Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Influence of phosphorus and nitrogen on photosynthetic parameters and growth in Vicia faba L.

The influence of phosphorus (P) and nitrogen (N) supply on biomass, leaf area, photon saturated photosynthetic rate (P_max), quantum yield efficiency (), intercellular CO₂ concentration (C_i), and carboxylation efficiency (CE) was investigated in Vicia faba. The influence of P on N accumulation, biomass, and leaf area production was also investigated. An increase in P supply was consistently associated with an increase in N accumulation and N productivity in terms of biomass and leaf area production. Furthermore, P increased the photosynthetic N use efficiency (NUE) in terms of P_max and . An increase in P supply was also associated with an increase in CE and a decrease in C_i. Under variable daily meteorological conditions specific leaf nitrogen content (N_L), specific leaf phosphorus content (P_L), specific leaf area (_L), root mass fraction (R_f), P_max, and remained constant for a given N and P supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L. We tested also the hypothesis that P supply positively affects both N demand and photosynthetic NUE by influencing the upper limit of the asymptotic values for P_max and CE, and the lower limit for C_i in response to increasing N.This revised version was published online in March 2005 with corrections to the page numbers.     

Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation

Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod⁻), or with ineffective nodules (nod⁺, fix⁻) were obtained, whilst in the other 4 hypernodulated mutants (nod⁺⁺) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod⁻), (nod⁺, fix⁻) and (nod⁺⁺, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.

Grafting experiments showed the (nod⁻) and (nod⁺, fix⁻) phenotypes are associated with the root genotypes and that (nod⁺⁺, nts) phenotype is associated with the shoot genotype.     

First report of non-mycorrhizal plant mutants (Myc) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc⁻, are characterized by aborted infections limited to one or two host cells. Expression of the myc⁻ character is associated with that of the nod⁻ character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc⁻ mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.     

Elevated CO2 enhances nitrogen fixation and growth in the marine cyanobacterium Trichodesmium

The increases in atmospheric pCO₂ over the last century are accompanied by higher concentrations of CO₂(aq) in the surface oceans. This acidification of the surface ocean is expected to influence aquatic primary productivity and may also affect cyanobacterial nitrogen (N)‐fixers (diazotrophs). No data is currently available showing the response of diazotrophs to enhanced oceanic CO₂(aq). We examined the influence of pCO₂ [preindustrial∼250 ppmv (low), ambient∼400, future∼900 ppmv (high)] on the photosynthesis, N fixation, and growth of Trichodesmium IMS101. Trichodesmium spp. is a bloom‐forming cyanobacterium contributing substantial inputs of ‘new N’ to the oligotrophic subtropical and tropical oceans. High pCO₂ enhanced N fixation, C : N ratios, filament length, and biomass of Trichodesmium in comparison with both ambient and low pCO₂ cultures. Photosynthesis and respiration did not change significantly between the treatments. We suggest that enhanced N fixation and growth in the high pCO₂ cultures occurs due to reallocation of energy and resources from carbon concentrating mechanisms (CCM) required under low and ambient pCO₂. Thus, in oceanic regions, where light and nutrients such as P and Fe are not limiting, we expect the projected concentrations of CO₂ to increase N fixation and growth of Trichodesmium. Other diazotrophs may be similarly affected, thereby enhancing inputs of new N and increasing primary productivity in the oceans.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Elevated CO2 stimulates associative N2 fixation in a C3 plant of the Chesapeake Bay wetland

In this study, the response of N₂ fixation to elevated CO₂ was measured in Scirpus olneyi, a C₃ sedge, and Spartina patens, a C₄ grass, using acetylene reduction assay and ¹⁵N₂ gas feeding. Field plants grown in PVC tubes (25 cm long, 10 cm internal diameter) were used. Exposure to elevated CO₂ significantly (P < 0·05) caused a 35% increase in nitrogenase activity and 73% increase in ¹⁵N incorporated by Scirpus olneyi. In Spartina patens, elevated CO₂ (660 ± 1 μ mol mol ^{− 1}) increased nitrogenase activity and ¹⁵N incorporation by 13 and 23%, respectively. Estimates showed that the rate of N₂ fixation in Scirpus olneyi under elevated CO₂ was 611 ± 75 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} compared with 367 ± 46 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} in ambient CO₂ plants. In Spartina patens, however, the rate of N₂ fixation was 12·5 ± 1·1 versus 9·8 ± 1·3 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} for elevated and ambient CO₂, respectively. Heterotrophic non-symbiotic N₂ fixation in plant-free marsh sediment also increased significantly (P < 0·05) with elevated CO₂. The proportional increase in ¹⁵N₂ fixation correlated with the relative stimulation of photosynthesis, in that N₂ fixation was high in the C₃ plant in which photosynthesis was also high, and lower in the C₄ plant in which photosynthesis was relatively less stimulated by growth in elevated CO₂. These results are consistent with the hypothesis that carbon fixation in C₃ species, stimulated by rising CO₂, is likely to provide additional carbon to endophytic and below-ground microbial processes.     

