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Plant Ecology, 180 (1), ISSN:1385-0237, ISSN:1573-5052

Trifolium repens L. was grown to test the following hypotheses: when P is deficient (i) N 2 fixation decreases as a result of the plant's adaptation to the low N demand, regulated by an N feedback mechanism, and (ii) the decrease in the photosynthetic capacity of the leaves does not limit N 2 fixation. Severe P deficiency prevented nodulation or stopped nodule growth when the P deficiency occurred after the plants had formed nodules. At low P, the proportion of whole-plant-N derived from symbiotic N 2 fixation decreased, whereas specific N 2 fixation increased and compensated partially for poor nodulation. Leaf photosynthesis was reduced under P deficiency due to low V c,max and J max . Poor growth or poor performance of the nodules was not due to C limitation, because (i) the improved photosynthetic performance at elevated P CO2 had no effect on the growth and functioning of the nodules, (ii) starch accumulated in the leaves, particularly under elevated P CO2 , and (iii) the concentration of WSC in the nodules was highest under P deficiency. Under severe P deficiency, the concentrations of whole-plant-N and leaf-N were the highest, indicating that the assimilation of N exceeded the amount of N required by the plant for growth. This was clearly demonstrated by a strong increase in asparagine concentrations in the roots and nodules under low P supply. This indicates that nodulation and the proportion of N derived from symbiotic N 2 fixation are down-regulated by an N feedback mechanism.

Accumulation of dry weight was measured in plant parts of meadow fescue (Festuca pratensis Huds.) that was grown at 16/11 °C or 26/21 °C and with 20 or 60 nE cm-2 s_1 photosynthetically active radiation. Plants reached anthesis about 3 weeks later at 16/11 °C than at 26/21 °C and had then a higher proportion of dry weight in inflorescences and less in leaf blades. Growth temperature had little effect on C02 exchange rate (CER) but plants grown at 60 nE cm-2 s_1 had higher CER than those grown at 20 nE cm-2 s-1. The concentration of water-soluble carbohydrates (WSC) at similar growth stages was usually higher at 16/11 °C than at 26/21 °C. High radiation also led to higher WSC in stem and leaf tissue. Root tissue changed least and WSC did not exceed 10% of dry weight during the experiment. In all tissues, when WSC was high, the fructans were distributed into a group with a high degree of polymerization (DP) and another with a low DP. The low DP group included sucrose, reducing sugars and fructans up to about 20 units long. An apparent threshold concentration of WSC was necessary for synthesis of the high DP fructans. This concentration was near 12% for leaf tissue, about 6% for stem base tissue, and 2-5% for root tissue. The average apparent DP of the high DP fructan group was 43 to 50 for leaf tissue, 31 to 93 for stem base tissue, and 27 to 31 for roots. These characteristics appeared to be mostly tissue dependent with less effect from temperature and radiation.

Lehnherr, B., Mächler, F. and Nösberger, J. 1985. Influence of temperature on the ratio of ribulose bisphosphate carboxylase to oxygenase activities and on the ratio of photosynthesis to photorespiration of leaves.—J. exp. Bot. 36: 1117-1125. Rates of net and gross photosynthesis of intact white clover leaves were measured by infrared gas analysis and by short term uptake of 14CO2 respectively. Ribulose bisphosphate carboxylase oxygenase (RuBPCO) was purified from young leaves and kinetic properties investigated in combined and separate assays. The ratio of carboxylase to oxygenase activities was compared with the ratio of photosynthesis to photorespiration at various temperatures and CO2 concentrations. The ratio of photosynthesis to photorespiration at 30 Pa p(CO2) was consistent with the ratio of carboxylase activity to oxygenase activity when each was measured above 20 °C. However, the ratio of photosynthesis to photorespiration increased with decreasing temperature, whereas the ratio of carboxylase to oxygenase activity was independent of temperature. This resulted in a disagreement between the measurements on the purified enzyme and intact leaf at low temperature. No disagreement between enzyme and leaf at low temperature occurred, when the ratio of photosynthesis to photorespiration was determined at increased CO2 concentrations. The results suggest an effect of low temperature and low CO2 concentration on the ratio of photosynthesis to photorespiration independent of the enzyme

Model projections suggest that although increased temperature and decreased soil moisture will act to reduce global crop yields by 2050, the direct fertilization effect of rising carbon dioxide concentration ([CO 2 ]) will offset these losses. The CO 2 fertilization factors used in models to project future yields were derived from enclosure studies conducted approximately 20 years ago. Free-air concentration enrichment (FACE) technology has now facilitated large-scale trials of the major grain crops at elevated [CO 2 ] under fully open-air field conditions. In those trials, elevated [CO 2 ] enhanced yield by ∼50% less than in enclosure studies. This casts serious doubt on projections that rising [CO 2 ] will fully offset losses due to climate change.

Summary • In grasslands, the loss of structural carbon (C) from nonharvested plant parts is a primary C source for the soil. The amount of input depends not only on the size of structural C pools but also on their loss rates. • In the field, we examined the effects of elevated atmospheric partial pressures of CO2 (pCO2) and nitrogen (N) supply on pool size and rates of structural C loss in stubble and roots of perennial ryegrass (Lolium perenne) by using multiple-pulse labelling and steady-state labelling. • Stubble retained structural C for roughly half the time it was retained in roots. Elevated pCO2 combined with low N supply enlarged the pools of roots and stubble. These conditions also stimulated the rate of structural C loss from stubble and, thus, the amounts available for further transformation. • The potential of multiple-pulse labelling as a field technique is highlighted. The stimulation of structural C loss from stubble by elevated pCO2 at low N provides a missing link between increased C assimilation and low yield response and indicates a potentially higher input of structural C into the soil.

The effects of elevated pCO2 on net ecosystem CO2 exchange were investigated in managed Lolium perenne (perennial ryegrass) and Trifolium repens (white clover) monocultures that had been exposed continuously to elevated pCO2 (60 Pa) for nine growing seasons using Free Air CO2 Enrichment (FACE) technology. Two levels of nitrogen (N) fertilization were applied. Midday net ecosystem CO2 exchange (mNEE) and night-time ecosystem respiration (NER) were measured in three growing seasons using an open-flow chamber system. The annual net ecosystem carbon (C) input resulting from the net CO2 fluxes was estimated for one growing season. In both monocultures and at both levels of N supply, elevated pCO2 stimulated mNEE by up to 32%, the exact amount depending on intercepted PAR. The response of mNEE to elevated pCO2 was larger than that of harvestable biomass. Elevated pCO2 increased NER by up to 39% in both species at both levels of N supply. NER, which was affected by mNEE of the preceding day, was higher in T. repens than in L. perenne. High N increased NER compared to low N supply. According to treatment, the annual net ecosystem C input ranged between 210 and 631 g C m - -2 year -1 and was not significantly affected by the level of pCO2. Low N supply led to a higher net C input than high N supply. We demonstrated that at the ecosystem level, there was a long-term stimulation in the net C assimilation during daytime by elevated pCO2. However, because NER was also stimulated, net ecosystem C input was not significantly increased at elevated pCO2. The annual net ecosystem C input was primarily affected by the amount of N supplied.

