Stimulation of nodulation in field peas (Pisum sativum) by low concentrations of ammonium in hydroponic culture

Although the inhibitory effects of high concentrations of mineral N (> 1.0 mM) on nodule development and function have often been studied, the effects of low, static concentrations of NH₄⁺ (< 1.0 mM) on nodulation are unknown. In the present experiments we examine the effects of static concentrations of NH₄⁺ at 0, 0.1 and 0.5 mM in flowing, hydroponic culture on nodule establishment and nitrogenase activity in field peas [Pisum sativum L. cv. Express (Svalöf AB)] for the initial 28 days after planting (DAP). Peas grown in the presence of low concentrations of NH₄⁺ had significantly greater nodule numbers (up to 4-fold) than plants grown without NH₄⁺. Nodule dry weight per plant was significantly higher at 14, 21 and 28 DAP in plants grown in the presence of NH₄⁺, but individual nodule mass was lower than in plants grown without NH₄⁺. The nodulation pattern of the plants supplied with NH₄⁺ was similar to that often reported for supernodulating mutants, however the plants did not express other growth habits associated with supernodulation. Estimates of N₂ fixation indicate that the plus-NH₄⁺ peas fixed as much or more N₂ than the plants supplied with minus-NH₄⁺ nutrient solution. There were no significant differences in nodule numbers, nodule mass or NH₄⁺ uptake between the plants grown at the two concentrations of NH₄⁺. Nodulation appeared to autoregulate by 14 DAP in the minus-NH₄⁺ treatment. Plant growth and N accumulation in the minus-NH₄⁺ plants lagged behind those of the plus-NH₄⁺ treatments prior to N₂ fixation becoming well established in the final week of the experiment. The plus-NH₄⁺ treatments appeared not to elicit autoregulation and plants continued to initiate nodules throughout the experiment.     

The stimulating effect of ammonium on nodulation in Pisum sativum L. is not long lived once ammonium supply is discontinued

Although mineral N generally has a negative effect on legume-rhizobia symbioses, experiments in hydroponic culture in our laboratory (Waterer et al., 1992) have shown that low concentrations of NH⁺ ₄ can stimulate nodulation in pea (Pisum sativum L.). The objectives of the current study were to determine the immediate and residual effects of NH⁺ ₄ on nodulation and N₂ fixation in pea in sand culture. Peas (cv. Express) were exposed to 0.0, 0.5, 1.0, and 2.0 mM of ¹⁵N-labelled (NH₄)₂SO₄ for 28 days after inoculation (DAI). From 28 to 56 DAI the plants were grown on a NH⁺ ₄-free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI and nitrogenase activity was measured by gas exchange at 28 and 56 DAI. Root, shoot, and nodule dry weight (DW) and total N content were obtained, in addition to nodule counts and ¹⁵N enrichment of plant composites. The 1.0 and 2.0 mM NH⁺ ₄ treatments consistently resulted in higher total plant DW accumulation than the control (0.0 mM NH⁺ ₄). At 28 DAI, plants exposed to 1.0 and 2.0 mM NH⁺ ₄ had 1.8 to 2.8 times more nodules plant^-1, respectively, and plants exposed to 2.0 mM NH⁺ ₄ had 1.7 fold higher specific nodulation (nodule number g^-1 root DW). However, individual nodule DW was greater in control plants, such that there were no differences in nodule DW per plant among treatments. Ammonium treatment resulted in more nitrogen derived from the atmosphere (NDFA) in peas early in the experiment, but by 28 DAI there were no treatment effects on NDFA. Whole plant and nodule specific nitrogenase activity (µmol H₂ g^-1 nodule DW h^-1) was higher in control plants at 28 DAI. However, by 56 DAI, after an additional 4 weeks of NH⁺ ₄-free nutrition, no differences in nitrogenase activity nor whole plant or specific nodulation were detectable. This study indicates that nodulation in pea is stimulated in sand culture while exposed to NH⁺ ₄. However, once NH⁺ ₄ is removed, relative growth rate, nodulation and nitrogenase activity becomes similar to plants that were never exposed to NH⁺ ₄.     

Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium‐induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH₄⁺ or NO₃^–) or for 3 weeks exposed to exogenous indole‐3‐acetic acid (IAA) or 6‐benzylaminopurine (BAP) at a range of concentrations (10^‐9?10^‐5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant^?1) and specific nodulation (nodules g^?1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4‐fold of the mineral‐N‐free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t‐zeatin (Z) and lower t‐zeatin riboside (ZR) than that the control or nitrate‐treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10^‐7?10^‐5 M strongly inhibited nodulation. However, 10^?9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

Interactions between Symbiotic Nitrogen Fixation, Combined-N Application, and Photosynthesis in Pisum sativum

The effect of photosynthetic photon flux density (PPFD) on nitrogen utilization was determined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum and treated with nutrient solutions containing no combined nitrogen, 16 mM NO₃^?, or 16 mM NH₄⁺. Plants were grown under controlled conditions at three PPFD values ranging from severely limiting to nearly saturating. Carboxylation efficiencies and CO₂-exchange rates were highest in the N₂-fixing plants and lowest in plants supplied with NH₄⁺, and they generally increased with increasing PPFD. Photoefficiencies increased with PPFD but did not differ appreciably with the form of nitrogen applied. Nitrogen fixation, calculated from C₂H₂-reduction and H₂-evolution data, was inhibited more by NH₄⁺ than by NO₃^?application. Inhibition was counteracted by increasing PPFD. Percentage nitrogen decreased with increasing PPFD in plants treated with combined nitrogen and increased in the plants dependent on N₂ fixation. The data reveal that photosynthetic efficiency and the capacity to fix N₂ in peas are functions of PPFD and the availability of combined nitrogen and that these two factors are interrelated.     

Growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] as affected by nitrogen source

A study was conducted to elucidate the effect of N form, either NH₄ ⁺ or NO₃ ^–, on growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] grown under 10 mM or 100 mM NaCl in hydroponics. Shoot biomass was not affected by N form, whereas NH₄ ⁺ compared to NO₃ ^– nutrition caused an almost 4-fold reduction in the root biomass at both salinity levels. Under NH₄ ⁺ nutrition, salinity had no effect on the biomass yield, whereas under NO₃ ^– nutrition, increasing salinity from 10 mM to 100 mM caused 23% and 36% reduction in the root and shoot biomass, respectively. The reduced root growth under NH₄ ⁺ nutrition was not attributable to impaired shoot to root C allocation since N form did not affect the overall root sugar concentration and the starch concentration was even higher under NH₄ ⁺ compared to NO₃ ^– nutrition. The low NH₄ ⁺ (2 mM) and generally higher amino-N concentrations in NH₄ ⁺- compared to NO₃ ^–-fed plants indicated that the grass was able to effectively detoxify NH₄ ⁺. Salinity had no effect on Ca²⁺ and Mg²⁺ levels, whereas their concentration in shoots was lower under NH₄ ⁺ compared to NO₃ ^– nutrition (over 66% reduction in Ca²⁺; over 20% reduction in Mg²⁺), but without showing deficiency symptoms. Ammonium compared to NO₃ ^– nutrition did not inhibit K⁺ uptake, and the K⁺-Na⁺ selectivity either remained unaffected or it was higher under NH₄ ⁺ than under NO₃ ^– nutrition. Results suggested that while NH₄ ⁺ versus NO₃ ^– nutrition substantially reduced root growth, and also strongly modified anion concentrations and to a minor extent concentrations of divalent cations in shoots, it did not influence salt tolerance of kallar grass.     

