Photosynthetic acclimation to elevated C0 2 in poplar grown in glasshouse cabinets or in open top chambers depends on duration of exposure

Journal of Experimental Botany, Vol. 48, No. 314, pp. 1681-1689, September 1997 Journal of Experimental Botany Photosynthetic acclimation to elevated C0 2 in poplar grown in glasshouse cabinets or in open top chambers depends on duration of exposure R. Ceulemans 1 ' 4, G. Taylor 2, C. Bosac 2, D. Wilkins 3 and R.T. Besford 35 1 Department of Biology, University of Antwerp, UIA, Universiteitsplein 1, B-2610 Wilrijk, Belgium 2 School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex BN19QG, UK 3 Horticulture Research International, Worthing Road, Littlehampton, West Sussex BN176LP, UK Received 15 January 1997; Accepted 10 May 1997 Abstract The effects of elevated CO 2 were studied on the photosynthetic gas exchange behaviour and leaf physiology of two contrasting poplar (Populus) hybrids grown and treated in open top chambers (OTCs in Antwerp, Belgium) and in closed glasshouse cabinets (GHCs in Sussex, UK). The CO 2 concentrations used in the OTCs were ambient and ambient +350 fimo\ mol \ while in the GHCs they were c. 360/miol mol 1 versus 719 //mol mol \ Measurements of photosynthetic gas exchange were made for euramerican and interamerican poplar hybrids in combination with measurements of dark respiration rate and Rubisco activity. Significant differences in the leaf anatomy and structure (leaf mass per area and chlorophyll content) were observed between the leaves grown in the OTCs and those grown in the GHCs. CO 2 stimulated net photosynthesis in the poplar hybrids after 1 month in the GHCs and after 4 months in the OTCs, and there was no evidence of downward acclimation (or downregulation) of photosynthesis when the plants in the two treatments were measured in their growth C0 2 concentration. There was also no evidence of downregulation of Rubisco activity and there were even examples of increases in Rubisco activity. Rubisco exerted a strong control over the light-saturated rate of photosynthesis, which was demonstrated by the close agreement between observed net photosynthetic rates and those that were predicted from Rubisco activities and Michaelis-Menten kinetics. After 17 months in elevated CO 2 in the OTCs there was a significant loss of Rubisco activity for one of the hybrid clones, i.e., but not for clone. The effect of the CO 2 measurement concentration (i.e. the short-term treatment effect) on net photosynthesis was always larger than the effect of the growth concentration in both the OTCs or GHCs (i.e. the longterm growth CO 2 effect), with one exception. For the interamerican hybrid dark respiration rates in the OTCs were not significantly affected by the elevated CO 2 concentrations. The results suggest that for rapidly growing tree species, such as poplars, there is little evidence for downward acclimation of photosynthesis when plants are exposed to elevated CO 2 for up to 4 months; longer term exposure reveals loss of Rubisco activity. Key words: CO 2, Populus, Rubisco, photosynthesis, chlorophyll content. Introduction Short-term exposure of C 3 plants to elevated atmospheric CO 2 concentrations often stimulates photosynthesis (Eamus and Jarvis, 1989; Gifford, 1992), producing major gains in biomass as a result of the improved competitiveness of CO 2 over O 2 as a substrate for the main C 3 photosynthetic enzyme, ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) (Bowes, 1993). Plants grown in elevated CO 2 can show a degree of photosynthetic acclimation (Besford et ai, 1990), i.e. an increase or more commonly a decrease in photosynthetic perform- * To whom correspondence should be addressed. Fax: +32 3 8202271. E-mail: rceulemeuia.ua.ac.be 5 Present address: Furlong, The Street, Patching, Worthing, West Sussex BN13 3XF, UK Oxford University Press 1997

1682 Ceulemans et al. ance as compared with plants grown in low (ambient) concentrations of CO 2, when measured under the same conditions, due to intrinsic changes in the photosynthetic machinery (Gunderson and Wullschleger, 1994). While the adaptive response of plants to elevated CO 2 can be positive, negative or indeed negligible (Ceulemans and Mousseau, 1994), in this paper the term acclimation will refer only to the negative aspects of plant response to elevated CO 2. Because photosynthesis more often than not increases with atmospheric CO 2 enrichment (Kalina and Ceulemans, 1997), the approach of comparing rates of net photosynthesis for leaves or plants measured at their respective growth CO 2 concentration (ambient or elevated) in many instances yields little information on acclimation per se if rates remain higher in elevated CO 2 (Long and Drake, 1991; Gunderson and Wullschleger, 1994). Therefore, the technique of measuring rates of net photosynthesis after plants grown at elevated CO 2 have been transferred to a similar ambient CO 2 atmosphere, can be used. If rates of net photosynthesis are comparable between leaves grown at elevated and ambient CO 2, then an acclimation response can generally be dismissed (Long et al., 1993; Gunderson and Wullschleger, 1994). This paper aims (1) to describe the effects of elevated CO 2 on photosynthetic gas exchange behaviour of poplar hybrids grown and treated both under open top chamber and glasshouse cabinet conditions, and (2) to relate the observed differences in photosynthetic CO 2 uptake to underlying biochemical characteristics, such as Rubisco activity. Materials and methods Two experimental methodologies were compared in this study: an open top chamber (OTC) experiment on the campus of the University of Antwerp (Belgium) and an indoor glasshouse cabinet (GHC) experiment at the University of Sussex in Brighton (UK) (Table 1). These