Tin and Tin-Resistant Microorganisms in Chesapeake Bay

Sediment and water samples from nine stations in Chesapeake Bay were examined for tin content and for microbial populations resistant to inorganic tin (75 mg of Sn liter⁻¹ as SnCl₄·5H₂O) or to the organotin compound dimethyltin chloride [15 mg of Sn liter⁻¹ as (CH₃)₂SnCl₂]. Tin concentrations in sediments were higher (3.0 to 7.9 mg kg⁻¹) at sites impacted by human activity than at open water sites (0.8 to 0.9 mg kg⁻¹), and they were very high (239.6 mg kg⁻¹) in Baltimore Harbor, which is impacted by both shipping and heavy industry. Inorganic tin (75 mg Sn liter⁻¹) in agar medium significantly decreased viable counts, but its toxicity was markedly reduced in liquid medium; it was not toxic in medium solidified with silica gel. Addition of SnCl₄·5H₂O to these media produced a tin precipitate which was not involved in the metal's toxicity. The data suggest that a soluble tin-agar complex which is toxic to cells is formed in agar medium. Thus, the toxicity of tin depends more on the chemical species than on the metal concentration in the medium. All sites in Chesapeake Bay contained organisms resistant to tin. The microbial flora was more sensitive to (CH₃)₂SnCl₂ than to SnCl₄·5H₂O. The elevated level of tin-resistant microorganisms in some aeas not containing unusually high tin concentrations suggests that factors other than tin may participate in the selection for a tin-tolerant microbial flora.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO₃⁻ medium were characterized. The in vivo NO₃⁻ reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO₂⁻ reductase as compared with the wild type. This coincided with higher accumulation of NO₂⁻ by the variant than by the wild type cells after transfer from alanine medium to NO₃⁻ medium. NO₂⁻ accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Charge Balance in NO(3)-Fed Soybean: Estimation of K and Carboxylate Recirculation

Soybeans (Glycine max L. Merr., cv Kingsoy) were grown on media containing NO₃⁻ or urea. The enrichments of shoots in K⁺, NO₃⁻, and total reduced N (N_r), relative to that in Ca²⁺, were compared to the ratios K⁺/Ca²⁺,NO₃⁻/Ca²⁺, and N_r/Ca²⁺ in the xylem saps, to estimate the cycling of K⁺, and N_r. The net production of carboxylates (R⁻) was estimated from the difference between the sums of the main cations and inorganic anions. The estimate for shoots was compared to the theoretical production of R⁻ associated with NO₃⁻ assimilation in these organs, and the difference was attributed to export of R⁻ to roots. The net exchange rates of H⁺ and OH⁻ between the medium and roots were monitored. The shoots were the site of more than 90% of total NO₃⁻ reduction, and N_r was cycling through the plants at a high rate. Alkalinization of the medium by NO₃⁻-fed plants was interrupted by stem girdling, and not restored by glucose addition to the medium. It was concluded that the majority of the base excreted in NO₃⁻ medium originated from R⁻ produced in the shoots, and transported to the roots together with K⁺. As expected, cycling of K⁺ and reduced N was favoured by NO₃⁻ nutrition as compared to urea nutrition.     

In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

Role of Chemotaxis in the Ecology of Denitrifiers

A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO₃⁻ and NO₂⁻ by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10⁻³ M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10⁻² M), tryptone, and a soil extract, but not to NH₄⁺, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO₃⁻, NO₂⁻, or a soil extract, and the chemoattraction to NO₃⁻ occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO₃⁻ was usually observed when the cells were grown aerobically with 10 mM NO₃⁻ in the growth medium. Capillary tubes containing 10⁻3 M NO₃⁻ submerged into soil-water mixtures elicited a significant chemotactic response to NO₃⁻ by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO₃⁻ and NO₂⁻, and which may facilitate the survival of naturally occurring populations of some denitrifiers.     

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO₃⁻ inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO₃⁻ biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO₃⁻ and synthetic, high-surface-area goethite. Results showed that the presence of NO₃⁻ inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO₃⁻ reduction, a 10-fold decrease in the rate of NO₂⁻ reduction, and a 20-fold increase in the amounts of N₂O produced. Nitrogen stable isotope experiments that utilized δ¹⁵N values of N₂O to distinguish between chemical and biological reduction of NO₂⁻ revealed that the N₂O produced during NO₂⁻ or NO₃⁻ reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO₃⁻ reduction produces NO₂⁻ and Fe(II), which then abiotically react to reduce NO₂⁻ to N₂O with the subsequent oxidation of Fe(II) to Fe(III).     

Regulation of NO(3) Influx in Barley : Studies Using NO(3)

Short-term (10 minutes) measurements of plasmalemma NO₃⁻ influx (_oc) into roots of intact barley plants were obtained using ¹³NO₃⁻. In plants grown for 4 days at various NO₃⁻ levels (0.1, 0.2, 0.5 millimolar), _oc was found to be independent of the level of NO₃⁻ pretreatment. Similarly, pretreatment with Cl⁻ had no effect upon plasmalemma ¹³NO₃⁻ influx. Plants grown in the complete absence of ¹³NO₃⁻ (in CaSO₄ solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO₃⁻. Based upon the documented effects of NO₃⁻ or Cl⁻ pretreatments on net uptake of NO₃⁻, these observations suggest that negative feedback from vacuolar NO₃⁻ and/or Cl⁻ acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl⁻ was without effect upon ¹³NO₃⁻ influx, but NH₄⁺ caused approximately 50% reduction of influx at this concentration.     

