Concentration-Dependent inhibition and enhancement of glutamine synthetase,glutamate dehydrogenase and nitrate reductase activities in pea roots by some respiratory inhibitors and uncouplers

The effect of 2,4-dinitrophenol (2,4-DNP), NaN₃, and iodoacetic acid (IDA) on glutamine synthetase (GS) and the effect of arsenate on GS, glutamate dehydrogenase (GDH) and nitrate reductase (NR) was studied in isolated pea roots. In sucrose supplied roots, GS level is depressed by higher concentrations of all the inhibitors tested and increased by lower (2 × and 3 × 10⁻ M) concentrations of 2,4-DNP; the decrease in GS level caused by sugar starvation is enhanced by all but IDA. GDH is enhanced by arsenate in a wider range of concentrations in sucrose-supplied roots than in roots cultivated without sucrose. NR is affected by arsenate similarly as GS.     

The effect of cultivation temperature on nitrate reductase,glutamine synthetase and glutamate dehydrogenase levels in isolatedPisum sativum roots

Nitrate reductase (NR), glutamine synthetase (GS), and glutamate dehydrogenase (GDH) levels are differently influenced by cultivation temperature, NR level being influenced most and GDH level least. The differences caused by cultivation temperature are more pronounced in roots cultivated without sucrose in which a slower decrease in NR and GS levels and a lower increase in GDH level occur at 14 and 18 °C than at 24 °C in comparison to roots cultivated with sucrose. Sugar consumption also tends to be slower at 14 °C than at 24 °C in roots cultivated without sucrose.     

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

The effects of exogenous sucrose, lactose, d -glucose, d (-)fructose, d -galactose, d -mannose, l -sorbose, l -arabinose and d -xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O₂ consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO₃. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O₂ consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

The effect of glycine,humus substances and sucrose on the growth of tomato rootsin vitro

The effect of glycine on the growth of isolated tomato roots in White’s nutrient solution was studied. It is obvious that the root tips grow better in the medium without glycine, because increasing rates of glycine produce inhibition. The growth of root explantates in nutrient media indicates a high synthetic ability of isolated roots. Optimal concentrations of humus acids in White’s nutrient solution without glycine and with glycine stimulate the growth of root explantates. It cannot be decided which of the two substances affects the root directly and which acts as a chelating agent in the medium. The effect of glycine varied, depending on the origin of sucrose used. It appears that in media containing beet sucrose, glycine is unsuitable but in media containing cane sucrose it is necessary.     

Nickel-induced depression of nitrogen assimilation in wheat roots

The influence of 50 and 100 μM Ni on the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) was studied in the wheat roots. Root fresh weight, tissue Ni, nitrate, ammonium, glutamate and protein concentrations were also determined. Exposure to Ni resulted in a marked reduction in fresh weight of the roots accompanied by a rapid accumulation of Ni in these organs. Both nitrate and ammonium contents in the root tissue were considerably enhanced by Ni stress. While protein content was not significantly influenced by Ni application, glutamate concentration was slightly reduced on the first day after treatment with the higher Ni dose. Treatment of the wheat seedlings with 100 μM Ni led to a decrease in NR activity; however, it did not alter the activation state of this enzyme. Decline in NiR activity observed after application of 100 μM Ni was more pronounced than that in NR. The activities of GS and NADH-GOGAT also showed substantial decreases in response to Ni stress with the latter being more susceptible to this metal. Starting from the fourth day, both aminating and deaminating GDH activities in the roots of the seedlings supplemented with Ni were lower in comparison to the control. While the activity of AspAT remained unaltered after Ni application that of AlaAT showed a considerable enhancement. The results indicate that exposure of the wheat seedlings to Ni resulted in a general depression of nitrogen assimilation in the roots. Increase in the glutamate-producing activity of AlaAT may suggest its involvement in supplying the wheat roots with this amino acid under Ni stress.     

