High nitrogen supply affects the metabolism of Matricaria chamomilla leaves

N-status of the two Matricaria chamomilla cultivars grown in the presence of high potassium nitrate concentration was evaluated and compared with ammonium nitrate supply. After 5 days of potassium nitrate treatment the visible increase of dry mass together with total chlorophyll accumulation were observed. In both cultivars, ammonium nitrate application led to increased accumulation of N-containing compounds in chamomile leaves. NH₄NO₃ nitrogen supply influenced activity of nitrate reductase positively. In vivo nitrate reductase activity reached maximum in lower nitrate supply and decreased in higher nitrate availability significantly. Among the most abundant leaf secondary metabolites, the high nitrate availability both KNO₃ and NH₄NO₃ significantly increased umbelliferone level. The highest potassium nitrate dose (60 mmol per plant) caused an osmotic stress accompanied with lower tissue water content and turgor loss. In such condition the decrease in (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid, herniarin and dicycloethers, as well as PAL activity was observed. On the other hand, strong increase of umbelliferone is likely a stress response and is related to its antioxidant activity.     

Effect of nitrates and sucrose on the induction of nitrate reductase in etiolated seedling leaves from selected barley genotypes in darkness

The parental genotypes, cv. Aramir and R567 line, as well as the selected DH lines C23, C47/1, C41 and C55, growing in darkness differed significantly in the level of NR activity in crude leaf extracts independently of nitrate concentration in the medium. The highest activity of the enzyme was found in the line C23. When plants grew on the medium with 0.5 mM KNO₃, NR activity in that genotype was almost 10-fold higher than in the parents and lines C41, C55 and also 3.5-fold higher than in the line C47/1. An increase of nitrate concentration in the medium to 10 mM caused a significant increase of NR activity in all the genotypes under study. In the line C23 this enzyme activity was only 20% lower than that found previously in the green leaves of that genotype in light. NR from the leaves of C23 and C41 lines was thermally unstable under in vitro conditions. This enzyme in the leaf extracts from the line C23 was characterized by a considerably lower unstability. The lines DH C23 and C41 growing in the dark on the medium with 0.5 mM KNO₃ did not differ in nitrate accumulation in leaves, whereas a larger nitrate content was found in the leaves of the line C41 when it grew on the medium with 10 mM KNO₃. Independently of nitrate concentration in the medium, leaves of the line C23 were found to have a higher sucrose content than those of the line C41. Excised, etiolated leaves of barley treated with 0.5 and 10 mM KNO₃ in dark under conditions favorable to transpiration had a low NR activity. Leaf treatment with a solution containing 10 mM KNO₃ + 0.2 M sucrose caused, on the average, a 13-fold increase of NR activity in comparison to leaves treated only with 10 mM KNO₃ and about a 6-fold increase of this enzyme in comparison to leaves treated with 0.5 mM KNO₃ + 0.2 M sucrose.     

Two effects of combined nitrogen on the adhesion of Rhizobium etli to bean roots

Combined forms of nitrogen negatively influence rhizobia-legume symbiosis. The effects of combined nitrogen are known for nodulation and dinitrogen (N₂) fixation, but little is known about the effect on preinfection events. Here, we studied the effects of combined nitrogen on the adhesion of Rhizobium etli to common bean (Phaseolus vulgaris L.) roots. When potassium nitrate (KNO₃) or sodium glutamate was added to an incubation mixture of rhizobia and plants that were previously grown in nitrogen-free solution, rhizobial adhesion to roots was stimulated. However, the rhizobial adhesion to bean roots that were previously grown with 10 mM KNO₃ was reduced by half. A fraction of the bean root exudates, which is thermolabile and has molecular mass larger than 12 kDa stimulated rhizobial adhesion, but this stimulatory activity was lost in root exudates obtained with 10 mM KNO₃. Thus, the inhibition of symbiosis in response to combined nitrogen may be controlled by the plant at the preinfection stage as well.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary The aim of this work was to investigate the effect of nitrogen starvation and subsequent fentilization with nitrate or ammonium on nitrate content and nitrate reductase activity of Rumex obtusifolius L. under natural conditions.When plants were transplanted to nitrate-poor media, endogenous nitrate was reduced within a few days. In parallel, nitrage reductase activities dropped to about 25% of the initial values. As a consequence of nitrate fertilization (1; 10 or 100 mmol KNO₃/l substrate), endogenous nitrate content of the plant abruptly increased within one day. In extreme cases, nitrate concentrations of up to 10% of plant dry weight could be observed without being lethal. High external nitrate concentrations caused an inhibition of nitrate reductase within the leaves, while low external concentrations provoked an increase in the enzyme activity of about 450% within one day. Ammonium fertilization (5 mmol (NH₄)₂SO₄/l substrate) also caused an increase in nitrate reductase activity and nitrate content within leaf blades. This observation indicates a rapid nitrification of ammonium in the substrate. When plants were fertilized with ammonium plus nitrate (2.5 mmol (NH₄)₂SO₄+ 5 mmol KNO₃/l substrate), an extremely high and long term increase in nitrate reduction could be observed. Due to an intensive enzymatic nitrate turnover, the nitrate content of leaf blades then remained relatively low. Our observations do not point to an inhibition of nitrate reductase activity in leaves of Rumex obtusifolius by ammonium. Despite temporarily high endogenous nitrate concentrations, Rumex obtusifolius may not be termed as a nitrate storage plant, since the accumulation of nitrate is a short term process only.     

