Activity Of Nitrogen Assimilation Enzymes Research Articles

Root-Derived Endophytic Diazotrophic Bacteria Pantoea cypripedii AF1 and Kosakonia arachidis EF1 Promote Nitrogen Assimilation and Growth in Sugarcane.

Excessive, long-term application of chemical fertilizers in sugarcane crops disrupts soil microbial flora and causes environmental pollution and yield decline. The role of endophytic bacteria in improving crop production is now well-documented. In this study, we have isolated and identified several endophytic bacterial strains from the root tissues of five sugarcane species. Among them, eleven Gram-negative isolates were selected and screened for plant growth-promoting characteristics, i.e., production of siderophores, indole-3-acetic acid (IAA), ammonia, hydrogen cyanide (HCN), and hydrolytic enzymes, phosphorus solubilization, antifungal activity against plant pathogens, nitrogen-fixation, 1-aminocyclopropane-1-carboxylic acid deaminase activity, and improving tolerance to different abiotic stresses. These isolates had nifH (11 isolates), acdS (8 isolates), and HCN (11 isolates) genes involved in N-fixation, stress tolerance, and pathogen biocontrol, respectively. Two isolates Pantoea cypripedii AF1and Kosakonia arachidis EF1 were the most potent strains and they colonized and grew in sugarcane plants. Both strains readily colonized the leading Chinese sugarcane variety GT42 and significantly increased the activity of nitrogen assimilation enzymes (glutamine synthetase, NADH glutamate dehydrogenase, and nitrate reductase), chitinase, and endo-glucanase and the content of phytohormones gibberellic acid, indole-3-acetic acid, and abscisic acid. The gene expression analysis of GT42 inoculated with isolates of P. cypripedii AF1 or K. arachidis EF1 showed increased activity of nifH and nitrogen assimilation genes. Also, the inoculated diazotrophs significantly increased plant nitrogen content, which was corroborated by the 15N isotope dilution analysis. Collectively, these findings suggest that P. cypripedii and K. arachidis are beneficial endophytes that could be used as a biofertilizer to improve plant nitrogen nutrition and growth of sugarcane. To the best of our knowledge, this is the first report of sugarcane growth enhancement and nitrogen fixation by Gram-negative sugarcane root-associated endophytic bacteria P. cypripedii and K. arachidis. These strains have the potential to be utilized as sugarcane biofertilizers, thus reducing nitrogen fertilizer use and improving disease management.

Ammonium detoxification mechanism of ammonium-tolerant duckweed (Landoltia punctata) revealed by carbon and nitrogen metabolism under ammonium stress

Abstract In this work, the ammonium-tolerant duckweed Landoltia punctata 0202 was used to study the effect of ammonium stress on carbon and nitrogen metabolism and elucidate the detoxification mechanism. The growth status, protein and starch content, and activity of nitrogen assimilation enzymes were determined, and the transcriptional levels of genes involved in ion transport and carbon and nitrogen metabolism were investigated. Under high ammonium stress, the duckweed growth was inhibited, especially when ammonium was the sole nitrogen source. Ammonium might mainly enter cells via low-affinity transporters. The stimulation of potassium transport genes suggested sufficient potassium acquisition, precluding cation deficiency. In addition, the up-regulation of ammonium assimilation and transamination indicated that excess ammonium could be incorporated into organic nitrogen. Furthermore, the starch content increased from 3.97% to 16.43% and 26.02% in the mixed-nitrogen and ammonium-nitrogen groups, respectively. And the up-regulated starch synthesis, degradation, and glycolysis processes indicated that the accumulated starch could provide sufficient carbon skeletons for excess ammonium assimilation. The findings of this study illustrated that the coordination of carbon and nitrogen metabolism played a vital role in the ammonium detoxification mechanism of duckweeds.