two experiments showed great similarity (same clonal material, identical measurements) as well as difference and complementarity (contrasting growth and exposure techniques, different duration of treatment, different number of replications, and time of measurements). This allowed the examination of the effects of the growth and exposure environment on poplar physiology. Plant materials In both Antwerp and Sussex, the fast growing, highly productive interamerican Populus trichocarpa (Torr. & Gray) x P. deltoides (Bartr. ex Marsh.) clone was compared with a slower growing euramerican P. deltoides (Bartr. ex Marsh.) x P. nigra (L.) clone. The clone Primo was chosen as the euramerican hybrid in Sussex, while in Antwerp, an older standard reference clone was used. Further details on these clones, such as parentage, sex, place of origin, and production performance can be found elsewhere (Ceulemans, 1990). All plants were grown from hardwood cuttings (25 cm long), which in Antwerp only were immersed in water for 24 h prior to planting. All measurements referred to in this paper were made on plants in their first or second growing season in elevated CO 2 (Table 1). Open top chambers in Antwerp Experiments in Antwerp were performed between April- November 1993 and March-November 1994 in open top chambers (OTCs) constructed on the campus of the University of Antwerp (Wilrijk, Belgium; 4 24'E, 51 10'N). Four decagonal OTCs with a floor area of 7 m 2 and a height of 4 m (1993) or 6 m (1994) were used. The walls (1 m wide) of the OTCs were made of acrylic Perspex and the distance between adjacent chambers was 8-10 m. Incoming air was supplied by large ventilators (S&P company, type CBM 320-65, Spain) at a rate of c. 2000 m 3 h" 1 resulting in nearly two air volume changes every minute. Air was passed through an aluminium pipe (with vertical adjustment) to a perforated, fibre-structured polythene annulus 1 m above the ground. Two of the OTCs received AMBIENT atmospheric CO 2 (c. 350 ^mol mol""') and two received ELEVATED atmospheric CO 2, i.e. ambient + 350/^mol mol" 1. The CO 2 was continuously supplied day and night from a 3 tonne large tank (L'Oxyhydrique Intern., Machelen, Belgium) throughout the entire growing season. Fifteen cuttings (7-8 plants from each clone) were planted on 22 April 1993 in each OTC in a circular planting pattern with an inter-plant distance of 0.6 m (representing a plant density of more than 21 200 plants ha" 1 ). Before planting the original, heavy loamy-clay soil within each OTC was excavated to a depth of 50 cm and replaced with a fertile sandy-loam horticultural soil. The first (or early) leaves appeared nearly 1 week after planting. Because of the rather late planting a number of cuttings from each clone did not survive and had to Table 1. Comparative table showing the similarity and complementarity of the experimental set-up and environmental conditions of the elevated CO 2 studies on poplar in Antwerp (Belgium) and Sussex (Brighton, UK) Location, site Antwerp (Belgium) Sussex (Brighton, UK) Coordinates of site Poplar clones Experimental set-up CO 2 treatments Number of replications/treatment Environmental conditions Date of planting Date of photosynthesis measurements Duration of CO 2 treatment Average height of plants at end of experiment 4 J 24'E and 51 "lcn and Open top chamber (c. 350fimol mol" ') and elevated (ambient + 350 ^mol mol ~ Two variable 21 April 1993 10-15 September 1993 April-November 1993 0"05Wand 50"52^ and Primo Glasshouse cabinet (c. 360 ^mol mol" ') and elevated (constant at 719^01 mol"') Two constant temperature 20-25 C 3 and 17 December 1993 10-16 January 1994 December 93-January 94 0.5-0.6 m

be replaced 2 weeks later. Two months after planting the soil in the OTCs was covered with a layer of woody chips to maintain soil moisture content and to control weeds. All plants were irrigated twice a day by a drip irrigation system. On 23 July 1993, 3.751 of a combined nutrient solution was supplied to each OTC, corresponding to a N supply of nearly 63.5 kg N ha" 1. A similar amount of N was added the second year. More details and a complete description of the OTC setup have been previously published (Ceulemans et al., 1995a, b). Photosynthetic photon flux density (PPFD), as measured at 30min intervals with a Sunfleck Ceptometer (Delta-T-Devices, Cambridge, UK), amounted to maximum values of 1340 fimol m~ 2 s" 1 at midday during the days of measurement. Closed glasshouse cabinets in Sussex Forty dormant cuttings of the clone Primo and 20 of the clone were planted in large, plastic pots (diameter 15 cm, height 50 cm) containing a 1:1 mix of compost and vermiculite. The pots were placed into each of four glasshouse cabinets (GHCs) at the University of Sussex at Brighton (0 05'W, 50 52'N) on 3 December 1993 (clone Primo) and 17 December 1993 (clone ), respectively. The four cabinets (length 0.86 m, width 0.56 m, height 1.56 m) in the university's glasshouse represented two experimental CO 2 treatments, i.e. AMBIENT atmospheric CO 2 (around 360 /xmol mop 1 ) and ELEVATED CO 2. The target CO 2 concentration of the elevated treatment was a constant 700ftmol mol" 1, but the mean concentration over the entire growth period was 719 + 6^mol mol" 1. The air flow to the cabinets was via an adapted air conditioning unit, and the constant CO 2 flow was supplied from a cylinder of pure CO 2 (CP grade; BOC Special Gases, Surrey, UK). Full details of the exposure and analysis system were given by Bosac et al. (1995) and Gardner et al. (1995). Air temperature in the GHCs was maintained at 20-25 C. Constant artificial light was supplied by high pressure sodium lamps from 07.00 h GMT until 21.00 h GMT, and the average PPFD in the glasshouse cabinets was c. 350fimol m" 2 s" 1 (daylight