Human activities are responsible for the strong and very fast increase in the concentration of atmospheric CO2 over the past 100 years; this trend will continue. An increase in CO2 is expected to affect significantly grassland vegetation, which covers approximately 70% of agricultural land world-wide and undoubtedly plays an important role in the global C cycle. This review summarises the information about the response of temperate, intensively managed grassland to elevated atmospheric CO2 under field conditions. 1. The yield response of grassland to elevated CO2 under field conditions was weaker than expected from short-term laboratory experiments. 2. Different functional types of plant species responded differently to elevated CO2, leading to changes in the proportion of plant species in grassland. 3. Adapted management of grassland was able to counteract—at least partly—CO2-induced changes in species proportion. 4. The availability of growth resources other than CO2 (e.g., N and P) strongly affected the plant’s response to elevated CO2. 5. Symbiotic N2 fixation and a strong sink for additionally fixed C (high growth rate, tillering, storage organs) were the functional traits that played a key role in the strong increase in yield under elevated CO2. 6. An increased input of N through symbiotic N2 fixation under elevated CO2 played an important role in maintaining the C/N balance of the legume plant and of entire fertile grassland ecosystems. 7. The yield response to elevated CO2 changed in the long-term indicating that processes in the soil that responded slowly (e.g., nutrient cycling and sequestration, activity and mass of micro-organisms) gradually adapted to the new environmental conditions. These results demonstrate that the response of ecosystems to elevated CO2 cannot be deduced from measurements based on individual plants in controlled environments. Long-term experiments in the field where the availability of growth resources is varied, the plants interact with each other and the soil interacts with the plants (feed-back mechanisms) are necessary for predicting the effect of elevated CO2.

Effects of free-air carbon dioxide enrichment (FACE, 60 Pa pCO2) on plant growth as compared with ambient pCO2 (36 Pa) were studied in swards of Lolium perenne L. (perennial ryegrass) at two levels of N fertilization (14 and 56 g m−2 a−1) from 1993 to 2002. The objectives were to determine how plant growth responded to the availability of C and N in the long term and how the supply of N to the plant from the two sources of N in the soil, soil organic matter (SOM) and mineral fertilizer, varied over time. In three field experiments, 15N-labelled fertilizer was used to distinguish the sources of available N. In 1993, harvestable biomass under elevated pCO2 was 7% higher than under ambient pCO2. This relative pCO2 response increased to 32% in 2002 at high N, but remained low at low N. Between 1993 and 2002, the proportions and amounts of N in harvestable biomass derived from SOM (excluding remobilized fertilizer) were, at high N, increasingly higher at elevated pCO2 than at ambient pCO2. Two factorial experiments confirmed that at high N, but not at low N, a higher proportion of N in harvestable biomass was derived from soil (including remobilized fertilizer) following 7 and 9 years of elevated pCO2, when compared with ambient pCO2. It is suggested that N availability in the soil initially limited the pCO2 response of harvestable biomass. At high N, the limitation of plant growth decreased over time as a result of the stimulated mobilization of N from soil, especially from SOM. Consequently, harvestable biomass increasingly responded to elevated pCO2. The underlying mechanisms which contributed to the increased mobilization of N from SOM under elevated pCO2 are discussed. This study demonstrated that there are feedback mechanisms in the soil which are only revealed during long-term field experiments. Such investigations are thus, a prerequisite for understanding the responses of ecosystems to elevated pCO2 and N supply.

The initial stimulation of photosynthesis observed on elevation of [CO 2 ] in grasslands has been predicted to be a transient phenomenon constrained by the loss of photosynthetic capacity due to other limitations, notably nutrients and sinks for carbohydrates. Legumes might be expected partially to escape these feedbacks through symbiotic N 2 fixation. The Free-Air Carbon dioxide Enrichment (FACE) experiment at Eschikon, Switzerland, has been the longest running investigation of the effects of open-air elevation of [CO 2 ] on vegetation. The prediction of a long-term loss of photosynthetic capacity was tested by analysing photosynthesis in Trifolium repens L. (cv. Milkanova) in the spring and autumn of the eighth, ninth and tenth years of treatment. A high and low N treatment also allowed a test of the significance of exogenous N-supply in maintaining a stimulation of photosynthetic capacity in the long-term. Prior work in this Free Air CO 2 Enrichment (FACE) experiment has revealed that elevated [CO 2 ] increased both vegetative and reproductive growth of T. repens independent of N treatment. It is shown here that the photosynthetic response of T. repens was also independent of N fertilization under both current ambient and elevated (600 μmol mol -1 ) [CO 2 ]. There was a strong effect of season on photosynthesis, with light-saturated rates (A sat ) 37% higher in spring than in autumn. Higher A sat in the spring was supported by higher maximum Rubisco carboxylation rates (V c,max ) and maximum rates of electron transport (J max ) contributing to RuBP regeneration. Elevated [CO 2 ] increased A sat by 37% when averaged across all measurement periods and both N fertilization levels, and decreased stomatal conductance by 25%. In spring, there was no effect of elevated [CO 2 ] on photosynthetic capacity of leaves, but in autumn both V c,max and J max were reduced by approximately 20% in elevated [CO 2 ]. The results show that acclimation of photosynthetic capacity can occur in a nitrogen-fixing species, in the field where there are no artificial restrictions on sink capacity. However, even with acclimation there was a highly significant increase in photosynthesis at elevated [CO 2 ].

