Effects of elevated [CO 2 ] on photosynthesis in European forest species: a meta-analysis of model parameters

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1 Plant, Cell and Environment (1999) 22, Effects of elevated [CO 2 ] on photosynthesis in European forest species: a meta-analysis of model parameters B. E. MEDLYN, 1 F. -W. BADECK, 2 D. G. G. DE PURY, 3 C. V. M. BARTON, 1 M. BROADMEADOW, 4 R. CEULEMANS, 3 P. DE ANGELIS, 5 M. FORSTREUTER, 6 M. E. JACH, 3 S. KELLOMÄKI, 7 E. LAITAT, 8 M. MAREK, 9 S. PHILIPPOT, 10 A. REY 1, J. STRASSEMEYER, 6 K. LAITINEN, 7 R. LIOZON, 2 B. PORTIER, 8 P. ROBERNTZ, 10 K. WANG 7 & P. G. JARVIS 1 1 IERM, University of Edinburgh, King s Buildings, Edinburgh, EH9 3JU, UK, 2 Laboratoire d Ecophysiologie Végétale, Université de Paris-Sud, France, 3 Department of Biology, University of Antwerpen, Wilrijk, Belgium, 4 Forestry Commission, Alice Holt Lodge, Farnham, Surrey, UK, 5 DISAFRI, University of Tuscia, Viterbo, Italy, 6 Institut für Ökologie, Technische Universität Berlin, Germany, 7 Faculty of Forestry, University of Joensuu, Finland, 8 Department de Biologie Vegétalé, Faculte des Sciences Agronomiques de Gembloux, Belgium, 9 Department of Ecological Physiology of Forest Trees, Institute of Landscape Ecology, Czech Academy of Sciences, Brno, Czech Republic, and 10 Department for Production Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden ABSTRACT The effects of elevated atmospheric CO 2 concentration on growth of forest tree species are difficult to predict because practical limitations restrict experiments to much shorter than the average life-span of a tree. Long-term, processbased computer models must be used to extrapolate from shorter-term experiments. A key problem is to ensure a strong flow of information between experiments and models. In this study, meta-analysis techniques were used to summarize a suite of photosynthetic model parameters obtained from 15 field-based elevated [CO 2 ] experiments on European forest tree species. The parameters studied are commonly used in modelling photosynthesis, and include observed light-saturated photosynthetic rates (A max ), the potential electron transport rate (J max ), the maximum Rubisco activity (V cmax ) and leaf nitrogen concentration on mass (N m ) and area (N a ) bases. Across all experiments, light-saturated photosynthesis was strongly stimulated by growth in elevated [CO 2 ]. However, significant down-regulation of photosynthesis was also observed; when measured at the same CO 2 concentration, photosynthesis was reduced by 10 20%. The underlying biochemistry of photosynthesis was affected, as shown by a down-regulation of the parameters J max and V cmax of the order of 10%. This reduction in J max and V cmax was linked to the effects of elevated [CO 2 ] on leaf nitrogen concentration. It was concluded that the current model is adequate to model photosynthesis in elevated [CO 2 ]. Tables of model parameter values for different European forest species are given. Key-words: down-regulation; elevated [CO 2 ]; forests; metaanalysis; photosynthesis Correspondence: Belinda Medlyn, Laboratoire d Ecophysiologie et Nutrition, Station de Recherches Forestières, INRA Pierroton, BP 45, Gazinet Cedex 33611, France. Fax: ; Belinda.Medlyn@pierroton.inra.fr INTRODUCTION Predicting the likely response of woody plant species to future increases in atmospheric CO 2 concentration is a difficult problem because of the longevity of these species (Eamus & Jarvis 1989). The longest-running experiments in which trees have been exposed to elevated [CO 2 ] now reach to just over 10 years (Idso & Kimball 1997; Pontailler et al. 1998). In general, practical and financial considerations limit the length of such experiments to no more than five years (e.g. Ceulemans et al. 1996; Johnson et al. 1996; Kellomäki & Wang 1996; Norby, Wullschleger & Gunderson 1996; Scarascia-Mugnozza et al. 1996; Rey & Jarvis 1997; Tissue, Thomas & Strain 1997). This length of time is a small fraction of the average tree life-span, or of the typical forest rotation length, which in Europe ranges from 30 to 150 years. The only practical approach to this mismatch of experimental and natural time scales is to build computer models, based on experimental observation, which can be used to extrapolate responses to the long term (e.g. Ågren et al. 1991; Comins & McMurtrie 1993; Medlyn & Dewar 1996; Friend et al. 1997; Kellomäki, Vaisanen & Kolstrom 1997). This approach has its own challenges, however. A primary difficulty is the synthesis of experimental observation. Increasing numbers of elevated [CO 2 ] experiments are being done, often with widely varying results, and the traditional method of synthesizing experiments, the narrative review paper, may struggle to deal adequately with the large amounts of information available (Lee, Overdieck & Jarvis 1998; Saxe, Ellsworth & Heath 1998). New techniques of quantitative synthesis of experimental results, known as meta-analysis, are therefore being applied in this field (Gurevitch & Hedges 1993; Arnqvist & Wooster 1995; Curtis & Wang 1998). A second methodological challenge is to ensure a strong information flow from experiments to models. The process of model parameterization may be flawed if modellers do not have adequate access to experimental information. Catalogues of model parameter values, 1999 Blackwell Science Ltd 1475