plus artificial light). Plants were irrigated twice per day with a nutrient solution according to a modified Lngestad approach as used and described by McDonald (1989) for the closely related genus, Salix. Gas exchange measurements For all CO 2 gas exchange measurements a portable, open gas exchange system (ADC, type LCA 3, Hoddesdon, UK) and a Parkinson leaf chamber (PLC type N) were used on intact, attached leaves. To discriminate between long-term effects (i.e. OTC or GHC growth effect) and short-term effects (i.e. measurement conditions) of CO 2 concentrations, measurements were made in ambient and elevated OTCs or GHCs at LOW (375 ^mol mol"') and HIGH (700 /xmol mol"') CO 2 concentrations. In Antwerp net photosynthetic rates were measured in the OTCs from 10-15 September, 1993 under saturating PPFD conditions of 1500fimol m" 2 s" 1 with an artificial light source (Philips sodium lamp). PPFD was monitored with a quantum photodiode (after Pontailler, 1990). Leaf temperature during the gas exchange measurements was 25 C. Mature, fully expanded leaves of leaf plastochron index (LPf) 7-10 of two plants were sampled; the relationship between leafage (expressed in LPI units) and photosynthetic rate, as well as the leaf longevity and optimal leaf age were known for the clones studied (Ceulemans and Impens, 1979). Three replicate measurements were made per CO 2 treatment and per clone. To measure the short-term effect, CO 2 was supplied from a gas cylinder (L'Oxyhydrique Internationale, Machelen, Belgium) and directly Photosynthetic responses to CO 2 in poplar 1683 injected into the leaf cuvette during the photosynthesis measurements. Dark respiration measurements were made prior to illumination for net photosynthesis measurements. In the Sussex experiment net photosynthetic rates were measured from 10 16 January 1994 (i.e. after 1 month in elevated CO 2 ) in the glasshouse. For the measurements, potted plants were removed from the growth cabinets and placed under two artificial light sources (400 W high pressure sodium lamps, Thermoforce Ltd., Maldon, UK) in the glasshouse. Saturated net photosynthesis of GHC plants was measured at a PPFD of 1500 ^onol m"" 2 s" 1 on mature leaves (LPI of 7-10) of three plants per clone and per growth cabinet under LOW (c. 350 ftmol mol" 1 ) and HIGH (700 /xmol mol" 1 ) atmospheric CO 2. The air flow for the gas exchange measurements was directly taken from the closed CO 2 controlled glasshouse cabinets. No attempt was made to control vapour pressure difference or temperature during the gas exchange measurements. Leaf temperature during the measurements was close to 29 C; however, a temperature correction was applied for the modelled A M (Long, 1991). Dark respiration rates were measured at the end of the dark period (between 05.45 h and 07.30 h GMT) on mature leaves of three plants per clone and per cabinet, i.e. on six plants per clone and per treatment. The end of night respiration (/? ) was assumed to be equal to the rate of CO 2 evolution from the alternative respiratory pathways in the light (i.e. day respiration or R^), but not to photorespiration in the light, which was calculated from Rubisco kinetics. The same assumptions were made in Besford et al. (1985). Therefore R^ is assumed to be equal to the minimum value of R a. This value of dark respiration was then used for the modelling exercise on Rubisco activity (Besford et al., 1985) as described below. Respiration data were collected for two reasons: (1) to look for short- and long-term effects of elevated CO 2 on respiration, and more importantly (2) to get a figure for respiration which could be used in the modelling of A ut. Modelling /!, from Rubisco values and comparing them to the measured values indicates where R n and net photosynthesis measurements are likely to be robust. Assays of Rubisco activity and chlorophyll content In Antwerp leaf discs were sampled for biochemical analysis around noon on 22 August 1993 and again on 20 July 1994 from recently matured leaves of 3-8 plants per clone and per OTC (a total of 12 replications per clone and per treatment). In Sussex samples for biochemical analysis were taken on the same days and time periods as the gas exchange measurements (see above). For clones and Primo 5 or 6 replicates, respectively, were taken per cabinet. In all cases (Antwerp and Sussex) discs of about 2.5 cm 2 (c. 50 mg FW) were harvested, and immediately plunged in liquid nitrogen. Rubisco was extracted as described by Paulilo et al. (1994) and Wilkins et al. (1994). After extraction samples were centrifuged at 15000g for 5 min and stored on ice until assayed (Wilkins et al., 1994). Rubisco activity was assayed by the coupled-enzyme method of Lilley and Walker (1974) at 20 C and ph 8.0 with a 20 min preincubation period (Besford, 1984). Rates of PPFD saturated photosynthesis (A M ) were predicted from the kinetics of Rubisco derived from Besford (1984) adjusted to 29 C as in Jordan and Ogren (1984), and based on the equations of Farquhar et al. (1980). In this modelling approach it was assumed that A al was limited by fully activated and RUBP-saturated Rubisco as in Besford et al. (1985, 1990). No convincing and consistent data exist to suggest a change in the degree of Rubisco activation in plants grown long term in