Beneficial Effects of Biochar-Based Organic Fertilizer on Nitrogen Assimilation, Antioxidant Capacities, and Photosynthesis of Sugar Beet (Beta vulgaris L.) under Saline-Alkaline Stress

The Songnen Plain, whose climatic conditions are perfectly suited to sugar beet growth, is located in northeastern China. Unfortunately, this region has a lot of saline-alkaline land, which is the most important factor limiting sugar beet production. This study was undertaken to determine whether biochar-based organic fertilizer could alleviate the negative effect of saline-alkaline soil on sugar beet yield and whether such an effect correlated with changes in nitrogen assimilation, antioxidant system, root activity, and photosynthesis. Three treatments were established: Chemical fertilizers were applied to neutral soil (CK), chemical fertilizers were applied to saline-alkaline soil (SA), and biochar-based organic fertilizer was applied to saline-alkaline soil (SA + B). Our results showed that saline-alkaline stress significantly inhibited the nitrogen assimilation and antioxidant enzymes activities in root, root activity, and photosynthesis, thus significantly reducing the yield and sugar content of sugar beet. Under saline-alkaline conditions, the application of biochar-based organic fertilizer improved the activities of nitrogen assimilation enzymes in the root; at the same time, the antioxidant enzymes activities of the root were significantly increased for improving root activity in this treatment. Moreover, the application of biochar-based organic fertilizer could improve the synthesis of photosynthetic pigments, PSII (Photosystem II) activity, stomatal opening, and photosynthesis of sugar beet under saline-alkaline conditions. Hence, the growth and yield of sugar beet were improved by applying biochar-based organic fertilizer to saline-alkaline soil. These results proved the significance of biochar-based organic fertilizer in alleviating the negative effect of saline-alkaline stress on sugar beet. The results obtained in the pot experiment may not be viable in field conditions. Therefore, in the future, we will verify whether biochar-based organic fertilizer could alleviate the adverse effects of saline-alkaline stress on sugar beets yield under field conditions.

Combination of Red and Blue Lights Improved the Growth and Development of Eggplant (Solanum melongena L.) Seedlings by Regulating Photosynthesis

The photosynthesis, photomorphogenesis, and photoperiod processes in plants are regulated according to light intensity and quality. The aim of this study was to investigate the effects of different light qualities on eggplant seedlings and determine the best light quality for growth. The seedlings of eggplant cultivar ‘Jingqiejingang’ were grown under light-emitting diodes (LEDs): white (W, the control), red (R), blue (B), and different ratios of B/R lights (B/R = 1/1, B/R = 1/3, B/R = 1/6, B/R = 1/9). The growth parameters, leaf morphology, photosynthetic performance, chlorophyll fluorescence, and the carbon and nitrogen metabolism in the leaves of eggplant seedlings under different LED light treatments were studied. The results showed that the plant height, leaf development, and photosynthetic characteristics were inhibited by red light but elevated by blue light compared with the control. Conversely, the contents of chlorophyll a, chlorophyll b, and carotenoids were all increased by red light, while decreased by blue light significantly. In addition, the contents of carbohydrates and the activities of nitrogen assimilation enzymes were not or little changed by the monochromatic blue and red light. The combined light of red and blue were more beneficial for growth than the monochromatic light, especially B/R = 1/3 light. Under B/R = 1/3 light, the parameter values of plant growth, leaf development, photosynthetic pigments and characteristics, and carbon and nitrogen metabolism were all maximum. Taken together, combined application lights of red and blue are good practice for the cultivation of eggplant seedlings, and LED B/R = 1/3 light was optimum.

