Artificial chalk grassland swards were exposed to either ambient air or air enriched to 600 μmol mol–1 CO2, using free-air CO2 enrichment technology, and subjected to an 8 week simulated grazing regime. After 14 months of treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco) activity (Vc,max) and electron transport mediated ribulose-1,5-bisphosphate (RuBP) regeneration capacity (Jmax), estimated from leaf gas exchange, were significantly lower in fully expanded leaves of Anthyllis vulneraria L. (a legume) and Sanguisorba minor Scop. grown in elevated CO2. After a change in source:sink balance brought about by defoliation, photosynthetic capacity was fully restored in A. vulneraria and S. minor, but acclimation continued in the grass Bromopsis erecta (Hudson) Fourr. Changes in net photosynthesis (Pn) with growth at elevated CO2 ranged from a 1·6% reduction in precut leaves of A. vulneraria to a 47·1% stimulation in postcut leaves of S. minor. Stomatal acclimation was observed in leaves of A. vulneraria (reduced stomatal density) and B. erecta (reduced stomatal conductance). The results are discussed in terms of whole-plant resource-use optimization and chalk grassland community competitive interactions at elevated CO2.
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Conclusions about the effects of increased atmospheric concentrations of CO2 (ca) on photosynthesis and growth are based, largely, on an extensive body of research which has utilized single plant species, very often crop species, grown individually in nonlimiting conditions (Sage 1994). In natural and seminatural plant communities, the magnitude and direction of photosynthetic and growth responses to elevated ca may depend critically on a number of factors, including the morphological and ecological characteristics of the individual species (Hunt et al. 1991, 1993), soil nutrient and water availability (Tissue & Oechel 1987; Arp & Drake 1991) and the degree of competition from other species (Bazzaz 1990).
When experiments have been carried out incorporating one or more of the above factors, results suggest that the short-term photosynthetic responses of C3 plant species to elevated ca are usually modified by some degree of longer term adjustment, or ‘acclimation’ of the photosynthetic apparatus (Oechel & Strain 1985). Photosynthetic acclimation to growth in elevated ca can be defined as the co-ordinated biochemical adjustment of processes such that the pool of resources available to the whole plant for investment into limiting processes is greater over the life of the plant (Sage 1994). Photosynthetic acclimation may therefore represent a beneficial shift in the resource-use optimum at elevated ca away from the nitrogen-costly Rubisco, which can account for 25% of total leaf nitrogen (Sage et al. 1989). Indeed, in an extensive survey of studies of plant responses to long-term CO2 enrichment, Long & Drake (1992) found that plants, both grown and measured at elevated ca, maintained an average net photosynthetic rate (Pn) 51% higher than plants grown and measured at ambient ca. These increases in Pn at elevated ca, in the majority of cases, were achieved in spite of downward adjustments in various components of the photosynthetic apparatus.
Photosynthetic acclimation appears to be most pronounced in plants which are sink limited (Arp 1991; Webber et al. 1994). Indeed, accumulation of soluble carbohydrates in source leaves has been related, experimentally, to a repression of the expression of specific photosynthetic genes, thus providing a mechanistic link between sink limitation and photosynthetic acclimation (Stitt 1991; Krapp et al. 1993; Van Oosten & Besford 1994).
The implications of such physiological and biochemical changes at the leaf level for the long-term competitive interactions between species are still unclear, given the complex relationships between photosynthesis, plant growth and community structure (Körner 1991; Stitt 1993). If observed leaf level acclimation does indeed represent an optimization of nitrogen use within the plant, then longer-term benefits related to increased nutrient uptake may be expected, particularly in low productivity ecosystems where competition for scarce nutrients ultimately limits plant growth (Tilman 1988). The more complex and biodiverse an ecosystem is, the more likely it is that differential physiological responses of component species will occur, leading to significant changes in ecosystem composition.
Here we report the photosynthetic responses of component species of model chalk grassland swards growing under free-air CO2 enrichment. Chalk grassland, which exists in fragments across Britain and continental Europe, is an extremely biodiverse assemblage of coexisting grass and forb species, which is traditionally managed by continuous summer livestock grazing (Grime 1990; Keymer & Leach 1990). We aimed to determine (i) the extent, and possible causes, of photosynthetic acclimation in three ecologically distinct species in the swards, and (ii) how a clipping treatment, aimed at simulating grazing, influenced photosynthetic acclimation by altering whole-plant source-sink balance.