1684 Ceulemans et al. elevated CO 2 (Van Oosten et al., 1995). It was further assumed that after a 20 min activation in Mg 2 + and HCOf the in vitro and in vivo activation states were the same. The maximum in vitro carboxylation rate in saturating CO 2 and RUBP (K c ) was measured by the Rubisco activity assay described above. For analysis of chlorophyll content leaf discs of about 50 mg FW (c. 2.5 cm~ 2 ) were harvested in Antwerp and about 100 mg FW in Sussex. For determination of chlorophyll content the DMF extraction method (Moran, 1982) was used for the samples in Antwerp, while the DMSO extraction protocol (after Hiscox and Israelstam, 1979) was used in Sussex. Chlorophyll absorption was measured spectrophotometrically at wavelengths of 663 and 654 nm. Leaf mass per area (LMA) was determined on leaf discs sampled from 5-10 leaves, and calculated as the ratio of leaf area to dry mass of the discs. Statistical analysis Results of Rubisco activity, net photosynthetic rates and leaf mass per area ratios were analysed for statistical differences between CO 2 treatments by a two sample Student's r-test. Results of chlorophyll analysis were subjected to an analysis of variance (ANOVA) to examine the effects of treatment and growth environment (GHC versus OTC), followed by a least significant differences (LSD) test to identify statisitical differences. _35 ^f30 f 20 8 15 c f 10 o I 5 cu a) Euramerican Open top chamber Glasshouse cabinet b) Interamerican Open top chamber Glasshouse cabinet Results Wef photosynthetic rate In general, PPFD saturated net photosynthetic rates and responses of photosynthesis were similar in plants grown in OTCs and GHCs. In both the OTCs in Antwerp and the GHCs in Sussex, net photosynthetic rates were significantly enhanced by the elevated CO 2 treatment (Fig. 1). This was evident for the euramerican (clones Primo and ) as well as for the interamerican (clone ) hybrids. For the ambient treatment no differences in absolute values of PPFD saturated net photosynthesis were found between OTCs and GHCs, but the photosynthesis of plants in the elevated treatment was always significantly higher (at P< 0.001) in the OTCs than in the GHCs. In both the OTCs and the GHCs, the shortterm (i.e. measurement) effect of high CO 2 on net photosynthesis was different from the long-term (i.e. growth) effect. When considering photosynthesis measured at high (700 ^mol mop 1 ) CO 2, values were significantly higher (at / > <0.01) for the elevated CO 2 treatment than for the ambient treatment in the OTC experiment. However, this was the converse in the GHC experiment of Sussex: lower net photosynthesis in the elevated treatment than in the ambient treatment when measurements were made in high CO 2 (Fig. 1), indicating some acclimation of photosynthesis in this case. However, net photosynthesis measured under high CO 2 (hatched bars on Fig. 1) was found to be at least twice that measured under low atmospheric CO 2 concentration (open bars on Fig. 1). This was true for both growth treatments (ambient and elevated) and for euramerican as well as for interamerican hybrid clones. Fig. 1. Net photosynthetic rates of euramencan (top figure) and interamencan (bottom figure) poplar hybrids grown under ambient (350-360 fimol mol" 1 ) and elevated atmospheric CO 2 concentrations in open top chambers (OTC, in Antwerp) or in controlled glasshouse cabinets (GHC, in Sussex). CO 2 concentrations were ambient + 350 ^mol mol~' in the OTCs, respectively, 719/xmol mol* 1 in the GHCs. Open bars represent measurements made in low CO 2 concentrations (350-375/xmol mol" 1 ) and hatched bars represent measurements made under high CO 2 concentrations (700 ^±mol mol" 1 ). All data are mean values of at least ten replications. Vertical bars represent single standard error of the mean. The actual measurement conditions were of more importance for net photosynthesis than the experimental growth (or treatment) conditions, and in this regard all clones had a very similar response. It should be noted here that measurements in the GHCs were made only 1 month after the onset of the elevated CO 2 treatment, while those in the OTCs were made after 4 months of CO 2 treatment. In previous work considerable genotypic differences in net photosynthesis were observed for the clones studied here (Ceulemans et al., 1987; Radoglou and Jarvis, 1990). However, in this study clonal differences both in the OTCs and the GHCs, were much smaller than observed previously (Fig. 1), but still significant (at P<0.01). The PPFD saturated net photosynthetic rate was higher in euramerican hybrids Primo and than in the interamerican hybrid clone. The short-term effects of elevated CO 2 on net photosynthesis were slightly

larger in the euramerican (c. + 160% for clone ) than in the interamerican hybrids (+140% for clone ; Fig. 1). Dark respiration Dark respiration rates were not significantly (at P<0.05) affected by growth in elevated CO 2, either in the OTC or in the GHC (Fig. 2). However, for the interamerican hybrid clone growth in elevated CO 2 in the OTC experiment significantly (at P<0.01) reduced the dark respiration rate by more than 60% (Fig. 2). For euramerican as well as for interamerican poplars, dark respiration rates in the GHCs were equal to, or a lot lower than those observed in the OTCs. Dark respiration rates were found to be higher in the interamerican clone than in the euramerican hybrid clones Primo and, but the differences were not significant. is o 34 I' Q a) Euramerican Open top chamber Glasshouse cabinet b) Interamerican Open top chamber 1 Glasshouse cabinet 1 Fig. 2. Dark respiration rates of euramerican (top figure) and interamerican (bottom figure) poplar hybrids grown under ambient (350-360 /irnol mol ~') and elevated atmospheric CO 2 concentrations in open top chambers (OTC, in Antwerp) or in controlled glasshouse cabinets (GHC, in Sussex). CO 2 concentrations were ambient + 350fimol mol"' in the OTCs, respectively, 719/nmolmol" 1 in the GHCs. Open bars represent respiration measurements made in low CO 2 concentrations (350-375^mol mol"') and hatched bars represent measurements made under high CO 2 concentrations (700 ^mol mol"') All data are mean values of at least ten replications. Vertical bars represent