Plant response to increasing atmospheric CO 2 partial pressure ( p CO 2 ) depends on several factors, one of which is mineral nitrogen availability facilitated by the mineralisation of organic N. Gross rates of N mineralisation were examined in grassland soils exposed to ambient (36 Pa) and elevated (60 Pa) atmospheric p CO 2 for 7 years in the Swiss Free Air Carbon dioxide Enrichment experiment. It was hypothesized that increased below-ground translocation of photoassimilates at elevated p CO 2 would lead to an increase in immobilisation of N due to an excess supply of energy to the roots and rhizosphere. Intact soil cores were sampled from Lolium perenne and Trifolium repens swards in May and September, 2000. The rates of gross N mineralisation ( m ) and NH 4 + consumption ( c ) were determined using 15 N isotopic dilution during a 51-h period of incubation. The rates of N immobilisation were estimated either as the difference between m and the net N mineralisation rate or as the amount of 15 N released from the microbial biomass after chloroform fumigation. Soil samples from both swards showed that the rates of gross N mineralisation and NH 4 + consumption did not change significantly under elevated p CO 2 . The lack of a significant effect of elevated p CO 2 on organic N turnover was consistent with the similar size of the microbial biomass and similar immobilisation of applied 15 N in the microbial N pool under ambient and elevated p CO 2 . Rates of m and c , and microbial 15 N did not differ significantly between the two sward types although a weak ( p p CO 2 by sward interaction occurred. A significantly larger amount of NO 3 − was recovered at the end of the incubation in soil taken from T. repens swards compared to that from L. perenne swards. Eleven percent of the added 15 N were recovered in the roots in the cores sampled under L. perenne , while only 5% were recovered in roots of T. repens . These results demonstrate that roots remained a considerable sink despite the shoots being cut at ground level prior to incubation and suggest that the calculation of N immobilisation from gross and net rates of mineralisation in soils with a high root biomass does not reflect the actual immobilisation of N in the microbial biomass. The results of this study did not support the initial hypothesis and indicate that below-ground turnover of N, as well as N availability, measured in short-term experiments are not strongly affected by long-term exposure to elevated p CO 2 . It is suggested that differences in plant N demand, rather than major changes in soil N mineralisation/immobilisation, are the long-term driving factors for N dynamics in these grassland systems.

Photosynthesis is commonly stimulated in grasslands with experimental increases in atmospheric CO 2 concentration ([CO 2 ]), a physiological response that could significantly alter the future carbon cycle if it persists in the long term. Yet an acclimation of photosynthetic capacity suggested by theoretical models and short-term experiments could completely remove this effect of CO 2 . Perennial ryegrass ( Lolium perenne L. cv. Bastion) was grown under an elevated [CO 2 ] of 600 m m m mol mol - 1 for 10 years using F ree A ir C O 2 E nrichment ( FACE ), with two contrasting nitrogen levels and abrupt changes in the source : sink ratio following periodic harvests. More than 3000 measurements characterized the response of leaf photosynthesis and stomatal conductance to elevated [CO 2 ] across each growing season for the duration of the experiment. Over the 10 years as a whole, growth at elevated [CO 2 ] resulted in a 43% higher rate of light-saturated leaf photosynthesis and a 36% increase in daily integral of leaf CO 2 uptake. Photosynthetic stimulation was maintained despite a 30% decrease in stomatal conductance and significant decreases in both the apparent, maximum carboxylation velocity ( V c,max ) and the maximum rate of electron transport ( J max ). Immediately prior to the periodic (every 4‐8 weeks) cuts of the L. perenne stands, V c,max and J max, were significantly lower in elevated than in ambient [CO 2 ] in the low-nitrogen treatment. This difference was smaller after the cut, suggesting a dependence upon the balance between the sources and sinks for carbon. In contrast with theoretical expectations and the results of shorter duration experiments, the present results provide no significant change in photosynthetic stimulation across a 10-year period, nor greater acclimation in V c,max and J max in the later years in either nitrogen treatment.

Plant competition strongly affects the species composition of managed grassland. To identify relevant processes, Festuca pratensis (Huds.) and Dactylis glomerata (L.) were grown as monocultures or mixtures in boxes placed in the field for two seasons and subjected to two cutting frequencies. Root and shoot competition effects were separated using soil and aerial partitions. Shoot competition was analysed by measuring the vertical distribution of the leaf area and root competition by analysing the absorption of tracers. Values of relative yield indicated that the two grasses fully competed for the same limiting resources under the experimental conditions. The competitive ability of F. pratensis was lower during both years relative to D. glomerata. This was mainly related to its consistently lower shoot competitive ability, which was associated with less leaf area in the upper layers of the canopy and shorter leaves. Root competitive ability of F. pratensis changed with season. It was similar to that of D. glomerata during spring and autumn, but less during summer. The lower root competitive ability in summer might be due to the lower root activity of F. pratensis, measured as rubidium (Rb) and strontium (Sr) absorption in July, 0·1 and 0·2 m below the ground. Cutting frequency did not greatly influence the relative importance of root and shoot competition during the first growing season. However, under infrequent defoliation, the competitive ability of F. pratensis decreased markedly in full competition during the second growing season. These results suggest that distinct differences in the canopy structure and root activity of different plant species can affect the relative importance of root and shoot competition during the season.

Journal of Experimental Botany, 53 (371), ISSN:1460-2431, ISSN:0022-0957

To test whether different nitrogen form (nitrate or ammonium) in substrate can alter the response to elevated partial pressure of CO2 (pCO2) plants of perennial ryegrass (Lolium perenne cv. Bastion) were grown from seeds in growth chambers under pCO2 of either 35 Pa (ambient, CA) or 70 Pa (elevated, CE) in a hydroponic system (with nutrient and pH control) for 24 d. Nitrogen was supplied as ammonium, nitrate or an equimolar mixture of both N forms. Under CE plants grew faster than their counterparts under CA during the first 14 d but after 23 d of cultivation stimulation disappeared. Despite the strong positive effect of mixed forms of N on plant growth, the beneficial effect of CE was similar to that in the other two N treatments. However, the almost alike final growth response to CE had different underlying mechanisms in different N treatments. Plants supplied with nitrate as a sole source of nitrogen had lower leaf mass ratio but much higher specific leaf area compared to plants supplied with ammonium. The decrease in the content of leaf organic N (per unit of structural dry mass) under CE was found only in leaves of plants supplied with ammonium on day 14. Nevertheless, the available form of N evidently contributes to changes of leaf N content under CE. The high levels of N and non-structural saccharides in plants supplied with ammonium at CE suggest that the CO2 response of these plants was controlled by factors other than amount of available carbon and nitrogen.

Although there are many indications that N cycling in grassland ecosystems changes under elevated atmospheric CO2 partial pressure (pCO2), most information has been obtained in short-term studies. Thus, N budgets were established for four years under ambient and 60 Pa pCO2 at two levels of N fertilization in two contrasting model ecosystems: Trifolium repens L. (white clover) and Lolium perenne L. (perennial ryegrass) were planted in soil in boxes in the Swiss FACE experiment. While T. repens showed an 80% increase in harvested biomass with no change in biomass allocation under elevated atmospheric pCO2 compared to ambient conditions, L. perenne showed an increase only in the biomass of the roots. During the four years of the experiment, the systems gained N both from N retained in the soil and from stubble/stolon and roots left after the final harvest; in total between 11 and 86 gN m−2. Nitrogen retention in the soil was between 4 and 64 g m2. The L. perenne system gained the most N and retained the most N in the soil at high N fertilization and elevated atmospheric pCO2. The input of new C and N into the soil correlated well in the L. perenne systems but not in the T. repens systems. Elevated atmospheric pCO2 led neither to an increase in N retention in the soil nor did it reduce the loss of N from the soil. In the L. perenne systems, N fertilization played the main role in both the retention of N and the sequestration of C, while in the T. repens systems symbiotic N2 fixation may have controlled N retention in the soil.