Toxicological effect of pendimethalin on some physiological parameters of the diazotrophic cyanobacterium Desmonostoc muscorum PUPCCC 405.10

Application of herbicides for the control of weeds in agri-ecosystem poses a great threat to natural inhabiting beneficial microbes. Toxicological impact of herbicides on the survivability of cyanobacteria has recently gained much attention. Thus, the present study was undertaken to understand the mode of action of pendimethalin on assimilation of carbon and nitrogen in diazotrophic cyanobacterium Desmonostoc muscorum PUPCCC 405.10. The lethal dose of pendimethalin for the test organism was 20 mg/l. Pendimethalin (4–12 mg/l) exhibits negative effect on the growth, photosynthetic pigments (8%–85%), photosynthesis (29%–58%), photochemical activities (16%–64%), and respiration (25%–62%). Nitrogen assimilation parameters such as nitrate (39%– 65%), nitrite (37%–83%), and ammonium (11%–37%) uptake and its enzymes such as nitrate reductase (27%– 40%), nitrite reductase (3%–18%), and glutamine synthetase (15%–39%) were also negatively affected by herbicide. Kinetic studies further revealed that the affinity of nitrate and nitrite uptake was unaffected by the herbicide, but the affinity of ammonium uptake was affected. Pendimethalin interacted non-competitively with activities of nitrogen assimilation enzymes.

Photoprotective role of UV-screening pigment scytonemin against UV-B-induced damages in the heterocyst-forming cyanobacterium Nostoc sp. strain HKAR-2

Scytonemin acts as a natural photoprotectant against high-intensity solar photosynthetically active radiation and harmful ultraviolet radiation, which has been reported in several cyanobacteria, and gets accumulated in their extracellular polysaccharide sheath. UV-B radiation (280–315 nm) has detrimental effect on physiological and biochemical processes of living organisms. In this study, scytonemin was extracted from dried cyanobacterial mats collected from the bark of mango trees and was partially characterized using high-performance liquid chromatography and electrospray ionization–mass spectrometry (retention time: 1.7 min; UVλmax: 386 nm; [M + H]+m/z: 545.1). Thereafter, photoprotective capabilities of scytonemin against UV-B radiation in a non-sheathed cyanobacterium Nostoc sp. strain HKAR-2 were assessed followed by studying its role in the recovery process. We found that UV-B radiation inhibited growth, survival, Chl a content and total protein concentration and caused an increase in total carotenoid content. The activities of nitrogen-assimilating enzymes, glutamine synthetase and nitrate reductase, were also affected. In vivo nitrate reductase activity exhibited a stimulatory response, while in vivo glutamine synthetase activity was adversely inhibited after UV-B exposure. Scytonemin with different concentrations exhibited efficient photoprotective ability by nullifying the deleterious effects of UV-B and also enhanced the recovery process. UV-screening effects of scytonemin in terms of growth, pigmentation, survival and nitrogen metabolism enzymes in the cyanobacterial strain have been reported for the first time in the present study. Our results suggest that scytonemin aids in the better survival and adaptability of cyanobacteria in stressed habitats facing harsh environmental conditions.

Manure Fertilization Gives High-Quality Earthworm Coprolites with Positive Effects on Plant Growth and N Metabolism

Humic substances (HS) are important soil components playing pivotal roles in guaranteeing long-term soil fertility. In this study, the chemical and biological properties of HS extracted from earthworm coprolites collected in soils subjected to different fertilization inputs (no fertilization, NF; fertilization with farmyard manure, FM; mineral input, M; mixed inputs, FMM, half farmyard manure plus half mineral input) were investigated. Results indicated a relationship between fertilization input and composition, molecular complexity and apparent molecular weight distribution of HS produced by earthworms. Coprolites from FM and FMM soils were the most enriched in organic carbon (OC), and HS from coprolites of FM soil were the highest in humic carbon (HC). Also, soil amendment with manure increased carboxylate and aromatic groups in HS, and the fraction with a high degree of polycondensation, thus indicating a positive impact of manure on plant residues’ degradation processes. These HS were the only to display hormone-like activity, which likely accounted for their most pronounced positive effects on plant growth and metabolism, including accumulation of chlorophylls, mineral nutrition, and activity of nitrogen assimilation enzymes, in oat (Avena sativa L.) plants growing in a soil-less system. We conclude that manure input favored the turnover of OC towards the humification process that led to the production of high-quality coprolites and HS with superior biological activity, and suggests that OC in coprolites and HC in HS from earthworms might be used as reliable indicators of soil fertility.

