Istituto di Biochimica ed Ecofisiologia Vegetale, Consiglio Nazionale delle Ricerche, via Salaria km , Monterotondo Scalo (Roma), Italy

Tree Physiology 19, 807--814 1999 Heron Publishing----Victoria, Canada Long-term effects of elevated carbon dioxide concentration and provenance on four clones of Sitka spruce (Picea sitchensis). II. Photosynthetic capacity and nitrogen use efficiency MAURO CENTRITTO 1 and PAUL G. JARVIS 2 1 Istituto di Biochimica ed Ecofisiologia Vegetale, Consiglio Nazionale delle Ricerche, via Salaria km 29.300, 00016 Monterotondo Scalo (Roma), Italy 2 Institute of Ecology and Resource Management, University of Edinburgh, Darwin Building, King s Buildings, Mayfield Road, Edinburgh, EH9 3JU, Scotland Received November 17, 1998 Summary Four clones of Sitka spruce (Picea sitchensis (Bong.) Carr.) from two provenances, at 53.2 N (Skidegate a and Skidegate b) and at 41.3 N (North Bend a and North Bend b), were grown for three growing seasons in ambient (~350 µmol mol 1 ) and elevated (~700 µmol mol 1 ) CO 2 concentrations. The clones were grown in stress-free conditions (adequate nutrition and water) to assess the effect of elevated [CO 2 ] on tree physiology. Growth in elevated [CO 2 ] significantly increased instantaneous photosynthetic rates of the clonal Sitka spruce saplings by about 62%. Downward acclimation of photosynthesis (A) was found in all four clones grown in elevated [CO 2 ]. Rubisco activity and total chlorophyll concentration were also significantly reduced in elevated [CO 2 ]. Provenance did not influence photosynthetic capacity. Best-fit estimates of J max (maximum rate of electron transport), V cmax (RuBP-saturated rate of Rubisco) and A max (maximum rate of assimilation) were derived from responses of A to intercellular [CO 2 ] by using the model of Farquhar et al. (1980). At any leaf N concentration, the photosynthetic parameters were reduced by growth in elevated [CO 2 ]. However, the ratio between J max and V cmax was unaffected by CO 2 growth concentration, indicating a tight coordination in the allocation of N between thylakoid and soluble proteins. In elevated [CO 2 ], the more southerly clones had a higher initial N use efficiency (more carbon assimilated per unit of leaf N) than the more northerly clones, so that they had more N available for those processes or organs that were most limiting to growth at a particular time. This may explain the initial higher growth stimulation by elevated [CO 2 ] in the North Bend clones than in the Skidegate clones. Keywords: acclimation, chlorophyll, electron transport, elevated [CO 2], photosynthesis, Rubisco. Introduction Predicted changes in atmospheric [CO 2 ] are expected to increase photosynthetic rates in C 3 plants both by increasing the rate of carbon fixation and by reducing photorespiratory loss of carbon. Generally, photosynthetic rates of woody plants are increased by a doubling in CO 2 concentration (Eamus and Jarvis 1989, Amthor 1995). Gunderson and Wullschleger (1994), in surveying studies of 39 tree species, reported an average increase of photosynthetic rates of 44% when measured at the growth [CO 2 ] concentrations. Ceulemans and Mousseau (1994) observed that, in short-term experiments, elevated [CO 2 ] stimulates photosynthesis on average about 40% in conifers and 61% in broad-leaved species. However, long-term growth in elevated [CO 2 ] often results in a variable decrease (depending on the species) in the amounts of photosynthetic pigments and enzymes, for instance, amount and activation state of Rubisco, and, thus, acclimation of the photosynthetic apparatus (Long and Drake 1992). Apparently, this decrease may occur even when the supply of nitrogen is adequate and rooting volume is large (Drake et al. 1997). Acclimatory depression of photosynthetic capacity has come to the fore as a process that regulates source--sink interactions (Sheen 1994). This study reports