Studies of the Regulation of Nitrate Influx by Barley Seedlings Using NO(3)

Using ¹³NO₃⁻, effects of various NO₃⁻ pretreatments upon NO₃⁻ influx were studied in intact roots of barley (Hordeum vulgare L. cv Klondike). Prior exposure of roots to NO₃⁻ increased NO₃⁻ influx and net NO₃⁻ uptake. This `induction' of NO<sub>3</sub><sup>−</sup> uptake was dependent both on time and external NO<sub>3</sub><sup>−</sup> concentration ([NO<sub>3</sub><sup>−</sup>]). During induction influx was positively correlated with root [NO<sub>3</sub><sup>−</sup>]. In the postinduction period, however, NO<sub>3</sub><sup>−</sup> influx declined as root [NO<sub>3</sub><sup>−</sup>] increased. It is suggested that induction and negative feedback regulation are independent processes: Induction appears to depend upon some critical cytoplasmic [NO<sub>3</sub><sup>−</sup>]; removal of external NO<sub>3</sub><sup>−</sup> caused a reduction of <sup>13</sup>NO<sub>3</sub><sup>−</sup> influx even though mean root [NO<sub>3</sub><sup>−</sup>] remained high. It is proposed that cytoplasmic [NO<sub>3</sub><sup>−</sup>] is depleted rapidly under these conditions resulting in `deinduction' of the NO₃⁻ transport system. Beyond 50 micromoles per gram [NO₃⁻], ¹³NO₃⁻ influx was negatively correlated with root [NO₃⁻]. However, it is unclear whether root [NO₃⁻] per se or some product(s) of NO₃⁻ assimilation are responsible for the negative feedback effects.     

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO₃⁻ and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO₃⁻, either by amplifying the effect of low endogenous NO₃⁻ levels, or by making NO₃⁻ available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO₃⁻ and benzyladenine, additive responses were obtained. The effects of NO₃⁻ and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO₃⁻, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO₃⁻ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO₃⁻ and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO₃⁻. At the time of the second exposure to benzyladenine, no NO₃⁻ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO₃⁻.     

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves

Corn seedlings (Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO₃⁻, with or without light and with NH₄Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO₃⁻. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO₃⁻ was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO₃⁻. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO₃⁻ was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH₄Cl⁻ was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO₃⁻ or by some other unidentified factor, and that higher levels of NO₃⁻ are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO₃⁻ to NO₂⁻.     

Comparative Induction of Nitrate Reductase by Nitrate and Nitrite in Barley Leaves

The comparative induction of nitrate reductase (NR) by ambient NO₃⁻ and NO₂⁻ as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO₃⁻ influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO₃⁻. As the ambient concentration of NO₃⁻ increased, the relative influences imposed by influx and reduction on NO₃⁻ accumulation changed with influx becoming a more predominant regulant. Significant levels of NO₃⁻ accumulated in NO₂⁻-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO₂⁻, about 60% more NO₃⁻ accumulated in the leaves than in the absence of the inhibitors. In NO₃⁻-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mm. No NR induction occurred in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 mm. In fact, NR induction from NO₂⁻ solutions was not seen until NO₃⁻ was detected in the leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NR as did equivalent amounts of NO₃⁻ accumulating from NO₃⁻-fed leaves. In all cases the internal concentration of NO₃⁻, but not NO₂⁻, was highly correlated with the amount of NR induced. The evidence indicated that NO₃⁻ was a more likely inducer of NR than was NO₂⁻.     

Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the main forms of nitrogen available in the soil for plants. Excessive NH₄⁺ accumulation in tissues is toxic for plants and exclusive NH₄⁺-based nutrition enhances this effect. Ammonium toxicity syndrome commonly includes growth impairment, ion imbalance and chlorosis among others. In this work, we observed high intraspecific variability in chlorophyll content in 47 Arabidopsis thaliana natural accessions grown under 1 mM NH₄⁺ or 1 mM NO₃⁻ as N-source. Interestingly, chlorophyll content increased in every accession upon ammonium nutrition. Moreover, this increase was independent of ammonium tolerance capacity. Thus, chlorosis seems to be an exclusive effect of severe ammonium toxicity while mild ammonium stress induces chlorophyll accumulation.     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Nitrate Reduction in Roots as Affected by the Presence of Potassium and by Flux of Nitrate through the Roots

Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO₃)₂, NaNO₃, or KNO₃; KNO₃ plus 50 or 100 millimolar sorbitol; and KNO₃ at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO₃⁻ transport had previously been induced by prior exposure to NO₃⁻ for 10 hours. The experimental system allowed direct measurements of net NO₃⁻ uptake and translocation, and calculation of NO₃⁻ reduction in the root. The presence of K⁺ resulted in small increases in NO₃⁻ uptake, but appreciably stimulated NO₃⁻ translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO₃⁻ that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO₃⁻ was reduced in the presence of K⁺. Results support the proposition that NO₃⁻ reduction in the root is reciprocally related to the rate of NO₃⁻ transport through the root symplasm.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean

Since NO₃⁻ availability in the rooting medium seriously limits symbiotic N₂ fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO₃⁻ and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M₂ generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant⁻¹) than the Williams parent (187 nodules plant⁻¹) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO₃⁻; each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO₃⁻ than did Williams at 1.5 millimolar NO₃⁻. The NO₃⁻ tolerance did not appear to be related to an altered ability to take up or metabolize NO₃⁻, based on solution NO₃⁻ depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M₅ generation in all three mutants.