2 1476 B. E. Medlyn et al. such as those compiled by Wullschleger (1993) and Ryan et al. (1994), are therefore invaluable for improving model predictions. These challenges were faced by the ECOCRAFT network, a group of laboratories conducting field-based experiments on the effects of elevated [CO 2 ] on European forest tree species. The network has existed since 1991, and the experimental results up to 1995 have been compiled by Jarvis (1998). Besford, Mousseau & Matteucci (1998) reviewed observations of photosynthetic rates in the ECOCRAFT experiments, and concluded that both up and down-regulation of photosynthesis has been found. Downregulation appears to be associated with either poor nutrient status or accumulation of starch, occurs more often late in the growing season and in the older needles of conifers. The group then faced the problem of quantifying these results in such a way that they could be included in models. This problem was addressed by establishing a central relational database of model parameters (Medlyn & Jarvis 1999). The parameters required, and the methods of deriving them from experimental data, were agreed upon by project working groups comprising both experimentalists and modellers. This paper reports on the photosynthesis parameters from the database, presenting them in formats useful for modelling. First, lists of photosynthesis parameters for different species, extracted from the database, are presented. This catalogue should provide a useful resource for modellers. Second, quantitative methods (i.e. meta-analysis) are used to estimate the effects of elevated [CO 2 ] on the parameters across experiments. Different hypotheses for the effects of long-term elevated [CO 2 ] on photosynthesis are examined. This analysis aids our understanding of photosynthetic responses to elevated [CO 2 ] and suggests ways in which we can improve model formulation. Photosynthesis is a key process when studying the effects of elevated [CO 2 ] on plants because it is directly affected by increasing [CO 2 ]. In the short term (hours), the response of photosynthesis to increasing [CO 2 ] is well understood: the rate of photosynthesis is increased, owing to the increased amount of substrate (Farquhar & von Caemmerer 1982). However, longer-term responses (months to years) are less well understood. In a number of early experiments with elevated [CO 2 ], down-regulation of photosynthesis was observed: that is, when photosynthesis was measured at the same CO 2 concentration, plants grown in elevated [CO 2 ] had lower photosynthetic rates than those grown in ambient [CO 2 ] (Eamus & Jarvis 1989; Gunderson & Wullschleger 1994; Sage 1994). However, it was suggested that this observation was an artefact resulting from growth of plants in small pots, and it was questioned whether downregulation would occur in field-grown plants (Arp 1991; Sage 1994). As a result of this criticism, the experimental focus subsequently shifted to field-based experiments. Down-regulation is less common in field-based experiments (Gunderson & Wullschleger 1994; Drake, Gonzalez- Meler & Long 1997), but, as noted by Besford et al. (1998), does sometimes occur (e.g. Van Oosten, Afif & Dizengremel 1992; Curtis et al. 1995; Marek, Kalina & Matoušková 1995; Epron, Liozon & Mousseau 1996). Debate continues about the significance of down-regulation of photosynthesis in the field, and the mechanism(s) causing down-regulation (see reviews by McGuire, Melillo & Joyce 1995; Drake et al. 1997; Wullschleger et al. 1997; Saxe et al. 1998; Norby et al. 1999). A number of alternative hypotheses have been proposed to explain down-regulation in elevated [CO 2 ]. One hypothesis is that down-regulation is the result of re-allocation of leaf nitrogen, away from the principal [CO 2 ]-fixing enzyme, Rubisco, and towards other nitrogenous compounds (Sage 1994). Such re-allocation would maximize plant nitrogenuse efficiency when carbon is present in excess. A second hypothesis is that increased C availability induces a nutrient limitation, leading to reduced leaf nutrient concentrations, and thereby reducing photosynthetic rates (Ceulemans & Mousseau 1994). Thirdly, it has been proposed that down-regulation results from a source sink imbalance; increased photosynthetic productivity is not matched by an increased demand for carbon, leading to a negative feedback on photosynthesis (Stitt 1991). Each of these hypotheses is expressed in terms of the photosynthesis model used in this paper, and meta-analysis is used to evaluate the likelihood of each. METHODS Experiments Experimental data were obtained from 15 separate elevated [CO 2 ] experiments. In each experiment, plants were grown in (at least) two atmospheric CO 2 concentrations, approximately 350 and 700 mmol mol -1. Brief details of each experiment are given in Table 1. More information on the experimental design of each experiment may be found in Pontailler et al. (1998) or the individual references given in Table 1. The experiments differed in a number of ways. They covered 12 different European forest tree species, including the most important commercial forestry species. Three main exposure facilities were employed: branch bag, open-top chamber, and mini-ecosystems. Some experiments included nutrient, drought, temperature, or ozone factorial treatments. However, there were two factors common to all experiments: they were all done on freely rooted plants, and all continued for at least two growing seasons. Photosynthesis and leaf nitrogen measurement protocols also differed between experiments. The key differences in methodology among experiments are outlined in Table 2. In most cases, photosynthesis measurements were performed in ambient conditions in the field, although sometimes with controlled temperature and vapour pressure deficit, and the Finnish partner made their measurements using detached shoots in the laboratory. The measurements were generally done as response curves of photosynthesis to intercellular [CO 2 ] (C i ) and/or incident photosynthetically active radiation (I), and results were expressed per unit leaf projected area (mmol m -2 s -1 ). The chief means