single standard error of the mean. Photosynthetic responses to CO 2 in poplar 1685 Significant genotypic differences between the euramerican and interamerican hybrids were observed in the response of dark respiration rate to the short-term CO 2 measurement conditions. In clone dark respiration was significantly lower when measurements were made under high CO 2 (700 ^mol mol" 1 ) than under low CO 2 (c. 350/^mol mol" 1 ), but no differences were observed between the measurement conditions with clones Primo or (Fig. 2). Rubisco activity After exposure to elevated CO 2 for up to 5 weeks in the GHCs there was no significant difference in the extractable Rubisco activity from Primo or. Also in the OTCs after exposure to elevated CO 2 for 4 months there was no significant difference in Rubisco activity obtained from (Table 2). However, there was an increase in Rubisco activity with elevated CO 2 in clone. After extended exposure to elevated CO 2 in the OTCs for 17 months both and contained less Rubisco activity (Table 2). There was a tendency for the photosynthesis rates of the OTC grown plants, when measured at low CO 2, to be higher after exposure to elevated CO 2 for 4 months (Fig. 1) which may have been related to the higher Rubisco activity, but only in clone. The PPFD saturated rate of photosynthesis was predicted from the extractable Rubisco activity assuming that the Rubisco in vivo is RuBP saturated and fully activated. With plants grown in the GHC, where photosynthesis measurements and Rubisco analyses were made on the same leaf, good predictions of photosynthetic performance were achieved (Table 3). Rubisco kinetics predicted accurately that leaves grown and measured in elevated CO 2 would double their PPFD saturated rate of photosynthesis compared with those grown and measured in ambient CO 2. Also the absolute rates were close to predicted rates (Table 3). With the OTC-grown plants modelling photosynthetic performance was poor, and in general photosynthetic rates were underestimated. However, measurements of Rubisco activity and photosynthesis were carried out on different leaves with a 3-week interval. Chlorophyll concentration and leaf mass per area ratio In the OTC experiment there was a significant (P<0.05) and consistent decrease in chlorophyll concentrations (Chi a, Chi b as well as total Chi) in the elevated treatment as compared to the ambient treatment (Table 4). However, in the GHC experiment some conflicting results were found: there was a non-significant decrease in Chi a concentration in clone, but for clone Primo the Chi concentrations significantly (at / > <0.01) increased under the elevated CO 2 treatment.

1686 Ceulemans et al. Table 2. Average Rubisco activity in euramerican (clones Primo and ) and interamerican (clone ) poplar hybrids exposed to ambient C0 2 or elevated C0 2 concentrations in glasshouse cabinets for 5 weeks (at Brighton) and in open top chambers for 4 months and 17 months (at Antwerp) All figures are expressed in ^mol CO 2 m ~ 2 s ~' and are mean values of 9-13 replications (± standard error). Levels of significance have been indicated. Hybrid Date CO 2 CO 2 Significance Glasshouse cabinets Pnmo and * January 1994 12.7(1 96) 14.8(2.13) Open top chambers August 1993 September 1994 August 1993 September 1994 17.6(3 58) 22.8 (2.27) 12.0(1.60) 14.3 (2.27) 180(2 12) 15.2(2.33) 16 1 (0.95) 122 (1.58) /><0.05 ns Values were meaned since no significant differences in extractable Rubisco activity were observed between the clones grown in either ambient or elevated CO 2. Table 3. Predicted and measured PPFD saturated net photosynthesis (A U J of interamerican and euramerican poplar hybrids grown and treated to ambient and elevated atmospheric CO 2 levels in open top chambers (OTC, Antwerp experiment) or glasshouse cabinets (GHC, Sussex experiment) Predicted net photosynthesis was obtained from in vitro Rubisco activity measurements (RUBP-saturated carboxylation rate) after Besford et al (1985). Predicted A al is based on means of at least nine Rubisco replications. Measured /(, is based on means of at least ten replications Hybrid Exposure system CO 2 treatment Predicted A M (fimol m~ 2 s"') Measured A ml (fimol m~ 2 s~') Primo and * Pnmo and * GHC GHC OTC OTC OTC OTC 9.9 19.3 8.3 16.9 12.3 19.2 10.6 19.8 12.0 29.5 11.9 28.5 "Mean values are used since no significant differences were observed in extractable Rubisco activity between the two clones grown in either ambient or elevated CO 2. Table 4. Results of chlorophyll analysis on interamerican (clone ) and euramerican (clones Primo and ) poplar hybrids grown under ambient and elevated CO 2 concentrations in glasshouse cabinets and in open top chambers All chlorophyll concentrations (Chi a, Chi b and total Chi) are expressed in jigcm~ 2 and are the means of at least nine replicates (standard error within brackets). Values followed by the same letter within the same column are not significantly different at P<0.0\. Hybrid CO 2 treatment Chi a Chi b Total Chi Chi a/b Glasshouse cabinets Primo Primo 30.6 (1.26)bc 29.6 (0.22)cd 27 3(2.93)d 33.5 (1.71)b 7.37 (0.18)b 7.11 (0.01)c 6.18 (0.60)d 7.60 (0.27)b 38.0 (1.41 Jc 36.7 (0.22)de 35 1 (3.77)e 41.1 (1.97)bc 4.14 (0.09)b 4.13 (004)b 4.39 (0.1 l)ab 4.39 (0.10)ab Open top chambers 42.5 (2.27)a 33.8 (3.27)b 42 0(6.80)a 29.7 (2.76)cd 1051 (1.26)a 8.53 (3.02)b 10 74 (3.02)a 7.67 (0.40)b 53.1 (3.20)a 42.3 (9.36)b 52.7 (9.36)a 37.4 (3.04)de 4.05 (0.39)c 3.96 (1 90)c 391 (1.90)c 3.87 (0.27)c For both poplar hybrids the values of chlorophyll concentrations (Chi a, Chi b and total Chi) differed significantly between the OTC and the GHC experiments (Table 4). Total chlorophyll (Chi a + Chi b) concentrations were between 35-41 ^g cm" 2 for the plants grown in the GHCs compared to 37-53 ^.g cm" 2 for plants grown in the OTCs (Table 4). In the ambient treatment of the GHC experiment there were also significant differences (at P <0.01) in the Chi a and Chi b concentrations between the two hybrid clones: hybrid clone Primo had lower Chi a and Chi b values than hybrid clone. No significant changes were observed in the Chi a:chl b ratio under the elevated CO 2 treatment. LMA significantly increased under elevated CO 2 for young, expanding leaves of clone Primo in the GHC experiment, and for mature leaves of both clones (