A critical experimental evaluation of methods for determination of NH4+ in plant tissue, xylem sap and apoplastic fluid

Ammonium (NH₄⁺) is a central intermediate in the N metabolism of plants, but the quantitative importance of NH₄⁺ in transporting N from root to shoot and the capability of plants to store NH₄⁺ in leaves are still matters of substantial controversy. This paper shows that some of these controversies have to be related to the use of inadequate analytical procedures used for extraction and quantification of NH₄⁺ in plants. The most frequently used methods for determination of NH₄⁺, viz. colorimetric methods based on the classical Berthelot reaction, suffered severely from interference caused by amino acids, amines, amides and proteins. For some of these metabolites the interference was positive, while for others it was negative, making correction impossible. Consequently, colorimetric analysis is inapplicable for determination of NH₄⁺ in plants. Results obtained by ion chromatography may overestimate the NH₄⁺ concentration due to co‐elution of NH₄⁺ with amines like methylamine, ethylamine, ethanolamine and the non‐protein amino acid Γ‐aminobutyric acid. Derivatization of NH₄⁺ with o‐phthalaldehyde at alkaline pH and subsequent quantification of NH₄⁺ by fluorescence spectroscopy was also associated with interference. However, when pH was lowered to 6.8 during derivatization and 2‐mercaptoethanol was used as reductant, NH₄⁺ could be determined with a high selectivity and sensitivity down to a detection limit of 3.3 μM in a 10‐μl sample volume. Derivatization was performed on‐line using a column‐less HPLC system, enabling rapid quantification of NH₄⁺ in a few minutes. Flow injection analysis with on‐line gas dialysis was, likewise, free from interference, except when applied on highly senescent plant material containing volatile amines. Labile N metabolites in leaf tissue extract, xylem sap and apoplastic fluid were degraded to NH₄⁺ during extraction and subsequent instrumental analysis if the samples were not stabilised. A simple and efficient stabilisation could be obtained by addition of 10 mM ice‐cold HCOOH to the plant extraction medium or to the samples of apoplastic fluid or xylem sap. We conclude that significant concentrations of NH₄⁺, exceeding 1 mM, may occur in xylem sap, leaf apoplastic fluid and leaf tissue water of nitrate‐grown tomato and oilseed rape plants. The measured NH₄⁺ concentrations were not a result of excessive N supplies, as even plants grown under mildly N‐deficient conditions contained NH₄⁺.     

Soybean [Glycine max (L.) merrill] embryogenic cultures: The role of sucrose and total nitrogen content on proliferation

Summary To improve proliferation of soybean cultures in liquid medium, the effects of sucrose; total inorganic nitrogen; content of No₃ ⁻, NH₄ ⁺, Ca²⁺, PO₄ ³⁻, K⁺; NH₄ ⁺/NO₃ ⁻ ratio; and medium osmotic pressure were studied using cv. Jack. Sucrose concentration, osmotic pressure, total nitrogen content, and ammonium to nitrate ratio were found to be the major factors controlling proliferation of soybean embryogenic cultures. Growth decreased linearly as sucrose concentration increased from 29.7 mM to 175.3 mM. A sucrose concentration of 29.2 mM, a nitrogen content of 34.9 mM, at 1 to 4 ammonium to nitrate ratio were found to be optimal for the fastest proliferation of soybean embryogenic cultures. There was no significant effect on proliferation of cultures when concentrations of NH₄ ⁺, Ca²⁺, PO₄ ³⁻, and K⁺ were tested in the range of 3.50 to 10.50, 1.02 to 3.06, 0.68 to 2.04, and 22.30 to 36.70 mM, respectively. The relative proliferation of embryogenic cultures of four soybean genotypes was evaluated in Finer and Nagasawa medium and in the new medium formulation. Despite genotype-specific differences in growth, the genotypes tested showed a biomass increase in the new formulation equal to 278, 269, 170, and 251% for Chapman, F138, Jack, and Williams 82, respectively, relative to their growth on standard FN medium. Due to its lowered sucrose and nitrogen content, we are referring to the new medium as FN Lite.     

