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1.
Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme 总被引:1,自引:0,他引:1
Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P
glucose-6-phosphate
- LDH
lactate dehydrogenase
- MDH
malate dehydrogenase
- PEP
phosphoenolpyruvate
- PEPCase
phosphoenolpyruvate carboxylase
- PVP
polyvinylpyrrolidone
- PPDK
pyruvate, orthophosphate dikinase
- U
unit of enzyme activity (mol/min) 相似文献
2.
Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene 总被引:1,自引:0,他引:1
Ken Ishimaru Yasunobu Ohkawa Teruo Ishige Dennis J. Tobias Ryu Ohsugi 《Physiologia plantarum》1998,103(3):340-346
Changes in carbon metabolism and δ13 C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO2 in the C4 photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r2 = 0.81), and was positively correlated with malate content (r2 = 0.88). A significant increase in the δ13 C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO2 . These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C4 ‐type carbon metabolism. However, since parameters associated with CO2 gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants. 相似文献
3.
C4 photosynthesis at low temperatures 总被引:4,自引:8,他引:4
S. P. LONG 《Plant, cell & environment》1983,6(4):345-363
Abstract. C4 plants grown in optimum conditions are, by comparison to C3 , capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C4 distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C4 process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C4 photosynthesis and the functioning of the individual steps in the C4 cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO2 which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO2 and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C4 species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C4 species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions. 相似文献
4.
Compatible solutes and their effects on phosphoenolpyruvate carboxylase of C4 -halophytes 总被引:1,自引:0,他引:1
Abstract Phosphoenolpyruvate carboxylase (PEPCase), extracted from two Poaceae (Cynodon dactylon and Sporobolus pungens) grown on saline soil, was affected physiologically by betaine and proline. Its affinity for phosphoenolpyruvate (PEP) was increased and full protection against NaCl inhibition was observed; enzymic activity was also stabilized when assayed at low PEP levels. Betaine has the same effects on PEPCase extracted from a Chenopodiaceae (Salsola soda), whereas proline behaves as a competitive inhibitor, i.e. it does not protect the enzyme against NaCl and it accelerates inactivation at low PEP levels. Betaine was only compatible with PEPCase extracted from Saisola kali, without any effect on activity, protection or stabilization, but proline was again inhibitory. The levels of free proline in the two salt-stressed Poaceae were high, whereas in the Chenopodiaceae the free proline was low, as in non-stressed plants. The above data indicate that osmoregulators could not only be compatible with cytoplasmic enzymes, but they could either promote or inhibit enzyme activity, depending on the source of enzyme. Coevolution of PEPCase with the osmolyte selected for, could also be inferred. 相似文献
5.
Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays) 总被引:1,自引:0,他引:1
Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C4 plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C4 photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP+ -malate dehydrogenase proceeded more rapidly than photosynthesis. 相似文献
6.
Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the Pi-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for Pi-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.Abbreviations BSA
bovine serum albumin
- G-6-P
glucose-6-phosphate
- MDH
malate dehydrogenase
- PCMB
p-chloromercuribenzoate
- PEP
phosphoenolpyruvate
- PEPCase
phosphoenol-pyruvate carboxylase
- PPDK
pyruvate, orthophosphate dikinase
- PVP
polyvinylpyrrolidone 相似文献
7.