MATERIALS AND METHODS
Growth conditions and FACE set-up
Between April 6th and 8th 1994, seeds of seven chalk grassland perennials (Table 1) (Emorsgate Seeds, King's Lynn, Norfolk) were sown together as a mixture in plastic tubs (dimensions: 35 cm length × 26 cm breadth × 19 cm depth) with an overall volume of 0·017 m3. The tubs were lined with a 2 cm layer of chalk/flint to aid drainage, the remainder being filled with a 50:50 mixture of chalk grassland rendzina soil collected from a site in East Sussex (Grid reference 078 385 – TQ 20/30) and potting compost (John Innes no. 2). Any germination from the soil seed bank was removed before the seeds were sown. The original target species composition of the tubs is indicated in Table 1. Overall seedling density was 26 374 m–2. All species sown became established.
Table 1. . The initial species composition of the chalk grassland turfs grown in the FACE facitlity, ETH-Eschikon, Switzerland
On April 28th the tubs were transferred to the free-air CO2 enrichment (FACE) facility at the Eschikon Experimental Station of the Swiss Federal Institute of Technology, Institute for Plant Sciences, Zurich, Switzerland. This particular FACE design, developed at Brookhaven National Laboratory, is distinguished from other free-air designs by the introduction of a prediluted fumigation gas at the periphery of an experimental plot and by relatively rapid, closed-loop control of treatment level based on gas concentration, wind speed and wind direction (Lewin et al. 1994). The experimental set-up consists of three replicates of blocked elevated (target, 600 μmol mol–1) and ambient plots. Data for the 1993 and 1994 seasons show that season-long average CO2 concentrations during treatment hours were 598 ± 2 μmol mol–1 measured at the control points at the centre of each ring at a height of 0·3 m (H Blum, personal communication). CO2 fumigation in the experimental plots was operational from the beginning of March until the end of November during all daylight hours.
Five replicate tubs were placed in each of the six experimental plots. The tubs were sunk into the ground so that the two soil surfaces were level. Once turfs had developed they were subjected to a cutting treatment at regular intervals: May, July and October 1994 and April, June and August 1995. The vegetation was clipped to a height of 5 cm above the soil surface. Above- and below-ground biomass responses are reported in Warwick et al. (1998).
Gas exchange measurements
Following 14 months of exposure to the CO2 treatments, steady-state leaf gas exchange measurements were made using an open gas-exchange system, incorporating a CO2 and water vapour infra-red gas analyser (CIRAS 1, PP Systems, Hitchin, Herts., UK). Air of the desired CO2 concentration was supplied by pressurized ‘Sparklets’ CO2 bulbs (ISI, Vienna, Austria) attached to an automatic CO2 flow regulation system. It was not considered possible to hold cuvette air temperature constant whilst working in the field. Prior to measurements, a stabilized quartz-iodide light source powered by a 12 V d.c. battery was clipped over the leaf chamber to provide saturating photosynthetic photon flux densities (700 μmol m–2 s–1). Boundary layer resistance (Rb) for the leaf cuvette was 0·3 m–2 s–1 mol–1.
The response of net carbon assimilation (A) versus the intercellular CO2 concentration (ci) was measured for three species of contrasting morphology –Anthyllis vulneraria (a leguminous dicotyledon), Sanguisorba minor (a tap-rooted, rosette-forming dicotyledon) and Bromopsis erecta (a tufted winter-green grass) – on nine days, approximately one week prior to the clipping treatment in June 1995, and on eight days, approximately 1–2 weeks after the clipping treatment in July 1995. Analysis of this A/ci response facilitates interpretation of the nature of the biochemical and stomatal processes controlling the long-term response of photosynthetic capacity to changing environmental conditions (Von Caemmerer & Farquhar 1981).