The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum

The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH₄NO₃ (1.25 mM) was the best nitrogen source but NH₄⁺ ions were more effectively utilized than NO₃⁻ ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH₄NO₃ (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants.     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

The influence of NO3 - and NH4 + nutrition on the carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) plants

The carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) grown hydroponically at a constant pH on either 4 mM or 12 mM NO₃ ^- or NH₄ ⁺ nutrition were investigated using either ¹⁴C or ¹⁵N techniques. Greater allocation of ¹⁴C to amino-N fractions occurred at the expense of allocation of ¹⁴C to carbohydrate fractions in NH₄ ⁺-compared to NO₃ ^--fed plants. The [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were 1.5-fold and 2.0-fold greater in shoots and roots respectively of 12 mM NO₃ ^--compared to 12 mM NH₄ ⁺-fed wheat. In both 4 mM and 12 mM N-fed maize the [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were approximately 1.7-fold and 2.0-fold greater in shoots and roots respectively of NO₃ ^--compared to NH₄ ⁺-fed plants. Similar results were observed in roots of wheat and maize grown in split-root culture with one root-half in NO₃ ^--and the other in NH₄ ⁺-containing nutrient media. Thus the allocation of carbon to the amino-N fractions occurred at the expense of carbohydrate fractions, particularly within the root. Allocation of ¹⁴N and ¹⁵N within separate sets of plants confirmed that NH₄ ^--fed plants accumulated more amino-N compounds than NO₃ ^--fed plants. Wheat roots supplied with ¹⁵NH₄ ⁺ for 8 h were found to accumulate ¹⁵NH₄ ⁺ (8.5 g ¹⁵N g^-1 h^-1) whereas in maize roots very little ¹⁵NH₄ ⁺ accumulated (1.5 g ¹⁵N g^-1 h^-1)It is proposed that the observed accumulation of ¹⁵NH₄ ⁺ in wheat roots in these experiments is the result of limited availability of carbon within the roots of the wheat plants for the detoxification of NH₄ ⁺, in contrast to the situation in maize. Higher photosynthetic capacity and lower shoot: root ratios of the C₄ maize plants ensure greater carbon availability to the root than in the C₃ wheat plants. These differences in carbon and nitrogen partitioning between NO₃ ^--and NH₄ ⁺-fed wheat and maize could be responsible for different responses of wheat and maize root growth to NO₃ ^- and NH₄ ⁺ nutrition.     

Extracellular and intracellular chemical environments in relation to embryogenesisin vitro

Summary Tissue culture isolated from the root ofDaucus carota readily produced embryos when they were grown on a medium (M_s) containing relatively large amounts of NH₄ ⁺ and nitrate. Embryos were never formed on a second medium (M_w) which contained only nitrate in a low concentration. The cells of M_s-cultures always had a high level of NH₄ ⁺ while it was scarcely detected in M_w-cultures. Addition of either nitrate or glutamate to M_w resulted in the formation of embryos and also in the accumulation of a detectable amount of NH₄ ⁺ in the cells of the tissues. Although the occurrence of NH₄ ⁺ in the medium is not necessary for embryo formationin vitro it appears that a certain level of intracellular NH₄ ⁺ is a prerequisite for this process. Since there is a positive correlation between embryo formation and the contents in the cultures of both soluble and insoluble organic nitrogen, it is probable that NH₄ ⁺ is important for embryo formation because it is an essential substrate for the synthesis of organic nitrogen compounds such as amino acids and proteins. The ability to synthesize organic nitrogen from NH₄ ⁺ decreased conspicuously in the tissues which were cultured on M_s for long periods of time and had lost the capacity to form embryos. A certain level of K⁺ in culture media is necessary for optimal production of embryos on media with NH₄ ⁺, while Na⁺ has no effect on embryo formation.The authors are indebted to Mr. E.Mahn, Miss S.Semenoff, and Mrs. E.Bock for their technical assistances.     

Further investigation of the roles of auxin and cytokinin in the NH4+-induced stimulation of nodulation using white clover transformed with the auxin-sensitive reporter GH3:gusA