The greater sensitivity of white clover (Trifolium repens L.) to low temperature compared with perennial ryegrass (Lolium perenne L.) is a major problem in sustaining the relative contributions to yield of the two species in mixed swards. The objectives of this investigation were to examine the dynamics of leaf development of two white clover cultivars, AberHerald and Grasslands Huia, under field conditions, and to determine the significance of leaf area in winter, and of competition by perennial ryegrass, for the overwintering and regrowth of white clover in spring. Undefoliated white clover plants developed 3·8–6·6 new leaves between late autumn and early spring, and stolon dry matter and total non-structural carbohydrates (TNC) content increased by 262 and by 68% respectively. In contrast, white clover plants that were defoliated frequently during the winter showed a 28% decrease in stolon dry matter and an 82% decrease in the content of TNC. Frequent defoliation in winter caused severe reduction in the rates of emergence of nodes (by 60%) and of buds (by 67%), and the rate of death of nodes and buds increased by a factor of 10, leading to small stolon systems of individual plants in spring. Competition had similar, but weaker, effects to those of winter defoliation, presumably caused by shading of white clover leaves. AberHerald had a higher cumulative leaf emergence (by 22%), a higher mean leaf number (by 23%), a higher stolon DM (by 36%) and a higher TNC content per plant (by 115%) than Grasslands Huia. Results demonstrate the crucial positive role of leaf area during winter, the negative effect of grass competition, and the importance of the clover cultivar, for the overwintering and subsequent spring regrowth of white clover., Annals of Botany, 88 (s1), ISSN:1095-8290, ISSN:0305-7364

International audience; A common experimental protocol was followed at 12 sites in Europe to study the development of two clover cultivars (AberHerald and Huia) grown in association with a ryegrass cultivar throughout their annual cycle. The duration of the experiment was between 1 and 3 years at each site. Detailed information about the morphogenesis and carbohydrate reserves of white clover during winter and the subsequent productivity of clover plants during the growing season was collected. Yield of the companion ryegrass was also measured at several harvests during the growing season and grass tiller density was recorded several Limes during winter, There was wide variation among sites and between years in climatic conditions, in the growth characteristics and chemical composition of the two cultivars, and in the tiller density of the ryegrass. The relative performance of the two cultivars varied among sites but AberHerald generally outperformed Huia, Major changes in plant characteristics (morphology, population size. chemical composition, etc,) occurred during overwintering. This paper presents a preliminary analysis of the effects of site. clover cultivar and sward age (years) on various plant characteristics. It is established that the data provide a unique basis for modelling the effects of a wide range of environmental conditions and plant properties on the performance of white clover in mixed swards. A modelling approach that seeks to replace site and year by climatic variables characterizing each site by year combination is developed in two companion papers.

Festuca pratensis Huds. is a forage grass with the ability to withstand harsh climatic conditions. However, its potential agronomic use is limited by its poor competitive ability, which can be traced to limitations in leaf growth. In order to characterize this process and to identify genes which might function as markers for leaf growth, three XET-related genes in the leaf elongation zone (LEZ) of F. pratensis are reported. A detailed expression analysis is presented of the three genes in two F. pratensis genotypes with contrasting leaf growth characteristics grown under two nitrogen levels. By means of a detailed spatial analysis of growth and XET encoding transcript pattern along the LEZ, a specific correlation is shown between FpXET1 expression and tissue elongation that is maintained under the different growth conditions, while the two other XETs expressed in the LEZ show different transcript dynamics. Tissue localization of FpXET1 and FpXET2 transcripts indicate an accumulation throughout young tissue, which is consistent with the encoded proteins playing roles in cell wall modification processes during growth. It is proposed that FpXET1 is a potential marker for tissue elongation and leaf growth in F. pratensis.

○ Plant response to elevated atmospheric CO 2 may depend on the carbon sink strength, determined by the availability of resources other than CO 2 , and the developmental stage. ○ In a 2-yr field experiment with model swards of Lolium perenne, the effect of CO 2 enrichment (FACE) on yield and allocation of dry mass (DM) and N were examined under three N fertilization treatments during vegetative and reproductive growth. ○ During vegetative growth, in the highest N treatment, the greatest increase in DM yield occurred at elevated CO 2 ; there was no change in DM allocation. By contrast, at low N, residual biomass, but not yield, increased under CO 2 enrichment, and the tillers were shorter. During reproductive growth, under CO 2 enrichment DM yield increased similarly across all N treatments; there was no change in DM and N partitioning. The mean weight and height of the reproductive tillers increased. ○ At high N availability, or during reproductive growth, L. perenne swards overcome carbon-sink limitation and show a strong yield response to elevated CO 2 . Biomass allocation and the height of the plants, in response to elevated CO 2 , clearly depend on N fertilization and developmental stage.

At elevated partial pressure of CO 2 (60 Pa pCO 2 ), field-grown perennial ryegrass (Lolium perenne L.) swards generally show a strong increase in photosynthesis of the youngest fully expanded leaves but a weak yield response. We investigated this discrepancy by determining the effects of elevated pCO 2 on the dynamics of regrowth. During the whole regrowth period, swards at elevated pCO 2 exhibited a greater dry mass of roots (68%), of pseudostems (34%), and of shoot necromass below cutting height (45%). During the first 4 wk of regrowth, when dry mass (DM) and the leaf area index increased strongly, elevated pCO 2 stimulated the yield, total DM, and leaf area by up to 46, 63, and 30%, respectively. Elevated pCO 2 also resulted in an increase of almost 60% in the number of tillers during this period when the transmission of photosynthetically active radiation to the tiller bases was high. Towards the end of regrowth, however, the leaf area, total DM, and yield showed no net increase; under elevated pCO 2 , the number of tillers decreased strongly. These results indicate that there was not a strong sink for additionally fixed C during the second half of regrowth. The gap between leaf photosynthesis and yield at elevated pCO 2 is suggested to be related to a loss of tillers when shading of tiller bases was strong and to a change in dry mass allocation.