高温对早籼稻花后剑叶氮代谢关键酶活性及籽粒蛋白质含量的影响

为探讨高温对不同类型早籼稻开花后剑叶中氮代谢关键酶活性及籽粒蛋白质含量的影响,利用人工气候室在籽粒灌浆成熟期进行高温(日均温31.5 ℃)和适温(日均温23.5 ℃)处理,对2个早籼稻品种(湘早籼24和株两优611)灌浆成熟期籽粒中蛋白质含量及剑叶中GS和GOGAT活性的动态变化进行了分析.结果表明:供试2个品种在高温...     

Growth responses of the perennial legume Sesbania sesban to NH4 and NO3 nutrition and effects on root nodulation

Preference for NH₄⁺ or NO₃⁻ nutrition by the perennial legume Sesbania sesban (L.) Merr. was assessed by supplying plants with NH₄⁺ and NO₃⁻ alone or mixed at equal concentrations (0.5 mM) in hydroponic culture. In addition, growth responses of S. sesban to NH₄⁺ and NO₃⁻ nutrition and the effects on root nodulation and nutrient and mineral composition of the plant tissues were evaluated in a hydroponic setup at a range of external concentration of NH₄⁺ and NO₃⁻ (0, 0.1, 0.2, 0.5, 2 and 5 mM). Seedlings of S. sesban grew equally well when supplied with either NH₄⁺ or NO₃⁻ alone or mixed and had high relative growth rates (RGRs) ranging between 0.19 and 0.21 d⁻¹. When larger plants of S. sesban were supplied with NH₄⁺ or NO₃⁻ alone, the RGRs and shoot elongation rates were not affected by the external concentration of inorganic N. At external N concentrations up to 0.5 mM nodulation occurred and contributed to the N nutrition through fixation of gaseous N₂ from the atmosphere. For both NH₄⁺ and NO₃⁻-fed plants the N concentration in the plant tissues, particularly water-extractable NO₃⁻, increased at high supply concentrations, and concentrations of mineral cations generally decreased. It is concluded that S. sesban can grow without an external inorganic N supply by fixing atmospheric N₂ gas via root nodules. Also, S. sesban grows well on both NH₄⁺ and NO₃⁻ as the external N source and the plant can tolerate relatively high concentrations of NH₄⁺. This wide ecological amplitude concerning N nutrition makes S. sesban very useful as a N₂-fixing fallow crop in N deficient areas and also a candidate species for use in constructed wetland systems for the treatment of NH₄⁺ rich waters.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Nitrogen rhizodeposition assessed by a NH3 shoot pulse-labelling of Lolium perenne L. grown on soil exposed to 9 years of CO2 enrichment

The effects of elevated CO₂ concentration upon rhizodeposition of nitrogen were investigated on field-grown Lolium perenne planted in soil cores set into the resident soil of a intensively managed ryegrass sward treated with elevated CO₂ for nine consecutive years, under two contrasted N fertilisation regimes (Swiss FACE Experiment). The planted cores were excavated from the ambiant (35 Pa pCO₂) and enriched (60 Pa pCO₂) rings at two dates during the growing season (spring and early autumn). The cores were brought back to the laboratory for a pulse-labelling of ryegrass shoots with ¹⁵NH₃, in order to quantify ¹⁵N-rhizodeposition.A recovery of 10–16% of the total ¹⁵N administred to the plant was recovered in the plant–soil system 48 h after the pulse-labelling; significant amounts of ¹⁵N were released into the soil adhering (adhering soil: AS) to the roots (0.44 μg ¹⁵N g AS⁻¹ and 0.60 μg g AS⁻¹ in the spring and the autumn samplings, respectively).In the spring sampling, there was no effect of atmospheric CO₂ concentration on N rhizodeposition. In the autumn sampling, elevated CO₂ stimulated N rhizodeposition that amounted to 7.2 and 5.2 mg ¹⁵N m⁻², under elevated and ambient CO₂, respectively. Nitrogen rhizodeposition was higher at high N (56 gN m⁻²) than at low N fertilisation (14 gN m⁻²), whatever the sampling date investigated.The mechanisms by which elevated atmospheric CO₂ leads to a stimulation of the net root-released N flux remains to be investigated: was it caused by a higher nitrogen immobilisation by the microbial biomass and a reduced re-assimilation of mineralized N and/or by a stimulation of N efflux from roots? Concomitant to the observed reduction of C rhizodeposition, the stimulation of net N efflux suggests that the quality of root released compounds was modified under elevated CO₂ concentration.     