Since air and leaf temperature varied between measurements, a temperature-dependent biochemical model of C3 photosynthesis was used to interpret the A/ci curves generated for the three species (Harley et al. 1992; McMurtrie & Wang 1993). This model uses the equations of Farquhar et al. (1980) to provide estimates, by the maximum-likelihood method, of the parameters Vc,max and Jmax as in vivo measures of Rubisco activity and electron transport capacity of the thylakoid, respectively.
In June 1994 leaf imprints were prepared for examination of stomatal parameters in the three species used for gas exchange measurements. A thin layer of clear nail varnish was painted on to the adaxial and then the abaxial surface of each leaf of each species. When this was dry, a strip of sticky tape was placed over the imprint, peeled off, taking the imprint with it, and mounted directly onto a microscope slide. The leaf replicas were examined under a light microscope to obtain the stomatal density and stomatal index. The numbers of stomata and of other epidermal cells were counted from five randomly selected half-fields of view per leaf surface from five leaves per experimental ring (one leaf per tub). The stomatal index was calculated, which relates the number of stomata per unit leaf area (S) to the number of epidermal cells (including guard cells) per unit leaf area (E), where SI = (S/E + S) × 100 (Salisbury 1927). Epidermal cell area of seven cells per leaf surface from five leaves per experimental ring (one leaf per tub) was measured using a camera lucida attached to a light microscope following the method of Ferris & Taylor (1994).
The photosynthetic variables, Vc,max and Jmax were tested for significance by two-way analysis of variance (CO2 treatment × block). Treatment effects were not considered valid if the CO2× block interaction term was significant. Stomatal characteristics were analysed by three-level nested analysis of variance (mixed model) (Sokal & Rohlf 1969) with a separate analysis for each leaf surface of each species. The design was hierarchical: five tubs nested within three blocks within two treatments. The data set was not transformed. Vc,max:Jmax ratios were arcsine transformed before testing for significance by two-way analysis of variance as above.
The photosynthetic response to growth at elevated CO2 was dependent both on species and on proximity to the cutting treatment. Downward adjustment of photosynthesis was apparent in all three species one week before the cutting treatment (seven weeks’ growth) and this resulted in significant reductions in both Vc,max and Jmax in leaves of A. vulneraria and S. minor grown in the FACE plots (Table 2). For A. vulneraria, photosynthetic rates were lower at all measured ci values in leaves of plants grown in FACE plots compared to ambient plots (Fig. 1a). However, 1 – 2 weeks after the cutting treatment, the photosynthetic capacity of the two forbs, A. vulneraria and S. minor, was restored to a level equivalent to that in the ambient grown plants, i.e. there were no significant differences in either Vc,max or Jmax between the two treatments (Table 2). This effect of cutting is illustrated in the A/ci plots for A. vulneraria and S. minor which show almost identical photosynthesis values at all ci values (Fig. 1a,b). The grass species, B. erecta, followed a similar, but nonsignificant, pattern of photosynthetic acclimation before the cut but, in contrast to the forbs, continued to exhibit a significant degree of downward adjustment after the cutting treatment (Fig. 1c & Table 2). All three species in both CO2 treatments had considerably higher absolute values of Vc,max and Jmax after defoliation than before it, with B. erecta showing the greatest difference (Table 2).
Table 2. . Mean values of Vc,max (μmol m–2 s–1) and Jmax (μmol m–2 s–1) for Anthyllis vulneraria, Sanguisorba minor and Bromopsis erecta, grown in either ambient or FACE plots, calculated both before and after a cutting treatment. Statistical significance: ***P<0·001; **P<0·01; *P<0·05; n.s., not significant
The ratio of Vc,max:Jmax, which provides an indication of the distribution of plant resources between Rubisco activity and the apparatus for regeneration of RuBP, appears to be reduced in all three species (not always statistically significant), both before and after the cutting treatment, with growth at elevated ca (Table 3). The values of Vc,max:Jmax were considerably higher in both CO2 treatments after the defoliation (Table 3).
