Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

Regulation of inorganic carbon acquisition by nitrogen and phosphorus levels in the Nannochloropsis sp.

Nannochloropsis sp. was grown to the exponential phase and transferred to the high CO₂ (2,800 μl l⁻¹) and irradiance (100 μmol photons m⁻² s⁻¹) condition with different levels of nitrate and phosphate for 72 h, then the photosynthetic activity and inorganic carbon acquisition of the alga were measured. The apparent photosynthetic efficiency (α) of Nannochloropsis sp. decreased with increasing NO₃ ⁻ concentration from 150 to 3,000 μM, and the high nitrate-grown cells showed the lowest levels of light-saturated photosynthetic rate (P _m), while the low nitrate-grown cells showed the highest levels of dark respiration rate (R _d). The maximal light-saturated photosynthetic rate and the minimal dark respiration rate were seen under the middle nitrate condition. When the nitrate concentration ranged from 150 to 3,000 μM, the affinity for inorganic carbons of Nannochloropsis sp. increased sharply with the increasing NO₃ ⁻ concentration to 300 μM and then decreased significantly. The middle phosphate-grown cells exhibited the highest light-saturated photosynthetic rate and apparent photosynthetic efficiency, however, the affinity for inorganic carbons of Nannochloropsis sp. was the maximum under the low phosphate condition. It was shown that the appropriate nitrogen and phosphorus levels were of vital importance to the photosynthesis of cells.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Effects of N supply on the accumulation of photosynthetic pigments and photoprotectors in <Emphasis Type="Italic">Gracilaria tenuistipitata</Emphasis> (Rhodophyta) cultured under UV radiation

We have studied the effects of nitrate supply under photosynthetic active radiation (PAR) plus ultraviolet radiation (UVR) exposure on photosynthetic pigments (chlorophyll a and carotenoids), photoprotective UV screen mycosporine-like amino acids (MAAs), and photosynthetic parameters, including the maximum quantum yield (F _v/F _m) and electron transport rate (ETR) on the red agarophyte Gracilaria tenuistipitata. Apical tips of G. tenuistipitata were cultivated under ten different concentrations of NO₃⁻ for 7 days. It has been shown that G. tenuistipitata cultured under laboratory conditions has the ability to accumulate high amounts of MAAs following a nitrate concentration-dependent manner under PAR + UVR. Two MAAs were identified, shinorine and porphyra-334. The relative concentration of the first increased under high concentrations of nitrate, while the second one decreased. The presence of antheraxanthin is reported for the first time in this macroalgae, which also contains zeaxanthin, lutein, and β-carotene. The accumulation of pigments, photoprotective compounds, and photosynthetic parameters of G. tenuistipitata is directly related to N availability. All variables decreased under low N supplies and reached constant maximum values with supplements higher than 0.5 mM NO₃⁻. Our results suggest a high potential to acclimation and photoprotection against stress factors (including high PAR and UVR) directly related to N availability for G. tenuistipitata.     

Photorespiration Process and Nitrogen Metabolism in Lettuce Plants (Lactuca sativa L.): Induced Changes in Response to Iodine Biofortification

Iodine is vital to human health, and iodine biofortification programs help improve human intake through plant consumption. There is no research on whether iodine biofortification influences basic plant physiological processes. Because nitrogen (N) uptake, utilization, and accumulation are determining factors in crop yield, the aim of this work was to establish the effect of the application of different doses (20, 40, and 80 μM) and forms of iodine (iodate [IO₃ ⁻] vs. Iodide [I⁻]) on N metabolism and photorespiration. For this study we analyzed shoot biomass and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), glutamate dehydrogenase (GDH), glycolate oxidase (GO), glutamate:glyoxylate aminotransferase (GGAT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HR) and catalase (CAT), nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), organic and total N, amino acids, proteins, serine (ser), malate, and α-ketoglutaric acid in edible lettuce leaves. Application of I⁻ at doses of at least 40 μM reduced the foliar concentration of NO₃ ⁻ with no decrease in biomass production, which may improve the nutritional quality of lettuce plants. In contrast, the application of 80 μM of I⁻ is phytotoxic for lettuce plants, reducing the biomass, foliar concentration of organic N and NO₃ ⁻, and NR and GDH activities. HR activity is significantly inhibited with all doses of I⁻; the least inhibition was at 80 μM. This may involve a decrease in the incorporation of carbonated skeletons from photorespiration into the Calvin cycle, which may be partially associated with the biomass decrease. Finally, the application of IO₃ ⁻ increases biomass production, stimulates NO₃ ⁻ reduction and NH₄ ⁺ incorporation (GS/GOGAT), and optimizes the photorespiratory process. Hence, this appears to be the most appropriate form of iodine from an agronomic standpoint.     

