Ueli A. Hartwig

Stimulation of Symbiotic N2 Fixation in Trifolium repens L. under Elevated Atmospheric pCO2 in a Grassland Ecosystem.

Symbiotic N2 fixation is one of the main processes that introduces N into terrestrial ecosystems. As such, it may be crucial for the sequestration of the extra C available in a world of continuously increasing atmospheric CO2 partial pressure (pCO2). The effect of elevated pCO2 (60 Pa) on symbiotic N2 fixation (15N-isotope dilution method) was investigated using Free-Air-CO2-Enrichment technology over a period of 3 years. Trifolium repens was cultivated either alone or together with Lolium perenne (a nonfixing reference crop) in mixed swards. Two different N fertilization levels and defoliation frequencies were applied. The total N yield increased consistently and the percentage of plant N derived from symbiotic N2 fixation increased significantly in T. repens under elevated pCO2. All additionally assimilated N was derived from symbiotic N2 fixation, not from the soil. In the mixtures exposed to elevated pCO2, an increased amount of symbiotically fixed N (+7.8, 8.2, and 6.2 g m-2 a-1 in 1993, 1994, and 1995, respectively) was introduced into the system. Increased N2 fixation is a competitive advantage for T. repens in mixed swards with pasture grasses and may be a crucial factor in maintaining the C:N ratio in the ecosystem as a whole.

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

Abstract 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 plants 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 plants 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.

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

Abstract 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.

Microbial community changes in the rhizospheres of white clover and perennial ryegrass exposed to Free Air Carbon dioxide Enrichment (FACE)

Abstract Increases in the global atmospheric concentration of CO 2 will not only directly affect the growth of plants, but might also alter the living conditions for soil biota. This could lead to shifts in the size and composition of the soil microbial communities. In this study we investigated the response of heterotrophic bacteria, NH 4 + -oxidising bacteria, and Rhizobium leguminosarum bv. trifolii populations to elevated atmospheric CO 2 concentrations in a model field-scale grassland ecosystem. The Free Air CO 2 Enrichment (FACE) facility in Eschikon, Switzerland, releases CO 2 -enriched air into three large circular areas, each of 18 m dia, to a final CO 2 concentration of 600 μmol mol −1 , while three control areas of the same size receive ambient CO 2 concentrations (∼350 μmol mol −1 ). For this study, white clover ( Trifolium repens L.) and perennial ryegrass ( Lolium perenne L.) were grown as replicated monocultures within the FACE rings. Soil samples were taken from 0–10 cm depth in May and November 1994 (the second year of CO 2 -enrichment), and rhizosphere soil was obtained from clover and ryegrass roots for enumeration of bacteria. While the total numbers of culturable heterotrophic bacteria (determined by plate counts) in the rhizospheres of both plant species were little affected by CO 2 -enrichment, the populations of R. leguminosarum bv. trifolii (enumerated by MPN) were increased two-fold in the rhizospheres of white clover exposed to elevated atmospheric CO 2 . There was no effect of the CO 2 concentration on the populations of R. leguminosarum bv. trifolii in the rhizospheres of perennial ryegrass, indicating that the increase of Rhizobium numbers is a host-related response to elevated atmospheric CO 2 . The numbers of autotrophic NH 4 + -oxidizing bacteria in the rhizospheres (enumerated by MPN) were unaffected by the atmospheric CO 2 concentration. There was also no effect of the CO 2 concentration on the amount of microbial biomass C in the bulk, non-rhizosphere soils in white clover or perennial ryegrass plots. These data indicate that under a legume crop, at least in terms of inoculum quality in the rhizosphere soil, symbiotic nitrogen-fixing organisms might be favoured by elevated atmospheric CO 2 concentrations.

Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa

Abstract. Open-flow assays of H2 evolution in Ar:O2 (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H2 evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial pressure of O2 in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual nodule mass, H2 evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule O2 permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule respiration and energetic metabolism is proposed.

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plants resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (15N enriched NH4NO3). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.

Due to symbiotic N2 fixation, five years of elevated atmospheric pCO2 had no effect on the N concentration of plant litter in fertile, mixed grassland

Experimental findings indicate that, in terrestrial ecosystems, nitrogen cycling changes under elevated partial pressure of atmospheric CO2 (pCO2). It was suggested that the concentration of N in plant litter as well as the amount of litter are responsible for these changes. However, for grassland ecosystems, there have been no relevant data available to support this hypothesis. Data from five years of the Swiss FACE experiment show that, under fertile soil conditions in a binary plant community consisting of Lolium perenne L. and Trifolium repens L., the concentration of litter N does not change under elevated atmospheric pCO2; this applies to harvest losses, stubble, stolons and roots as the sources of litter. This is in strong contrast to the CO2 response of L. perenne swards without associated legumes; in this case the above-ground concentration of biomass N decreased substantially. Increased symbiotic N2 fixation in T. repens nodules and a greater proportion of the N-rich T. repens in the community are regarded as the main mechanisms that buffer the increased C introduction into the ecosystem under elevated atmospheric pCO2. Our data also suggest that elevated atmospheric pCO2 results in greater amounts of litter, mainly due to increased root biomass production. This study indicates that, in a fertile grassland ecosystem with legumes, the concentration of N in plant litter is not affected by elevated atmospheric pCO2 and, thus, cannot explain CO2-induced changes in the cycling of N.