Tropical food legumes are grown in a wide range of environments, and water stress is considered the principal environmental factor limiting growth and yield. Potassium fertilizer mitigates the impact of water stress in plants. However, the benefits of potassium in overcoming stress in tropical food legumes have not been investigated in comparative studies. The purpose of this study was to determine the benefits of potassium in overcoming water stress in mungbean and cowpea, two important tropical food legumes with different adaptabilities to soil moisture regimes. The experiment carried out under controlled conditions placed emphasis on vegetative growth and selected physiological parameters. The impact of potassium was different in the two legumes grown at optimal and suboptimal soil moisture. Potassium increased shoot growth of mungbean to a greater extent than in cowpea under suboptimal moisture conditions. The roots of cowpea showed a greater response to potassium fertilizer than in mungbean under suboptimal soil moisture. The plant water relations and photosynthetic rates of mungbean were improved to a greater extent by potassium under suboptimal soil moisture than those of cowpea. Although differences were observed in the responses of the vegetative growth of these species to moisture and potassium, in overall terms potassium promoted growth of both species when subject to suboptimal soil moisture. While field studies are required to validate the results, the application of potassium fertilizer can be considered a significant factor in overcoming soil moisture stress in these legumes commonly grown in tropical cropping systems.

Ryegrasses, like many C3 plants, accumulate fructan, which plays an important role in assimilate partitioning, as the major non-structural storage carbohydrate. The present study describes the transformation of a Bacillus subtilis sacB gene, with vacuolar targeting signal sequences and driven by constitutive promoters, into Italian ryegrass (Lolium multiflorum Lam.) by microprojectile bombardment of embryogenic suspension cells. The expression of the chimeric sacB genes in transgenic ryegrass plants and the concomitant accumulation of low levels of bacterial levan were found to substantially distort the native grass fructan synthesis pattern. High-molecular-weight native fructan was depleted, and the pattern of accumulation of oligosaccharides in the range of 5–35 degree of polymerization was altered. The levan-accumulating sacB-transgenic ryegrass plants had a lower level of total fructose, unchanged sucrose levels, and slightly reduced hexose levels compared to the isogenic controls. Growth of the levan-accumulating sacB-transgenic ryegrass plants slowed down with the onset of the reproductive phase. Flowering plants were stunted and had narrower leaves and poorly developed roots. The association between the manipulated fructan metabolism and the phenotype of the levan-accumulating sacB-transgenic ryegrass plants is discussed.

Grasses contain a number of genes encoding both α- and β-expansins. These cell wall proteins are predicted to play a role in cell wall modifications, particularly during tissue elongation. We report here on the characterisation of five α- and three vegetative β-expansins expressed in the leaf elongation zone (LEZ) of the forage grass, Festuca pratensis Huds. The expression of the predominant α-expansin (FpExp2) was localised to the vascular tissue, as was the β-expansin FpExpB3. Expression of another β-expansin (FpExpB2) was not localised to vascular tissue but was highly expressed in roots and initiating tillers. This is the first description of vegetative β-expansin gene expression at the organ and tissue level and also the first evidence of differential expression between members of this gene family. In addition, an analysis of both α- and β-expansin expression along the LEZ revealed no correlation with growth rate distribution, whereas we were able to identify a novel xyloglucan endotransglycosylase (FpXET1) whose expression profile closely mimicked leaf growth rate. These data suggest that α- and β-expansin activities in the grass leaf are associated with tissue differentiation, that expansins involved in leaf growth may represent more minor components of the spectrum of expansin genes expressed in this tissue, and that XETs may be useful markers for the analysis of grass leaf growth.

The effect of different rates of potassium (K + ) on shoot water potential, photosynthesis and carbon movement (using 14 C) at the V3/4 growth stages was studied in mungbean (Vigna radiata L. Wilczek), a drought-susceptible legume, and cowpea (Vigna unguiculata L. Walp), a drought-tolerant legume, grown under low- and high-irrigation regimes under controlled conditions. Soil moisture and K + affected all measured parameters in the two species. The rate of photosynthesis was higher at reduced water stress when K + was applied. The impact was greater in cowpea, which had an inherently high rate of carbon assimilation. Mungbean and to a lesser extent cowpea allocated greater quantities of carbon to roots under dry conditions, especially with added K + . The distribution of 14 C into other plant parts was also increased at higher rates of K + application under both soil moisture regimes. Thus, application of K + seems to have a beneficial effect in overcoming soil moisture stress and increasing physiological parameters and carbon partitioning in these two important tropical food legumes.

Symbiotic N2 fixation may be an important source of N for legumes in alpine ecosystems, though, this has hardly been investigated. Symbiotic N2 fixation in nine legume species in permanent grassland over an altitudinal gradient (from 900 up to 2600 m a.s.l.) was investigated in the Swiss Alps on strictly siliceous soils. To assess symbiotic N2 fixation, an enriched 15N isotope dilution method was established for low N input, permanent grasslands and was evaluated with the 15N natural abundance method. The non-N2-fixing reference species used in both methods differed significantly in their 15N atom%-excess. However, when several reference species were combined, the enriched 15N isotope dilution method was reliable and led to the conclusion that up to their altitudinal limit, legumes may acquire from 59% to more than 90% of their N through symbiotic N2 fixation depending on the species. These findings were confirmed by the 15N natural abundance method. Even at the legumes’ altitudinal limit all plants investigated showed apparently active nodules. Moreover, a clear host-microsymbiont specificity between plant and rhizobia was evident at high altitudes. This suggests that symbiotic N2 fixation is well adapted to the climatic and acidic soil conditions in the Alps and contributes, up to the altitudinal limit, a significant amount of N to the N nutrition of legumes.

Summary Although legumes showed a clearly superior yield response to elevated atmospheric pCO2 compared to nonlegumes in a variety of field experiments, the extent to which this is due to symbiotic N2 fixation per se has yet to be determined. Thus, effectively and ineffectively nodulating lucerne (Medicago sativa L.) plants with a very similar genetic background were grown in competition with each other on fertile soil in the Swiss FACE experiment in order to monitor their CO2 response. Under elevated atmospheric pCO2, effectively nodulating lucerne, thus capable of symbiotically fixing N2, strongly increased the harvestable biomass and the N yield, independent of N fertilization. In contrast, the harvestable biomass and N yield of ineffectively nodulating plants were affected negatively by elevated atmospheric pCO2 when N fertilization was low. Large amounts of N fertilizer enabled the plants to respond more favourably to elevated atmospheric pCO2, although not as strongly as effectively nodulating plants. The CO2-induced increase in N yield of the effectively nodulating plants was attributed solely to an increase in symbiotic N2 fixation of 50–175%, depending on the N fertilization treatment. N yield derived from the uptake of mineral N from the soil was, however, not affected by elevated pCO2. This result demonstrates that, in fertile soil and under temperate climatic conditions, symbiotic N2 fixation per se is responsible for the considerably greater amount of above-ground biomass and the higher N yield under elevated atmospheric pCO2. This supports the assumption that symbiotic N2 fixation plays a key role in maintaining the C/N balance in terrestrial ecosystems in a CO2-rich world.

