Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Nitrogen fixation and assimilation efficiency in Ethiopian and German pea varieties

Dinitrogen (N₂) fixation and assimilation efficiency in a German and two Ethiopian varieties of Pisum sativum L. was studied in a pot experiment during vegetative and reproductive growth. The objective of the study was to assess whether genotypes having contrasting growth habits showed differences in physiological processes that affect the efficiency of N₂ fixation and assimilation. Dry matter formation, nodulation and nitrogen assimilation were compared between two treatments where one depended solely on N₂ fixation while the other was nourished with nitrate. Moreover, carbon (C) costs of N₂ fixation and the capacity of different respiratory chains in roots and nodules were determined at vegetative and reproductive growth. As compared to the Ethiopian cultivars, the German variety displayed a more rapid vegetative growth with intensive N₂ fixation and assimilation and highly efficient individual nodules. However, during reproductive growth, N₂ fixation in the German variety declined sharply, while continuing in the Ethiopian varieties. Lowest C costs of N₂ fixation coincided with most efficient individual nodules in both growth intervals. C costs of N₂ fixation were lower during reproductive growth in all varieties which was accompanied by a shift in root/nodule respiratory capacity towards more C efficient respiratory pathways. The results provide further evidence that unreliable nitrogen fixation rates during reproductive growth of pea can be connected with restricted C supply to nodules. One strategy of pea plants to adapt to critical C availability is an increase in capacity of more C efficient root/nodule respiration.     

Photosystem II Cyclic Heterogeneity and Photoactivation in the Diazotrophic, Unicellular Cyanobacterium Cyanothece Species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N₂-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O₂-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (q_N) of PSII increased during N₂ fixation and persisted after treatments known to induce transitions to state 1. The q_N was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, ε, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N₂ fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O₂ evolution, altered dark stability of PSII centers, and substantial changes in q_N.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

Nodulation and biological nitrogen fixation of 80 soybean cultivars in symbiosis with indigenous rhizobia

Eighty soybean cultivars were assessed for their potential for nodulation and nitrogen fixation with indigenous rhizobia in a Nigerian soil. Seventy-six days after planting (DAP) 87%, 3% and 10% of the soybean cultivars had from 0 to 30, 31 to 60 and over 61 nodules/plant, respectively. Only 8% had a nodule dry weight of 600 to 1100 mg/plant. At 84 DAP the proportion of nitrogen derived from the atmosphere (Ndfa) ranged from 0 to 65% 16% of the cultivars derived 51 to 65% of their N₂ from the atmosphere. The diversity of soybean germplasm and the variation in nodulation and N₂ fixation permitted the selection of the five best cultivars in terms of their compatibility with indigenous rhizobia, % Ndfa and the amount of N₂ which they fixed.     

The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N₂ fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1-3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven-day-old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28-day-old plants were treated with 15 mM NO₃^?, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO₃^?, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate-(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short-term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g^?1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g^?1 fresh weight in NOD1-3. Increase in levels of free Asn and in total free amino acids in the shoot following the short-term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate-tolerant mutant NOD1-3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial-nitrate tolerance of nodulation in the hypernodulated NOD1-3 mutant is associated with a lesser change in tissue N following nitrate treatment.     

Identification and characterization of common bean (Phaseolus vulgaris L.) lines well nodulated in the presence of high nitrate

Common bean,Phaseolus vulgaris L., is known to be ‘inefficient’ in nodulation and N₂ fixation although it responds to applied nitrogen. An experiment was conducted to identify and to characterize bean cultivars nodulating in the presence of a high level of nitrogen. Sixteen cultivars and a check for inefficient nodulation, OAC Seaforth, were inoculated and grown for 40 days in replicated pots supplied with zero, 3.5 and 10.5 mM combined nitrogen as NO ₃ ⁻ and NH ₄ ⁺ . Seven traits relating to nodulation and N₂ fixation were all significantly affected by N level (N), cultivar (Cv) and N × Cv interactions (except for root dry weight), indicating that cultivars responded differently to the N treatments. Total dry weight (W) and shoot to root ratio (S/R) increased with the increased N levels. Nodule dry weight (Wn), visual nodulation score (Nv) and nodulation index (Nx) decreased as the N increased. Percent N and N content per plant increased with the increased N level. Plant weight (W) was positively correlated with Wn, Nv and N content and negatively correlated with %N. Nodulation score was positively associated with Wn and plant N content. Genotypes superior in nodulation and N₂ fixation in the presence of N were identified. Cultivars Italian Barlotti, California Light Red Kidney, Kentucky Wonder A and Pueblo 152 were selected for further testing and use in improving the nitrate tolerant nodulating characteristic of bean.     