Changes in the activity of enzymes involved with primary nitrogen metabolism due to ectomycorrhizal symbiosis on jack pine seedlings

Jack pine (Pinus banksiana Lamb.) seedlings were inoculated with either one of the ectomycorrhizal (ECM) fungi, Laccaria bicolor (Maire) Orton or Pisolithus tinctorius (Pers.) Coker and Couch, and grown for 16 weeks in a growth chamber along with non-ECM controls. Five enzymes involved with the assimilation of nitrogen or the synthesis of amino acids were measured in the 3 jack pine root systems as well as in the pure fungal cultures. Pisolithus tinctorius in pure culture had no detectable activity of nitrate reductase (NR. EC 1.6.6.1), glutamate dehydrogenase (GDH. EC 1.4.1.2), glutamate decarboxylase (GDCO. EC 4.1.1.15) or glutamate oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13) but did have some glutamine synthetase (GS, EC 6.3.1.2) activity. Laccaria bicolor in pure culture had no NR activity, small levels of GDCO activity, and high GS, GDH and GOGAT activity. The high levels of enzymatic activity present in L. bicolor indicate that it may play a greater role in the nitrogen metabolism of its host plant than P. tinctorius. ECM infection clearly altered the enzymatic activity in jack pine roots but the nature of these changes depended on the fungal associate. Non-ECM root systems had higher specific activities than ECM root systems for NR, GS, GDH and GDCO but GOGAT activites were the same for both the ECM and non-ECM roots. Root systems infected with L. bicolor had significantly greater NR and GDCO activity than those infected with P. tinctorius. Differences in the GS activity of the two fungi in pure culture corresponded to the GS activity of jack pine roots in symbiotic association with these fungi. While the free amino acid profiles in roots were significantly affected by ECM infection, the profile of free amino acids exported to the stem was the same for all treatments. High asparagine and low glutamine in roots infected with P. tinctorius indicates that asparagine synthetase (EC x.x.x.x) activity should be higher within this symbiotic association than in the L. bicolor association or in the non-mycorrhizal roots.     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

不同浓度的NO^—3和NH^＋4对小麦根谷氨酰胺合成酶及其相关酶的影响

利用酶活性测定和 Northern分子杂交等技术 ,研究了小麦幼苗根在不同浓度的 Na NO3 和(NH4) 2 SO4的供应下 ,其谷氨酰胺合成酶 (GS)、天冬酰胺合成酶 (AS)、谷氨酸脱氢酶 (GDH)、硝酸还原酶 (NR)以及 GS- m RNA的变化。结果表明 :NH 4 处理的小麦 ,其根部 GS活性比 NO-3 处理的高 ;高浓度处理的比低浓度处理的高 ;Northern杂交结果说明 GS- m RNA转录量与 GS活性一致 ;3mmol/ L NO-3促进了 AS的活性。AS酶活性变化与 GS酶活性变化无明显依赖关系。在实验的条件下 ,没能测出 GDH的活性 ,不同浓度的 NO-3 和 NH 4 处理对 NR活性没有明显的规律。     

Influence of Different Concentrations of NO-3 and NH+4 on the Activity of Glutamine Synthetase and Other Relevant Enzymes of Nitrogen Metabolism in Wheat Roots

Influence of different concentrations of NO3 and NH+ on the activity of glutamine synthetase (GS), asparagine synthetase (AS), glutamate dehydrogenase (GDH), nitrate reductase (NR) and the changes of GS-mRNA in wheat roots have been studied with enzymes activity assay and Northern blot. The results showed that the higher GS activity was found in roots of wheat when NH+4-N was the sole nitrogen source than when NO3-N was the sole nitrogen source. GS-mRNA of Northern blot was simillar to GS activity. 3 mmol/L NO3- promoted the activity of AS. The change of AS was independent of the change of GS. GDH activity was not been detected, and change in regulation of NR activity was not found.     

Nitrogen metabolism of Plantago lanceolata as dependent on the supply of mineral nutrients

Various aspects of nitrogen metabolism of Plantago lanceolata L., a grassland species from a relatively nutrient poor habitat, were investigated under high nutrient conditions or low nutrient conditions. In addition the responses after switching the plants from high nutrient conditions to low nutrient conditions and vice versa were studied. The activities of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamine synthetase (GS) were determined. In the roots they were correlated with the level of the nutrient supply. Not only NR but also GDH and GS increased upon a switch from low nutrients to high nutrients and decreased upon a switch from high nutrients to low nutrients. Reduced nitrogen content of both roots and shoots was also correlated with the nutrient supply and changed rapidly after a switch. The flexibility of the nitrogen metabolism of Plantago lanceolata is discussed in relation to its ecological habitat.     