Lolium perenne, a main component species in managed grassland, is well adapted to defoliation, fertilization, and regrowth cycles; and hence, to changes in the assimilatory carbon source-sink ratio. In the Swiss Free Air CO2 Enrichment experiment the source-sink ratio is (i) increased by elevated partial pressure of CO2 (pCO2), (ii) decreased by enhanced carbon use under high N fertilization, and (iii) gradually increased during regrowth after defoliation. Since sucrose synthesis plays a central role in leaf carbohydrate metabolism in this fructan-accumulating species, we investigated how sucrose-phosphate synthase (SPS) responds to the differing assimilatory carbon fluxes and source-sink ratios in the field. Assimilatory carbon flux, as estimated by leaf gas exchange, strongly depended on pCO2. Surprisingly, the SPS content per leaf area did not increase with pCO2, but increased with N fertilization. During later regrowth, when a dense canopy had formed, the SPS content decreased; in particular, SPS was decreased at high N under elevated pCO2. Further, the higher assimilatory carbon flux through SPS at elevated pCO2 was accompanied by a higher activation state of SPS. The SPS content correlated very strongly with the ratio of free sucrose to free amino acid in leaves, which represents the carbon source-sink ratio. Hence, SPS content in L. perenne appears to be regulated by the current, strongly nitrogen-dependent, source-sink relation.

The effect of elevated (60 Pa) atmospheric carbon dioxide partial pressure (pCO2) and N fertilisation on the availability of mineral N and on N transformation in the soil of a Lolium perenne L. monoculture was investigated in the Swiss FACE (Free Air Carbon dioxide Enrichment) experiment. The apparent availability of nitrate and ammonium for plants was estimated during a representative, vegetative re-growth period at weekly intervals from the sorption of the minerals to mixed-bed ion-exchange resin bags at a soil depth of 5 cm. N mineralisation was measured using sequential coring and in situ exposure of soil cores in the top 10 cm of the soil before and after the first cut in spring 1997. High amounts of mineral N were bound to the ion exchange resin during the first week of re-growth. This was probably the combined result of the fertiliser application, the weak demand for N by the newly cut sward and presumably high rates of root decay and exudation after cutting the sward. During the first 2 weeks after the application of fertiliser N at the first cut, there was a dramatic reduction in available N; N remained low during the subsequent weeks of re-growth in all treatments. Overall, nitrate was the predominant form of mineral N that bound to the resin for the duration of the experiment. Apparently, there was always more nitrate than ammonium available to the plants in the high N fertilisation treatment for the whole re-growth period. Apparent N availability was affected significantly by elevated pCO2 only in the first week after the cut; under high N fertilisation, elevated pCO2 increased the amount of mineral N that was apparently available to the plants. Elevated pCO2 did not affect apparent net transformation of N, loss of N or uptake of N by plants. The present data are consistent with earlier results and suggest that the amount of N available to plants from soil resources does not generally increase under elevated atmospheric pCO2. Thus, a possible limiting effect of N on primary production could become more stringent under elevated atmospheric pCO2 as the demand of the plant for N increases.

To gain insight into the molecular processes occurring in root nodule metabolism after stress, we used a mRNA differential display (DDRT-PCR) approach to identify cDNAs corresponding to genes whose expression is enhanced in nodules of decapitated Medicago truncatula plants. Two full-length cDNAs of plant origin were isolated (MTD1 and MTD2). Sequence analysis revealed that MTD1 is identical to an EST clone (accession number AW559774) expressed in roots of M. truncatula upon infection with Phytophthora medicaginis, while MTD2 is highly homologous to an Arabidopsis thaliana gene (accession number AL133292) coding for a RNA binding-like protein. The two mRNAs started to accumulate in root nodules at 4 h after plant decapitation and reached even higher transcript levels at 24 h from the imposition of the treatment. MTD1 and MTD2 mRNAs were mainly induced in nodules, with very little induction in roots. The abundance of the two transcripts did not change in response to other perturbations known to decrease nitrogenase activity, such as nitrate and Ar/O2 treatments. Our results suggest that MTD1 and MTD2 represent transcripts that accumulate locally in nodules and may be involved in changes in nodule metabolism in response to decapitation.

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.

The significance of symbiotic N2 fixation in legumes (Trifolium alpinum L., T. nivale Sieber, T. pratense L., T. badium Schreber, T. thalii Vill., T. repens L., Lotus alpinus [DC.] Schleicher, L. corniculatus L., Vicia sativa L.) and other N sources for the N budget of grassland ecosystems was studied along an altitudinal gradient in the Swiss Alps. The total annual symbiotic N2 fixation was compared with other sources of N for plant growth of the total plant community (mineralisation and wet deposition). The contribution of symbiotically fixed N to total above-ground N yield of the swards decreased from at least 16% to 9% with increasing altitude where legumes were present. This decrease was due to a decrease in the yield proportion of legumes from 15% at 900 and 1380 m a.s.l. to 5% at 2100 and 2300 m a.s.l. (no legumes were found above 2750 m a.s.l.) and not to a decline in the activity of symbiotic N2 fixation. With increasing altitude legumes are more patchily distributed. The high symbiotic N2 fixation of individual plants up to their altitudinal limit is not primarily the result of low mineral N availability since an addition of NH4 + or NO3 − fertiliser at 2300 m a.s.l. led either to no decrease or only to a minor decrease in symbiotic N2 fixation. At 1380 m a.s.l., N mineralisation (13.45 g N m−2 yr−1) appeared to be the main source of N for growth of the sward; N from symbiosis (at least 1.0 g to 2.6 g N m−2 yr−1) and wet deposition (0.4 g to 0.6 g m−2 yr−1) was not a significant N source for plant growth at this altitude. At 2100 m a.s.l., the combined amounts of N from symbiotic N2 fixation (at least 0.1 g N m−2 yr−1) and wet deposition (0.3 g N m−2 yr−1) appeared to be similarly important for plant growth as soil N mineralisation (0.47 g N m−2 yr−1). At high altitudes, wet N deposition and symbiotic N2 fixation together represent a significant source of N for the grassland ecosystem while at low altitudes these N inputs appear to be much less important.

The mechanisms responsible for fluctuations in species composition of semi-natural grassland are not well understood. To identify plant traits that determine the poor competitive ability of Festuca pratensis compared to Dactylis glomerata especially during summer, the growth of both grasses was monitored over time and at different temperatures and photoperiods. Plants of both grasses were grown from seed with non-limiting nutrient supply at three day/night temperatures (11/6, 18/13 and 25/20°C) and two photoperiods (16 and 12 h). F. pratensis had a significantly lower relative growth rate than D. glomerata, mainly due to its lower specific leaf area and reduced nitrogen productivity. At high temperature, F. pratensis had a considerably lower root weight ratio than D. glomerata leading to substantially slower root growth. F. pratensis responded to a shorter photoperiod with an increase in the net assimilation rate, whereas D. glomerata responded with an increase in specific leaf area. The low competitive ability of F. pratensis compared to D. glomerata was mainly associated with its lower specific leaf area and nitrogen productivity. The stronger decline of its competitive ability during summer was probably related to the decreased allocation of dry matter to the roots at higher temperatures which leads to slower root growth compared to D. glomerata.