Diurnal change in psychological and physiological responses to consistent relative humidity

Diurnal changes in physiological and psychological responses to consistent relative humidity (RH) conditions were investigated in the present study. Lightly clothed six male and six female subjects participated in the first experiment at 40% and 50% RH, and seven male and seven female subjects participated in the second experiment at 60%, 70%, and 80% RH. Both experiments were conducted at 28 °C air temperature (T_a) from 9:00–18:30. Skin temperatures, local heat flux rates and tympanic temperature (T_ty) were monitored at 2-min intervals throughout the experimental period. Body weight loss and oxygen consumption rate were measured during the 9:30–10:30, 13:30–14:30, and 17:30–18:30 periods. Thermal sensation and thermal comfort responses were recorded at the same periods. The amount of heat loss was greater than metabolic heat production (M) in the male subjects but was well balanced with M in the female subjects. A morning increase in T_ty at 50%–80% RH was observed, and mean skin temperature (${\bar{\text{T}}}_{\text{sk}}$) at 70% and 80% RH was significantly higher (p < 0.05) than T_sk at 40% and 50% RH in both subject groups. Although difference in the relationship between thermal sensation and ${\bar{\text{T}}}_{\text{sk}}$ based on sex was confirmed, diurnal changes in thermal sensation were observed in both subject groups based on the responses of “warm” in the morning but “neutral” or “slightly warm” in the evening at 70% and 80% RH. This result demonstrates that high RH may be acceptable in the late afternoon and evening at 28 °C and indicates that dynamic control of RH during the daytime (e.g., low RH in the morning and high RH in late afternoon) may be beneficial to save energy when using air-conditioning.     

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N₂) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N₂ fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N₂ fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N₂ fixation and photosynthesis commonly observed. However, N₂ fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.     

Effects of water stress on N2 fixation and grain yield of Phaseolus vulgaris L.

Most dry bean production in Mexico is under non-irrigated conditions in the semi-arid highlands. One of the most limiting factors is insufficient moisture during the reproductive stage and sometimes during the vegetative stage. The objective of this experiment was to study the effect of drought on nodulation, N₂ fixation and grain yield of beans. The cultivars evaluated were: Flor de Mayo Bajio, Bayocel, Bat-477 and Honduras-35. The treatments were water stress treatments during vegetative or reproductive stage and a control of minimal stress. To measure N₂ fixation, ¹⁵N-labelled fertilizer was used. Data for soil moisture, nodule number and nodule dry weight, as well as, straw and grain yield and total N were taken. The results indicated that water stress during vegetative stage depressed nodulation temporarily, but after watering regularly plants not only recovered completely but were nodulated better than the control. Water stress during the reproductive stage depressed nodulation and after watering there was no recovery. Grain yield was not affected by water stress during vegetative stage but it was reduced when water stress was imposed during the reproductive stage. The percentage of N derived from fixation varied among cultivars but was not affected by water stress treatment. The highest N₂ fixation occurred in Bayocel and Bat-477 and the lowest in Honduras-35 and F.M. Bajio, although the amounts were not as low as in some other reports.     