3 Table 1. The ECOCRAFT experiments Institution Age of Length of Number of Experiment name code 1 Latitude Longitude Species Nutrients 2 Water 2 plants 3 exposure 4 Stocking 5 replicates Reference Branch bags Flakaliden SLU N E Picea abies Low, high Medium Roberntz & Stockfors (1998) Glencorse BB UE N 3 12 W Picea sitchensis Medium High Barton, Lee & Jarvis (1993) Mekrijärvi BB JOY N E Pinus sylvestris Low, high High Kellomäki & Wang (1997) Mini-ecosystems Berlin 1 TUB N E Fagus sylvatica High High Forstreuter (1995) Berlin 2 TUB N E Fagus sylvatica High High Forstreuter (1996) Orsay UPS N 2 09 E Fagus sylvatica High Medium Badeck et al. (1997) Open-top chambers Glencorse OTC UE N 3 12 W Betula pendula Medium Medium Rey & Jarvis (1998) Headley mixed FC N W Pinus sylvestris, Low Low, high Crookshanks, Taylor & Quercus robur, Broadmeadow (1998) Fraxinus excelsior Headley oak FC N W Quercus petraea, Low Low Quercus rubra Macchia UVT N E Quercus ilex, Low Low De Angelis & Scarascia- Pistacia lentiscus, Mugnozza (1998) Phillyrea angustifolia Mekrijärvi OTC JOY N E Pinus sylvestris Low Medium Wang, Kellomäki & Laitinen (1996) Bily Kriz ILE N E Picea abies Medium Medium Marek, Kalina & Matoušková (1995) UIA Pine UIA N 4 24 E Pinus sylvestris Medium Medium Jach & Ceulemans (1999) UIA Poplar UIA N 4 24 E Populus Robusta, High High Ceulemans et al. (1996) Populus Beaupre UIA Poplar Coppice 6 UIA N 4 24 E Populus Robusta, High High 2-year-old Will & Ceulemans (1997) Populus Beaupre coppice Vielsalm FUSAG N 5 55 E Picea abies Low High Laitat, Loosveldt & Boussard (1994) Vielsalm Fert 6 FUSAG N 5 55 E Picea abies Low, high High Laitat, Chermanne & Portier (1999) Details of experiments from which photosynthesis parameters were obtained. In all experiments trees were freely rooted. 1 Institution Codes: SLU, Swedish University of Agricultural Sciences; UE, University of Edinburgh; JOY, University of Joensuu; TUB, Technical University of Berlin; UPS, Universite de Paris-Sud; FC, UK Forestry Commission; UVT, University of Viterbo; ILE, Academy of Sciences of the Czech Republic; UIA, Universitaire Instelling Antwerpen; FUSAG, Faculté Universitaire des Sciences Agronomiques de Gembloux. 2 The levels of nutrient and water availability in each experiment were classified by the persons responsible for the experiment. 3 Age of the plants (years) at the beginning of the experiment. 4 Length of exposure in growing seasons. 5 Stocking is in stems ha 1 for branch bag experiments, otherwise in plants per chamber. 6 Indicates experiments continuing on from previous experiments, after a major change in conditions. At Vielsalm, a mature overstory was removed and a fertilization treatment applied to half of each OTC. At UIA, the poplars were coppiced and allowed to re-grow. Photosynthetic acclimation to elevated CO2 1477

4 Table 2. The photosynthesis measurement protocols used in the ECOCRAFT experiments. The details of the experiments are given in Table 1 Experiment Field / Measurement Foliar nitrogen Foliar nitrogen name Species Equipment Laboratory type(s) Climatic conditions Sampling measurement sampling Branch bags Flakaliden Picea abies LI-COR 6200 Field A/C i Ambient T (10 24 C); RH ~ 50%; Current shoot from C:N analyser Needles used for gas PAR > 1000 mmol m -2 s -1 3rd 4th whorl exchange Glencorse BB Picea sitchensis ADC-LCA3 Field A/C i, A/I T 23 ± 3 C, VPD 0 6 ± 0 3 kpa, Shoots of three age Flow injection Shoots of three age PAR 800 mmol m -2 s -1 (for A/C i ) classes from 3rd whorl analyser classes from 3rd whorl Mekrijärvi BB Pinus sylvestris Automatic open Laboratory A/C i, A/I T 20 ± 0 5 C, VPD < 0 6 kpa Current shoot from Kjeldahl method Current shoot from system (Wang 3rd whorl 3rd whorl 1996) Mini-ecosystems Berlin 1 Fagus sylvatica Walz CMS-400 Field A/I T 20 C, VPD 0 75 to 0 94 kpa, Leaves at various C:N analyser Leaves at various CO 2 350/700 mmol mol -1 depths in canopy depths in canopy Berlin 2 Fagus sylvatica Walz CMS-400 Field A/C i at VPD 1 3 kpa, PAR > Leaves at various C:N analyser Leaves used for gas varying T 1200 mmol m -2 s -1 depths in canopy exchange Orsay Fagus sylvatica LI-COR 6400 Field A/C i, A/I T C, VPD kpa, Leaves at various C:N analyser Leaves used for gas PAR = 1000 mmol m -2 s -1 depths in canopy exchange (for A/C i ), CO 2 = 1500 ppm (for A/Q) Open-top chambers Glencorse OTC Betula pendula LI-COR 6200 Field A/C i Ambient T, VPD; PAR = Leaves at middle- Flow injection Recent fully 1200 mmol m -2 s -1 bottom of canopy analyser expanded leaves from middle crown Headley Mixed Pinus sylvestris, LCA3 Field A/C i, A/I T 25 C; PAR 1675 mmol m -2 s -1 Leaves / shoots at Kjeldahl method Youngest fully Quercus robur, (Quercus), (for A/C i ); growth [CO 2 ] (for A/Q) top of canopy expanded leaves in Fraxinus excelsior A max full sun (Fraxinus) Headley Oak Quercus petraea, CIRAS Field A/C i T 25 C; PAR 1675 mmol m -2 s -1 Leaves at top of Kjeldahl method Leaves used for gas Quercus rubra canopy exchange Macchia Quercus ilex, Walz CMS-400 Field A/C i T 25 C; VPD 1 3 kpa; PAR Sun leaves at top C:N analyser Sun leaves at top of Pistacia lentiscus, 1200 mmol m -2 s -1 of canopy canopy Phillyrea angustifolia Mekrijärvi OTC Pinus sylvestris Automatic open Laboratory A/C i, A/I at VPD < 0 6 kpa Current shoot from Kjeldahl method Measurements made system (Wang varying T lower third of canopy after harvest on 1996) three age classes in three layers Bily Kriz Picea abies LI-COR 6250 Field A/C i, A/I T 20 ± 2 C; RH 55 ± 3% Current and 1-year-old Amylase Current shoots from or CIRAS shoots from 4th 5th protocol 3rd whorl whorl UIA Pine Pinus sylvestris CIRAS Field A/C i, A/I Ambient T; RH 80%; 1 2 fascicles from Dynamic flush Needles used for PAR 970 mmol m -2 s -1 (for A/C i ) top of canopy combustion gas exchange method UIA Poplar Populus Robusta ADC-LCA3 Field A max T C; RH 65%; light Mature, fully Kjeldahl method Mature, fully Populus Beaupre saturation; growth [CO 2 ] expanded leaves from expanded leaves upper canopy from upper canopy UIA Poplar Populus Robusta ADC-LCA3 Field A max T C; RH 65%; PAR > 2 3 ramets per clone Coppice Populus Beaupre 1200 mmol m -2 s -1 ; [CO 2 ] 350/700 ppm Vielsalm Picea abies Binos 100 IRGA Field A/C i, A/I T 18 C; VPD < 0 8 kpa; Current-year shoots Kjeldahl method Random samples of PAR 1100 mmol m -2 s -1 (for A/C i ); all age classes growth [CO 2 ] (for A/Q) Vielsalm Fert Picea abies Binos 100 IRGA Field A/C i, A/I T 18 C; VPD < 0 8 kpa; 2nd-order shoots from Kjeldahl method Shoots of all ages PAR 1100 mmol m -2 s -1 (for A/C i ); 2nd whorl from 4th whorl growth [CO 2 ] (for A/Q) 1478 B. E. Medlyn et al.

