Effect of plant growth regulators and different nitrogen sources on NADP-isocitrate dehydrogenase activity of radish cotyledons

NADP-isocitrate dehydrogenase (NADP-ICDH) activity of radish (Raphanus sativus L.) seedlings pretreated with various plant growth regulators: KiN, GA, and ABA and different nitrogen sources viz. KNO₃, NH₄Cl and NH₄NO₃ in light and dark was investigated. ICDH activity was significantly higher in light than in dark; addition of different nitrogen sources reduced it to a greater extent in NO₃ supplementation. Among hormonal treatment only KiN showed slight promotion with KNO₃ and NH₄NO₃. On the other hand in light KNO₃ and/or NH₄NO₃ promoted ICDH activity and among hormones, KiN significantly promoted the activity in KNO₃ and NH₄NO₃ supplemented seedlings while ABA was effective in NH₄CL. It is suggested that in non-photosynthetic tissues, NADP-ICDH provides both reductant and carbon skeleton for glutamate synthesis.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.     

INORGANIC NITROGEN NUTRITION OF THE SEEDLINGS OF THE ORCHID,CATTLEYA

When grown in vitro in a medium containing NH₄NO₃ as the sole source of nitrogen, seeds ro the orchid, Cattleya (C. labiata ‘Wonder’ X C. labiata ‘Treasure'), germinated readily and proceeded to form small plantlets. Development of the embryos was accompanied by an increase in their total nitrogen and a decline in the percent dry weight. Growth responses of the seedlings in other ammonium salts like (NH₄)₂SO₄, (NH₄)₂HPO₄, NH₄Cl, ammonium acetate and ammonium oxalate were similar to that in NH₄NO₃. However, when grown in a medium containing NaNO₃, development of the seedlings was drastically inhibited; KNO₃, Ca(NO₃)₂, KNO₂ and NaNO₂ also were poor nitrogen sources. Attempts to grow the seedlings in NaNO₃ by changing the pH or by addition of kinetin, molybdenum or ascorbic acid as supplements were completely unsuccessful. When seedlings growing in NH₄NO₃ for varying periods were transferred to NaNO₃, it was found that those plants allowed to grow for 60 or more days in NH₄NO₃ could resume normal growth thereafter in NaNO₃. Determination of the nitrate reductase activity in seedlings of different ages grown in NaNO₃, after NH₄NO₃, showed that the ability of the seedlings to assimilate inorganic nitrogen was paralleled by the appearance of the enzyme.     

Effect of nitric oxide and other nitrogen compounds on the adhesion and penetration of nodule bacteria into root tissues and on growth of etiolated pea seedlings

The action of sodium nitroprusside, a nitric oxide donor, and other nitrogen compounds (KNO₃, KNO₂, and (NH₄)2SO₄) on adhesion and penetration of nodule bacteria into root tissues of etiolated pea seedlings was studied. Only nitroprusside displayed a clearly negative effect on rhizobium adhesion and penetration and seedling growth. This effect was not observed with other nitrogen compounds even at high (20 mM) concentrations. Hemoglobin attenuated the negative effect of nitroprusside on bacteria and seedlings. The results are discussed in the context of the role of nitric oxide in the life of plants and nodule bacteria.     

Nitrate reductase activity of radish cotyledons as affected by phytohormones and different nitrogen sources

Radish (Raphanus sativus L.) seedlings pretreated with different hormones viz. kinetin, gibberellic acid and abscisic acid were subjected to different N-forms. The seedlings were treated with different concentrations of KNO₃, NH₄Cl and NH₄NO₃ and the changes in nitrate reductase activity were seen in light and dark conditions in the cotyledons. Nitrate reductase activity was affected differently by hormone application. Nitrate increased and ammonia decreased nitrate reductase activity; in both light and dark-grown seedlings KNO₃ induced more in vitro nitrate reductase activity. NH ₄ ⁺ when combined with NO ₃ ⁻ , however, could level up to some extent, with KNO₃ in light, except in kinetin. A transient response of induction of NR activity was evident with decreased levels after a certain specific ambient N-concentration, despite the presence of high N in the medium. However, the pattern of transition varied with the hormones applied. Further, hormones are found to affect induction of different isoforms of nitrate reductase by NH ₄ ⁺ and NO ₃ ⁻ . NH ₄ ⁺ induced isoform was prominently promoted by kinetin treatment in dark. The data documents a particular kind of interaction between controlling factors (light, N-source and phytohormones) which affect nitrate reductase levels.     

