Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Ammonium repression of the nitrite-nitrate ( nasAB ) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Received: 4 December 1996 / Accepted: 27 March 1997     

Map order and linkage distances of molecular markers close to the supernodulation ( nts-1 ) locus of soybean

The molecular characteristics of markers in the chromosome region surrounding the supernodulation gene (nts-1) of soybean (Glycine max L. Merr.) were investigated in 187 F₂ plants from a cross of G. max cv. Bragg (nts) and G. soja PI468.397 (wild-type nodulation). RFLP marker pUTG-132a, linked tightly (0.7±0.5 cM) to nts-1, was converted to a PCR marker. The polymorphism resides within a 1.72 kb PstI fragment and consists of an 832 bp insertion in G. max relative to the wild progenitor G. soja. The insertion is flanked by a 35 bp direct duplication that was found only once in G. soja. Data suggest that the pUTG-132a sequence exists only once in the genome, which is compatible with the recessive nature of nts-1. Accordingly, pUTG-132a is a valuable marker for map-based cloning. Another RFLP marker, pA-381, was mapped 4.8 cM distal to nts-1. Marker order, established by Maximum Likelihood Analysis, placed nts-1 between pUTG-132a and pA-381. To generate additional molecular markers, a segregating F₂ population was analysed using bulked segregant analysis (BSA) and single oligonucleotide primer-based PCR (DNA amplification fingerprinting; DAF). PCR marker pcr5-4L was mapped to soybean linkage group H and sequenced. The data revealed (i) recombination events and marker order in the nts-1 region; (ii) the molecular nature and cause of polymorphisms in linked molecular markers; (iii) a low density of polymorphisms around nts-1, and (iv) diploidy of the distal region of linkage group H of soybean. Received: 18 January 1996 / Accepted: 9 October 1996     

Does nitrogen nutrition restrict the CO2 response of fertile grassland lacking legumes?

The extent of the response of plant growth to atmospheric CO₂ enrichment depends on the availability of resources other than CO₂. An important growth-limiting resource under field conditions is nitrogen (N). N may, therefore, influence the CO₂ response of plants. The effect of elevated CO₂ (60 Pa) partial pressure (pCO₂) on the N nutrition of field-grown Lolium perenne swards, cultivated alone or in association with Trifolium repens, was investigated using free air carbon dioxide enrichment (FACE) technology over 3 years. The established grassland ecosystems were treated with two N fertilization levels and were defoliated at two frequencies. Under elevated pCO₂, the above-ground plant material of the L. perenne monoculture showed a consistent and significant decline in N concentration which, in general, led to a lower total annual N yield. Despite the decline in the critical N concentration (minimum N concentration required for non-N-limited biomass production) under elevated pCO₂, the index of N nutrition (ratio of actual N concentration and critical N concentration) was lower under elevated pCO₂ than under ambient pCO₂ in frequently defoliated L. perenne monocultures. Thus, we suggest that reduced N yield under elevated pCO₂ was evoked indirectly by a reduction of plant-available N. For L. perenne grown in association with T. repens and exposed to elevated pCO₂, there was an increase in the contribution of symbiotically fixed N to the total N yield of the grass. This can be explained by an increased apparent transfer of N from the associated N₂-fixing legume species to the non-fixing grass. The total annual N yield of the mixed grass/legume swards increased under elevated pCO₂. All the additional N yielded was due to symbiotically fixed N. Through the presence of an N₂-fixing plant species more symbiotically fixed N was introduced into the system and consequently helped to overcome N limitation under elevated pCO₂. Received: 11 November 1996 / Accepted: 20 May 1997     

Net nitrogen mineralization and net nitrification rates in soils following deforestation for pasture across the southwestern Brazilian Amazon Basin landscape