Photosynthetic responses to CO 2 in poplar 1687 and ) in the OTC experiment (Table 5). Differences in LMA between ambient and elevated CO 2 treatments were not significant in the GHC experiment for clone. Significant differences in LMA were observed between plants in the OTCs and those in the GHCs as also evidenced in Table 5. LMA values were always lower, thus thinner leaves, in the GHCs than in the OTCs. Discussion Although there is strong evidence in many tree species for acclimation (or down-regulation) of photosynthesis and Rubisco activity when grown long term in elevated CO 2 (Sage et al., 1989; Van Oosten et al., 1992; Tissue et al., 1993; Wilkins et al., 1994), this paper shows two examples where negative acclimation to elevated CO 2 was not seen after lengthy periods of exposure, although in one case there was an indication of slight acclimation. These observations were made on clonal poplar plants grown for 1 month under elevated CO 2 in GHCs or for more than 4 months in OTCs. Because acclimation in crop plants and in trees is often observed by 30 d if not sooner, the lack of early acclimation in poplar may imply an unusually high sink strength (Gaudillere and Mousseau, 1988). Moreover nutrients were most probably not limiting in these experiments (Ceulemans et al., 19956; Gardner et al., 1995). Evidence was also found for a strong control of Rubisco on PPFD saturated photosynthesis since observed and predicted rates of net photosynthesis based on Rubisco activity were close when the same leaves were used for analysis. Another technique used in assessing photosynthetic acclimation, was to measure rates of net photosynthesis after plants grown at ambient and elevated CO 2 had been transferred to a similar ambient CO 2 atmosphere (Gunderson and Wullschleger, 1994). With plants in the OTCs the rates of PPFD saturated net photosynthesis were comparable between leaves of both clones grown at ambient or elevated CO 2 ; thus, acclimation during the first few months can be dismissed. However, there were some indications of loss of photosynthetic capacity in plants grown in the GHC experiment since photosynthesis measured in high CO 2 was lower for trees grown in elevated CO 2 compared with those grown in ambient CO 2. It does not seem very likely that this slight acclimation of photosynthesis might be due to root restriction in the pots in the GHCs since pot volume was far above the 12.5 dm 3 suggested by Arp (1991). After 17 months in elevated CO 2 there was significant loss of Rubisco activity in the OTCs, i.e. acclimation to elevated CO 2 concentrations. This is one of the very few studies in which a good prediction of photosynthesis has been made from Rubisco activities in a tree species. As far as is known, there is no other example in the literature where both net photosynthesis and maximum V c were measured (as opposed to calculated) and where there was a good agreement between predicted and measured photosynthesis (Table 3). When the standard errors are included in Table 3 it becomes clear that there is indeed no significant difference between predicted and measured rates in half of the cases. Based on the data it seems evident that the PPFD saturated rate of photosynthesis at ambient CO 2 concentrations is strongly controlled by carboxylase activity, e.g. in GHC ambient CO 2 -grown clones Primo and. There is other evidence in the literature for this. The degree to which photosynthesis is controlled by Rubisco activity is known as the Rubisco control coefficient (Kacser and Porteous, 1987). In low light the control coefficient is low (implying low control of photosynthesis by Rubisco), but in saturating light (as in the measurements made in this study) the control coefficient approaches 1, implying almost total control over photosynthesis by Rubisco (see also the findings of Krapp et al. (1994), who found very high control coefficients in genetically engineered tobacco plants in saturating light). Further literature evidence has been provided for tomato (Besford et al., 1985, 1990) and for rice (Makino et al., 1984). Initial Rubisco activities were not measured for practical reasons. Furthermore, there are also conflicting reports on the effect of elevated CO 2 on initial activities, Table 5. Leaf mass per area (gm 2 ) of euramerican (clones and Primo) and interamerican (clone ) poplar hybrids grown under ambient CO 2 or elevated CO 2 concentrations in open top chambers and in glasshouse cabinets All figures are mean values of 9-13 replications and standard erros are within parentheses. ns = non significant; * = significantly different at ^ = 0.05. Hybrid Type of leaf CO 2 CO 2 Significance Glasshouse cabinets Beauprc Primo Young, expanding Young, expanding 30.7 (2.4) 24.6(1.3) 32.2(4 8) 28.7(0.7) ns * Open top chambers Beauprc Young, expanding Mature, fully expanded Young, expanded Mature, fully expanded 40.0(2.6) 61.7(4.6) 34.5(7.2) 65.4 (4.7) 45.0(3.4) 82.0 (6.0) 33.9 (2.0) 87.0(6.1) ns ns