Table 3. . The mean ratio of Vc,max : Jmax for A. vulneraria, S. minor and B. erecta grown in either ambient or FACE plots. Ratios were transformed (arcsine) before two-way (CO2× block) ANOVA. Differences between ambient and FACE (n=5) are shown:
As a result of these adjustments to the photosynthetic apparatus, net photosynthesis (Pn) (measured at the CO2 concentration to which the plants were exposed during growth) of A. vulneraria declined by 1·6% whilst in S. minor and B. erecta it increased by 22·2% and 33·7%, respectively, with growth at elevated CO2 prior to a cutting treatment (Table 4). Defoliation results in large stimulations of Pn in A. vulneraria (41·6%) and S. minor (47·1%) with growth at elevated CO2, but for B. erecta the stimulation of Pn by high CO2 was lower (30·7%) than before the cutting (Table 4).
Table 4. . Mean rates (±SE) of net photosynthesis (Pnμmol m–2 s–1), stomatal conductance (gs mmol m–2 s–1) and transpiration (E mmol m–2 s–1) for Anthyllis vulneraria, Sanguisorba minor and Bromopsis erecta grown in either ambient or FACE plots and measured at the growth ca both before and after a cutting treatment
The ci:ca ratio, which directly reflects changes in the relationship between stomatal conductance and the biochemical capacity for CO2 fixation (Ball & Berry 1982; Tissue et al. 1995), varied significantly between species and measurement time. Before the cutting treatment ci:ca was higher in FACE than in ambient plots in leaves of all three species (Fig. 2a,c,e). This difference between treatments was, however, only statistically significant in A. vulneraria between 150 and 900 μmol mol–1 measurement ca. This was the species which also showed the largest reduction in inferred Rubisco activity (Vc,max: Table 2). After the cutting treatment, this effect of growth at elevated CO2 disappeared in A. vulneraria and S. minor, so that the ci:ca ratios were almost identical in both treatments (Fig. 2b,d,f). However, in B. erecta the effect of growth at elevated CO2 was reversed by the defoliation; the ci:ca ratio of FACE grown plants was significantly lower than that of plants grown in ambient conditions at 900 and 1200 μmol mol–1 measurement ca, thus indicating a possible stomatal limitation on photosynthesis (Fig. 2f). In addition to this reduction in ci:ca, there was also a 38·5% reduction in mean gs of B. erecta grown in elevated CO2 after the cutting treatment (Table 4). In fact, gs was reduced by growth in elevated CO2 in all three species both before and after defoliation, with the exception of S. minor before the cut, which showed a 19·7% increase (Table 4).
Few significant effects on stomatal numbers and distribution were observed after 3 months of exposure to elevated CO2. However, a significant increase in epidermal cell area on the abaxial leaf surface of A. vulneraria appeared to lead to increased spacing between individual stomata and, hence, to reduced stomatal density (Table 5).
Table 5. . The mean (n=75) stomatal density (mm–2), stomatal index and epidermal cell area (μm2) of the abaxial leaf surface of Anthyllis vulneraria, Sanguisorba minor and Bromopsis erecta grown in either ambient or FACE plots. Treatment effects from a nested ANOVA are indicated: *P<0·05; n.s., P<0·1
In this study we have observed differing acclimatory responses of photosynthesis in three chalk grassland perennial species of contrasting morphology, which coexist in this biodiverse plant community: Sanguisorba minor, a tap-rooted, rosette forming herb; Bromopsis erecta, a tufted winter-green grass; and Anthyllis vulneraria, a leguminous herb. Sage (1994) examined the results from over 30 studies which used A/ci analysis to investigate photosynthetic acclimation, and identified six general response patterns. In our study, before a cutting treatment, all three species behaved similarly to Solanum melongena (Sage et al. 1989), i.e. with a reduction both in the initial slope of the A/ci response (inferred Rubisco activity) and in the asymptote and plateau (electron transport-mediated rate of RuBP regeneration). However, when A/ci analysis was carried out after a cutting treatment, the pattern of acclimation changed in A. vulneraria and S. minor to one consistent with the behaviour of Lonicera japonica, where elevated CO2 had little or no effect on the A/ci response. Bromopsis erecta, in contrast, was unaffected by the cutting and continued to show S. melongena-type acclimation. However, even with these significant downward adjustments in photosynthetic capacity, Pn, and, hence, carbon fixation, of plants grown at elevated ca never fell significantly below Pn of plants grown at current ambient ca. In addition, the reduction in the ratio of Vc,max:Jmax in all three species demonstrates the increasing control that the electron transport-mediated rate of RuBP regeneration exerts over photosynthesis at elevated ca (Farquhar et al. 1980). Unlike the absolute values of Vc,max and Jmax for A. vulneraria and S. minor, this shift in the Vc,max:Jmax ratio was still evident after a change in the source:sink balance, indicating a more permanent resource reallocation within the photosynthetic apparatus. This ‘within chloroplast’ acclimation has also been shown to occur when plants are grown under different irradiances (Evans 1989), when there is considerable plasticity between light absorption potential and electron transport capacity.