Current Nitrogen Fixation Is Involved in the Regulation of Nitrogenase Activity in White Clover (Trifolium repens L.)

Previous studies have shown that nitrogenase activity decreases dramatically after defoliation, presumably because of an increase in the O2 diffusion resistance in the infected nodules. It is not known how this O2 diffusion resistance is regulated. The aim of this study was to test the hypothesis that current N2 fixation (ongoing flux of N2 through nitrogenase) is involved in the regulation of nitrogenase activity in white clover (Trifolium repens L. cv Ladino) nodules. We compared the nitrogenase activity of plants that were prevented from fixing N2 (by continuous exposure of their nodulated root system to an Ar:O2 [80:20] atmosphere) with that of plants allowed to fix N2 (those exposed to N2:O2, 80:20). Nitrogenase activity was determined as the amount of H2 evolved under Ar:O2. An open flow system was used. In experiment I, 6 h after complete defoliation and the continuous prevention of N2 fixation, nitrogenase activity was higher by a factor of 2 compared with that in plants allowed to fix N2 after leaf removal. This higher nitrogenase activity was associated with a lower O2 limitation (measured as the partial pressure of O2 required for highest nitrogenase activity). In experiment II, the nitrogenase activity of plants prevented from fixing N2 for 2 h before leaf removal showed no response to defoliation. The extent to which nitrogenase activity responded to defoliation was different in plants allowed to fix N2 and those that were prevented from doing so in both experiments. This leads to the conclusion that current N2 fixation is directly involved in the regulation of nitrogenase activity. It is suggested that an N feedback mechanism triggers such a response as a result of the loss of the plants N sink strength after defoliation. This concept offers an alternative to other hypotheses (e.g. interruption of current photosynthesis, carbohydrate deprivation) that have been proposed to explain the immediate decrease in nitrogenase activity after defoliation.

Nitrogen plays a major role in leaves when source-sink relations change: C and N metabolism in Lolium perenne growing under free air CO2 enrichment

Swards of Lolium perenne L. were grown in the field in a long-term free air CO2 enrichment (FACE) facility. The CO2 treatment was combined with two levels of N fertilization and regular defoliation, which resulted in plants with a wide range of source-sink relations. C and N metabolism were investigated to assess the role of carbohydrate and nitrogenous compounds in leaves in indicating source-sink relations. Sucrose exhibited the largest changes in contents during the day-night cycle; therefore, it was identified as the main short-term storage compound for night-time export. Fructan accumulation indicated the degree of surplus C supply in the source compared to C use in sinks. Nitrate content depended mainly on N fertilization, and was reduced under elevated pCO2. Nitrate appeared to indicate a current surplus of available N relative to the need for growth. Amino acid content responded strongly to N fertilization but decreased only slightly under elevated pCO2. Protein content, however, decreased significantly under elevated pCO2. The patterns of diurnal changes of C or N compounds did not differ between CO2 treatments. Down-regulation of photosynthesis appeared to occur when plants were extremely N-limited as under elevated pCO2, low N and at a late regrowth stage.

Symbiotic N2 fixation increases under elevated atmospheric pCO2 in the field

Abstract Plant growth is stimulated by elevated atmospheric pCO2, and hence demand for nutrients increases. In this context, nitrogen is a very prominent element; it can either be supplied from the limited available soil N or through biological (e.g. symbiotic) nitrogen fixation. In this study, the effect of elevated pCO2 (60 Pa) on symbiotic N2 fixation (15N-isotope dilution method) was investigated using Free-Air-CO2-Enrichment (FACE) technologh over a period of two growing seasons. Trifolium repens L. was cultivated either alone or in mixed swards together with Lolium perenne L. (non-fixing reference crop). In T. repens, percentage of plant N derived from symbiotic N2 fixation (%Nsym) increased from 59 to 66% under elevated pCO2. The major part of the additionally assimilated N was derived from symbiotic N2 fixation. In the mixed swards, increased N yield was entirely due to increased symbiotic N2 fixation. It is suggested that increased N2 fixation is an important factor in the satisfaction of increased N demand in both clover and the associated grass under elevated pCO2.