It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne ( Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.

A 3-year experiment was conducted on an extensively managed hay meadow to assess the effect of management intensity and sward density on growth of meadow fescue. Two management treatments, intensive (6 cuts, 240 kg N ha−1 year−1) and extensive (3 cuts, 75 kg N ha−1 year−1), with and without gaps around the meadow fescue plants, were applied. The highest yields were obtained under frequent defoliation. The yield of meadow fescue when grown in gaps increased four- to fivefold on an area basis in the first growing season, and reached a factor of 17 the following year. However, the difference between the treatments with and without gaps decreased strongly during the third growing season. The contribution of meadow fescue to the total yield remained high and constant in swards with gaps combined with extensive management for the 3 years. In contrast, in swards with gaps combined with intensive management, the yield contribution of meadow fescue decreased, and in swards without gaps it remained very low under both management regimes. The results show that meadow fescue was a weak competitor. Its response to management intensity was strongly affected by light intensity and soil water content. Low light intensity and dry top soils enhanced the suppression of meadow fescue.

The application of biotechnology to forage grass has the potential to complement and speed up conventional breeding. To ascertain the suitability of regenerants for improvement programs, primary regenerants of tall fescue (Festuca arundinacea Schreb.) cv Tacuabe and their progenies were investigated in a field experiment located in Eschikon near Zurich (Switzerland). Thereby, the following two aspects were focused on. First, do regenerated plants and their progenies perform similar to seed-grown plants; and second, do different regeneration systems have an effect on the performance of the regenerants ? Two sets of plants, regenerated from different single-genotype derived embryogenic suspension cultures (ESC) of tall fescue, were evaluated for agronomic traits in a replicated field experiment for three growing seasons. Embryogenic suspension cultures were either routinely subcultured or cryopreserved and re-established. Seed from the primary regenerated and seed-grown plants were harvested to evaluate morphological and phenological traits of corresponding half-sib progenies in a further field experiment. When compared with seed-grown plants of the same cultivar, primary regenerants showed reduced vegetative growth and fertility. The performance of plants regenerated from cryopreserved-reestablished ESC was not inferior to corresponding plants regenerated from routinely subcultured ESC. The performance of progenies of regenerated plants did not differ from those of seed-grown plants and was within the range of the cultivar. There was no effect of the regeneration system on the performance of corresponding progenies; thus, suggesting that the weak growth of the primary regenerants was most likely of epigenetic origin. Therefore regenerated plants can be integrated into breeding programs without major restrictions.

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH4+ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O2-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O2-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.

Three widely used cultivars of each of the species Festuca pratensis Huds., Lolium perenne L., and Dactylis glomerata L. were investigated by means of randomly amplified polymorphic DNA (RAPD) markers and vegetative growth traits in order to investigate genetic variability within each cultivar and to compare the level of diversity among cultivars and species. RAPD markers allowed a clear separation of the three species. Genetic variability based on RAPD markers was considerably lower for F. pratensis cultivars than for L. perenne and D. glomerata cultivars which showed similar levels of variability. The proportion of variability due to variation within cultivars, determined by an analysis of molecular variance, was lower in F. pratensis (64.6%) than in L. perenne (82.4%) and D. glomerata (85.1%). A comparison of F. pratensis and L. perenne, based on vegetative growth traits, confirmed the differences in genetic variability within cultivars. F. pratensis showed lower coefficients of genetic variation for eight of ten traits when compared to L. perenne. This study demonstrates considerable differences in genetic variability which may have consequences for the adaptability and persistency of individual cultivars.

The growth response of white clover (Trifolium repens L.) to the expected increase in atmospheric partial pressure of CO2 (pCO2) may depend on P availability. A decrease in the rate of transpiration due to increased pCO2 may reduce the amount of P transported to the shoot, thereby causing a change in the partitioning of P between the root and shoot. To test these hypotheses, four concentrations of P in the nutrient solution, combined with two pCO2 treatments, were applied to nodulated white clover plants. Compared to ambient pCO2 (35 Pa), twice ambient pCO2 (70 Pa) reduced the rate of transpiration but did not impair the total P uptake per plant. However, at twice ambient pCO2 and a moderate to high supply of P, concentrations of structural P and soluble P (Pi) were lower in the leaves and higher in the roots. The activity of root acid phosphatase was lower at twice ambient pCO2 than at ambient pCO2; it depended on the Pi concentration in the roots. At the highest P concentration, twice ambient pCO2 stimulated photosynthesis and the growth rate of the plant without affecting the concentration of nonstructural carbohydrates in the leaves. However, at the lower P concentrations, plants at twice ambient pCO2 lost their stimulation of photosynthesis in the afternoon, they accumulated nonstructural carbohydrates in the leaves and their growth rate was not stimulated; indicating C-sink limitation of growth. P nutrition will be crucial to the growth of white clover under the expected future conditions of increased pCO2.

Permanent pastures and meadows are species-rich vegetation systems that play an important role in the ecology and agriculture of temperate climates. Intensive management reduces species diversity and may also influence the genetic diversity within individual species and populations. The objective of this study was to assess genetic variability of meadow fescue, an important component of species-rich grasslands, and to determine whether fertilization and defoliation frequency influence genetic variability within natural populations. Genetic diversity of six natural populations and three cultivars of Festuca pratensis was investigated using randomly amplified polymorphic DNA (RAPD) markers and agronomic traits. Samples of natural populations were taken from two unrelated long-term experiments, where treatments had been applied for 11–38 years. RAPD analysis detected a clear genetic distinction of the cultivars from the natural populations. Genetic variability within cultivars was lower than within natural populations. Analysis of molecular variance (AMOVA) showed a significant effect of management on genetic variability. Fertilization and frequent defoliation led to a reduction in genetic variability within natural populations. Analysis of agronomic traits was only partially congruent with the results of RAPD analysis. This study shows that significant genetic variability exists within cultivars and natural populations of Festuca pratensis and can be reduced by intensive management.

The objective of this investigation was to examine the effect of an elevated atmospheric CO 2 partial pressure (pCO 2 ) on the N-sink strength and performance of symbiotic N 2 fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient pCO 2 in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated pCO 2 (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 pCO 2 evoked a significant increase in biomass production from day 12 after the start of CO 2 enrichment. For plants supplied with 7.5 mol m -3 N, the relative contribution of symbiotically fixed N (%N svm ) as opposed to N assimilated from mineral sources ( 15 N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N sym was unaffected by atmospheric pCO 2 over the entire experimental period. In plants fully dependent on N 2 fixation, the increase in N yield reflects a stimulation of symbiotic N 2 fixation that was the result of the formation of more nodules rather than of higher specific N 2 fixation. These results are discussed with regard to physiological processes governing symbiotic N 2 fixation and to the response of symbiotic N 2 fixation to elevated pCO 2 in field-grown T. repens.