1688 Ceulemans et al. e.g. Woodrow (1994) versus Rowland-Bamford et al. (1991) and Tissue et al. (1993). These authors found very different effects of high CO 2 on activation state (decreased, unchanged or increased, respectively). In many studies initial activation state has been found to be very high (80-90%); so, the error in assuming a 100% activation state in the modelling probably has not affected the results very much. Major and significant differences were observed in the morphological characteristics of the leaves that were produced in the GHCs as compared to the OTCs. Possibly because of the lower growth irradiance, higher air temperature and higher relative humidity in the growth cabinets, leaves formed in the GHCs were more like shade-type leaves with lower leaf mass per area, lower chlorophyll concentrations and generally lower Rubisco activities than leaves formed in the OTCs (more sun-type leaves). This was true for the two contrasting hybrids. Lower growth irradiance decreases photosynthetic capacity and Rubisco activity (Besford, 1984; Besford et al., 1990). In general chlorophyll concentrations are reduced under elevated CO 2, although there is also one report of an increased chlorophyll concentration in elevated CO 2 in citrus (Koch et al., 1983). The significant difference in the chlorophyll concentrations between plants in OTCs and those in GHCs might be explained (1) either by the different technique that was used in Antwerp and in Sussex (DMSO technique versus DMF analysis method), but most likely by (2) the significant difference in leaf structure and anatomy between the two growth systems (OTCs and GHCs). The latter is also reflected in their leaf mass per area ratios, which reflect leaf density and/or thickness. The lower Chi a and Chi b concentrations in clone Primo as compared to clone (in the ambient treatment of the GHC experiment) were confirmed by lower chlorophyll fluorescence values observed for clone Primo (Kalina and Ceulemans, 1997). The experimental results presented here for two contrasting poplar hybrids indicate that the elevated CO 2 treatment significantly stimulated net photosynthesis in the leaves of the OTC experiment in Antwerp (sunadapted, heavier or thicker leaves grown under high light) and in those grown in the cabinets in Brighton (shadeadapted, lighter or thinner leaves grown under low light conditions). The magnitude of the response of net photosynthesis to the elevated CO 2 treatment differed between OTCs and GHCs, which might be explained by the lower light intensity in the glasshouse (compare with Visser et al., 1993). After exposure to elevated CO 2 for up to 4 months there was no evidence of a loss in the rate of net photosynthesis or Rubisco activity which supports the view that fast growing tree species would show no sign of acclimation to elevated CO 2. However, after extended exposure to elevated CO 2 in OTCs acclimation of Rubisco was observed. Acknowledgements This research was financially supported at the University of Antwerp (Belgium) by the EC through its Environment R&D programme (contract No. EV57-CT92-0127 as the ECOCRAFT research network) and at the University of Sussex (UK) by the Biotechnology and Biological Sciences Research Council (BBSRC grant No. PG085/0524). The support from the British-Flemish Academic Research Collaboration Programme (British Council grant No. 26/94) allowed us to travel easily between our two laboratories and to perform a series of joint experimental pulse studies. Further financial support was provided by the Fund for Scientific Research- Flanders (FWO). We gratefully acknowledge SDL Gardner, XN Jiang and BY Shao for help with data collection, N Calluy and F Kockelbergh for technical assistance, as well as the associate editor and two anonymous reviewers for their critical comments and useful suggestions on earlier drafts of the manuscript. RC is a Senior Research Associate of the FWO (Brussels). References Arp WJ. 1991. Effects of source-sink relations on photosynthetic acclimation to elevated CO 2. Plant, Cell and Environment 14, 869-75. Besford RT. 1984. Some properties of ribulose bisphosphate carboxylase activity and photosynthesis during leaf development in tomato. Journal of Experimental Botany 35, 495-504. Besford RT, Ludwig LJ, Withers AC. 1990. The greenhouse effect: acclimation of tomato plants grown in high CO 2, photosynthesis and ribulose-1,5-bisphosphate carboxylase protein. Journal of Experimental Botany 41, 925-31. Besford RT, Withers AC, Ludwig LJ. 1985. Ribulose bisphosphate carboxylase activity and photosynthesis during leaf development in tomato. Journal of Experimental Botanv 36, 1530-41. Bosac C, Gardner SDL, Taylor G, Wilkins D. 1995. CO 2 and hybrid poplar: a detailed investigation of root and shoot growth and physiology of P. euramericana 'Primo'. Forest Ecology and Management 74, 103-16. Bowes G. 1993. Facing the inevitable: plants and increasing atmospheric CO 2. Annual Review of Plant Physiology and Molecular Biology 44, 309-22. Ceulemans R. 1990. Genetic variation in functional and structural productivity determinants in poplar. Amsterdam' Thesis Publishers^ 101 pp. Ceulemans R, Impens I. 1979. Study of CO 2 exchange processes, resistances to carbon dioxide and chlorophyll content during leaf ontogenesis in poplar. Biologia Plantaruni 21, 302-6. Ceulemans R, Impens I, Steeoackers V. 1987. Variations in photosynthetic, anatomical and enzymatic leaf traits and correlations with growth in recently selected Populus hybrids. Canadian Journal of Forest Research 17, 273-83. Ceulemans R, Jiang XN, Shao BY. 1995a. Growth and physiology of one-year old poplar (Populus) under elevated atmospheric CO 2 levels. Annals of Botany 75, 609-17. Ceulemans R, Jiang XN, Shao BY. 1995ft" Effects of elevated atmospheric CO 2 on growth, biomass production and nitrogen allocation of two Populus clones. Journal of Biogeography 22, 261-8. Ceulemans R, Mousseau M. 1994. Tansley Review No. 71: Effects of elevated atmospheric CO 2 on woody plants. New Phytologist 127, 425-46.