Changes in Net O(2) Exchange Induced by Inorganic Nitrogen in the Blue-Green Alga Anacystis nidulans

The response of net O₂ exchange to light intensity by intact Anacystis nidulans cells in the presence of saturating NaHCO₃ concentrations followed a curve with an inflection near the light-compensation point. Addition of either KNO₃ or NH₄Cl stimulated O₂ uptake in the dark and at light intensities below the light-compensation point. This resulted in steeper slopes of the curve calculated below and above the light-compensation point. At O₂ concentrations limiting dark respiration, addition of inorganic nitrogen had no effect on either dark respiration or O₂ exchange in the light. The apparent changes in photosynthetic yield observed under normal O₂ concentration disappeared when respiration was limited by O₂ availability, indicating that the effects of inorganic nitrogen on O₂ exchange at low light intensities are due to stimulation of respiration rather than to increases in photosynthetic yield.     

Enhanced In Vitro Plantlet Regeneration from Mature Embryo-derived Primary Callus of a Basmati Rice Cultivar Through Modification of Nitrate-nitrogen and Ammonium-nitrogen Concentrations

Mature-embryo derived primary calli of the basmati rice (Oryza sativa L.) cv Karnal Local showed significant enhancement in in vitro green-plantlet regeneration efficiency through modification of nitrogen content of the callusing medium. Using KNO₃ as the source of nitrate nitrogen and (NH₄)₂SO₄ as the source of ammonium nitrogen, forty-five media combinations involving 9 levels of KNO₃ (0–40 mM) and 5 concentrations (0–6.5 mM) of (NH₄)₂SO₄ were examined. The highest frequency of plantlet regeneration (100%) and a maximum number of green-plantlets (～ 7) per embryo-derived primary callus was obtained in calli derived from the medium having 35 mM KNO₃ and 5 mM (NH₄)₂SO₄. Higher concentrations of KNO₃ and/or (NH₄)₂SO₄ showed a decline in the regeneration efficiency. It was also observed that although the nitrogen content of the callus induction medium had a profound effect on the regenerability of the callus, the nitrogen composition of the regeneration medium also affected it significantly.     

Assay of molybdenum cofactor of barley

Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N₂MoO₄) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO₃ or 15 mM NH₄NO₃. Growth in the presence of 15 mM (NH₄)₂SO₄ did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO₃ and NH₄NO₃-treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH₄)₂SO₄ did not affect the ‘free’ Mo-cofactor level.     

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.     

Carbohydrate and nitrogen sources affect respectively in vitro germination of immature ovules and early seedling growth of Impatiens platypetala Lindl.

In vitro germination of 20-day old immature ovules of Impatiens platypetala Lindl. was inhibited at concentrations as low as 50 mM sucrose or mannitol and 100 mM glucose. Younger ovules (12, 14, and 16 days old) were similarly inhibited at 100 mM sucrose.Inorganic nitrogen concentration did not affect germination regardless of ovule age, but seedling fresh weight was significantly less and abnormal development of seedlings was significantly increased by total inorganic nitrogen concentrations higher or lower than 30 mM (at a ratio of 20: 10 mM NO₃ ^-: NH₄ ⁺) in the culture medium.     