5 Photosynthetic acclimation to elevated CO used to measure leaf nitrogen concentration were the Kjeldahl method and C:N analysis, although some groups also used flow injection analysis, an amylase protocol, or the dynamic flush combustion method (Table 2). In most cases, specific leaf area was also measured, using either a leaf area meter or leaf dimensions, allowing leaf nitrogen to be expressed both on a mass (g g -1 ) and an area (g m -2 ) basis. Database The parameters stored in the database cover a wide range of plant processes such as photosynthesis, respiration, stomatal conductance and carbon allocation. The parameters for each process were decided upon by project working groups comprising both experimentalists and modellers. The list of parameters used in compiling this review is given in Table 3; it includes photosynthetic capacity, the parameters of the Farquhar & von Caemmerer (1982) model of photosynthesis, leaf chemistry, and specific leaf area. To ensure comparability of parameters obtained from different experiments, it was necessary to define the parameters clearly, and, in the case of the photosynthesis model, to specify the equations used in their derivation. Definitions of the parameters covered in this review are given in Table 3.The agreed form of the Farquhar & von Caemmerer (1982) model is given in Appendix 1. The temperature relations used to correct the parameters of this model to 25 C are also given in Appendix 1. Parameter values were obtained from data by each partner and transmitted to the central database. A common Excel spreadsheet was used for fitting the Farquhar & von Caemmerer (1982) model by a number of partners; other partners implemented the same equations in other statistical computer packages. Statistical analysis The chief statistical technique employed was meta-analysis. The meta-analysis techniques used were those described by Curtis & Wang (1998) and implemented in the statistical software MetaWin (Rosenberg, Adams & Gurevitch 1997). The mean, standard deviation, and number of observations for each parameter value were required. The standard deviation was taken to be the between-chamber standard deviation, and the number of observations was taken as the number of chamber replicates, although the total standard deviation of all measurements is also stored in the database. The standard deviation is used in the meta-analysis to weight each observation. Some observations in the dataset had no corresponding standard deviation because there was only one chamber replicate. These observations were included conservatively, by assigning to them the smallest of the weights of the other experiments. The meta-analysis was done on the natural logarithm of the response ratios, as described by Curtis & Wang (1998). A mixed-model analysis was assumed (Gurevitch & Hedges 1993). Each observation used in the meta-analysis is required to be independent. Parameter values from different nutrient, drought or temperature treatments, or from different species, in the same experiment, were assumed to be independent and therefore included separately in the analysis, following the precedent of Curtis & Wang (1998). However, where parameter values were given for different measurement periods (different seasons or different years), only one value was used. The value from the middle of the growing season in the final year of the experiment was chosen where possible. The effects of elevated [CO 2 ] on photosynthetic rates over time were considered separately from the meta-analysis (see Results). Additionally, if values were given for different canopy layers or age classes of foliage, the value for current-year foliage from the top of the canopy was selected. The meta-analysis was used to determine the effect of elevated [CO 2 ] on model parameters. In addition, the effect on the relationships between model parameters (such as V cmax and N a ) was examined using a simple statistical test for coincidence of these relationships in ambient and elevated [CO 2 ] (Kleinbaum et al. 1998; p 329). Table 3. Definitions of parameters used in compiling the meta-analysis. These definitions are taken from the ECOCRAFT database Parameter name Units Definition A max mmol m -2 s -1 The light-saturated rate of photosynthesis, measured at the growth CO 2 concentration A 350 mmol m -2 s -1 The light-saturated rate of photosynthesis, measured at a CO 2 concentration of 350 mmol mol -1 A 700 mmol m -2 s -1 The light-saturated rate of photosynthesis, measured at a CO 2 concentration of 700 mmol mol -1 J max mmol m -2 s -1 The potential electron transport rate at 25 C a J mmol m -2 s -1 The intercept of the relationship between J max and N a b J mmol g -1 Ns -1 The slope of the relationship between J max and N a V cmax mmol m -2 s -1 The maximum rate of Rubisco activity at 25 C a V mmol m -2 s -1 The intercept of the relationship between V cmax and N a b V mmol g -1 Ns -1 The slope of the relationship between V cmax and N a N m gng -1 leaf The leaf nitrogen concentration (dry mass basis) N a gnm -2 leaf The leaf nitrogen concentration (leaf area basis) Chl a g Chl m -2 leaf The leaf chlorophyll content per unit area Stch g starch g -1 leaf The leaf starch concentration (dry mass basis) SLA m 2 kg -1 The specific leaf area (projected) on a dry mass basis

6 1480 B. E. Medlyn et al. RESULTS Photosynthesis meta-analysis The results of the meta-analysis of photosynthetic rates are shown in Table 4. Across the 15 experiments, the light-saturated photosynthetic rate at the growth CO 2 concentration, A max, was increased significantly, by 51%, in elevated [CO 2 ]. Despite this increase, down-regulation of photosynthetic rates in elevated [CO 2 ] was observed consistently in the experiments. When measured at the same CO 2 concentration, the photosynthetic rate was significantly reduced in the elevated compared to the ambient [CO 2 ] treatment. When measured at [CO 2 ] = 350 mmol mol -1, the photosynthetic rate (A 350 ) was reduced by 19%; when measured at [CO 2 ] = 700 mmol mol -1, the photosynthetic rate (A 700 ) was reduced by 9% (Table 4). The reductions in A 350 and A 700 may be partially caused by increased stomatal limitation in elevated [CO 2 ]. In this paper, we are principally concerned with the effects of elevated [CO 2 ] on the underlying biochemistry of photosynthesis; we will address the issue of the effects of elevated [CO 2 ] on stomatal conductance in a separate paper (Medlyn et al., in preparation).the effects of changes in stomatal conductance can be eliminated from the analysis by considering the A/C i response curve, and, in particular, the parameters of the Farquhar & von Caemmerer (1982) photosynthesis model,namely the maximum rate of electron transport,j max, and the maximum rate of Rubisco activity, V cmax. The meta-analysis