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.     

Energy metabolism of Plantago major ssp. major as dependent on the supply of mineral nutrients

Plantago major L. ssp. major , a grassland species from a relatively nutrient-rich habitat, was grown in nutrient-rich and nutrient-poor culture solutions. Half of the plants were transferred from high to low or from low to high nutrient conditions. The rate of dry matter accumulation in both shoots and roots decreased slowly upon transfer of plants to low nutrient conditions and the shoot to root ratio was unaffected. The rate of structural growth of both roots and shoots increased upon transfer from low to high nutrient conditions and the shoot to root ratio, if calculated from non-structural-carbohydrate-free dry weights, increased.
Photosynthesis was largely independent of the nutrient supply. Root respiration, particularly the activity of the alternative oxidative pathway, decreased with increasing age. This decrease was ascribed to a decreased shoot to root ratio, which reduced the relative amount of carbohydrates translocated to the roots and thus the amount available for the alternative pathway. It is calculated that in young as well as in old plants grown in full nutrient solution 48% of the daily produced photosynthates was translocated to the roots.
This is at variance with data on P. lanceolata , where a decreasing proportion of the daily produced photosynthates was translocated to the roots when the plants grew older. It is concluded that shoot growth plus shoot respiration consumed a constant amount of the daily produced photosynthates in P. major and that the rest was left for translocation. It is further calculated that in P. major plants grown in full nutrient solution c . 25% and c . 2% of the daily produced photosynthates in young and old plants, respectively, was respired in a way that is not involved in production of energy that is utilized in growth and maintenance ('inefficient root respiration').
The results are discussed in comparison with those of P. lanceolata , a species from a relatively nutrient-poor habitat.     

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

Plantago lanceolata L., a grassland species from a relatively nutrient-poor habitat, was grown in nutrient-rich and in nutrient-poor culture solutions. Half of the plants were trensferred from high to low or from low to high nutrient conditions. Shoot growth was immediately reduced upon transfer to low nutrient conditions, whilst it reacted more slowly upon transfer of plants to high nutrient conditions. Root growth was less dependent on the supply of nutrients, but it was slightly reduced upon transfer of plants to high nutrient conditions.
Photosynthesis was largely independent of the nutrient supply, apart from an initial increase upon transfer of plants to low nutrient conditions. Photosynthesis decreased with age in all treatments, and this decrease was not due to mutual shading. The decrease of photosynthetic rate was not accompanied by a decreased relative growth rate: it was compensated by a more efficient root respiration, since the activity of the alternative nonphosphorylating pathway continuously decreased in plants grown in a high nutrient environment.
It is concluded that the alternative pathway was of significance in removal of carbohydrates, which could not be utilized for growth, energy production, etc. , due to a temporary or structural imbalance between assimilate production and requirement. The alternative pathway also appeared to allow P. lanceolata plants to adapt to a changed environment as regards mineral nutrition.
The experimental value for root growth respiration of P. lanceolata grown under high nutrient conditions was compared with a theoretical value, calculated from the biochemical composition of plant dry matter and the known energy costings for biosynthetic and transport processes. A good correlation between the experimental and theoretical value of root growth respiration was found if it was assumed that ion uptake required c . 1.0 molecule of ATP per ion per membrane passage.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

The effects of mineral nutrition and benzyladenine on the plasmalemma ATPase activity from roots of wheat and Plantago major ssp. pleiosperma

There is an increasing awareness of the possibilities of mineral nutrition as regulator of growth substance action and vice versa. The present paper focuses on the effects of mineral nutrition and benzyladenine at the level of the plasma membrane. Seedlings of wheat ( Triticum aestivum L. cv. Drabant) and juvenile plants of Plantago major L. ssp. pleiosperma (Pilger) were grown hydroponically at different mineral levels with or without benzyladenine. Purified plasmalemma preparations from roots of wheat and P. major ssp. pleiosperma were obtained by the two phase partitioning method, using 6.5% (w/w) of each of Dextran T-500 and polyethylene glycol 3350. The Mg²⁺ and (Mg²⁺+ K⁺) dependent ATPase activities of the root plasmalemma in both species and the (Mg²⁺+ Cl⁻) one in P. major ssp. pleiosperma increased with increasing mineral levels, but the ionic strength did not influence the substrate specificity, the sensitivity to inhibitors or the pH optima.
The addition of 10⁻⁸ M benzyladenine to a nutrient solution increased the ATPase activities. The pH optima and the sensitivity to several inhibitors were not affected by benzyladenine, but the substrate specificity for ATP decreased, except for the K⁺ stimulation. In conclusion, benzyladenine mimics the effects of a higher mineral level than actually applied. Data from this and previous experiments indicate that benzyladenine exerts its effects by increasing the endogenous cytokinin concentrations and by modulating membrane components.     