Although mineral N (nitrate and ammonium) is believed to have generally negative effects on nodulation in legume–rhizobia symbioses, previous studies have shown that low, static concentrations of ammonium stimulate nodulation in pea, and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium (0.0, 0.1, 0.5 and 2.5 mM) on nodulation and auxin levels were investigated in wild‐type (WT) white clover (Trifolium repens cv. Haifa) and its transformants (lines 38 and 41) which contain the auxin‐sensitive reporter gene (GH3:gusA). The effects of exogenous application (10^?10, 10^?9 and 10^?8 M) of the cytokinin 6‐benzylaminopurine (BAP) were also assessed. Whole‐plant nodulation (nodules plant^?1) and dry weight (DW)‐specific nodulation (nodules g^?1 root DW) were stimulated (up to 49%) in all white clover lines by 0.1 mM NH₄⁺. This represents the first confirmation of an NH₄⁺‐induced stimulation of DW‐specific nodulation in a species other than pea. At 2.5 mM NH₄⁺, the effect was lost on whole‐plant nodulation and was inhibitory on DW‐specific nodulation. Rhizobial inoculation resulted in a decline in the expression of GH3:gusA in root tips as expected; however, ammonium treatment did not affect GH3 expression in any root zones. Exogenous application of BAP at 10^?9 and 10^?8 M stimulated whole‐plant and DW‐specific nodulation in wild‐type white clover to a similar degree as treatment with 0.1 mM NH₄⁺. These results support our previous hypothesis that the stimulation of nodulation by low concentrations of ammonium involves the alteration of the ratio of cytokinin to auxin, specifically by increasing cytokinin.     

Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress

Summary Callus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na⁺ and Cl⁻ were greatly increased by the treatment with NaCl. Uptake of K⁺ was also enhanced and was accompanied by increasing levels of Na⁺ and Cl⁻ so that the Na⁺/K⁺ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg²⁺ and Ca²⁺ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH ₄ ⁺ , NO ₃ ⁻ and SO ₄ ²⁻ ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of ¹⁵NH ₄ ⁺ into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of ¹⁵NO ₃ ⁻ into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings

We investigated the influence of an increased inorganic carbon supply in the root medium on NO^?₃ uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO^?₃ (group A) or 0.2 mM NH⁺₄ (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol^?1 CO₂. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO₃, or to a medium containing 2 mM NO^?₃ and 5 mM NaHCO₃. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO^?₃ uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with ¹⁴C by a 1-h pulse of H¹⁴CO^?₃ which was added to a 5 mM NaHCO₃ solution containing 0 or 100 mM NaCl and 0 or 2 mM NO^?₃, and ¹⁴C-assimilates were extracted and fractionated. The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO^?₃ was absent from the root medium. Both a high level of root malate and aeration with CO₂-enriched air stimulated NO^?₃ uptake. Analysis of ¹⁴C-assimilates indicated that with no NO^?₃ in the medium, the ¹⁴C was present mainly in organic acids, whereas with NO^?₃, a large proportion of ¹⁴C was incorporated into amino acids. Transport of root-incorporated ¹⁴C to the shoot was enhanced by NO^?₃, while the amino acid fraction was the major ¹⁴C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH⁺₄ resulting from NO₃ reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO^?₃ from the cytoplasm of the root cells may in turn increase NO^?₃ uptake.     

Asparagine as a major factor in the N‐feedback regulation of N2 fixation in Medicago truncatula

The objective of this study was to assess whether a whole plant N‐feedback regulation impact on nitrogen fixation in Medicago truncatula would manifest itself in shifts of the composition of the amino acid flow from shoots to nodules. Detected shifts in the phloem amino acid composition were supposed to be mimicked through artificial phloem feeding and concomitant measurement of nodule activity. The amino acid composition of the phloem exudates was analyzed from plants grown under the influence of treatments (limiting P supply or application of combined nitrogen) known to reduce nodule nitrogen fixation activity. Plants in nutrient solution were supplied with sufficient (9 µM) control, limiting (1 µM) phosphorus or 3 mM NH₄NO₃ (downregulated nodule activity). Low phosphorus and the application of NH₄NO₃ reduced per plant and specific nitrogenase activity (H₂ evolution). At day 64 of growth, phloem exudates were collected from cuts of the shoot base. The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity. The increase in the downregulated treatments was almost exclusively the result of a higher proportion of asparagine in both phloem exudates and nodules. Leaf labeling with ¹⁵N showed that nitrogen from the leaves is retranslocated to nodules. An artificial phloem feeding with asparagine resulted in an increased concentration of asparagine in nodules and a decreased nodule activity. A possible role of asparagine in an N‐feedback regulation of nitrogen fixation in M. truncatula is discussed.