shows that J max and V cmax were both significantly reduced by approximately 10% by growth in elevated [CO 2 ] across all the experiments (Table 4). The reduction in these parameters indicates that growth in elevated [CO 2 ] did cause some adjustment in photosynthetic capacity at the biochemical level. It appears that growth in elevated [CO 2 ] affected J max and V cmax equally, since both were reduced by a similar amount, and the relationship between J max and V cmax did not change in elevated [CO 2 ] (Fig. 1). The values of J max and V cmax obtained from the experiments are listed in Table 5. Simple inspection of Tables 1 and 5 shows that no single experimental factor can be used to separate those experiments in which photosynthesis was down-regulated from those in which it was not. Meta-analysis was used to test this conclusion, using the methodology of Curtis & Wang (1998). Experiments were categorized according to water availability (high, or low or intermediate), nutrient availability (high, or low or intermediate), age of plants (young or mature), functional group (coniferous, deciduous or broadleaf evergreen) and experiment type (branch bag, open-top chamber or mini-ecosystem) (Table 1). Each grouping was tested to determine whether the response of A max, J max and V cmax to elevated [CO 2 ] differed between any categories. None of the tests showed any significant difference between categories (P > 0 14 in all cases), suggesting that down-regulation cannot easily be attributed to any one single experimental factor. Effects of elevated [CO 2 ] over time One limitation of the meta-analysis procedure is that only one observation from each experiment can be included, because of the requirement that observations be independent. Observations were taken, where possible, from the middle of the growing season in the final year of the experiment. However, the effect of elevated [CO 2 ] on photosynthesis may change over time. Photosynthetic rates change as foliage ages and senesces, and this process may be hastened or delayed by growth in elevated [CO 2 ] (Curtis & Teeri 1992; Lewis, Tissue & Strain 1996; Murthy, Zarnoch & Dougherty 1997). Additionally, feedbacks to photosynthesis from growth in elevated [CO 2 ], such as nutrient limitation, may take time to develop (Gifford et al. 1996). Such feedbacks may only become apparent after several growing seasons of exposure. Thus, the effect of elevated [CO 2 ] on photosynthesis may change during the course of a year, or from year to year. It is therefore necessary to examine the Mean effect 95% confidence Different from 1 Parameter size interval n (at 5% level) Table 4. Output from the meta-analysis of photosynthesis parameters A max Yes A Yes A Yes J max Yes V cmax Yes N m Yes SLA Yes N a No Chl a No Stch Yes For each parameter, the estimate of the mean effect size (value in elevated [CO 2 ]:value in ambient [CO 2 ]) and the confidence interval on the effect size are given. n is the number of observations, which varies between parameters as not all parameters were measured in all experiments

7 Photosynthetic acclimation to elevated CO Figure 1. Relationship between the mean values of J max and V cmax given in Table 5. Filled symbols, values for trees grown in ambient [CO 2 ] (350 mmol mol -1 ); open symbols, values in elevated [CO 2 ] (700 mmol mol -1 )., : values for conifers;, : values for broadleaves. The solid regression line is fitted to all ambient values; the dotted regression line is fitted to all elevated values. The regression equations are: ambient, J max = 2 39 V cmax 14 2, r 2 = 0 80; elevated, J max = 2 5 V cmax 14 3, r 2 = time course of the [CO 2 ] response, in addition to conducting the meta-analysis on snapshot observations. The effects of elevated [CO 2 ] on photosynthesis at different times during the growing season were measured in seven of the experiments: Glencorse OTC, Glencorse BB, Bily Kriz, UIA Pine, UIA Poplar Coppice, Headley Oaks and Orsay. There was some evidence for increasing downregulation of photosynthesis with foliage age (Fig. 2). In the Glencorse OTC (birch) experiment, the stimulation of photosynthesis by elevated [CO 2 ] declined steadily from June to September. Similar patterns were observed in beech at Orsay (cf. Table 5) and in both poplar species in the UIA Poplar Coppice experiment. The one exception among the deciduous species was the Headley Mixed Oaks experiment, in which stimulation of photosynthesis by elevated [CO 2 ] increased over the period May to early September. Foliage age influenced the photosynthetic response to elevated [CO 2 ] in conifers also. Down-regulation of photosynthesis increased dramatically between April and October in P. abies at Bily Kriz. It should be noted, however, that in this experiment, [CO 2 ] fumigation ceased during winter and was only re-started in April of each year. Stimulation of photosynthesis by elevated [CO 2 ] was also less in one-year-old needles, compared to current-year needles, in both P. sylvestris (UIA Pine) and P. sitchensis (Glencorse BB). The effects of elevated [CO 2 ] on photosynthesis in different years were also measured in several of the experiments (Fig. 3). In the experiments with mature trees, the effect of elevated [CO 2 ] on photosynthesis did not change with increasing exposure length. In the branch bag experiments at Glencorse (Picea sitchensis) and at Flakaliden (P. abies), photosynthesis was stimulated by elevated [CO 2 ] to the same degree in the third or fourth growing season as it was in the first. The mature P. abies growing in open-top chambers at Bily Kriz showed down-regulation of photosynthesis, but the degree of down-regulation did not increase between the first and third years of the experiment. In young beech trees growing in the Berlin miniecosystems, photosynthesis continued to be stimulated to the same extent by elevated [CO 2 ] for four years. In three other experiments with young trees, however, photosynthesis was stimulated less by elevated [CO 2 ] in the later years of the experiment. In the UIA Pine experiment, down-regulation of photosynthesis occurred in the second year, but not in the first. In the Orsay experiment, photosynthesis was up-regulated in the second year but not in the third. In the Headley Mixed experiment, down-regulation became apparent in the third year of the experiment. Thus, growth in elevated [CO 2 ] can cause long-term feedbacks on photosynthesis which take several years to develop. In our experiments, such feedbacks were most evident in young, rapidly growing plants. Interaction with nitrogen availability Photosynthetic rates are generally related to leaf nitrogen content (Field & Mooney 1986), and relationships with leaf nitrogen (Eqns A14 & A15) are commonly used to estimate the parameters J max and V cmax (Field 1983; Harley et al.