Abiotic immobilization of nitrate in two soils of relic <Emphasis Type="Italic">Abies pinsapo</Emphasis>-fir forests under Mediterranean climate

Evidence for abiotic immobilization of nitrogen (N) in soil is accumulating, but remains controversial. Identifying the fate of N from atmospheric deposition is important for understanding the N cycle of forest ecosystems. We studied soils of two Abies pinsapo fir forests under Mediterranean climate seasonality in southern Spain—one with low N availability and the other with symptoms of N saturation. We hypothesized that biotic and abiotic immobilization of nitrate (NO₃ ⁻) would be lower in soils under these forests compared to more mesic temperate forests, and that the N saturated stand would have the lowest rates of NO₃ ⁻ immobilization. Live and autoclaved soils were incubated with added ¹⁵NO₃ ⁻ (10 μg N g⁻¹ dry soil; 99% enriched) for 24 h, and the label was recovered as total dissolved-N, NO₃ ⁻, ammonium (NH₄ ⁺), or dissolved organic-N (DON). To evaluate concerns about possible iron interference in analysis of NO₃ ⁻ concentrations, both flow injection analysis (FIA) and ion chromatography (IC) were applied to water extracts, soluble iron was measured in both water and salt extracts, and standard additions of NO₃ ⁻ to salt extracts were analyzed. Good agreement between FIA and IC analysis, low concentrations of soluble Fe, and 100% (±3%) recovery of NO₃ ⁻ standard additions all pointed to absence of an interference problem for NO₃ ⁻ quantification. On average, 85% of the added ¹⁵NO₃ ⁻ label was recovered as ¹⁵NO₃ ⁻, which supports our hypothesis that rates of immobilization were generally low in these soils. A small amount (mean = 0.06 μg N g⁻¹ dry soil) was recovered as ¹⁵NH₄ ⁺ in live soils and none in sterilized soils. Mean recovery as DO¹⁵N ranged from 0.6 to 1.5 μg N g⁻¹ dry soil, with no statistically significant effect of sterilization or soil type, indicating that this was an abiotic process that occurred at similar rates in both soils. These results demonstrate a detectable, but modest rate of abiotic immobilization of NO₃ ⁻ to DON, supporting our first hypothesis. These mineral soils may not have adequate carbon availability to support the regeneration of reducing microsites needed for high rates of NO₃ ⁻ reduction. Our second hypothesis regarding lower expected abiotic immobilization in soils from the N-saturated site was not supported. The rates of N deposition in this region may not be high enough to have swamped the capacity for soil NO₃ ⁻ immobilization, even in the stand showing some symptoms of N saturation. A growing body of evidence suggests that soil abiotic NO₃ ⁻ immobilization is common, but that rates are influenced by a combination of factors, including the presence of plentiful available carbon, reduced minerals in anaerobic microsites and adequate NO₃ ⁻ supply.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Spatial and temporal patterns of net nitrate uptake regulation and kinetics along the tap root of <Emphasis Type="Italic">Citrus aurantium</Emphasis>