Elevated CO2-induced changes in cytokinin and nitrogen metabolism are associated with different responses in the panicle architecture of two contrasting rice genotypes

The yield response of Yangdao6 (YD6, Oryza sativa L. ssp. indica) to an enriched CO2 concentration was at least 30% higher, an approximately twofold increase, compared with that of Wuyunjing23 (WYJ23, Oryza sativa L. ssp. japonica). The differences in grain yield response levels between these two contrasting rice genotypes were mainly attributed to the disparities in the number of spikelets per panicle in response to elevated CO2. The number of spikelets per panicle was significantly increased by ~ 11.3% for YD6, but that of WYJ23 was decreased by 6.0% under elevated CO2 conditions. The increase in the differentiated spikelet number and decrease in the percentage of degenerated spikelet number on secondary branches were the key factors accounting for this phenomenon. To clarify the underlying physiological mechanisms corresponding to the above phenotype, the cytokinin content and nitrogen assimilation enzymes were assayed in the panicles and roots of YD6 and WYJ23 under ambient and elevated CO2 conditions (+ 200 µmol mol− 1). The results showed that locally synthesized cytokinins in panicles under elevated CO2 increased the differentiated spikelet number on secondary branches for YD6. Elevated CO2 also caused the enhanced or stabilized catalytic activity of most nitrogen assimilation enzymes in the panicles of YD6 but caused only a slight increase, or even a significant decrease, in the panicles of WYJ23. The increased activity of nitrogen assimilation enzymes in the panicles may reduce the degenerated spikelet number of YD6. These findings have significant implications for the utilization of germplasm resources in ensuring food security under future elevated CO2 conditions.

The effects of phenolic acid on nitrogen metabolism in Populus × euramericana ‘Neva’

The declines in soil fertility and productivity in continuously cropped poplar plantations are related to phenolic acid accumulation in the soil. Nitrogen is a vital life element for poplar and whether the accumulation of phenolic acid could influence nitrogen metabolism in poplar and thereby hinder continuous cropping is not clear. In this study, poplar cuttings of Populus × euramericana ‘Neva’ were potted in vermiculite, and phenolic acids at three concentrations (0X, 0.5X and 1.0X) were added according to the actual content (1.0X) in the soil of a second-generation poplar plantation. Each treatment had eight replicates. We measured gas exchange parameters and the activities of key enzymes related to nitrogen metabolism in the leaves. Leaf photosynthetic parameters varied with the concentration of phenolic acids. The net photosynthetic rate (PN) significantly decreased with increasing phenolic acid concentration, and non-stomatal factors might have been the primary limitation for PN. The activities of nitrate reductase (NR), glutamine synthetase (GS) and glutamate synthase (GOGAT), as well as the contents of nitrate nitrogen, ammonium nitrogen, and total nitrogen in the leaves decreased with increasing phenolic acid concentration. This was significantly and positively related to PN (P < 0.05). The low concentration of phenolic acids mainly affected the transformation process of NO3− to NO2−, while the high concentration of phenolic acids affected both processes, where NO3− was transferred to NO2− and NH4+ was transferred to glutamine (Gln). Overall, phenolic acid had significant inhibitory effects on the photosynthetic productivity of Populus × euramericana ‘Neva’. This was probably due to its influence on the activities of nitrogen assimilation enzymes, which reduced the amount of amino acids that were translated into protein and enzymes. Improving the absorption and utilization of nitrogen by plants could help to overcome the problems caused by continuous cropping.