8 Table 5. Values of J max and V cmax at 25 C for trees grown in ambient and elevated [CO 2 ] conditions, obtained from ECOCRAFT experiments Ambient [CO 2 ] Elevated [CO 2 ] Experiment Years of name Date exposure Species Note Mean Replicate SD Total SD Mean Replicate SD Total SD Values of J max Glencorse OTC 1-Jun-94 4 Betula pendula (6) (6) Glencorse OTC 1 1-Aug-94 4 Betula pendula (4) (4) Glencorse OTC 1-Sep-94 4 Betula pendula (6) (6) Berlin 2 1-Aug-94 1 Fagus sylvatica (13) (12) Berlin 2 1-Aug-95 2 Fagus sylvatica (24) (24) Berlin 2 25-Jul-96 3 Fagus sylvatica (9) (10) Berlin Aug-97 4 Fagus sylvatica (13) (14) Orsay 15-Sep-96 2 Fagus sylvatica (3) (5) Orsay 18-May-97 3 Fagus sylvatica (4) (4) Orsay 20-Jul-97 3 Fagus sylvatica (4) (4) Orsay 1 30-Aug-97 3 Fagus sylvatica (4) (4) Bily Kriz 1-Apr-93 1 Picea abies (4) (12) (4) 9 5 (12) Bily Kriz 1-Oct-93 1 Picea abies (4) 6 8 (12) (4) 5 44 (12) Bily Kriz 1-Apr-95 3 Picea abies (4) (12) (4) (12) Bily Kriz 1 1-Oct-95 3 Picea abies (4) (13) (4) 6 93 (12) Flakaliden 1 2-Sep-95 4 Picea abies Unfertilized (6) (6) Flakaliden 1 5-Sep-95 4 Picea abies Fertilized (6) (6) Vielsalm Aug-93 5 Picea abies (2) (6) (2) (6) Vielsalm Aug-96 8 Picea abies Unfertilized (2) (6) Vielsalm Aug-96 8 Picea abies Fertilized (3) (2) (5) Glencorse BB 1 14-Aug-94 4 Picea sitchensis (6) (6) Mekrijärvi OTC 1 28-Jul-94 3 Pinus sylvestris Ambient T (4) (4) Mekrijärvi OTC 1 28-Jul-94 3 Pinus sylvestris Elevated T (4) (4) UIA Pine 1-Aug-96 1 Pinus sylvestris (2) (6) (2) (8) UIA Pine 15-May-97 2 Pinus sylvestris C + 1 needles (2) (8) (2) (8) UIA Pine 15-Jul-97 2 Pinus sylvestris C needles (2) (8) (2) (7) UIA Pine 15-Jul-97 2 Pinus sylvestris C + 1 needles (2) (8) (2) (6) UIA Pine 1 15-Sep-97 2 Pinus sylvestris C needles (2) (8) (2) (8) UIA Pine 15-Sep-97 2 Pinus sylvestris C + 1 needles (2) (8) (2) (8) Macchia 1 1-Jun-94 3 Phillyrea angustifolia (2) 51 (4) (2) Macchia 1 1-Jun-94 3 Pistacia lentiscus (2) 46 (4) (2) 10 (4) Macchia 1 1-Jun-94 3 Quercus ilex (2) 58 (4) (2) 6 (4) Headley Mixed 1 1-Jul-96 3 Quercus petraea (2) 28 6 (10) (2) 19 6 (12) Headley Oak 1-May-97 1 Quercus petraea (4) 20 (8) (4) 15 (8) Headley Oak 1-Jun-97 1 Quercus petraea (4) 33 (8) (4) 30 (8) Headley Oak 1-Jul-97 1 Quercus petraea (4) 39 (8) (4) 35 (8) Headley Oak 1 1-Aug-97 1 Quercus petraea (4) 34 (8) (4) 39 (8) Headley Oak 1 1-Jul-97 1 Quercus rubra (4) 24 (8) (4) 20 (8) 1482 B. E. Medlyn et al.

9 Values of V cmax Glencorse OTC 1-Jun-94 4 Betula pendula (6) (6) Glencorse OTC 1 1-Aug-94 4 Betula pendula (4) (4) Glencorse OTC 1-Sep-94 4 Betula pendula (6) (6) Berlin 2 1-Aug-94 1 Fagus sylvatica (13) (12) Berlin 2 1-Aug-95 2 Fagus sylvatica (24) (24) Berlin 2 25-Jul-96 3 Fagus sylvatica (9) (10) Berlin Aug-97 4 Fagus sylvatica (13) (13) Orsay 15-Sep-96 2 Fagus sylvatica (3) (5) Orsay 18-May-97 3 Fagus sylvatica (4) (4) Orsay 20-Jul-97 3 Fagus sylvatica (4) (4) Orsay 1 30-Aug-97 3 Fagus sylvatica (4) (4) Bily Kriz 1-Apr-93 1 Picea abies (4) 8 19 (12) (4) 4 13 (12) Bily Kriz 1-Oct-93 1 Picea abies (4) 18 9 (12) (4) 7 21 (12) Bily Kriz 1-Apr-95 3 Picea abies (4) 6 97 (12) (4) 9 83 (12) Bily Kriz 1 1-Oct-95 3 Picea abies (4) 4 73 (12) (4) (12) Flakaliden 1 1-Sep-95 4 Picea abies Unfertilized (6) (6) Flakaliden 1 4-Sep-95 4 Picea abies Fertilized (6) (6) Vielsalm Aug-93 5 Picea abies (2) 13 6 (6) (2) 2 91 (6) Vielsalm Aug-96 8 Picea abies Unfertilized (2) (6) Vielsalm Aug-96 8 Picea abies Fertilized (3) (2) (5) Glencorse BB 1 14-Aug-94 4 Picea sitchensis (6) (6) Mekrijärvi OTC 1 28-Jul-94 3 Pinus sylvestris Ambient T (4) (4) Mekrijärvi OTC 1 28-Jul-94 3 Pinus sylvestris Elevated T (4) (4) UIA Pine 1-Aug-96 1 Pinus sylvestris (2) (6) (2) (8) UIA Pine 15-May-97 2 Pinus sylvestris C + 1 needles (2) 9 35 (8) (2) (8) UIA Pine 15-Jul-97 2 Pinus sylvestris C needles (2) (18) (2) (12) UIA Pine 15-Jul-97 2 Pinus sylvestris C + 1 needles (2) (16) (2) 6 71 (11) UIA Pine 1 15-Sep-97 2 Pinus sylvestris C needles (2) (12) (2) (12) UIA Pine 15-Sep-97 2 Pinus sylvestris C + 1 needles (2) (12) (2) (12) Macchia 1 1-Jun-94 3 Phillyrea angustifolia (2) (4) (2) Macchia 1 1-Jun-94 3 Pistacia lentiscus (2) (4) (2) 5 65 (4) Macchia 1 1-Jun-94 3 Quercus ilex (2) (4) (2) 3 3 (4) Headley Mixed 1 1-Jul-96 3 Quercus petraea (2) 8 75 (10) (2) 8 47 (12) Headley Oaks 1-May-97 1 Quercus petraea (4) 14 (8) (4) 15 (8) Headley Oaks 1-Jun-97 1 Quercus petraea (4) 14 (8) (4) 10 (8) Headley Oaks 1-Jul-97 1 Quercus petraea (4) 8 (8) (4) 9 (8) Headley Oaks 1 1-Aug-97 1 Quercus petraea (4) 8 (8) (4) 9 (8) Headley Oaks 1 1-Jul-97 1 Quercus rubra (4) 6 (8) (4) 8 (8) All values were derived from experimental data using the equations given in Appendix 1. Values are given as means and standard deviations. Two different standard deviations (SD) are given: the between-chamber (replicate) SD and the SD of all measurements (total SD). The number of observations is given in brackets for each SD. Values for conifer species are expressed on a projected area basis. 1 Observation included in meta-analysis. Photosynthetic acclimation to elevated CO2 1483