K. MATSUBA N. IMAIZUMI S. KANEKO M. SAMEJIMA R. OHSUGI 《Plant, cell & environment》1997,20(2):268-274
Photosynthetic rates, the activities of key enzymes associated with the C4 cycle and ribulose-1,5-bisphosphate carboxylase (RuBPCase), and the levels of metabolites involved in the C4 cycle were compared between the two phosphoenolpyruvate carboxykinase (PCK) type C4 species Spartina anglica, which is cold-tolerant, and Zoysia japonica, which is cold-sensitive, during exposure to low temperature. Plants of both species grown outside in summer were placed in a growth chamber at 27/20 °C day/night temperatures. After 1 week, plants were exposed to 20/17 °C for 1 week and then to 10/7 °C for 2 weeks. Photosynthetic rates in Z. japonica decreased progressively to about 25% during the chilling treatments. In contrast, S. anglica exhibited a 43% increase in photosynthetic rates after exposure to 20 °C for 1 week, which remained relatively constant thereafter. Consistent with these observations, most of the C4 enzymes and RuBPCase in Z. japonica declined. Phosphoenolpyruvate carboxylase (PEPC) and PCK activities declined particularly drastically during the treatments. However, the activities of these enzymes in S. anglica showed either a slight increase or decrease upon a mild cold treatment, and remained relatively constant during further chilling treatments. There was a sharp decline in phosphoenolpyruvate in Z. japonica after exposure to 10 °C. On the other hand, metabolite levels in S. anglica were largely unaffected by the chilling treatments. These results suggest that the drastic declines of both PEPC and PCK activities may be important limiting factors responsible for cold sensitivity in C4 photosynthesis of Z. japonica. 相似文献
8.
Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms. 相似文献
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Previous studies have indicated that the rate of photorespiration in C4 plants is low or negligible. In this study, wild-type and mutant leaves of the C4 plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO2. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C4 plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO2 assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C3 plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO2 assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C4 plants, which can play an important role in photosynthetic metabolism in these plants. 相似文献
11.
When exposed to osmotic stress, Mesembryanthemum crystallinum plants switch from C3 to CAM photosynthesis. Phosphoenolpyruvate carboxylase (PEPCase) is a key enzyme in CAM plants, being responsible for the initial fixation of CO2. In C3 plants the enzyme has been shown to be involved in the replenishing of TCA cycle intermediates and in the operation of stomatal guard cells. Multiple PEPCase isoforms were observed in C3-performing leaves with four isoelectric points of 5.2, 5.5, 5.6 and 5.9 and four apparent subunit molecular masses of 105, 108, 113 and 116 kDa. In some instances, subunits of different size possessed exactly the same pI. The induction of CAM led to the predominance of a new isoform of pI 6.5 with subunit molecular mass of 108 kDa, but in addition, changes were observed in some of the isoforms present in the C3 plant. PEPCase subunits were purified from the C3 and CAM forms of M. crystallinum and subjected to pep-tide mapping. Two distinct though similar sets of maps were obtained, one from the CAM isoform (pI 6.5) and C3-associated subunits of pi 5.9 and another for C3 subunits of pI 5.2 and 5.5. It was inferred from these data that the C3 isoforms expressed in the leaf were derived from at least two genes. The C3 isoform (pI 5.9) showing greatest similarity to the CAM isoform in terms of peptide mapping also increased in response to salt stress. It is speculated that the CAM isoform may have evolved from this enzyme. 相似文献
12.
Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates 总被引:1,自引:0,他引:1
Jean-Pierre Simon 《Physiologia plantarum》1987,69(2):205-210
Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m−2 s−1 ). The activities of four C4 enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, Pi dikinase (PPDK; EC 2.7.9.1) and NADP+ -malic enzyme (NADP+ -ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP+ -malate dehydro-genase (NADP+ -MDH; EC 1.1.1.82) and NADP+ -ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C. 相似文献
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Activation of pyruvate,Pi dikinase by light was studied in leaf discs of maize which were illuminated for 1 h at light intensities ranging from approximately 3% to 50% of full sunlight and at temperatures of 10, 22.5, and 35°C. At the highest light intensity the degree of activation was similar and relatively independent of temperature between 10 and 35°C. Under low light the degree of activation was high at 10°C but decreased rapidly with increasing temperature. There was a similar effect of light and temperature on the activation of NADP-malate dehydrogenase.At low temperature, the rate of activation of pyruvate,Pi dikinase was relatively low and independent of the light intensity used and the rate of inactivation in the dark was extremely low. At high temperature, the rate of activation was high and dependent on the light intensity used while the rate of dark inactivation was also relatively high. The degree of activation is discussed in relation to the possible influence of light and temperature on the turnover between the active and inactive forms of pyruvate,Pi dikinase during illumination.This research was supported by the Japan-U.S. Cooperative Research Program (The Japan Society for the Promotion of Science, NFS Grant INT 78-17245), NSF Grant PCM 77-09284, by the Japanese Ministry of Education and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin. 相似文献
15.