Mechanisms underlying acclimation
It has frequently been suggested that for individual plants growing in small pots, observed photosynthetic acclimation is merely an artefact caused by the limited rooting volume preventing increased sink development (Robbins & Pharr 1988; Arp 1991). Sage (1994) also retrospectively demonstrated this pot-size effect where 11 out of 15 studies which used small pots (<0·005m3) produced Aelevated: Aambient ratios of less than one. However, in this study, where multiple individuals of several species were growing together in a turf, the size of the container is not relevant. It is probable that the low nutrient status (particularly N and P) of native rendzina soils (Rorison 1990), and competition among roots for these nutrients, are the primary factors in initiating the chain of events which lead to photosynthetic acclimation in these three chalk grassland perennials. Pettersson & McDonald (1994) have suggested that whole-plant growth response to nitrogen supply strongly affects the ability of a plant to utilize any additional carbohydrate that may be produced under conditions of elevated CO2, and that this may largely determine the degree of photosynthetic acclimation. Build-up of soluble carbohydrates has been linked experimentally with decreases in photosynthetic rate (Azcon-Bieto 1983; Plaut et al. 1987), with decreases in various photosynthetic enzymes and chlorophyll (Krapp et al. 1991) and, more recently, with decreases in steady-state mRNA transcript levels coding for various photosynthetic proteins (Van Oosten et al. 1994; Nie et al. 1995). In this experiment, we have found that the soluble sugar content of leaves was highest whenever photosynthetic acclimation occurred (Bryant & Taylor, unpublished results). This before-cut sink limitation is especially surprising in A. vulneraria, whose association with Rhizobium might be expected to act as a significant carbohydrate sink as well as providing additional nitrogen for photosynthetic protein formation.
Interpretation of photosynthetic acclimation
Bloom et al. (1985) define acclimation as ‘the short-term physiological adjustment by a plant to achieve both a similar benefit-to-cost ratio for each resource and an optimal allocation among processes’. In other words, an individual plant faces a trade-off in its allocation of protein (nitrogen) to producing efficient photosynthetic mechanisms versus efficient nutrient uptake and retention mechanisms in order to maximize its competitive ability (Tilman 1988). In this study, the maintenance of higher rates of net photosynthesis at elevated ca together with the reduced investment of resources in the photosynthetic machinery, are facts strongly suggestive of a CO2-induced increase in photosynthetic nitrogen-use efficiency. Such a whole-plant resource reallocation away from photosynthesis would seem logical in nutrient-poor chalk soils, where, ultimately, it is not light capture which limits plant survival, but competition for scarce nutrients (Tilman 1982). Indeed, Stitt (1993), using a model system of nitrogen-limited tobacco seedlings, demonstrated that ‘excess’ photosynthetic nitrogen is reallocated to root growth following carbohydrate-induced acclimation. Additional support for this hypothesis in our experiment comes from Warwick et al. (1998), who have shown that overall root biomass in the chalk grassland swards increased by 89% with growth at elevated CO2 accompanied by a 77% increase in root length.
After defoliation, competition for light capture and therefore the rapid re-establishment of a leaf canopy become primary to plant productivity (Monteith 1977). Thus, immediately after the cutting treatment, photosynthetic capacity (Vc,max and Jmax) was fully restored in A. vulneraria and S. minor to the level seen in ambient grown plants. As a result, Pn was greatly stimulated in both these species growing in elevated CO2. However, despite the continued acclimation observed in B. erecta, Pn remains significantly stimulated by elevated CO2 and, most importantly, the absolute values of Pn, Vc,max and Jmax are considerably higher at both ambient and elevated CO2 after the cut than in the other two species. It is possible, therefore, that B. erecta is able to continue acclimating at elevated CO2 (with all the benefits which that may bring in terms of nitrogen-use optimization) without apparently foregoing any advantage in terms of carbon fixation after cutting. On the other hand, it would appear that A. vulneraria and S. minor may re-invest costly nitrogen in increasing Vc,max and Jmax in order to maintain Pn at a competitive rate.