Previous studies of the effect of tropical forest conversion to cattle pasture on soil N dynamics showed that rates of net N mineralization and net nitrification were lower in pastures compared with the original forest. In this study, we sought to determine the generality of these patterns by examining soil inorganic N concentrations, net mineralization and nitrification rates in 6 forests and 11 pastures 3 years old or older on ultisols and oxisols that encompassed a wide variety of soil textures and spanned a 700-km geographical range in the southwestern Brazilian Amazon Basin state of Rondônia. We sampled each site during October-November and April-May. Forest soils had higher extractable NO₃ ^?-N and total inorganic N concentrations than pasture soils, but substantial NO₃ ^?-N occurred in both forest and pasture soils. Rates of net N mineralization and net nitrification were higher in forest soils. Greater concentrations of soil organic matter in finer textured soils were associated with greater rates of net N mineralization and net nitrification, but this relationship was true only under native forest vegetation; rates were uniformly low in pastures, regardless of soil type or texture. Net N mineralization and net nitrification rates per unit of total soil organic matter showed no pattern across the different forest sites, suggesting that controls of net N mineralization may be broadly similar across a wide range of soil types. Similar reductions in rates of net N transformations in pastures 3 years old or older across a range of textures on these soils suggest that changes to soil N cycling caused by deforestation for pasture may be Basin-wide in extent. Lower net N mineralization and net nitrification rates in established pastures suggest that annual N losses from largely deforested landscapes may be lower than losses from the original forest. Total ecosystem N losses since deforestation are likely to depend on the balance between lower N loss rates from established pastures and the magnitude and duration of N losses that occur in the years immediately following forest clearing.     

Why do leaves and leaf cells of N-limited barley elongate at reduced rates?

The objective of the present study was to assess whether, in barley, nitrogen supply limits the rate of leaf elongation through a reduction in (relative) cell elongation rate and whether this is attributable to a reduced turgor, a reduced availability of osmolytes or, by implication, changed wall properties. Plants were grown on full-strength Hoagland solution (“Hoagland”-plants), or on N-deficient Hoagland solution while receiving N at a relative addition rate of 16 or 8% N · plant-N⁻¹ · d⁻¹ (“16%-” and “8%-plants”). Hoagland-plants were demand-limited, whereas 16%- and 8%-plants were supply-limited in N. Third leaves were analysed for leaf elongation rate and final epidermal cell length, and, within the basal growing region, for the spatial distribution of relative segmental elongation rates (RSER, pin-pricking method), epidermal cell turgor (cell-pressure probe), osmotic pressure (OP, picolitre osmometry) and water potential (Ψ). During the development of the third leaf, plants grew at relative growth rates (relative increase in fresh weight ) of 18.2, 15.6 and 8.1% · d⁻¹ (Hoagland-, 16%- and 8%-plants, respectively). Final leaf length and leaf elongation rate were highest in Hoagland plants (ca. 34.1 cm and 2.33–2.60 mm · h⁻¹, respectively), intermediate in 16%- plants (31.0 cm and 1.89–1.96 mm · h⁻¹) and lowest in 8%-plants (29.4 cm and 1.41–1.58 mm · h⁻¹). These differences were accompanied by only small differences in final cell length, but large differences in cell-flux rates (146, 187 and 201 cells · cell-file⁻¹ · d⁻¹ in 8%-, 16%- and Hoagland-plants, respectively). The length of the growth zone (32–38 mm) was not much affected by N-levels (and nutrient technique). A decrease in RSER in the growth zone distal to 10 mm produced the significant effect of N-levels on leaf elongation rate. In all treatments, cell turgor was almost constant throughout the growing region, as were cell OP and Ψ in 16%- and 8%-plants. In Hoagland-plants, however, cell OP increased by ca. 0.1 MPa within the zone of highest elongation rates and, as a consequence, cell Ψ decreased simultaneously by 0.1 MPa. Cell Ψ increased considerably where elongation ceased. Within the zone where differences in RSERs were highest between treatments (10–34 mm from base) average turgor was lowest, OP highest and Ψ most negative in Hoagland- compared to 8%- and 16%-plants (P < 0.001), but not significantly different between 8%- and 16%-plants. Received: 9 January 1997 / Accepted: 6 March 1997     

Nutrient dynamics of the highly productive C4 macrophyte Echinochloa polystachya on the Amazon floodplain

1. Echinochloa polystachya forms extensive monotypic stands on the lower levels of the Amazon floodplains. During its annual growth cycle c. 100t (dry mass) ha^–1 of biomass is formed as the floodplain is being submerged (December–September) and a phase of death and decomposition occurs when the water has retreated (October–November). This study examines the mineral nutrient dynamics of this plant and its potential significance to the nutrient status of the floodplain.
2. Echinochloa polystachya was sampled monthly from a study site in the central Amazon. N, P and K contents for different plant organs were determined and net uptake calculated from concurrent measurements of dry matter production and turnover.
3. Leaf N, P and K contents were c. 20, 1·7 and 19gkg^–1, values typical of nutrient-replete stands of C₄ plants. Stem concentrations were c. 12% of those of the leaves. Net N and P uptake followed the rise in the river level, whilst K appeared independent of water level.
4. The vegetation accumulated 377, 51 and 1136kgha^–2 of N, P and K, respectively, during the growth phase. Over a possible 5000km² of these stands in the Várzea, this represents a massive sequestration of nutrients in the flood phase and a high release during the following low-water period. It is suggested that the E. polystachya stands could have a role in maintaining the nutrient status of the Amazon floodplain.     