Exogenous GABA application improves the NO 3 − -N absorption and assimilation in Ca(NO 3 ) 2 -treated muskmelon seedlings

Abstract The potential of exogenous γ-aminobutyric acid (GABA) application to improve the NO 3 − -N absorption and assimilation in Ca(NO 3 ) 2 -treated muskmelon seedlings was investigated in ‘Yipintianxia 208’, a salt-sensitive melon cultivar grown in a deep-flow hydroponic system. Plants were treated under control or 80 mM Ca(NO 3 ) 2 stress conditions with or without foliar spraying 50 mM GABA. We found that under Ca(NO 3 ) 2 stress, the activities of nitrate reductase (NR), glutamate synthetase (GS), and glutamate amino-transferase (GOGAT) in muskmelon seedlings were significantly reduced, while the activities of glutamate dehydrogenase (GDH), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate aminotransferase (GPT) were enhanced. These changes lead to a decrease in the content of NO 3 − -N and an increase in that of NH 4 + -N in muskmelon roots and leaves, which further severely inhibited the plant growth. Exogenous GABA application effectively improved the absorption of NO 3 − -N in muskmelon roots and leaves under Ca(NO 3 ) 2 stress and the NH 4 + -N assimilation by enhancing the activities of nitrogen assimilation enzymes (NR, GS, and GOGAT) in muskmelon seedlings. Exogenous GABA also reduced the NH 4 + release by limiting the GDH deamination, thus further alleviating the ammonia toxication induced by Ca(NO 3 ) 2 stress. Our results suggest that exogenous GABA applications can relieve the nitrogen metabolic disorders caused by Ca(NO 3 ) 2 stress and eventually promote plant growth. In addition, Exogenous GABA regulated the transaminase (GOT and GPT) activities, maintaining the balance of the amino acid metabolism. Furthermore, the accumulation of the functioning as a nitrogen source endogenous GABA was promoted, eventually improving the resistance of muskmelon seedlings to Ca(NO 3 ) 2 stress.

Effect of Nitrogen Source and Concentration on Growth and Activity of Nitrogen Assimilation Enzymes in Roots of a Moroccan Sorghum Ecotype

Carbon and nitrogen metabolism pathways are regulated by complex mechanisms in order to optimize growth and development of plants. This study was conducted to contribute to the determination of roles of some key enzymes of carbon and nitrogen metabolism in sorghum roots. Sorghum were grown under nitrate (NO3-), ammonium (NH 4+) or nitrate combined to ammonium at different concentrations. Growth parameters and protein content were evaluated. In addition, we analyzed key enzymes activities involved in nitrogen and carbon metabolism such as: nitrate reductase (NR), glutamine synthetase (GS), aspartate aminotransferase (AAT) and phosphoenolpyruvate carboxylase (PEPC). At high nitrogen levels (ammonium or nitrate), the sorghum roots showed an increase in biomass accompanied by an increase in protein content. Likewise, high concentrations induced accumulation of the non-photosynthetic isoform of phosphoenolpyruvate carboxylase. Glutamine synthetase and aspartate aminotransferase activities were also increased, especially at high levels of ammonium. These results showed that sorghum plants have an efficient system for nitrogen assimilation in their roots, allowing this plant to tolerate excessive concentration of this nutrient.

Effect of pretilachlor on nitrogen uptake and assimilation by the cyanobacterium Desmonostoc muscorum PUPCCC 405.10