Nitrate Uptake during Recovery from Nitrogen Deficiency

Two-week-old nitrogen-deficient wheat plants attained a high rate of nitrate uptake on the first day of exposure to nutrient solutions supplemented with KNO₃. Ammonium uptake from similar solutions supplemented with NH₄NO₃ was also high during the first day of exposure, but nitrate uptake from this solution was lower than from the KNO₃ treatment. During the next two to three days there was a progressive decrease in uptake of both nitrogen ions. A steady increase in uptake then occurred as the plants fully recovered from the nitrogen-deficient state. The transient low nitrate uptake after three or four days of exposure to KNO₃ was not due to an excessive accumulation of nitrate in the tissue, nor to a failure in nitrate reduction as indicated by the rate of nitrate accumulation relative to the uptake rate. Nitrogen supplied as ¹⁵N-nitrite during the low uptake period was effectively incorporated into organic forms and effectively translocated to the shoots. Failure of the root tissue to increase in soluble carbohydrates during illumination was characteristic of the low uptake period. This contrasted with an increase in root soluble carbohydrates in the light during rapid uptake associated with full recovery from the nitrogen-deficient state. It is concluded that carbohydrate translocation to the root system was insufficient during the intermediate recovery period for optimal nitrate uptake, although it was sufficient for effective reduction and translocation of nitrate and reduced nitrogen. Ammonium uptake from NH₄NO₃ was restricted during darkness by the third day whereas there was little difference between light and dark periods in nitrate uptake from KNO₃ until about the sixth day of recovery. The extent to which ammonium restricted nitrate uptake increased progressively for two or three days following which a lessening influence seemed evident, and the effects were not directly associated with the rate of ammonium uptake.     

Seed germination in Chenopodium album L.: further evidence for the dependence of the effects of growth regulators on nitrate availability

Abstract Chenopodium album L. plants, grown under controlled environmental conditions on different levels of soil nitrate, produced seeds with proportionately different NO^?₃ contents. Regardless of the endogenous NO^?₃ content, few seeds germinated in water or upon treatment with KNO₃. Ethylene promoted germination, and the extent of germination was positively correlated with the endogenous seed NO^?₃ content. Combined application of ethylene and KNO₃ in the dark had a synergistic effect on NO^?₃ -deficient seed. The synergism between ethylene and KNO₃ was attributable to the NO^?₃ moiety of the nitrate salt. Ethylene and light showed moderate synergism in seeds with low or high endogenous nitrate. Addition of nitrate, however, masked the interaction between ethylene and light. Gibberellic acid₄₊₇ (GA₄₊₇) or red light, each alone or combined with KNO₃, had little effect on germination. When applied together in the dark, ethylene and GA₄₊₇ synergistically enhanced the germination of NO^?₃-deficient seed. The combined effects of the two hormones on this seed were further enhanced by the addition of KNO₃. There was no synergism between ethylene and GA₄₊₇ in NO^?₃-rich seed. These interactions among GA₄₊₇, ethylene and KNO₃ were not affected by light. The results confirm and further elaborate our earlier finding that the sensitivity of C. album seeds to ethylene may depend on nitrate availability.     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

Role of nitrogen metabolism in the development of salt tolerance in barley plants