Spatial–temporal variation of the regulation and the kinetics of net nitrate (NO₃ ⁻) uptake rate (NNUR) along the tap root of Citrus aurantium L. were analysed. Suberin incrustation in the peripheral cell layers and plasma membrane (PM) H⁺-ATPase localisation, anatomical and physiological factors involved in NO₃ ⁻ uptake were also investigated. The results clearly indicated a spatially uniform distribution of the regulation process, accompanied by a temporal heterogeneous pattern of the kinetics of NO₃ ⁻ uptake along citrus tap root. In particular, kinetic analysis had a biphasic pattern, saturating (high affinity transport system) and linear (low affinity transport system), in response to increasing external NO₃ ⁻ concentrations in each root region, where 200 μM NO₃ ⁻ represented the threshold separating these two systems. Kinetic parameters, K _m and V _max, clearly indicated that apical segments reached the maximum value of induction before basal segments. Hence, the apical root zones, early exhibiting the maximum of potential capacity to absorb the NO₃ ⁻, could be considered more efficient than basal root segments for acquiring NO₃ ⁻ from external solution. Suberin incrustations in the hypodermal cell layer, characterised by uniform fluorescence intensity among the root segments, could be responsible for the unchanged NNUR, while the PM H⁺-ATPase could explain the temporal pattern of NNUR.     

Denitrification,dissimilatory nitrate reduction to ammonium,and nitrogen fixation along a nitrate concentration gradient in a created freshwater wetland

Wetlands are often highly effective nitrogen (N) sinks. In the Lake Waco Wetland (LWW), near Waco, Texas, USA, nitrate (NO₃⁻) concentrations are reduced by more than 90% in the first 500 m downstream of the inflow, creating a distinct gradient in NO₃⁻ concentration along the flow path of water. The relative importance of sediment denitrification (DNF), dissimilatory NO₃⁻ reduction to ammonium (DNRA), and N₂ fixation were examined along the NO₃⁻ concentration gradient in the LWW. “Potential DNF” (hereafter potDNF) was observed in all months and ranged from 54 to 278 μmol N m⁻² h⁻¹. “Potential DNRA” (hereafter potDNRA) was observed only in summer months and ranged from 1.3 to 33 μmol N m⁻² h⁻¹. Net N₂ flux ranged from 184 (net denitrification) to −270 (net N₂ fixation) μmol N m⁻² h⁻¹. Nitrogen fixation was variable, ranging from 0 to 426 μmol N m⁻² h⁻¹, but high rates ranked among the highest reported for aquatic sediments. On average, summer potDNRA comprised only 5% (±2% SE) of total NO₃⁻ loss through dissimilatory pathways, but was as high as 36% at one site where potDNF was consistently low. Potential DNRA was higher in sediments with higher sediment oxygen demand (r ² = 0.84), and was related to NO₃⁻ concentration in overlying water in one summer (r ² = 0.81). Sediments were a NO₃⁻ sink and accounted for 50% of wetland NO₃⁻ removal (r ² = 0.90). Sediments were an NH₄⁺ source, but the wetland was often a net NH₄⁺ sink. Although DNRA rates in freshwater wetlands may rival those observed in estuarine systems, the importance of DNRA in freshwater sediments appears to be minor relative to DNF. Furthermore, sediment N₂ fixation can be extremely high when NO₃⁻ in overlying water is consistently low. The data suggest that newly fixed N can support sustained N transformation processes such as DNF and DNRA when surface water inorganic N supply rates are low.     

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO₂⁻) or nitrate (NO₃⁻) as the sole nitrogen source and aerated with air or with CO₂-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 μM NaNO₃ as nitrogen source. Accordingly, NO₂⁻ concentration reached 10.4 μM after 95 h at the low CO₂ condition (aerated with air); while the maximum NO₂⁻ concentration was only around 2.0 μM at the high CO₂ condition (aerated with 5% CO₂ in air), furthermore, after 30 h it decreased to no more than 1.0 μM. NO₂⁻ was almost assimilated in 80 h when C. muelleri was cultured at the high CO₂ condition with 100 μM NaNO₂ as sole nitrogen source. At the high CO₂ condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO₂ condition. It was indicated that enriched CO₂ concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO₂ might be an effective way to control nitrite content in aquaculture systems.     