Tolerance limit, kinetics of nitrogen uptake and activity of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) enzymes of cyanobacterium Desmonostoc muscorum PUPCCC 405.10 were studied under the regime of chloroacetanilide herbicide pretilachlor. The organism was isolated from the paddy field under application of the herbicide and tolerated pretilachlor up to 10 ppm under laboratory conditions. The incubation of the cyanobacterium in 2.5 ppm pretilachlor did not cause significant effect on N uptake while supplementation of 5–10 ppm pretilachlor in medium reduced the rate of nitrate and nitrite uptake by 35–73 % with 50 % decrease in V max and no change in K m. These results indicated that herbicide did not affect the affinity of uptake system to nitrate and nitrite. In presence of herbicide, the activity of nitrogen assimilation enzymes was inhibited by 16 % for NR and 18 % for NiR. Unchanged K m and decreased V max (50–60 %) in presence of herbicide indicated non-competitive-type inhibition of the enzymes. A 50 % decrease in V max and K m of ammonium uptake indicated un-competitive type inhibition of the ammonium transport system by the herbicide. GS activity also exhibited non-competitive inhibition in presence of pretilachlor with 21 % decrease in activity with unchanged K m and 40 % decrease in V max. Likewise, decreases in nitrogenase activity by 43 % and heterocyst formation by 40 % were recorded in presence of herbicide. Results indicated that pretilachlor affected nitrogen assimilation at uptake level and interacted with nitrogen-assimilating enzymes in a non-competitive manner.

Elevated CO&lt;sub&gt;2&lt;/sub&gt; concentrations alter nitrogen metabolism and accelerate senescence in sunflower (Helianthus annuus L.) plants

Elevated CO&lt;sub&gt;2&lt;/sub&gt; concentrations were found to cause early senescence during leaf development in sunflower (Helianthus annuus L.) plants, probably by reducing nitrogen availability since key enzymes of nitrogen metabolism, including nitrate reductase (NR); glutamine synthetase (GS) and glutamate dehydrogenase (GDH), were affected. Elevated CO&lt;sub&gt;2&lt;/sub&gt; concentrations significantly decreased the activity of nitrogen assimilation enzymes (NR and GS) and increased GDH deaminating activities. Moreover, they substantially rose the transcript levels of GS1 while lowering those of GS2. Increased atmospheric CO&lt;sub&gt;2&lt;/sub&gt; concentrations doubled the CO&lt;sub&gt;2&lt;/sub&gt; fixation and increased transpiration rates, although these parameters decreased during leaf ontogeny. It can be concluded that elevated atmospheric CO&lt;sub&gt;2&lt;/sub&gt; concentrations alter enzymes involved in nitrogen metabolism at the transcriptional and post-transcriptional levels, thereby boosting mobilization of nitrogen in leaves and triggering early senescence in sunflower plants.

The combined effects of salinity and nitrogen forms on Catharanthus roseus: The role of internal ammonium and free amino acids during salt stress

AbstractCatharanthus roseus is widely cultivated at the seaside of Hainan island of China for production of vinblastine with anticancer activity. Improving growth of this plant and vinblastine content under saline conditions is one of the agronomic challenges. In this study, we compare the responses of C. rosesus upon nitrate (N1) or ammonium nitrate (N2) nutrition to salinity. Biomass accumulation, reproductive growth, photosynthetic gas exchange, and the concentrations of soluble sugars (SS), free amino acids (FAA), ammonium (NH$ _4^+ $), sodium (Na+), and vinblastine were determined during 3 weeks of treatment in N1‐control, N1‐salt, N2‐control, and N2‐salt plants. The retarded growth caused by salt stress was largely ameliorated by the N2 nitrogen form. One of the reasons for this improved salt resistance in N2‐salt plants was related to the competitive uptake of NH$ _4^+ $ and Na+. The N2‐salt plants displayed a more rapid response to salinity, leading to decreased transpiration rate, well‐kept water relations, and reduced uptake of Na+. The N2‐control plants rapidly increased the concentrations of soluble sugars and free amino acids, such as sucrose, glucose, proline, and threonine in the first week. These compounds may act as compatible solutes to adjust osmotic stress and retain ion homeostasis in salt‐stressed plants. The increased FAA was found to be at the expense of glutamate accumulation, and the activities of nitrogen assimilation enzymes were tightly linked with this process. The medical alkaloid vinblastine in C. roseus was significantly increased in N2‐salt plants in the first week and in N1‐salt ones in the third week compared to their controls. It is concluded that the supplied nitrogen regime modified salinity‐induced vinblastine production in a time‐dependent way, and this process was tightly related to NH$ _4^+ $ status.