The 7- to 8-day-old barley (Hordeum vulgare L.) seedlings grown in KNO3 solutions (1-40 mM) were characterized by the substrate activation of nitrate reductase (NR) in the apical leaf segments (1–2 cm in length), as well as by stimulated growth, broadened leaf blades, and by vigorously developed system of shortened roots. When the seedlings were grown in the presence of 20 mM KNO₃, the ability of leaf segments to generate superoxide anion radical remained at the level typical of control plants grown in water. The content of 5-aminolevulinic acid (ALA) in plants grown in the presence of 20 mM KNO₃ was 2.2–2.4 times higher than in control plants. The plants grown in the presence of nitrate had an elevated content of chlorophylls a and b, heme, and protein (by 42%). At the same time, the proline content was almost twofold lower than in control plants, which was due to substantial reduction (by 40%) in activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS). It is concluded that the substrate activation of NR by KNO₃ under normal growth conditions results in predominant utilization of glutamic acid (the primary product of inorganic nitrogen assimilation) for biosynthesis of tetrapyrroles and protein amino acids at the expense of inhibition of proline synthesis. When barley seedlings were grown in 150 mM NaCl solution, the plant growth and the root system development were suppressed to the levels of 63 ± 6% and 61 ± 11% of the control values, respectively. In the apical leaf tissues of plants adapted to NaCl, there was a slight decrease in the total NR activity (by 10%), a significant reduction in protein content (by 32%), and a parallel increase in the content of ALA (by a factor of 4.3), chlorophylls, heme, carotenoids, proline (2.2-fold) and P5CS (1.6-fold) with respect to the control values. It is proposed that the accumulation of ALA and proline under salinity-induced suppression of nitrogen assimilation results from the predominant allocation of glutamate for biosyntheses of ALA and proline at the expense of inhibition of growth-related processes requiring intense protein synthesis. The substrate activation of NR by KNO₃ under salinity conditions was associated with prevailing allocation of the assimilated nitrogen for synthesis of proline and protein amino acids, which reinforced plant cell protection against salinity and stimulated plant growth.     

Effect of light and nitrates on nitrate reductase activity and stability in seedling leaves of selected barley genotypes

Parental genotypes (cv. Aramir and line R567) and the selected doubled haploid (DH) lines C23, C47/1, C41, C55 did not differ in NR activity when they grew on a nutrient solution containing 10 mM KNO₃ and were illuminated with light at 124 μmol·m⁻²·s⁻¹ intensity. A decrease of nitrate content in the nutrient medium to 0.5 mM at 44 μmol·m⁻²·s⁻¹ light intensity caused a significant reduction of NR activity in the parental genotypes as well as in the lines C41 and C55. An increase in light intensity to 124 μmol·m⁻²·s⁻¹ raised NR activity in the leaf extracts of these genotypes. However, independently of light intensity, a high level of this enzyme activity was maintained in the line C23 growing on the nutrient medium with 10 mM and 0.5 mM KNO₃. The NR activity in that line dropped only when nitrate content in the medium decreased to 0.1 mM. NR in the leaves of the line C23, as compared to C41, was characterized by a higher thermal stability in all experimental combinations. An increase in light intensity had no significant influence on NR thermal stability in the leaves of the line C41, but induced a significant increase of this enzyme stability in the line C23. The lines C23 and C41 growing on the nutrient medium with 0.5 mM KNO₃ differed appreciably by nitrate concentration in leaves. A higher accumulation of nitrates was detected in the leaves of the line C41.     

Role of potassium and malate in nitrate uptake and translocation by wheat seedlings

Wheat seedlings (Triticum vulgare) treated with 1 mm KNO₃ or NaNO₃, in the presence of 0.2 mm CaSO₄, were compared during a 48-hour period with respect to nitrate uptake, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO₃ absorbed and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO₃. Within seedlings of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO₃-treated seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO₃-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO₃- and NaNO₃-treated seedlings. The effects of K⁺ and Na⁺ on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO₃ treatment, and increased only slightly in the KCl control.     

Relationships between nutrients and plant density in liquid media during micropropagation and acclimatization of turmeric

To improve micropropagation and acclimatization of turmeric (Curcuma longa L.), we evaluated the effects of media volume, plant density, macronutrient ion concentrations, cationic ratios [NH₄ ⁺]/[K⁺], and sucrose concentration. Multiplication was highest with low bud density. Yield of new plants was highest with high bud density, the most sucrose, the highest concentration of macronutrients, and the greatest volume of medium. However, maximum plant size required low-density, reduced sucrose and elimination of NH₄ ⁺. The largest plants grew quickest during greenhouse acclimatization when macronutrients were lowered to 20 mM. In a follow-up experiment, media volume was set at 40 mL with 5% sucrose with NH₄ ⁺ reduced to 5 mM, and the effects of varying P, Ca, Mg, KNO₃, and bud densities were assessed. The largest plants were produced at low density. More importantly, at high density the optimal concentrations of P, Ca, Mg, and KNO₃ predicted plant size that was nearly equal to the maximum value from low-density cultures in the prior experiment. Growth of plants during greenhouse acclimatization was increased by modifications of in vitro medium with plants cultured with 3.32 mM P, 4.5 mM Mg, and 37 mM KNO₃ predicted to grow most rapidly. The effect of starter fertilizer in the greenhouse mix was much less than the effects of P, Mg, and KNO₃ in vitro. These results showed (1) optimal media formulae for different stages of micropropagation and (2) process-related factors such as plant density and media volume affected the optimal nutrient concentrations.     