Effect of form and level of applied nitrogen on nitrogenase and nitrate reductase activities in faba beans

The effects of nitrogen applied at increasing levels of 0, 4, 8, 16 and 32 mM N (KNO₃ or NH₄Cl) were studied in faba bean (Vicia faba) nodulated byRhizobium leguminosarum bv.viceae RCR lool. Nitrogenase activity was higher at 4 and 8 mM N than the zero N treatment (control), but 16 and 32 mM N significantly reduced the efficiency of nodule functions. Nitrate reductase activities (NRA) of leaves, stems, roots, nodules and nodule fractions (bacteroid and cytosol) were increased with rising the NO₃ ^? or NH₄ ⁺ levels. NRA decreased in the order of nodules>leaves>stems>roots. Cytosolic NR was markedly higher than that recorded in the bacteroid fractions. Nitrate levels were linearly correlated to NRA of nodules. Accumulation of NO₂ ^? within nodules suggests that NO₂ ^? inhibits nodule’s activity after feeding plants with NO₃ ^? or NH₄ ⁺.     

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.     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue

This paper seeks to calarify conflicting reports on the nitrogen requirements for in vitro embryogenesis in Daucus carota. Tissue derived from petiole explants of the wild strain of this species were tested with a variety of sources of cellular nitrogen under conditions otherwise favorable for in vitro embryogenesis. The use of very small, sieved and well-washed inocula reduced the carry-over of soluble materials with the inoculum. Embryo yield was quantified by direct counting of samples. Nitrate at concentrations ranging from 5 to 95 mM KNO₃ supportes only weak growth and very low embryogenesis under the exacting conditions of these experiments. As little as 0.1 mM NH₄Cl added to a nitrate medium allows some embryogenesis and 10 mM NH₄Cl is near optimal when KNO₃ is in the range of 12 to 40 mM concentration. Glutamine, glutamic acid, urea and alanine can individually partially replace NH₄Cl as a supplement to KNO₃. Glutamine, alanine, and possibly glutamic acid can serve as sole sources of nitrogen supporting both good growth and embryogenesis. It was concluded that a reduced nitrogen source is required, at least as a supplement to nitrate, for rapid growth and for in vitro embryogenesis of cultured wild carrot tissue. The relationship of pH of the culture medium to growth and embryogenesis was explored and optima observed at approximately pH 5.4 for both processes.     

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

CULTIVATION OF ARTHROSPIRA (SPIRULINA) PLATENSIS (CYANOPHYCEAE) BY FED‐BATCH ADDITION OF AMMONIUM CHLORIDE AT EXPONENTIALLY INCREASING FEEDING RATES1

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was cultivated under light‐limited conditions in 5‐L open tanks by daily supplying NH₄Cl as nitrogen source. Exponentially increasing feeding rates were adopted to prevent ammonia toxicity. The total feeding time (T) was varied between 12 and 20 days, and the starting (m₀) and total (m_T) quantities of the nitrogen source per unit reactor volume were varied in the ranges 0.19–1.7 mM and 2.3–23.1 mM, respectively. This intermittent addition of the nitrogen source prevented ammonia from reaching inhibitory levels and ensured final cell concentrations (X_m) and cell productivities (P_x) comparable with those of batch runs with KNO₃. Moreover, the lower nitrogen addition due to the use of NH₄Cl rather than KNO₃ allowed for higher nitrogen‐to‐cell conversions (Y_x/n). These results were evaluated using three‐factor, five‐level, central composite experimental planning, combined with the response surface methodology, selecting T, m₀, and m_T as the independent variables and X_m, P_x, and Y_x/n as the response variables. This approach allowed us to identify, through the simultaneous optimization of the variables, T=16 days, m₀=1.7 mM, and m_T=21.5 mM as the best conditions for A. platensis cultivation at 72 μmol photons·m^?2·s^?1. Under these conditions, a maximum cell concentration of 1239 mg ·L^?1 was obtained, which is a value comparable with that obtained using KNO₃ as nitrogen source and nearly coincident with the theoretical one estimated by the response surface methodology.