Changes in biomass allocation and phenolic compounds accumulation due to the effect of light and nitrate supply in <Emphasis Type="Italic">Cecropia peltata</Emphasis> plants

Cecropia peltata is popularly known as “guarumbo” in Mexico and is used in traditional medicine for treatment of diabetes mellitus. C. peltata plants were cultivated in a hydroponic system under controlled conditions. Gradients of light (20, 30 and 100 μmol m⁻² s⁻¹) and nitrate concentrations (13, 2 and 0.2 mM) were applied to estimate their effect on biomass allocation and accumulation of bioactive (chlorogenic acid and isoorientin) phenolic compounds over a 28-day period. According to carbon nutrient balance (CNB) hypothesis predictions, biomass accumulation in foliage was stimulated by the highest irradiance (100 μmol m⁻² s⁻¹); similarly, at highest irradiance in combination with lowest nitrate concentration (0.2 mM), root growth was stimulated (root-to-shoot ratio increased twofold with respect to the control). In these conditions, total phenolics (TP) and chlorogenic acid (CGA) contents were higher in aerial parts than in roots, with a 3.8-fold increase in TP and a 7.7-fold increase in CGA in foliage with respect to the control plants. Isoorientin was accumulated at very low levels. Antioxidant activity and total phenolic content showed a strong positive correlation. Phenylalanine ammonia-lyase activity (PAL) in aerial parts exhibited significant changes (>twofold) by highest irradiance. C. peltata plants allocate biomass and/or phenolic compounds to compensate the oxidative damage (increase in MDA levels) due to changes in light and nitrate restriction. The results are the basis for the establishment of a system of C. peltata culture in view of the potential use of C. peltata in therapeutic preparations for the treatment of diabetes mellitus.     

Differential growth response of <Emphasis Type="Italic">Ulva lactuca</Emphasis> to ammonium and nitrate assimilation

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH₄⁺; NO₃⁻; and the combination NH₄NO₃) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NH₄Cl and NaNO₃ rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day⁻¹ and 9.4 ± 0.72% day⁻¹, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m⁻²·day⁻¹. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NH₄NO₃. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

The effect of the osmotic potential and specific ion concentration of the nutrient solution on the uptake and reduction of nitrate by barley seedlings

Summary Short-term absorption experiments were conducted with intact barley (Hordeum vulgare L.) seedlings to observe the effects of the osmotic potential (Ψ^π) and salt species on nitrate uptake andin vivo nitrate reduction. The experiments consisted of growing barley seedlings for 5 days in complete nutrient solutions salinized to (Ψ^π) levels of −0.6, −1.8, −3.0, −4.2, and −5.4 bars with NaCl, CaCl₂ or Na₂SO₄. After the absorption period, the seedlings were separated into shoots and roots, weighed, then analyzed for NO₃. The nutrient solutions were sampled for NO₃ analysis each day immediately before renewing the solutions. The accumulative loss of NO₃ from the solutions was considered to be uptake whereas NO₃ reduction was the difference between uptake and seedling content. Lowering the (Ψ^π) of the nutrient solutions resulted in decreased concentrations of NO₃ in the plant, little or no effect (except at the lowest (Ψ^π) level) on uptake, and increased nitrate reductase activity. Increased rates of NO₃ reduction were in particular associated with the Cl concentration of the nutrient solution.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

Whole-stream nitrate addition affects litter decomposition and associated fungi but not invertebrates

We assessed the effect of whole-stream nitrate enrichment on decomposition of three substrates differing in nutrient quality (alder and oak leaves and balsa veneers) and associated fungi and invertebrates. During the 3-month nitrate enrichment of a headwater stream in central Portugal, litter was incubated in the reference site (mean NO₃-N 82 μg l⁻¹) and four enriched sites along the nitrate gradient (214–983 μg NO₃-N l⁻¹). A similar decomposition experiment was also carried out in the same sites at ambient nutrient conditions the following year (33–104 μg NO₃-N l⁻¹). Decomposition rates and sporulation of aquatic hyphomycetes associated with litter were determined in both experiments, whereas N and P content of litter, associated fungal biomass and invertebrates were followed only during the nitrate addition experiment. Nitrate enrichment stimulated decomposition of oak leaves and balsa veneers, fungal biomass accrual on alder leaves and balsa veneers and sporulation of aquatic hyphomycetes on all substrates. Nitrate concentration in stream water showed a strong asymptotic relationship (Michaelis–Menten-type saturation model) with temperature-adjusted decomposition rates and percentage initial litter mass converted into aquatic hyphomycete conidia for all substrates. Fungal communities did not differ significantly among sites but some species showed substrate preferences. Nevertheless, certain species were sensitive to nitrogen concentration in water by increasing or decreasing their sporulation rate accordingly. N and P content of litter and abundances or richness of litter-associated invertebrates were not affected by nitrate addition. It appears that microbial nitrogen demands can be met at relatively low levels of dissolved nitrate, suggesting that even minor increases in nitrogen in streams due to, e.g., anthropogenic eutrophication may lead to significant shifts in microbial dynamics and ecosystem functioning. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Effect of nitrite on growth and microcystins production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC7806