the effects of stress-free (adequate nutrition, water, pot space) growth in a CO 2 concentration of 700 µmol mol 1 on gas exchange, and carbon and nitrogen relationships of four clones of Sitka spruce (Picea sitchensis (Bong.) Carr.) of two provenance after three years of CO 2 exposure. It has been shown in a companion paper (Centritto et al. 1999) that the dry mass of all four clones was increased by elevated [CO 2 ], but that the more northerly clones, despite being grown at a latitude close to their latitudinal provenance, were significantly less responsive than the more southerly clones. To understand whether these differences in growth resulted from a direct effect of elevated [CO 2 ] on photosynthesis, the photosynthetic capacity of the clones was analyzed in relation to leaf nitrogen concentration. Clones from two provenances were used because in the temperate and northerly regions the contribution of clones to the fitness of the populations is relevant (Callaghan et al. 1992). However, climate change is thought to give rise to mass migration of plants, thereby modifying composition of plant communities by changing the intraspecific as well as interspecific competitive performances. Understanding specific ways in which different

808 CENTRITTO AND JARVIS genotypes respond to elevated [CO 2 ] is necessary to predict the likely impact of global change on vegetation. Materials and methods Saplings of four clones of Sitka spruce (Picea sitchensis (Bong.) Carr.) from two provenances, at 53.2 N (Skidegate a and Skidegate b) and at 41.3 N (North Bend a and North Bend b), were grown for three growing seasons in open top chambers (OTCs) with ambient [CO 2 ] (~350 µmol mol 1 ) or elevated [CO 2 ] (ambient + ~350 µmol mol 1 ). The OTCs were located at the Institute of Terrestrial Ecology (ITE), Bush Estate, near Edinburgh, U.K. The saplings were potted in standard potting compost, watered every other day to pot water capacity and regularly fertilized in both growing seasons following Ingestad s principles (Ingestad and Ågren 1992). Full details of the growth conditions, and of the statistical analysis used to test the data are described in a companion paper (Centritto et al. 1999). Gas exchange measurements were made in a glasshouse at the University of Edinburgh, on the central section of currentyear branches with a portable gas exchange system (ADC- LCA-3, Analytical Development Co. Ltd., Hoddesdon, U.K.) equipped with a conifer leaf chamber (PLC-3, Analytical Development Co. Ltd., Hoddesdon, U.K.). To enable measurements of PPFD-saturated photosynthetic rates, illumination of the leaf cuvette by natural sunlight was supplemented with artificial light (provided by a white fluorescent lamp) to maintain PPFD incident on the needles above 1700 µmol m 2 s 1. The planar area of needles on each sample was determined by removing all the needles enclosed by the leaf cuvette and passing them through a leaf area meter (LI 3000, Li-Cor, Inc., Lincoln, NE). Instantaneous leaf CO 2 assimilation rates (A) were measured between 1100 and 1300 h at the end of July 1993 on 20 saplings (five plants per clone) per [CO 2 ] treatment, at the growth CO 2 concentrations. Rates of short-term (10 min) PPFD-saturated (1700--2000 µmol m 2 s 1 ) CO 2 assimilation were measured at leaf internal CO 2 concentrations (A--C i ) ranging from 40 to 1200 µmol mol 1. Observations were made in August 1993 between 1000 and 1700 h, on twelve saplings (three per clone) per [CO 2 ] treatment. The initial slope of the A--C i curves is an estimate of the carboxylation efficiency (V cmax, RuBP-saturation rate of Rubisco), whereas the maximum rate of assimilation (A max ) (the net CO 2 assimilation rate under conditions of PPFD and CO 2 saturation) is indicative of the role of RuBP regeneration, and is related to electron transport under conditions of PPFD-saturation (J max ). Parameters J max, V cmax, and