Effects of CaSO4 and NaCl on growth and nitrogen fixation ofLeucaena leucocephala

Effects and interactions of varying CaSO₄ and NaCl levels on growth and nitrogen fixation ofLeucaena leucocephala K8 were examined. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM) in randomized blocks with five replications. While NaCl significantly reduced plant growth, additions of CaSO₄ increased plant height, leaf number, and biomass of salt treated plants. For the nonsaline treatments, high CaSO₄ levels slightly depressed growth, which contradicts suggestions that Leucaena has a high calcium requirement. A significant calcium/sodium interaction was not seen for nodule number or weight. Nodule number was significantly depressed by 100 mM NaCl and nodule weight of the salt stressed plants significantly increased as CaSO₄ concentration increased from 0.5 to 2.5 mM. Effects of NaCl and CaSO₄ on nitrogen content of plant parts were inconclusive. The promotion of Leucaena salinity tolerance by addition of CaSO₄ may be attributed to the effect of calcium in maintaing the selective permeability of membranes.     

Influence of host genotypes on growth,symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N₂ fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.Abbreviations ARA acetylene reduction activity - NADH-GOGAT NADH-dependent glutamate synthase - GS glutamine synthetase     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

Isolation and characterisation of a Bam HI element in psam 3 a gene of Pisum sativum L. induced during early stages of arbuscular mycorrhiza development

An Alu-like element was identified in the 5′-UTR of psam 3, a Pisum sativum L. gene which shows enhanced expression during early events of AM formation. Two sets of primers specific for the 5′-UTR of the gene psam 3 and for the Alu-like element, respectively, were designed to study psam 3 gene organisation by targeted Alu PCR carried out on pea genomic DNA. PCR products free of Alu-like sequences were produced. A 1.0 kb DNA fragment showing up to 65 percnt; similarity to a Bam HI repeated sequence of Vicia faba was isolated in both wild-type and mycorrhiza-resistant pea. Southern blot analysis revealed the presence of other Bam HI related sequences in pea. The possibility that Bam HI repeated sequences might constitute complex repeating units together with an Alu-like element in the P. sativum genome is discussed.     

Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination

Summary Whole leaves and guard-cell protoplasts of the C₃ plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), V_max and K_{m (PEP · Mg)}, were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mm glucose-6-P_free (Glc-6-P, an activator), and the presence of 5 mm malate_free (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or - and less likely - that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically.The values of V_max were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC V_max were restricted to a small range (52.4 ± 5.9 (SD) to 64.4 ± 4.8 (SD) mol · g fresh mass^-1 · h^-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC V_max was also restricted to a small range: 7.48 ± 0.89 (SD) pmol · guard-cell pair^-1 · h^-1 (pH 8.1, light, +Glc-6-P) to 5.79 ± 0.60 (SD) pmol · guard-cell pair^-1 · h^-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in K_{m (PEP · Mg}) were calculated (whole-leaf PEPC: 0.03 to 3.84 mm; guard-cell PEPC: 0.06 to 3.43 mm). For both extracts, the low values were obtained at pH 8.1, +Glc-6-P, and the high values at pH 7.0, +malate. Although the ranges of K_m values were broadly similar, the PEPCs reacted differently to individual changes in assay components. In very general terms, whole-leaf PEPC was relatively more efficient at pH 8.1, whereas at pH 7.0, the enzymes behaved more similarly.An effect of in vivo pre-illumination on guard-cell PEPC was not detected. A leaf pre-illumination effect on whole-leaf PEPC was highly statistically significant when assayed under control conditions at pH 7.0. The effect was small - typically a 26% decrease in K_{m (PEP · Mg}) this typical decrease was less than the range of values in replicate experiments. Such a small pre-illumination effect (even if real) could, therefore, easily go undetected. Whether such a small change could have physiological relevance is an open question. Neither with the whole-leaf PEPC nor with the guard-cell PEPC was the IC₅₀ (malate) or A_0.5 (Glc-6-P) determined for any condition. These kinetic parameters are a focus of present work.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .