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1.
Our results show that AtNRT2.1 expression has a positive effect on the NH4+ ion influx, mediated by the HATS, as also occurs with AtAMT1.1 expression on the NO3 ion influx. AtNRT2.1 expression plays a key role in the regulation of AtAMT1.1 expression and in the NH4+ ion influx, differentiating the nitrogen source, and particularly, the lack of it. Nitrogen starvation produces a compensatory effect by AtAMT1.1 when there is an absence of the AtNRT2.1 gene. Our results also show that, in the atnrt2 mutant lacking both AtNRT2.1 and AtNRT2.2, gene functions present different kinetic parameters on the NH4+ ion influx mediated by the HATS, according to the source and availability of nitrogen. Finally, the absence of AMT1.1 also produces changes in the kinetic parameters of the NO3 influx, showing different Vmax values depending on the source of nitrogen available.  相似文献   

2.
Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO3, NH4+ or NO3 in conjunction with NH4+, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO3 nutrition, lowest with NH4+ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO3-fed plants than in either NH4+- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO3-deprived than in NO3-sufficient plants. Root malate accumulation was high in NO3-fed plants, but decreased significantly in plants that were fed with NH4+. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO3-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO3- and (NO3 + NH4+)-fed plants, but was undetectable in those administered NH4+. Both the nitrate and the ammonium contents were significantly reduced in NO3- and (NO3 + NH4+)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH4+-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.  相似文献   

3.
Plants of Sorghum bicolor (C4 species) were grown at different nitrate or ammonium concentrations (0.5, 5, 20 and 50 mM) in order to examine the effect of nitrogen nutrition on growth, phosphoenolpyruvate carboxylase (PEPC) and antioxidant enzymes activities in both roots and leaves of 30-day-old plants. At high NO3? levels (20 and 50 mM) the fresh weight was significantly higher. When the nitrogen source was in ammonium form, the leaf and root mass increased drastically at low concentration 5 mM and significantly at 20 mM, however similar fresh weight was found at high level of ammonium (50 mM). The leaves catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), and glutathione S-transferase (GST) activities and the roots glutathione reductase and glutathione S-transferase activities were significantly higher in the NH4+-fed plants than those grown in the nitrate medium. Activity and proteins levels of phosphoenolpyruvate carboxylase in both leaves and roots of sorghum plants were increased progressively with increasing external nitrogen concentration. This increase was more pronounced at high level of ammonium (50 mM), being 2-fold at 50 mM of NO3? and 3-fold at 50 mM of NH4+. Our results suggested that antioxidant enzymes activities and PEPC play a key role in ammonium detoxification and tolerance in sorghum plants.  相似文献   

4.
Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO2 assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in 15N-labeled NH3, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The 15N-enrichment of various amino acids derived from these 15N-substrates were examined. The amido-N of glutamine was the first 15N-labeled product in leaves incubated with 15NH4Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly 15N-labeled products during incubation with [15N]glycine. In contrast, aspartate and alanine were the first 15N-labeled products when [15N] glutamate was used. These results indicate that NH3 was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.  相似文献   

5.
The rate of NH4+ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH4+ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H14CO3 into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C3 organism.  相似文献   

6.
The effects of urea, cations (K+, NH4, Na+, Cs+, Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle, were analyzed in two anuran amphibians, an estivating species, the spadefoot toadScaphiopus couchii, and a semi-aquatic species, the leopard frogRana pipiens. Urea, which accumulates naturally to levels of 200–300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes. Rather, we found that urea is largely a non-perturbing solute for anuran enzymes (I50 values were>1 M for both PK and PFK in both species) and we propose that its accumulation in high concentrations during estivation helps to minimize the increase in cellular ionic strength that would otherwise occur during desiccation and to alleviate the accompanying negative effects of high salt on individual enzyme activities and overall metabolic regulation.Abbreviations PFK 6-phosphofructo-1-kinase - PK pyruvate kinase  相似文献   

7.
We compared C3 and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2nd leaf pair and non-inoculated 3rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C3 plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C3 and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C3 plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C3- and CAM-specific.  相似文献   

8.
To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO2 into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C3 plants possessing a C4-like photosynthesis pathway worked by expression of PEPC.  相似文献   

9.
It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4+ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.  相似文献   

10.
Substantial concentrations of NH4 + are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with 15NH4 + revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH4 + transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH4 + as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH4 + uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH4 +. Withdrawal of NH4 + supply for up to 96 h had little effect on mRNA expression or NH4 + uptake; however, plants grown continuously at high NH4 + levels exhibited decreased mRNA expression. BnAMT1;2mRNA expression was highest when NH4 + was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2may be regulated by similar mechanisms to GS2 in leaves.  相似文献   

11.
Photosynthetic14CO2 assimilation, ribulose 1, 5-bisphosphate carboxylase (RuBPC), phosphoenol pyruvate carboxylase (PEPC) and dry matter (DM) production were examined in wheat under varying levels and forms of nitrogen.14CO2 assimilation increased gradually after germination reaching a peak value at anthesis, followed by a sharp decline. A similar pattern was observed for both the carboxylases, RuBPC and PEPC activities. Increase in nitrogen levels, in general, brought about a significant increase over the control (zero-nitrogen) in14CO2 assimilation, RuBPC, PEPC activities and DM production. There were no significant differences in RuBPC activity and14CO2 assimilation with respect to the forms of nitrogen. Significantly higher PEPC activity and DM was observed in plants supplied with nitrate-nitrogen (NO3-N), as compared to those supplied with ammonium-nitrogen (NH4-N). The significance of PEPC activity in C3 photosynthesis is discussed in relation to DM distribution.  相似文献   

