate synthesis by CO 2 dark fixation and malate accumulation in the vacuoles of leaf slices of Kalanchoë daigremontiana Hamet et Perrier, a plant performing crassulacean acid metabolism, occurs only in external solutions where the osmotic pressure difference between the cells and the medium is low. Conversely, malate loss from the vacuoles depends on a high osmotic pressure difference between the cells and the medium and is observed in media of low osmotic pressure. This suggests that the diurnal oscillations of malate levels in crassulacean acid metabolism leaf cells are regulated by osmotic gradients. These findings support a model which is introduced to explain how the rhythm of crassulacean acid metabolism may function in the intact plant. 相似文献
15.
Long term feeding of acetate-2- 14C, 14CO 2, citrate-1,5- 14C, fumarate-2,3- 14C, and succinate-2,3- 14C to mung bean ( Phaseolus aureus L. var. Mungo) leaves in the dark gave labeling predominantly in tricarboxylic acid cycle intermediates. Kinetics of the intermediates during dark/light/dark transitions showed a light-induced interchange of 14C between malate and aspartate, usually resulting in an accumulation of 14C in malate and a decrease of it in aspartate. 14C-Phosphoenolpyruvate also showed a marked decrease during illumination. Changes in other intermediates of the tricarboxylic acid cycle were relatively minor. The kinetic data have been analyzed using the Chance crossover theorem to locate control points during the dark/light/dark transitions. The major apparent control points are located at malate and isocitrate dehydrogenases, and less frequently at citrate synthase and fumarase. These findings are explained in terms of the light-induced changes in adenine nucleotides and nicotinamide adenine dinucleotides. 相似文献
16.
Exposure of the leaf canopy of corn seedlings ( Zea mays L.) to atmospheric CO 2 levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO 2 in the dark and maintained in an atmosphere containing 100 μl/l CO 2 accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO 2, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO 2 was high. Plants exposed to increasing CO 2 levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO 2) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate. 相似文献
17.
Kinetics and osmoregulation of cotton ( Gossypium hirsutum L.) fiber growth (primarily extension) have been studied. Growth is dependent on turgor pressure in the fiber. It is inhibited when a decrease in the water potential of the culture medium due to an addition of Carbowax 6000, equals the turgor pressure of the fiber. Potassium and malate accumulate in the fiber and reach peak levels when the growth rate is highest. Maximum concentrations of potassium and malate reached in the fiber can account for over 50% of the osmotic potential of the fiber. As growth slows down, levels of potassium and malate decrease and turgor pressure declines. Cotton ovules are capable of fixing H 14CO 3− in the dark, predominantly into malate. Fiber growth is inhibited by the absence of potassium and/or atmospheric CO 2. We suggest that potassium and malate act as osmoregulatory solutes and that malate, at least in part, arises from dark CO 2 fixation reactions. 相似文献
18.
Phosphoenolpyruvate (PEP) carboxylation was measured as dark 14CO 2 fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO 2 fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO 2-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO 2 fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM
crassulacean acid metabolism
- PEP
Phosphoenolpyruvate
- PEPCase
phosphoenolpyruvate carboxylase
- PP
protein phosphatase
- PK
protein kinase
This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged. 相似文献
19.
The use of mesophyll protoplast extracts from various C 4 species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C 4 photosynthesis. Conditions regulating the formation of the C 4 acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C 4 species, Eleusineindica, an NAD-malic enzyme C 4 species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C 4 species. Light-dependent induction of CO 2 fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO 2 fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C 4 mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO 2 fixation was not. 相似文献
20.
Malate efflux from leaf cells of the Crassulacean acid metabolism plant Kalanchoë daigremontiana Hamet et Perrier was studied using leaf slices submerged in experimental solutions. Leaves were harvested at the end of the dark phase and therefore contained high malate levels. Water potentials of solutions were varied between 0 and −5 bar using mannitol (a slowly permeating solute) and ethylene glycol (a rapidly permeating solute), respectively. Mannitol solutions of water potentials down to −5 bar considerably reduced malate efflux. The slowly permeating solute mannitol reduces both water potential and turgor potential of the cells. The water potential of a mannitol solution of −5 bar is just above plasmolyzing concentration. Malate efflux in ethylene glycol at −5 bar was only slightly smaller than at 0 bar, and much higher than in mannitol at −5 bar. Tissues in rapidly permeating ethylene glycol would have turgor potentials similar to tissues in 0.1 m m CaSO 4. The results demonstrate that malate efflux depends on turgor potential rather than on water potential of the cells. 相似文献
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