Physiological adaptations of glutamate dehydrogenase isozyme activities and other nitrogen-assimilating enzymes in the macroalga Bryopsis maxima

The activities of nitrogen-assimilating enzymes in the marine macroalga, Bryopsis maxima Okamura ex Segawa (Chlorophyta) were studied. Changes in these enzymatic activities were observed during cultivation in a laboratory setting, and after the addition of chemicals. The following results were obtained. (1) The chloroplastic NADP-glutamate dehydrogenase (NADP-GDH; EC 1.4.1.4) increased by 49%, while the cytosolic/mitochondrial NADP-GDH decreased to 23% of its original level, showing organelle-specificity. Chloroplastic and cytosolic glutamine synthetases (GS; EC 6.3.1.2) showed 46% increases in 3 days, followed by decreases to their original levels. Chloroplastic NADPH-glutamate synthase (NADPH-GOGAT; EC 1.4.1.13) showed an increase. Cytosolic NADH-nitrate reductase (NADH-NR; EC 1.6.6.1) decreased to about half its original level in 7 days. (2) A compensatory enhancement of NADP-GDH activity was not observed when the GS activity was completely inhibited by methionine sulfoximine. (3) The chloroplastic NADP-GDH decreased to 82% while cytosolic/mitochondrial NADP-GDH increased to 250% with ATP treatment. These results suggest that the activities of nitrogen-assimilating enzymes are regulated independently in this alga.

Growth characteristics, nitrogen uptake and enzyme activities of the nitrate-utilising ectomycorrhizal Scleroderma verrucosum

The growth characteristics and uptake capacities for NO3− and NH4+, as well as activities of nitrogen assimilating-enzymes have been determined in Scleroderma verrucosum. Biomass, grown on agar or liquid MMN media, was higher on nitrate. When S. verrucosum was grown in the presence of equal amounts of ammonium and nitrate, uptake rate for NH4+ was considerably higher than that of NO3−. In addition, it was found that a limitation in ammonium concentration was a prerequisite for nitrate uptake. Nitrate reductase was stimulated when the fungus was transferred from ammonium to nitrate containing media, indicating that the enzyme was inducible by nitrate and repressed by ammonium. Glutamine synthetase and NAD-glutamate synthase activities were clearly detected in S. verrucosum, while the NADP-glutamate dehydrogenase was almost undetectable. This is consistent with the view that ammonium assimilation occurs through the GS/GOGAT cycle in S. verrucosum.

Effects of nitrogen source on growth and activity of nitrogen-assimilating enzymes in Douglas-fir seedlings

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown for 68 days in a growth chamber in nutrient solutions with ammonium, nitrate or ammonium nitrate as the nitrogen source. Among the nitrogen sources tested, whole-seedling biomass, relative growth rate (RGR), root and shoot elongation, and number of lateral roots, were greatest in seedlings grown with ammonium. In the absence of nitrogen, plant growth and formation of lateral roots were poor. Initially, glutamine synthetase, NAD-glutamate dehydrogenase and aspartate aminotransferase activities were high in young roots and shoots, but all three enzymatic activities decreased after one month of culture. In root apices, glutamine synthetase and aspartate aminotransferase activities were higher than NAD-glutamate dehydrogenase activity. Enzymatic activities were often higher in ammonium-fed seedlings than in seedings supplied with the other forms of nitrogen. Activities of all three enzymes were significantly reduced in seedlings grown in the absence of nitrogen. The beneficial effect of ammonium is discussed on the basis of its involvement in the assimilation pathways of Douglas-fir.

Nitrogen Metabolism in Canavalia ensiformis (L.) DC. : II. Changing Activities of Nitrogen-Assimilating Enzymes during Growth

Summary The ontogeny of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), asparagine synthetase (EC 6.4.5.4) and glutamate dehydrogenase (EC 1.4.1.2) has been followed in cotyledons, roots, stems and leaves of young plants of Canavalia ensiformis . The four enzymes were found in all regions. Glutamine synthetase activity increased in the leaves with time and remained low in the cotyledons. Initially glutamate synthase activity in the cotyledons was NADH-dependent but later became Fd-dependent. Asparagine synthetase specific activity was highest in the roots until day 5 after which activity was similar in all regions. While glutamate dehydrogenase specific activity decreased with time in the leaves it increased in the cotyledons and roots. We suggest that in the early stages of seedling growth asparagine synthetase and glutamate dehydrogenase both play a fundamental role in the reassimilation of ammonium released from canavanine. When the plant acquires full photosynthetic capacity the glutamine synthetase/glutamate synthase pathway becomes the principal pathway of reassimilation in the aerial parts of the plant.

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

Chenopodium rubrum cells were grown in suspension as a photoautotrophic culture with a 16 hour day. Cell growth had three phases: a 3-day lag, a 3-week logarithmic phase, and a 10-day stationary phase. Chlorophyll content increased steadily during log phase and reached a level of 0.5 to 0.6 mg Chl g(-1) fresh weight. Soluble protein of the cells increased more rapidly from day 4 to day 12 than during midlog phase. Initially, ammonium was taken up in preference to nitrate. However, during the second two weeks of growth, ammonium and nitrate were taken up simultaneously; this period of growth was the time of highest rates of N uptake by the cultured cells. Glutamine synthetase had a high specific activity (17 mumol.hour(-1) mg(-1) protein) in day 1 cells, and this level was sustained until midlog phase when it increased by 20%. Methyl viologen-dependent glutamate synthase specific activity increased rapidly in lag phase cells (day 4 = 10 mumol.hour(-1) mg(-1) protein), but decreased by day 9 to about 50% of the peak and remained constant. NADH:nitrate reductase specific activity increased rapidly in lag phase cells and reached a plateau that lasted from day 4 to 14 (1 mumol.hour(-1) mg(-1) protein). Methyl viologen-dependent nitrite reductase specific activity was high when assayed on day 5 and increased to a maximum on day 15 to 16 (12 mumol.hour(-1) mg(-1) protein). NADPH- and NADH-dependent glutamate dehydrogenase specific activities remained rather constant throughout the growth cycle. The cells appeared to have developed photosynthetic competence and to have leaf-like activities of nitrogen assimilation enzymes.

Changing Activities of Nitrogen-assimilating Enzymes During Growth and Senescence of Dwarf Beans (Phaseolus vulgaris L.)

Nitrate reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase were measured in extracts of leaves, pods and seeds of Phaseolus vulgaris L. grown hydroponically with 16 mM nitrate as nitrogen source. Nitrate reductase activity per leaf was highest in young expanding and well illuminated leaves, and decreased rapidly in the lower leaves after full expansion. Low nitrate reductase activity was found in senescing leaves and pods. No nitrate reductase activity was detectable in seeds. Glutamine synthetase and methylviologen dependent glutamate synthase both increased in activity with leaf expansion, remained active longer than nitrate reductase and decreased during leaf nitrogen mobilization. Glutamine synthetase and glutamate synthase activities were low in pods, and in seeds, only glutamine synthetase was measurable with the methods used. Glutamate dehydrogenase was present in all organs analyzed. In leaves, glutamate dehydrogenase activity increased in fully expanded leaves and remained fully active during leaf senescence. Nitrate reductase activity was always lowest of the enzymes measured. The time courses of extractable nitrate reductase activity suggest that nitrate assimilation within the shoot was most active in developing and well illuminated leaves. Since glutamine synthetase and glutamate synthase remained active longer than nitrate reductase in the individual leaves, these two enzymes could continue to reassimilate ammonia released from mitochondria during photo-respiration. The high glutamate dehydrogenase activity in senescing leaves is consistent with its possible function in the oxidation of glutamate.