Improved production of <Emphasis Type="Italic">Oroxylum indicum</Emphasis> (L.) Kurz by altering the salts of the medium

The present study was conducted to test the effects of KNO₃, KH₂PO₄, and CaCl₂ on shoot multiplication, root proliferation, and accumulation of phytochemicals in in vitro cultures of Oroxylum indicum. The results indicate that modifying the MS salt formulation in relation to particular inorganic nutrients highly affected shoot multiplication, root proliferation, and accumulation of flavonoids in in vitro cultures. A concentration of 0.60 g L^?1 CaCl₂ resulted in the highest frequency of shoot regeneration (5.6 shoots per explant). A concentration of 0.40 g L^?1 CaCl₂ resulted in the highest frequency of root regeneration (7.8 roots per shoot). Modifications of the concentrations of inorganic salts were also found to be advantageous for production media for both multiple shoots and shoot-derived root in vitro cultures. Multiple shoots generated on shoot induction medium with a concentration of 0.60 g L^?1 CaCl₂ and roots generated on root induction medium with a concentration of 1.5 g L^?1 KNO₃ yielded about a five times higher flavonoid level than cultures generated on control medium respectively.     

Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni (Asteraceae)

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH₄NO₃ is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO₃, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3–72.1 mM NH₄NO₃ and 9.4–65.8 mM KNO₃ and generated six different media with altered nitrogen. Similarly, six different concentrations of KH₂PO₄, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH₂PO₄. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g^?1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g^?1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g^?1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC–MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles.     

Optimization of cell growth and bacoside-A production in suspension cultures of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst. using response surface methodology

Plant secondary metabolites have emerged as potential raw materials, which are used in the pharmaceutical, food, chemical, and cosmetic industries. Bacoside-A, a secondary metabolite produced by Bacopa monnieri, is known for its memory-facilitating properties. In recent years, various strategies have been developed to enhance biomass accumulation and synthesis of secondary compounds in cultures. In the present investigation, various factors affecting the production of biomass and bacoside-A in the cell suspension cultures of B. monnieri were optimized using the statistical experimental design approach. Preliminary screening by Plackett–Burman’s design revealed that among the tested factors, glucose, KNO₃, KH₂PO₄, and inoculum density significantly influenced cell growth and bacoside-A production. Furthermore, using response surface methodology (RSM), glucose, KNO₃, and KH₂PO₄ at a concentration of 5.67, 0.313, and 0.29%, respectively, and an inoculum density of 0.66% in basal MS medium were found to be optimal for cell growth and bacoside-A production. After optimization, the biomass yield increased about twofold (from 5.52 to 12.58 g L^?1 fresh cell weight) and bacoside-A production about 1.7-fold (5.56 to 9.84 mg g^?1 dry weight). The present study results show the successful application of RSM to enhance the production of biomass and accumulation of bacoside-A content in cell suspension cultures of B. monnieri.     

Nitrate Uptake by Roots as Regulated by Nitrate Reduction Products of the Shoot

A mechanism is proposed by which secondary products of nitrate reduction in the shoot control the uptake of nitrate by the roots. KNO₃ enters the roots and is translocated to the shoot where nitrate is reduced and, at the same time, malate is produced. The reduction of nitrate is stoichiometric to the synthesis of malate (1). Part of the K-malate moves down to the root system in which malate is oxidized, yielding KHCO₃ which exchanges for KNO₃. Nitrate reduction in the shoot promotes the synthesis of malate which, after its translocation to the root, allows the preferential uptake of nitrate. Thus, plants reducing large amounts of nitrate may take up the anion without a superfluous accumulation of the cation. Furthermore, the utilization of nitrate by the shoot regulates its uptake by the root.     

Influence of the nitrogen source and concentration on N fractions and free amino acid levels of grape vine explants

The influence of the source of inorganic nitrogen (KNO₃, (NH₄)₂SO₄ and NH₄NO₃) and its concentration (5, 10, 20 and 30 mM N) on total N incorporation, as well as on N distribution into different fractions (amminiacal, amino, amide and protein) and on free amino acid levels has been determined in grape vine explants cultured in vitro.Increasing concentrations of the nitrogen source resulted in increased total N content in tissues. This effect was small for KNO₃, higher for (NH₄)₂SO₄ and maximal for NH₄NO₃. In addition, nitrate promoted an increase in amino-N only, whereas ammonium increased both the ammoniacal-N and the amino-N fractions. Incorporation of N into amide-N and protein-N were not affected significantly by the N sources tested.The application of increasing quantities of N enhanced the accumulation of most free amino acids, especially arginine, alanine and proline, but to different extents, depending on both the N source and its concentration. The combination of ammonium and nitrate resulted in a higher accumulation of amino acids than that observed with either one of the two forms alone.