The effects of photon flux density and temperature on net photosynthesis and transpiration rates of mature and immature leaves of three-year-old Japanese larch Larix kaempferi (Lamb.) Sarg. trees were determined with an infrared, differential open gas analysis system. Net photosynthetic response to increasing photon flux densities was similar for different foliage positions and stage of maturity. Light compensation was between 25 and 50 μmol m−2 s−1 . Rates of photosynthesis increased rapidly at photon flux densities above the compensation level and became saturated between 800 and 1000 μmol m−2 s−1 . Transpiration rates at constant temperature likewise increased with increasing photon flux density, and leveled off between 800 and 1000 μmol m−2 s−1 . Photosynthetic response to temperature was determined in saturating light and was similar for all foliage positions; it increased steadily from low temperatures to an optimum range betweeen 15 and 21°C and then decreased rapidly above 21°C. Transpiration rate, however, increased continuously with rising temperature up to the experimental maximum. CO2 compensation concentrations for mature foliage varied between 58 and 59 μl l−1 ; however, foliage borne at the apex of the terminal leader compensated at 75 μl l−1 . None of these data support the claim that Japanese larch possesses C4 photosynthetic characteristics. 相似文献
16.
Stomatal function mediates physiological trade‐offs associated with maintaining a favourable H2O balance in leaf tissues while acquiring CO2 as a photosynthetic substrate. The C3 and C4 species appear to have different patterns of stomatal response to changing light conditions, and variation in this behaviour may have played a role in the functional diversification of the different photosynthetic pathways. In the current study, we used gain analysis theory to characterize the stomatal conductance response to light intensity in nine different C3, C4 and C3‐C4 intermediate species Flaveria species. The response of stomatal conductance (gs) to a change in light intensity represents both a direct (related to a change in incident light intensity, I) and indirect (related to a change in intercellular CO2 concentration, Ci) response. The slope of the line relating the change in gs to Ci was steeper in C4 species, compared with C3 species, with C3‐C4 species having an intermediate response. This response reflects the greater relative contribution of the indirect versus direct component of the gs versus I response in the C4 species. The C3‐C4 species, Flaveria floridana, exhibited a C4‐like response whereas the C3‐C4 species, Flaveria sonorensis and Flaveria chloraefolia, exhibited C3‐like responses, similar to their hypothesized position along the evolutionary trajectory of the development of C4 photosynthesis. There was a positive correlation between the relative contribution of the indirect component of the gs versus I response and water use efficiency when evaluated across all species. Assuming that the C3‐C4 intermediate species reflect an evolutionary progression from fully expressed C3 ancestors, the results of the current study demonstrate an increase in the contribution of the indirect component of the gs versus I response as taxa evolve toward the C4 extreme. The greater relative contribution of the indirect component of the stomatal response occurs through both increases in the indirect stomatal components and through decreases in the direct. Increases in the magnitude of the indirect component may be related to the maintenance of higher water use efficiencies in the intermediate evolutionary stages, before the appearance of fully integrated C4 photosynthesis. 相似文献
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Abstract Shifts in ?13C of the graminaceous C3 halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO2-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C3 plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm3 m?3 CO2, the CO2-enrichment enhanced the effects of salinity on ?13C. This is consistent with a biophysical explanation for salt-induced shifts in ?13C, whereby there is a steepening of the CO2 diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO2 concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced. 相似文献