10 1484 B. E. Medlyn et al. Figure 2. Effect size of elevated [CO 2 ] on A max, expressed as a ratio of the elevated [CO 2 ] value to the ambient [CO 2 ] value, as a function of foliage age (calculated from January of year of initiation). Open symbols, values for conifers; filled symbols, values for broadleaves. Key to experiments: Bily Kriz; Glencorse BB; UIA Pine; Glencorse; UIA Poplar (Beaupré); UIA Poplar (Robusta). 1992). Thus, if photosynthesis is down-regulated in elevated [CO 2 ],it is pertinent to ask whether this is caused by a reduction in leaf nitrogen content or a shift in the relationship between the photosynthetic parameters and leaf nitrogen. Meta-analysis was used to analyse whether growth in elevated [CO 2 ] leads to changes in leaf nitrogen concentration (Table 4). When expressed on a per unit mass basis, leaf nitrogen (N m ) was significantly reduced, by approximately 15%, in elevated [CO 2 ]. However, this was compensated for by a similar decrease in specific leaf area, so that leaf nitrogen per unit area (N a ) was unchanged in elevated [CO 2 ]. The general reduction in leaf nitrogen concentration may thus be thought of as a dilution effect, caused by increased leaf mass per unit area. The increase in leaf mass per area was at least partly caused by an accumulation of starch; starch content per unit area increased significantly in elevated [CO 2 ] (Table 4). The dilution effect does not account for the down-regulation of photosynthesis on an area basis, since leaf nitrogen per unit area was unchanged. Leaf chlorophyll per unit area was also not significantly reduced in elevated [CO 2 ] (Table 4). However, although the meta-analysis shows no general reduction in leaf nitrogen per unit area, it does not necessarily follow that leaf nitrogen plays no role in causing photosynthetic down-regulation. Figure 4 compares the ratio of values of V cmax measured in elevated and ambient [CO 2 ] to the ratio of the values of N a in elevated and ambient [CO 2 ]. There is a clear correlation between the effect of elevated [CO 2 ] on V cmax and that on N a. Thus, although there was no general reduction in N a in elevated [CO 2 ] across all experiments, those experiments in which there was a reduc- tion in N a also tended to show a reduction in V cmax.the correlation implies that there is a link between effects of elevated [CO 2 ] on leaf nitrogen and its effects on photosynthesis. We also investigated whether the relationships between the photosynthetic parameters, J max and V cmax, and leaf nitrogen concentration, changed in elevated [CO 2 ]. A down-regulation in photosynthesis could be explained by a downward shift in these relationships in elevated [CO 2 ]. These relationships were examined in six of the experiments (Table 6). The source of variation in leaf nitrogen content differed between the different experiments; data from different measurement periods, years, canopy layers and nutrient treatments were combined to obtain these relationships. In the case of deciduous species, it was appropriate to combine data from foliage of different ages, but foliage of different age classes in pine behaved sufficiently differently to warrant fitting separate relationships. For foliage less than one year old, the strength of the relationships between J max or V cmax and N a varied across the experiments and treatments, with values of r 2 ranging from 0 08 to 0 94, but in most cases good correlations were found. The probabilities that the relationships do not differ between ambient and elevated [CO 2 ] treatments are given in Table 6.The relationships between V cmax and N a are illustrated in Fig. 5. The relationships are statistically different (P < 0 05) between ambient and elevated [CO 2 ] treatments for UIA Pine, Mekrijärvi OTC and Headley Oaks experiments. However, Fig. 5 demonstrates that in only one of these experiments, UIA Pine, did the difference take the form of a reduction in the photosynthetic parameters per

11 Photosynthetic acclimation to elevated CO Figure 3. Effect size of elevated [CO 2 ] on A max, expressed as a ratio of the elevated [CO 2 ] value to the ambient [CO 2 ] value, as a function of years of [CO 2 ] treatment. (a) Values from experiments with mature trees. Key: Flakaliden BB (fertilized); Flakaliden BB (unfertilized); Bily Kriz; Glencorse BB. (b) Values from experiments with young trees. Key: UIA Pine; Berlin 2; Orsay; Headley mixed (Quercus petraea). unit leaf nitrogen. We conclude that, for current foliage, down-regulation which is related to a downward shift in the photosynthetic parameters nitrogen relationship can occur, but is perhaps less likely than down-regulation caused by reduced leaf nitrogen, since the former only occurred in one of six experiments. The relationships between photosynthetic parameters and leaf nitrogen differed considerably between different age classes of needle in pine (Table 6), with the relationships shifting downwards with increasing needle age. The downward shift in older needles was more pronounced in elevated than in ambient [CO 2 ] (Table 6), which may indicate that needles aged more rapidly in elevated [CO 2 ]. DISCUSSION Meta-analysis Meta-analysis provides a very useful methodology for quantifying the effects of elevated [CO 2 ] on woody plant