CO_2倍增对紫花苜蓿碳、氮同化与分配的影响

本文简要报道ＣＯ２倍增下紫花苜蓿碳素积累、氮素的吸收与生物固氮及其产物在地上、地下部分配的特性１　材料和方法在北京香山中国科学院植物研究所植物园试验区,建立了两个高２．８ｍ、直径２．２ｍ的钢管支撑的圆柱状开顶式薄膜培养室,由底部向室内连续通气,保证培养室内每分钟换气３次。对照室通入正常空气（３５０×１０－６,１×ＣＯ２）,处理室通入ＣＯ２加倍的空气（７００×１０－６,２×ＣＯ２）。室内ＣＯ２浓度经红外ＣＯ２分析仪（ＱＧＤ－０７型,北京分析仪器厂产品）测定,２４ｈ内均可保持在３５０×１０－６及７０…     

Net primary production and light use efficiency in a mixed coniferous forest in Sweden

Simple light use efficiency (ɛ) models of net primary production (NPP) have recently been given great attention (NPP = ɛ × absorbed photosynthetically active radiation). The underlying relationships have, however, not been much studied on a time step less than a month. In this study daily NPP was estimated as the sum of net ecosystem exchange (NEE) and heterotrophic respiration (R_h) of a mixed pine and spruce forest in Sweden. NEE was measured by eddy correlation technique and R_h was estimated from measurements of forest floor respiration (R_f) and the root share of R_f. The total yearly NPP was on average 810 g C m⁻² year⁻¹ for 3 years and yearly ɛ was between 0.58 and 0.71 g C MJ⁻¹, which is high in comparison with other studies. There was a seasonal trend in ɛ with a relatively constant level of approximately 0.90 g C MJ⁻¹ from April to September Daily NPP did not increase for daily intercepted radiation above 6 MJ m⁻² d⁻¹, indicating that between-years variation in NPP is not directly dependent on total Q_i. The light was most efficiently used at an average daytime temperature of around 15 °C. At daytime vapour pressure deficit above 1400 Pa ɛ was reduced by approximately 50%.     

Nitrogen concentration and δ15N signature of ombrotrophic Sphagnum mosses at different N deposition levels in Europe

Alteration of the global nitrogen (N) cycle because of human‐enhanced N fixation is a major concern particularly for those ecosystems that are nutrient poor by nature. Because Sphagnum‐dominated mires are exclusively fed by wet and dry atmospheric deposition, they are assumed to be very sensitive to increased atmospheric N input. We assessed the consequences of increased atmospheric N deposition on total N concentration, N retention ability, and δ¹⁵N isotopic signature of Sphagnum plants collected in 16 ombrotrophic mires across 11 European countries. The mires spanned a gradient of atmospheric N deposition from about 0.1 up to about 2 g m^?2 yr^?1. Mean N concentration in Sphagnum capitula was about 6 mg g^?1 in less polluted mires and about 13 mg g^?1 in highly N‐polluted mires. The relative difference in N concentration between capitulum and stem decreased with increasing atmospheric N deposition, suggesting a possible metabolic mechanism that reduces excessive N accumulation in the capitulum. Sphagnum plants showed lower rates of N absorption under increasing atmospheric N deposition, indicating N saturation in Sphagnum tissues. The latter probably is related to a shift from N‐limited conditions to limitation by other nutrients. The capacity of the Sphagnum layer to filter atmospheric N deposition decreased exponentially along the depositional gradient resulting in enrichment of the mire pore water with inorganic N forms (i.e., NO₃^?+NH₄⁺). Sphagnum plants had δ¹⁵N signatures ranging from about ?8‰ to about ?3‰. The isotopic signatures were rather related to the ratio of reduced to oxidized N forms in atmospheric deposition than to total amount of atmospheric N deposition, indicating that δ¹⁵N signature of Sphagnum plants can be used as an integrated measure of δ¹⁵N signature of atmospheric precipitation. Indeed, mires located in areas characterized by greater emissions of NH₃ (i.e., mainly affected by agricultural activities) had Sphagnum plants with a lower δ¹⁵N signature compared with mires located in areas dominated by NO_x emissions (i.e., mainly affected by industrial activities).