Two sets of plants (Lb and Lc), regenerated from different single-genotype-derived embryogenic suspension cultures of Lolium perenne cv Citadel, were evaluated for agronomic traits in a modified polycross design in the field. Seed from the primary regenerated plants was harvested to evaluate morphological and phenological traits of corresponding progenies in a replicated field experiment. When compared to seed-grown plants of the same cultivar, primary regenerants of the Lb set showed a significant delay in ear emergence and a more-erect growth habit, while primary regenerants from the Lc set showed a significantly higher seed yield. However, progenies of regenerated plants did not differ from those of seed-grown plants. Embryogenic suspension cells of L. perenne have the potential for producing fertile, well-performing, material which can be integrated into breeding programs.

The effect of regeneration of Lolium perenne and Festuca rubra from embryogenic suspension cells and protoplasts on fertility and growth was evaluated. Embryogenic suspension cultures were either routinely subcultured or cryopreserved and re-established. Phenology, morphology and fertility of regenerated plants were studied for two growing seasons in a replicated field experiment. Most regenerated L. perenne and F. rubra plants showed a delay in inflorescence emergence, a reduced seed yield and differences in morphological traits when compared with seed-grown plants. For L. perenne, performance of plants regenerated from cryopreserved suspension cultures and protoplasts was similar to that of respective plants regenerated from routinely maintained suspension cultures. However, differences in performance were observed for respective regenerants in F. rubra. The phenotypic deviation observed was partly reflected in the randomly amplified polymorphic DNA (RAPD) analysis performed. However, regenerants of both species showing similar, or even superior performance to the seed-grown plants were also found. Embryogenic suspension cells and corresponding protoplasts of L. perenne and F. rubra have the potential for producing fertile, well-performing plants which can be integrated in breeding programs.

To test inter- and intraspecific variability in the responsiveness to elevated CO2, 9–14 different genotypes of each of 12 perennial species from fertile permanent grassland were grown in Lolium perenne swards under ambient (35 Pa) and elevated (60 Pa) atmospheric partial pressure of CO2 (pCO2) for 3 years in a free air carbon dioxide enrichment (FACE) experiment. The plant species were grouped according to their functional types: grasses (L. perenne, L. multiflorum, Arrhenatherum elatius, Dactylis glomerata, Festuca pratensis, Holcus lanatus, Trisetum flavescens), non-legume dicots (Rumex obtusifolius, R. acetosa, Ranunculus friesianus), and legumes (Trifolium repens, T. pratense). Yield (above a cutting height of 4.5 cm) was measured three times per year. The results were as follow. (1) There were highly significant differences in the responsiveness to elevated pCO2 between the three functional types; legumes showed the strongest and grasses the weakest yield increase at elevated pCO2. (2) There were differences in the temporal development of responsiveness to elevated pCO2 among the functional types. The responsiveness of the legumes declined from the first to the second year, while the responsiveness of the non-legume dicots increased over the 3 years. During the growing season, the grasses and the non-legume dicots showed the strongest response to elevated pCO2 during reproductive growth in the spring. (3) There were no significant genotypic differences in responsiveness to elevated pCO2. Our results suggest that, due to interspecific differences in the responsiveness to elevated pCO2, the species proportion within fertile temperate grassland may change if the increase in pCO2 continues. Due to the temporal differences in the responsiveness to elevated pCO2 among species, complex effects of elevated pCO2 on competitive interactions in mixed swards must be expected. The existence of genotypic variability in the responsiveness to elevated pCO2, on which selection could act, was not found under our experimental conditions.

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.

In plagiotropic plants, axillary buds on the stolon can be exposed to low red:far-red (R:FR) ratios, while the leaves may be positioned in the uppermost layer of the sward where they are exposed to a high R:FR ratio. We tested whether the light environment of unfolded leaves influences outgrowth of the axillary buds and the formation of nodal roots of Trifolium repens. Single plants were grown in a growth cabinet with high photosynthetic photon flux rate (PPFR) and a high R:FR ratio (FHRH, control), low PPFR and high R:FR (FLRH) or low PPFR and low R:FR (FLRL). In an additional treatment (SS), only stolons were shaded so that developing leaves grew into light conditions similar to the control treatment. Neutral shading (FLRH) had a minor effect on branching and did not influence root formation. A reduction in the R:FR ratio (FLRL) significantly delayed the outgrowth of axillary buds so that, compared to the control plants, the percentage of branched phytomers was reduced by 43% on the parent axis and by 75% on primary branches. Furthermore, the number of nodal roots per plant was reduced by about 30%. When only the stolons were shaded (SS), the percentage of branched and rooted phytomers was similar to that of the control plants. Extension of petioles and leaves was very variable, increasing the values in the FLRL treatment at least 2.5-fold compared with the control plants. It was concluded that the light environment of the unfolded leaves had a significant influence on the regulation of the outgrowth of axillary buds and that the high plasticity in petiole growth allows the positioning of the leaves in a light environment conducive to the stimulation of branch outgrowth.

The effect of an elevated partial pressure of CO2 (pCO2) on carbohydrate concentrations in source leaves and pseudo-stems (stubble) of Lolium perenne L. (perennial ryegrass) during regrowth was studied in a regularly defoliated grass sward in the field. The free air carbon dioxide enrichment (FACE) technology enabled natural environmental conditions to be provided. Two levels of nitrogen (N) supply were used to modulate potential plant growth. Carbohydrate concentrations in source leaves were increased at elevated pCO2, particularly at low N supply. Elevated leaf carbohydrate concentrations were related to an increased structural carbon (C) to N ratio and thus reflected an increased C availability together with a N-dependent sink limitation. Immediately after defoliation, apparent assimilate export rates (differences in the carbohydrate concentrations of young source leaves measured in the evening and on the following morning) showed a greater increase at elevated pCO2 than at ambient pCO2; however, replenishment of carbohydrate reserves was not accelerated. Distinct, treatment-dependent carbohydrate concentrations in pseudo-stems suggested an increasing degree of C-sink limitation from the treatment at ambient pCO2 with high N supply to that at elevated pCO2 with low N supply. During two growing seasons, no evidence of a substantial change in the response of the carbohydrate source in L. perenne to elevated pCO2 was found. Our results support the view that the response of L. perenne to elevated pCO2 is restricted by a C-sink limitation, which is particularly severe at low N supply.