12 1486 B. E. Medlyn et al. Figure 4. Effect size of elevated [CO 2 ] on V cmax, expressed as a ratio of the elevated [CO 2 ] value to the ambient [CO 2 ] value, versus the effect on leaf nitrogen content per unit area. Key: values included in the meta-analysis; other values. growth. The chief advantage of meta-analysis is that it provides a rigorous quantitative summary of large sets of data. A second advantage is the increased statistical power obtained by pooling a number of datasets, enabling small trends, such as the 10% decrease in leaf nitrogen concentration, to be rendered significant. Meta-analysis techniques were particularly appropriate for this study, for which an unbiased set of comparable parameter values was available. Clear definitions of the parameters required, and methods for their derivation, were supplied to all project partners. The parameter values are therefore directly comparable, avoiding one problem faced by meta-analysis based on published literature, that parameters may be derived in quite different ways by different authors. A second criticism of meta-analysis based on published literature is that known as the desk drawer problem (Cooper & Hedges 1994), i.e. the outcome of a meta-analysis may be biased because of the tendency to publish only significant results, and to consign non-significant results to the desk drawer. This bias is also not present in the current study because all available data from ECOCRAFT experiments with plants rooted in the ground were transmitted to the database. However, the results from the current study are generally comparable with results from previous meta-analyses based on published literature (Curtis 1996; Curtis & Wang 1998). The results of the meta-analysis of leaf nitrogen content in the current study agree closely with those of Curtis (1996): both studies conclude that leaf nitrogen per unit mass and specific leaf area are reduced, but leaf nitro- Table 6. Relationships between photosynthetic parameters J max and V cmax (mmol m -2 s -1 ) and leaf N concentration (N a,gm -2 ) in ambient and elevated [CO 2 ], obtained from ECOCRAFT experiments Experiment Species Ambient r 2 n Elevated r 2 n P Combined r 2 Regressions of J max against N a Berlin 2 Fagus sylvatica 55 6 N a N a N a Orsay Fagus sylvatica 40 1 N a N a N a Headley Oaks Quercus petraea 75 9 N a N a N a Flakaliden Picea abies 16 2 N a N a N a Mekrijärvi OTC Pinus sylvestris 23 8 N a N a N a UIA Pine Pinus sylvestris C 53 1 N a N a < N a Pinus sylvestris C N N < N a Regressions of V cmax against N a Berlin 2 Fagus sylvatica 32 6 N a N a N a Orsay Fagus sylvatica 17 6 N a N a N a Headley Oaks Quercus petraea 12 7 N a N a N a Flakaliden Picea abies 20 N a N a N a Mekrijärvi OTC Pinus sylvestris 11 4 N a N a N a UIA Pine Pinus sylvestris C 13 5 N a N a < N a Pinus sylvestris C N N < N a P, probability that regression lines from ambient and elevated [CO2] treatments are coincident. Regression lines are illustrated in Fig. 5. Values from different years, different fertilization treatments, and different canopy layers were combined to obtain these relationships

13 Photosynthetic acclimation to elevated CO Figure 5. Relationships between the photosynthetic parameter V cmax and leaf nitrogen concentration obtained from ECOCRAFT experiments: (a) Orsay; (b) Berlin 2; (c) Headley Oaks; (d) Flakaliden BB; (e) Mekrijärvi OTC; (f) UIA Pine. The equations for the regression lines are given in Table 6. Key: filled symbols and solid lines, ambient [CO 2 ]; open symbols and dashed lines, elevated [CO 2 ]. gen per unit area is unaffected by growth in elevated [CO 2 ]. With respect to photosynthetic rates, Curtis & Wang (1998) also found that A max was strongly stimulated by growth in elevated [CO 2 ], by 52%. Interestingly, however, Curtis & Wang (1998) found no reduction in photosynthetic rates measured at a common [CO 2 ] across their dataset, whereas this study found a significant 18% reduction in A 350.The two studies are based on quite different sets of experiments: while our study includes only long-term, field-based experiments, Curtis & Wang s (1998) study included short-term and indoor pot experiments as well. Curtis & Wang (1998) also used meta-analysis techniques to test for differences in effect size between categorical groupings of experiments. We followed the same procedure in the current study, but found no differences in effect size between categories in any grouping, in contrast to a number

14 1488 B. E. Medlyn et al. Figure 5. Continued of other studies. Ceulemans & Mousseau (1994), for example, reported a difference in the CO 2 response of growth between coniferous and deciduous species, while McGuire et al.(1995) found an effect of nutrition on the CO 2 response of photosynthesis. It has also been suggested that responses in branch-bag experiments should differ those in whole-tree exposures, as feedbacks which operate at the whole-tree level would not be observed (Saxe et al. 1998). In this study, none of these differences between categories were observed, indicating that the CO 2 response was somewhat independent of experimental technique. Unfortunately, this conclusion is not strong, because the small number of experiments limited the utility of the testing procedure. It was not possible to carry out a two-level categorization (e.g. nutrient level functional grouping) because some categories then contained too few experiments. A further limitation to the meta-analysis is that only one time point from each experiment can be included. It was helpful to examine time series of effect sizes as an adjunct to the meta-analysis. Comparison of effect sizes at different time points showed that down-regulation of photosynthesis in elevated [CO 2 ] tended to increase with increasing