Relationship between transpiration and respiration in plants during the dark period

Energetic aspects of the relation between transpiration and respiration during the dark period were evaluated. One-year old seedlings of three trees, one bush and one annual plant were grown in controlled conditions. Experiments were performed under uniform environment during the day and two regimes of air relative humidity (RH) during the night, low (50 - 65 %) and high (95 %). For all investigated plant species the dark transpiration rate (E), the free energy of respiratory substrate, the entropy production and the free energy balance (FEB) of the dark respiration were higher at low than at high RH. E was linearly related to the FEBr ² ranged between 0.63 and 0.90)     

Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.)

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO₂ (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N₂ fixation were largely unaffected by pO₂ from 10 to 40%. Decrease in fixation at pO₂ below 5% was due to lower nodulation and nodule mass and, at pO₂ above 60%, to a fall in specific N₂-fixing activity of nodules. Root:shoot ratios were significantly lower at pO₂ below 2.5%. The effect of pO₂ on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O₂. H₂ evolution showed a sharp optimum at 20% O₂ but was at most 4% of total nitrogenase activity. The ratio of CO₂ evolved to substrate (C₂H₂+H⁺) reduced by crown root nodulation zones was constant (6 moles CO₂ per mole substrate reduced) from 2.5 to 60% O₂ but at levels below 2.5 and above 80% O₂ reached values between 20 and 30 moles CO₂ per mole substrate reduced. Effects of long-term growth with nonambient pO₂ on adaptation and efficiency of functioning of nodules are discussed.     

A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation

N₂ fixation in lines of Phaseolus vulgaris was measured by ¹⁵N-isotope dilution to determine whether a programme of crossing and recurrent selection had resulted in enhanced nitrogen fixation. In field experiments on an isohyperthermic Aquic Hapludoll soil the amount of N₂ fixed by the different lines ranged from 18 to 36 kg ha^–1 (32 to 47% of plant N) in 56 days. The quantity of N₂ fixed and the proportion of plant N derived from fixation was not significantly greater in the lines selected for N₂ fixation (RIZ lines) than parental lines. Total shoot N ranged from 53 to 77 kg ha^–1 and partitioning of N to pods differed from 28 to 52% among the lines which all had similar growth habit and duration. Nodulation patterns were also distinct. Nodules formed early (10 to 15 plant^–1 at 13 days) in many lines, and smallest amounts of fixation were observed in those lines which nodulated slowly and did not form substantial nodule mass until after 40 days. The screening criteria used in the selection of the RIZ lines had been largely indirect with other factors such as disease resistance also being included. Progress for increasing N₂ fixation over good-fixing parental lines such as BAT76 was not significant and it is recommended that more attention be paid to early nodulation, to the use of soils with lower available N and to inter-crossing of lines having different good N₂ fixation traits in order to further enhance the potential for N₂ fixation in beans.     

Seasonal variation in nitrogen fixation and effects of climate change in a subarctic heath

Background and aims

Nitrogen fixation associated with cryptogams is potentially very important in arctic and subarctic terrestrial ecosystems, as it is a source of new nitrogen (N) into these highly N limited systems. Moss-, lichen- and legume-associated N₂ fixation was studied with high frequency (every second week) during spring, summer, autumn and early winter to uncover the seasonal variation in input of atmospheric N₂ to a subarctic heath with an altered climate.

Methods

We estimated N₂ fixation from ethylene production by acetylene reduction assay in situ in a field experiment with the treatments: long- vs. short-term summer warming using plastic tents and litter addition (simulating expansion of the birch forest).

Results

N₂ fixation activity was measured from late April to mid November and 33 % of all N₂ was fixed outside the vascular plant growing season (Jun–Aug). This substantial amount underlines the importance of N₂ fixation in the cold period. Warming increased N₂ fixation two- to fivefold during late spring. However, long-term summer warming tended to decrease N₂ fixation outside the treatment (tents present) period. Litter alone did not alter N₂ fixation but in combination with warming N₂ fixation increased, probably because N₂ fixation became phosphorus limited under higher temperatures, which was alleviated by the P supply from the litter.

Conclusion

In subarctic heath, the current N₂ fixation period extends far beyond the vascular plant growing season. Climate warming and indirect effects such as vegetation changes affect the process of N₂ fixation in different directions and thereby complicate predictions of future N cycling.     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.