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.     

Nitrogen metabolism in leaves of a tank epiphytic bromeliad: characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO₃⁻/NH₄⁺ or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary, GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH₄⁺ assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad's leaves.     

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N₂-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.     

Effects of cadmium on the co-ordination of nitrogen and carbon metabolism in bean seedlings

The effect of cadmium (Cd) was investigated on the in vitro activities of leaf and root enzymes involved in carbon (C) and nitrogen (N) metabolism of bean (Phaseolus vulgaris L. cv. Morgane). Cd induced a high increase in maximal extractable activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). Cd promoted ammonium accumulation in leaves and roots, and a tight correlation was observed between ammonium amount and GDH activity. Changes in GDH activity appear to be mediated by the increase in ammonium levels by Cd treatment. Cd stress also enhanced the activities of phosphoenolypyruvate carboxylase (PEPC, EC 4.1.1.31) and NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH, EC 1.1.1.42) in leaves while they were inhibited in roots. Immuno-titration, the PEPC sensitivity to malate and PEPC response to pH indicated that the increase in PEPC activity by Cd was due to de novo synthesis of the enzyme polypeptide and also modification of the phosphorylation state of the enzyme. Cd may have modified, via a modulation of PEPC activity, the C flow towards the amino acid biosynthesis. In leaves, Cd treatments markedly modified specific amino acid contents. Glutamate and proline significantly accumulated compared to those of the control plants. This study suggests that Cd stress is a part of the syndrome of metal toxicity, and that a readjustment of the co-ordination between N and C metabolism via the modulation of GDH, PEPC and ICDH activities avoided the accumulation of toxic levels of ammonium.     

Effects of vesicular-arbuscular mycorrhizal infection and phosphate on Plantago major ssp. pleiosperma in relation to internal cytokinin concentrations

Relations between cytokinin concentrations and effects of P and vesicular-arbuscular mycorrhizal (VAM) infection were investigated in Plantago major L. ssp. pleiosperma Pilger. Both mycorrhizal infection by Glomus fasciculatum (Thaxt. sensu Gerdemann) Gerdemann and Trappe and P addition increased the shoot to root ratio, specific leaf area (SLA), and P concentrations of shoot and roots, and decreased the percentage of dry matter in the shoot during the experiment. In general, P concentration in the shoot and roots of each treatment correlated positively with the shoot to root ratio and specific leaf area, and negatively with the percentage of dry matter in the shoot. Cytokinin concentrations in the tissue of shoots and roots were determined using an enzyme-linked immunosorbent assay. Concentrations of zeatin and zeatin-ribosides in the free base and nucleotide fractions had increased more after P addition than in the case of mycorrhizal infection in both shoot and roots, whereas the P concentrations had increased less. It is suggested that zeatin and zeatin-ribosides are not the primary growth-substances involved in mediating VAM effects.     

Effect of Light Quality on Nitrogen Metabolism of Radish Plants

The long-term action of blue or red light on nitrogen metabolism was studied in radish (Raphanus sativus L.) plants. The potential activity of nitrate reductase (NR) in vivo and its maximum activity in vitro, the content of soluble protein and free amino acids were determined in the course of the growth of a third leaf of radish plants. The effect of light quality on NR activity was found to depend significantly on the stage of leaf development. Blue light (BL) stimulated NR activity in leaves, when their areas were about 11–13% of the fully developed leaves. The efficiency of red light (RL) was significantly lower, because the maximum NR activity was observed in the leaves developed to the stage, when their areas were 38–40% of the final one. The comparative analysis of the pool of free amino acids in expanding leaves of BL- or RL-grown plants revealed significant changes in the contents of individual amino acids. Despite a higher accumulation of two amino acids in the leaves of BL-grown plants, namely, Asp (27% as compared to 13–16% in the RL-grown leaves) and Gly (5% against 2.5% in RL-grown leaves), the BL-grown leaves also demonstrated a significant decrease in Ala (10% as compared to 23% in the RL-grown leaves) and some decrease in the amounts of Ser and Gly. The content of soluble protein in a juvenile BL-grown leaf was observed to decrease gradually during leaf development. However, the protein content in the BL-grown leaf was always higher than in the RL-grown leaf of the same age. We concluded that the photoregulatory action of BL on NR activity determined the different rates of nitrogen assimilation in BL- and RL-grown plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 349–356.Original Russian Text Copyright © 2005 by Maevskaya, Bukhov.     

Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype

Nitrogen (N) availability can impact plant resistance to pathogens by the regulation of plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered to be the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or nitrate‐deficient medium. N deficiency led to enhanced or reduced susceptibility depending on the plant genotype. Focusing on four genotypes displaying contrasting responses, we determined the impact of N deficiency on plant growth and shoot N concentration, and performed expression analyses on N‐ and defence‐related genes, as well as the quantification of soluble phenolics and different amino acids in roots. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation or to mechanisms previously identified to be involved in plant resistance. Furthermore, our studies highlight a role of glutamine in mediating the susceptibility to A. euteiches in M. truncatula.     

Effects of vesicular-arbuscular mycorrhizal infection and phosphate on Plantago major ssp. pleiosperma in relation to the internal phosphate concentration

Two experiments were carried out to study physiological effects of vesicular-arbuseular mycorrhizal infection on Plantago major L., ssp. pleiosperma (Pilger). In the first experiment, infection by the Glomus fasciculatum (Thaxt. sensu Gerdemann) Gerdemann and Trappe increased growth, shoot to root ratio, P concentrations in both shoot and roots and total uptake of P per plant. The percentages of dry matter in both shoot and roots were lower in mycorrhizal plants.
In the second experiment different P treatments were applied to both mycorrhizal and non-mycorrhizal P. major plants to separate any effects of mycorrhizal infection from increased uptake of P. In addition to the effects found in the first experiment, mycorrhizal, P, and mycorrhizal x P interaction effects were found on root respiration rate and the concentration of soluble sugars in the roots. No clear effects on total dry weight, N and starch concentrations in shoot and roots and sugar concentraion in the shoot were found. Irrespective of the mycorrhizal treatment, increased P concentration in the shoot correlated with an increased shoot to root ratio and root respiration rate, and a decreased percentage dry matter and sugar concentration in the roots. However, the root respiration rate and the P concentration in the roots of mycorrhizal plants were enhanced more than expected from the increased P concentrations in the shoots of these plants.     

Effect of water stress on the enzymes of nitrate metabolism in two Brassica species

Brassica juncea (drought susceptible) and B. campestris (drought tolerant) were germinated under simulated water stress created by polyethylene glycol (MW 6000). The two species showed characteristic differences in dry weight, nitrate reductase, aspartate amino transferase, alanine aminotransferase, glutamate dehydrogenase and free proline accumulation in the embryo axis under water stress. Stress resulted in the decreased activities of these enzymes and the decrease was more in B. juncea than in B. campestris. In both species, protein content was higher under stress. In B. juncea, a 12-fold increase in free proline occurred as compared to a 7-fold increase in B. campestris at ?6 atm osmotic potential.     

Effect of NO3- supply on N metabolism of potato plants (Solanum tuberosum L.) with special focus on the tubers

The response of the tubers to NO₃^– was studied in comparison to the other organs of Solanum tuberosum var. Sava, with special focus on: (a) whether tubers are capable of primary N assimilation; (b) whether N assimilation is stimulated by NO₃^–; and (c) whether primary N assimilation in tubers is important for tuber growth. NO₃^– reduction via nitrate reductase (NR; EC 1.6.6.1) and NH₄⁺ assimilation via glutamine synthetase (GS; EC 6.3.1.2) occurred predominantly in the shoots, but up to 20% was contributed by the tubers under low‐NO₃^– conditions. NR activation was highest in tubers (up to 90%) and declined in all organs with increasing NO₃^– supply. NR and GS activity responded with a decline in tubers and roots as opposed to an increase in the shoots. This corresponded to relative organ growth: growth of tubers and roots was stimulated relative to that of shoots at a limiting NO₃^– supply. Absolute growth of all organs was stimulated by NO₃^–, whereas tuber number declined. The concentration of N compounds increased with NO₃^– supply in all organs: NO₃^– increased most dramatically in the shoots (81‐fold), free amino acids most markedly in the tubers (three‐fold). The amount of patatin and of the 22 kDa protein complex in the tuber reached a minimum when the amount of Rubisco in the shoot reached maximum as a response to NO₃^– supply. Tuber sucrose and starch increased by 40%, whereas glucose and fructose declined two‐fold as plant N status increased. It is concluded that tubers are potentially N autotroph organs with capacity for de novo synthesis of amino acids. Primary N assimilation in tubers, however, declines with increasing NO₃^– supply and is not of major importance for tuber growth.     

Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles

Four-year-old seedlings of Scots pine ( Pinus sylvestris L.) were exposed to filtered air (FA), and to FA supplemented with NH₃ (60 and 240 μg m⁻³) in controlled-environment chambers for 14 weeks. Exposure to the higher NH₃ concentration resulted in an increased activity of glutamine synthetase (GS, EC 6.3.1.2), and an increase in the concentrations of soluble proteins, total nitrogen, free amino acids and leaf pigments in the needles. The GS activity (μmol g⁻¹ fresh weight h⁻¹) in the needle extract increased to levels 69% higher than in FA and the soluble protein concentration to levels 22% higher. Total nitrogen concentration in the needles was 42% higher than in FA, while the free amino acid concentration was 300% higher, which was caused by an increase in arginine, glutamate, aspartate and glutamine. Chlorophyll a , chlorophyll b and carotenoid concentrations were 29, 38 and 11% higher, respectively. Neither the glutamate dehydrogenase (GDH, EC 1.4.1.2) activity nor the concentrations of free NH₄⁺ and glucose in the needles were affected by exposure to NH₃. After NH₃ fumigation at 240 μg m⁻³ the starch concentration decreased by 39% relative to the FA. The results indicate that the metabolism of Scots pine acclimates to concentrations of NH₃ which are 3 to 10 times higher than the average concentration in areas with intensive stock farming. The possible mechanisms underlying acclimation to NH₃ are discussed.     

Significance of Ni supply for growth,urease activity and the concentrations of urea,amino acids and mineral nutrients of urea-grown plants

The effect of Ni supply on growth, N metabolism and leaf urease activity of six plant species (rye, wheat, soybean, rape, zucchini and sunflower) grown on urea-based nutrient solutions was investigated. Activity of urease, which is activated by Ni, was hardly detectable in plants of all six species grown without supplementary Ni. As a consequence Ni-deprived plants accumulated considerable amounts of urea, showed a reduced dry matter production and reduced total N concentrations. The lack of urease activation in low-Ni plants made them metabolically N deficient, which is illustrated by the chlorotic appearance of these plants. The soluble amino acid N concentration was reduced by inadequate Ni supply. The amide concentrations (glutamine, asparagine) were considerably affected in all species. The same applied to the concentrations of the urea cycle intermediates arginine, ornithine, and citrulline. These results stress the necessity of Ni for urease activation and thus for the growth of plants on urea-based media.     

Applications of mineral nutrients to heavily N-fertilized Scots pine trees: Effects on arginine and mineral nutrient concentrations

The aim of the investigation was to study if improved nutrient status in Scots pine (Pinus sylvestris L) trees would be reflected in decreased concentrations of arginine in the needles. The studies trees had imbalanced mineral nutrient composition and elevated needle arginine concentrations caused by long-term fertilization with N. Concentrations of arginine and mineral nutrients in needles were followed over three consecutive years of additional fertilization with N alone or with P, K, Mg and micronutrients in combination with and without N.Analysis of needle mineral concentrations suggested that there were deficiencies only in K and Mg. The N concentration increased both in trees fertilized with N alone and in trees fertilized with N in combination with mineral nutrients. In the control treatment and in trees fertilized with mineral nutrients other than N the N concentration remained fairly constant. The highest Ca/N, K/N and P/N ratios were found in trees fertilized with mineral nutrients other than N while the lowest ratios were found in trees fertilized with N alone. Arginine concentrations in needles from trees fertilized with N alone remained at a high level throughout the experiment while arginine concentrations in trees given the other treatments decreased.The results show that the mineral nutrient balance can be improved with appropriate fertilization and that this improvement is reflected in decreasing arginine levels. Furthermore the study demonstrates that when N supply is reduced the arginine concentration also decreases also as an effect of reduced N supply per se. The study also indicates that arginine may be a better measure of the N status in pine trees than total N.