As a common pollutant, nitrite concentrations can approach 15 mg NO₂⁻-N L⁻¹ in some aquatic systems. Microcystis aeruginosa blooms are common and widespread in eutrophic freshwater bodies. In this study, M. aeruginosa was exposed to nitrite concentrations ranging from 0 to 15 mg NO₂⁻-N L⁻¹, and the responses of M. aeruginosa were investigated. The specific growth rates, maximum cell densities, light-saturated photosynthetic rates (P_m ^chla ), dark respiration rates (R_d ^chla ), and apparent photosynthetic efficiencies (α^chla ) showed a significant decline with nitrite concentrations increasing. Electrical conductivity and malondialdehyde contents investigation revealed cell membrane damage and apparent leakage of intracellular contents under high nitrite level conditions due to oxidative stress enhancement. Intracellular microcystin (MC)-LR content reached the highest value at 10 mg NO₂⁻-N L⁻¹; however, extracellular MC-LR contents showed a continuous increase until 15 mg NO₂⁻-N L⁻¹ owing to the increasing leakage of intracellular contents. These results elucidated that the high-level nitrite inhibited M. aeruginosa growth by rising oxidative stress, damaging cell membrane, and reducing photosynthesis. However, the moderate increase in nitrite concentrations promoted toxin production and release of toxin.     

Annual sediment respiration in estuarine sandy intertidal flats in the Seto Inland Sea, Japan

To quantify organic matter mineralization at estuarine intertidal flats, we measured in situ sediment respiration rates using an infrared gas analyzer in estuarine sandy intertidal flats located in the northwestern Seto Inland Sea, Japan. In situ sediment respiration rates showed spatial and seasonal variations, and the mean of the rates is 38.8 mg CO₂-C m⁻² h⁻¹ in summer. In situ sediment respiration rates changed significantly with sediment temperature at the study sites (r ² = 0.70, p < 0.05), although we did not detect any significant correlations between the rates and sediment characteristics. We prepared a model for estimating the annual sediment respiration based on the in situ sediment respiration rates and their temperature coefficient (Q ₁₀ = 1.8). The annual sediment respiration was estimated to be 92 g CO₂-C m⁻² year⁻¹. The total amount of organic carbon mineralization for the entire estuarine intertidal flats through sediment respiration (43 t C year⁻¹) is equivalent to approximately 25% of the annual organic carbon load supplied from the river basin of the estuary.     

Non-photosynthetic enhancement of growth by high CO2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)

The effects of increased CO₂ levels (10,000 μl l⁻¹) in cultures of the green nitrophilic macroalga Ulva rigida C. Agardh were tested under conditions of N saturation and N limitation, using nitrate as the only N source. Enrichment with CO₂ enhanced growth, while net photosynthesis, gross photosynthesis, dark respiration rates and soluble protein content decreased. The internal C pool remained constant at high CO₂, while the assimilated C that was released to the external medium was less than half the values obtained under ambient CO₂ levels. This higher retention of C provided the source for extra biomass production under N saturation. In N-sufficient thalli, nitrate-uptake rate and the activity of nitrate reductase (EC 1.6.6.1) increased under high CO₂ levels. This did not affect the N content or the internal C:N balance, implying that the extra N-assimilation capacity led to the production of new biomass in proportion to C. Growth enhancement by increased level of CO₂ was entirely dependent on the enhancement effect of CO₂ on N-assimilation rates. The increase in nitrate reductase activity at high CO₂ was not related to soluble carbohydrates or internal C. This indicates that the regulation of N assimilation by CO₂ in U. rigida might involve a different pathway from that proposed for higher plants. The role of organic C release as an effective regulatory mechanism maintaining the internal C:N balance in response to different CO₂ levels is discussed. Received: 27 March 2000 / Accepted: 9 October 2000