12.
We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4 pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca2+ chelator, a Ca2+ channel inhibitor, and a hydrogen peroxide (H2O2) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (PN) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the PN of rice plants. The treatments with phospholipase inhibitors and a Ca2+ chelator decreased the PN of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca2+ both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca2+ level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca2+.  相似文献   

13.
《Phytochemistry》1986,25(3):601-606
Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H14CO3 incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent Km (PEP) and Vmax values compared more favourably with those obtained from a C3-species (spinach) than from a C4-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C4 photosynthetic enzyme.  相似文献   

14.
15.
16.
OsAMT is a high-affinity ammonium transporter responsible for NH 4 + uptake by rice plants. To investigate the expression patterns of OsAMT in different genotypes in relation to nitrogen accumulation, we measured the expression of OsAMT1.1, OsAMT1.2, and OsAMT1.3 using Real-Time PCR (RT-PCR) in GD (higher N accumulation) and NG (lower N accumulation) seedlings of the Oryza sativa L. cultivar treated with 0.1 mM NH4NO3 and 2 mM NH4NO3. We found that the expression level of OsAMT1.1 was significantly higher than those of OsAMT1.2 and OsAMT1.3 in the roots treated with 0.1 mM NH4NO3, suggesting that OsAMT1.1 contributed the most to N accumulation among the three genes. In GD root, OsAMT1.1 had significantly higher expression levels when it was up-regulated by 0.1 mM NH4NO3 than when down-regulated by 2 mM NH4NO3. OsAMT1.1 was mainly found in GD roots treated with 0.1 mM NH4NO3. We conclude that the OsAMT1.1 in GD roots, which was significantly up-regulated by low N and down-regulated by high N, was the dominating factor in determining the higher N acquisition in GD than in NG at 0.1 mM NH4NO3.  相似文献   

17.
18.
Many plants develop toxicity symptoms and have reduced growth rates when supplied with ammonium (NH4+) as the only source of inorganic nitrogen. In the present study, the growth, morphology, NH4+ uptake kinetics and mineral concentrations in the tissues of the free-floating aquatic plant Salvinia natans (water fern) supplied exclusively with NH4+–N at concentrations of 0.25–15 mM were investigated. S. natans grew well, with relative growth rates of c. 0.25 g g?1 d?1 at external NH4+ concentrations up to 5 mM, but at higher levels growth was suppressed and the plants had small leaves and short roots with stunted growth. The high-affinity transport system (HATS) that mediate NH4+ uptake at dilute NH4+ levels was downregulated at high NH4+ concentrations with lower velocities of maximum uptake (Vmax) and higher half-saturation constants (K1/2). High NH4+ levels also barely affected the concentrations of mineral cations and anions in the plant tissue. It is concluded that S. natans can be characterized as NH4+-tolerant in line with a number of other species of wetland plants as growth was unaffected at NH4+ concentrations as high as 5 mM and as symptoms of toxicity at higher concentrations were relatively mild. Depolarization of the plasma membrane to the equilibrium potential for NH4+ at high external concentrations may be a mechanism used by the plant to avoid excessive futile transmembrane cycling. S. natans is tolerant to the high NH4+ levels that prevail in domestic and agricultural wastewaters, and the inherent high growth rate and the ease of biomass harvesting make S. natans a primary candidate for use in constructed wetland systems for the treatment of various types of nitrogen-rich wastewaters.  相似文献   

19.
The carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) grown hydroponically at a constant pH on either 4 mM or 12 mM NO3 - or NH4 + nutrition were investigated using either 14C or 15N techniques. Greater allocation of 14C to amino-N fractions occurred at the expense of allocation of 14C to carbohydrate fractions in NH4 +-compared to NO3 --fed plants. The [14C]carbohydrate:[14C]amino-N ratios were 1.5-fold and 2.0-fold greater in shoots and roots respectively of 12 mM NO3 --compared to 12 mM NH4 +-fed wheat. In both 4 mM and 12 mM N-fed maize the [14C]carbohydrate:[14C]amino-N ratios were approximately 1.7-fold and 2.0-fold greater in shoots and roots respectively of NO3 --compared to NH4 +-fed plants. Similar results were observed in roots of wheat and maize grown in split-root culture with one root-half in NO3 --and the other in NH4 +-containing nutrient media. Thus the allocation of carbon to the amino-N fractions occurred at the expense of carbohydrate fractions, particularly within the root. Allocation of 14N and 15N within separate sets of plants confirmed that NH4 --fed plants accumulated more amino-N compounds than NO3 --fed plants. Wheat roots supplied with 15NH4 + for 8 h were found to accumulate 15NH4 + (8.5 g 15N g-1 h-1) whereas in maize roots very little 15NH4 + accumulated (1.5 g 15N g-1 h-1)It is proposed that the observed accumulation of 15NH4 + in wheat roots in these experiments is the result of limited availability of carbon within the roots of the wheat plants for the detoxification of NH4 +, in contrast to the situation in maize. Higher photosynthetic capacity and lower shoot: root ratios of the C4 maize plants ensure greater carbon availability to the root than in the C3 wheat plants. These differences in carbon and nitrogen partitioning between NO3 --and NH4 +-fed wheat and maize could be responsible for different responses of wheat and maize root growth to NO3 - and NH4 + nutrition.  相似文献   

20.
Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that is the recruited substrate, but the uncharged form NH3 is conducted. AtAMT2 partially co-localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell.  相似文献   

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