Contributions from stomata
In addition to this photosynthetic acclimation we have also observed acclimation to growth at elevated CO2 in some stomatal characteristics. Both before and after the cutting treatment, ci:ca mirrored the changes seen in the photosynthetic data: it was higher in all three species grown in FACE plots, significantly so in A. vulneraria, demonstrating their reduced biochemical capacity for CO2 fixation and also an increase in stomatal conductance in S. minor. This is an unusual response to elevated CO2, since in the majority of studies, including those on the other two species measured here, lower stomatal conductance is observed (Reining 1994). After the cut, as the photosynthetic capacity of A. vulneraria and S. minor was restored, so ci:ca of FACE-grown plants returns to the values seen in ambient-grown plants. However, the significant reductions observed in ci:ca of FACE-grown B. erecta, along with reductions in both gs and E, would appear to indicate that a stomatal limitation to photosynthesis is contributing to the continued acclimation observed in this species.
From current evidence, it is unclear whether future elevated atmospheric CO2 concentrations will produce changes in stomatal numbers (Woodward 1987; Körner 1988). However, a recent comprehensive survey by Woodward & Kelly (1995) of 100 species has shown an average reduction in stomatal density of 14% with growth in elevated atmospheric ca. In this study we also observed a reduction in the stomatal density on the abaxial leaf surface of A. vulneraria, which appeared to be caused by a significant increase in epidermal cell area. However, there were no significant effects on the other two species studied. Ferris & Taylor (1994) observed a similar reduction of abaxial stomatal density in A. vulneraria growing in controlled environments but, in contrast, this was thought to result from a reduced stomatal index. These contrasting results, between controlled environment and field, highlight the importance of the experimental growth conditions when studying the effects of elevated CO2 on stomata. In the field, it may be that other micrometeorological factors are of greater importance in determining stomatal initiation and development than CO2 concentration.
Implications for competitive interactions in chalk grasslands
The ability to maintain a nitrogen-efficient photosynthetic apparatus must confer considerable competitive advantage upon a species exposed to elevated atmospheric ca and growing in nitrogen-poor soil. Our results have shown that all three species measured in the sward will carry out photosynthesis more efficiently at elevated ca, particularly when the ratio of source:sink is high. However, only B. erecta maintained this efficiency when the source:sink balance decreased after defoliation. Given that these changes in source:sink balance, brought about by defoliation, occur frequently in chalk grasslands, we suggest that B. erecta, and possibly other grasses, could increase as a component of this plant community at future atmospheric CO2 concentrations. However, these conclusions are not supported by the results of Warwick et al. (1998), who have detected a significant shift in species composition in the same swards. Following two seasons’ exposure to elevated ca, the growth and proportion of biomass in the sward of the nitrogen-fixing legume species, A. vulneraria and Lotus corniculatus, were increased. Similar findings were recently reported by Hebeisen et al. (1997) for a mixed grass–clover sward. These results suggest that initial nitrogen acquisition may be more important than its subsequent efficiency of use in determining competitive ability of chalk grassland plants growing in CO2-enriched environments.
In conclusion, this research is one of the few studies that has demonstrated differing degrees of photosynthetic acclimation in species growing in competition, under natural conditions of limited nutrient availability, in a long-term CO2-enrichment experiment. It is likely that these differences contribute, albeit indirectly, to the changes which occur in species composition at the community level. Studies such as this can only help to broaden our understanding of the complex relationships which exist between photosynthesis, plant growth, community composition and environment.
We thank Herbert Blum for assistance during our stay at Eschikon and also the FACE team from Brookhaven National Laboratory, led by George Hendrey, who played a major part in the construction of the ETH-FACE facility. We would also like to thank Kevin Warwick for setting up and transporting the swards, and the NERC TIGER programme and Sussex University for funding.