Biochar improved nodulation and nitrogen metabolism of soybean under salt stress

To investigate salt stress and biochar application effects on nodulation and nitrogen metabolism of soybeans (Glycine max cv. M7), an experiment was conducted under the control condition. The treatments comprised three biochar rates (non, 50 and 100 g kg^?1 soil) and three salinities (0, 5 and 10 dS m^?1 NaCl), with four replications of treatments. Salt stress diminished the number of nodules and their weights in the soybean roots. Nitrogen content and metabolism decreased in nodules, roots and shoots, while reducing the activity of glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT) and nitrate reductase (NR). Also, salinity brought down root and shoot weight, total plant biomass, chlorophyll content, leaf area (LA) and rubisco activity in the soybean. On the other hand, application of biochar improved nodulation, nitrogen content, rubisco activity, GDH, GS, GOGAT and NR activities in different parts of the soybean and nodules under salt stress, and consequently improved chlorophyll content, LA, root and shoot weight. Both the 50 and 100 g kg^?1 biochar rates showed similar effects in improving nitrogen metabolism and plant performance under salt stress. Generally, biochar increased nodulation and nitrogen metabolism of the soybean under saline conditions.     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

Boron alleviates aluminum toxicity in pea (Pisum sativum)

One important target of boron (B) deficiency and aluminum (Al) toxicity is cell wall. Thus we studied the hypothesis that B is capable of alleviating Al toxicity in pea (Pisum sativum). Short-term and prolonged Al exposure to pea roots at different B levels was carried out on uniform seedlings pre-cultured at a low B level. When seedlings with a low B level were supplied with or without B for 1 and 2 days before 24 h Al exposure, roots were longer while root diameter was thinner after B addition especially for 2 days even with exposure to Al; root elongation was inhibited while root diameter was enlarged by Al exposure. Callose induction by Al toxicity was higher with B added, but this was reversed after the removal of the cotyledons. Hematoxylin staining was lighter in the root tips given B, and Al content in the root tips and cell walls dropped after exposure to B. This indicates that B alleviated Al toxicity in the root tips during short-term Al exposure by decreasing Al binding in root cell walls. An increase in chlorophyll and biomass and reduced chlorosis were found at the higher level of B during prolonged Al treatment, which was coincided with the decreased Al contents, indicating that B alleviated Al toxicity to shoots. B supplementation alleviates some of the consequences of Al toxicity by limiting some Al binding in cell walls, resulting in less injury to the roots as well as less injury to the shoots.     

Root hydrotropism of an agravitropic pea mutant, ageotropum

We have partially characterized root hydrotropism of an agravitropic pea mutant, ageotropum (from Pisum sativum L. cv. Weibull's Weitor), without interference of gravitropism. Lowering the atmospheric air humidity inhibited root elongation and caused root curvature toward the moisture-saturated substrate in ageotropum pea. Removal of root tips approximately 1.5 mm in length blocked the hydrotropic response. A computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to a greater inhibition of elongation on the humid side than the dry side of the roots. Similarly, gravitropic curvature of Alaska pea roots resulted from inhibition of elongation on the lower side of the horizontally placed roots, while the upper side of the roots maintained a normal growth rate. Gravitropic bending of Alaska pea roots was apparent 30 min after stimulation, whereas differential growth as well as curvature in positive root hydrotropism of ageotropum pea became visible 4–5 h after the continuous hydrostimulation. Application of 2,3,5-triiodobenzoic acid or ethyleneglycol-bis-( β -aminoethylether)-N,N,N',N'-tetraacetic acid was inhibitory to both root hydrotropism of ageotropum pea and root gravitropism of Alaska pea. Some mutual response mechanism for both hydrotropism and gravitropism may exist in roots, although the stimulusperception mechanisms differ from one another.     

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels. This revised version was published online in July 2006 with corrections to the Cover Date.