Eamus D, Jarvis PG. 1989. The direct effects of increase in the global atmospheric CO 2 concentration on natural and commercial temperate trees and forests. Advances in Ecological Research 19, 1-55. Farquhar CD, von Caemmerer S, Berry JA. 1980. A biochemical model of photosynthetic CO 2 assimilation in leaves of C 3 species. Planta 149, 78-90. Gaudillere JP, Mousseau M. 1989. Short-term effect of CO 2 enrichment on leaf development and gas exchange of young poplars (Populus euramericana cv. I 214). Oecologia Plantarum 10, 95-105. Gardner SDL, Taylor G, Bosac C. 1995. Leaf growth of hybrid poplar following exposure to elevated CO 2. New Phytologist 131, 81-90. Gilford RM. 1992. Interaction of carbon dioxide with growth limiting environmental factors in vegetation productivity: implications for the global carbon cycle. Advances in Bioclimatology 1, 24-58. Gunderson CA, Wullschleger SD. 1994. Photosynthetic acclimation in trees to rising atmospheric CO 2 : A broader perspective. Photosynthesis Research 39, 369-88. Hiscox JD, Israelstam GF. 1979. A method for extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany 57, 1332-4. Jordan DB, Ogren WL. 1984. The COJ/OJ specificity factor of ribulose-l,5-bisphosphate carboxylase/oxygenase. Planta 161, 308-13. Kacser M, Porteous JW. 1987. Control of metabolism: what do we have to measure? Trends in Biochemical Sciences 12, 5-14. Kalina J, Ceulemans R. 1997. Clonal differences in the response of dark and light reactions of photosynthesis to elevated atmospheric CO 2 in poplar. Photosynthetica 33, 51-61. Koch KE, White DW, Jones PH, Avigne WR, Hartwell Jr LH. 1983. CO 2 enrichment of Carrizo Citrange and Swingle Citrumelo rootstocks. Proceedings Florida State Horticultural Society 96, 37-40. Krapp A, Chaves MM, David MM, Rodriques ML, Pereira JS, Stitt M. 1994. Decreased ribulose-l,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with 'antisense' rbcs. VIII. Impact on photosynthesis and growth in tobacco growing under extreme irradiance and high temperature. Plant, Cell and Environment 17, 945-53. Lilley RC, Walker DA. 1974. An improved spectrophotometric assay for ribulose bisphosphate carboxylase. Biochimica and Biophysica Ada 358, 245-50. Long SP. 1991. Modification of the response of photosynthetic productivity to rising temperature by atmospheric CO 2 concentrations: Has its importance been underestimated? Plant, Cell and Environment 14, 729-39. Long SP, Drake BG. 1991. Effect of the long-term elevation of CO 2 concentration in the field on the quantum yield of photosynthesis of the C 3 sedge, Scirpus olneyi. Plant Physiology 96, 221-6. Photosynthetic responses to CO 2 in poplar 1689 Long SP, Baker NR, Raines CA. 1993. Analysing the responses of photosynthetic CO 2 assimilation to long-term elevation of atmospheric CO 2 concentration. Vegetatio 104/105, 33-45 Makino A, Mae T, Ohira K. 1984. Relation between nitrogen and ribulose-1,5-bisphosphate carboxylase in rice leaves from emergence through senescence. Plant and Cell Physiology 25, 429-37. McDonald AJS. 1989. Nitrate availability and shoot area development in small willow (Salix viminalis). Plant, Cell and Environment 12, 417-24. Moran R. 1982. Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Phvswlogy 69, 1376-81. Paulilo MTS, Besford RT, Wilkins D. 1994. Rubisco, PEP carboxylase and acclimation to irradiance in a Brazilian Cerrado tree species, Qualea grandiflora Mart. (Vochysiaceae). Tree Physiology 14, 165-77. Pontailler JY. 1990. A cheap sensor using a gallium arsenide photodiode. Functional Ecology 4, 591-6. Radoglou KM, Jarvis PG. 1990. Effects of CO 2 enrichment on four poplar clones. I. Growth and leaf anatomy. Annals of Botany 65, 617-26. Rowland-Bamford AJ, Baker JT, Allen LH, Bowes G. 1991. Acclimation of rice to changing atmospheric carbon dioxide concentration. Plant, Cell and Environment 14, 577-83. Sage RF, Sharkey TD, Seeman JR. 1989. Acclimation of photosynthesis to elevated CO 2 in five C 3 species. Plant Physiology 89, 590-6. Tissue DT, Thomas RB, Strain BR. 1993. Long-term effects of elevated CO 2 and nutrients on photosynthesis and Rubisco in loblolly pine seedlings. Plant, Cell and Environment 16, 859-65. Van Oosten JJ, Afif D, Dizengremel P. 1992. Long-term effects of a CO 2 -enriched atmosphere on enzymes of the primary carbon metabolism of spruce trees. Plant Physiology and Biochemistry 30, 541-7. Van Oosten JJ, Wilkins D, Besford RT. 1995. Acclimation of tomato plants after transfer to different carbon dioxide concentrations. Impact on the relationships between the biochemistry and gas exchange during leaf development. New Phvtologist 130, 357-67. Visser AJ, Coelho AN, Rozema J. 1993. The effect of CO 2 enrichment on growth and photosynthesis in springwheat (Triticum aestivum cv. Minaret). Comparison between open top chambers and glasshouses. San Miniato, Italy: Abstract International Workshop on Carbon Dioxide Springs and Their Use in Biological Research, Academia dei Georgofili. Wilkins D, Van Oosten JJ, Besford RT. 1994. Effects of elevated CO 2 on growth and chloroplast proteins in Prunus avium. Tree Physiology 14, 769-79. Woodrow IE. 1994. Control of steady-state photosynthesis in sunflowers growing in enhanced CO 2. Plant, Cell and Environment 17, 277-86.