A max, were estimated by fitting the C 3 photosynthesis model of Farquhar et al. (1980) to individual A--C i response curves following the procedure outlined by Wullschleger (1993). Total Rubisco activity was assayed spectrophotometrically by a coupled enzyme method (Besford 1984, Van Oosten et al. 1995). Five needles from one plant from each clone (i.e., 100 needles per [CO 2 ] treatment) were sampled in July 1993 for Rubisco activity assays. The needles were removed from midway along current-year branches. Needle mass and area were rapidly measured before plunging the needles in liquid nitrogen. Needle concentrations of chlorophylls a, b, and a + b were measured on different needles sampled as described above. The concentration of chlorophylls was measured in intact leaf tissues by immersion in N,N-dimethylformamide (DMF) following the techniques described by Porra et al. (1989). The saplings were sampled once a month from March to October in the second growing season, and from February to October in the third growing season. Needles were removed from midway down a current-year branch and their area rapidly measured before they were plunged in liquid nitrogen. Samples for determination of sugar and starch concentrations of roots and current-year needles were taken at each harvest during the second and third growing seasons (see Centritto et al. 1999). The numbers of root and leaf samples taken were 24 per [CO 2 ] treatment (six per clone) on Day 381, 40 per [CO 2 ] treatment (10 per clone) on Day 551, and 20 per [CO 2 ] treatment (five per clone) on Days 719 and 972. Soluble sugars were measured by high pressure liquid chromatography (HPLC). Five cm 3 of NaOH solution (0.0025 mol m 3 ) was added to freeze-dried, ground tissue of the sapling samples ranging in mass between 0.049 and 0.051 g. The samples were incubated for 15 minutes in a water bath at 30 C. The solutions were then centrifuged for 15 minutes at 3000 g, and the supernatant collected and vacuum filtered using a 0.2 µm nitrocellulose filter (Whatman International Ltd., Maidstone, U.K.). The amount of soluble sugars present in the sample was determined by HPLC (Dionex DX 500, Dionex Corp., Sunnyvale, CA). Starch concentration was determined by the iodometric method (Allen 1989). Results Elevated CO 2 significantly (P < 0.001) increased CO 2 assimilation rate of Sitka spruce saplings by about 62% at the end of July of the third growing season, when measured at the growth CO 2 concentration (Table 1). Stimulation occurred in all clones, and was higher, though not significantly, in the Skidegate a and b clones (about 95% and 76%, respectively) than in the North Bend clones (about 43%) (Table 2). The relationship between PPFD-saturated CO 2 assimilation rate and leaf internal CO 2 concentration was used to ascertain the Table 1. Total Rubisco activity (on a leaf area basis), measured with saturating CO 2 in vitro, and assimilation rate (A) of the Sitka spruce saplings (all clones) in ambient or elevated [CO 2 ]. Data are means of 20 plants per [CO 2 ] treatment ± 1 SEM. Measurements of A were made at the growth CO 2 concentrations, with a mean temperature of 24.9 ± 0.28 C (1 SEM) in saturating PPFD (> 1700 µmol m 2 s 1 ) between 1100 and 1300 h. Rubisco (µmol m 2 s 1 ) A (µmol m 2 s 1 ) Elevated 27.88 ± 1.54 16.83 ± 0.44 Ambient 43.72 ± 3.13 10.09 ± 0.37 Significance: CO 2 P < 0.001 P < 0.001 TREE PHYSIOLOGY VOLUME 19, 1999

EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY 809 Table 2. Assimilation rate (A) measured at the growth CO 2 concentrations, A max (from A--C i curves), and total Rubisco activity in vitro (on a leaf area basis), of the four Sitka spruce clones. Data are means of three to five plants per treatment ± 1 SEM. The significance levels (* = P < 0.05, ** = P < 0.01, *** = P < 0.001) apply to the within-clone difference in response to [CO 2 ]. A (µmol m 2 s 1 ) A max (µmol m 2 s 1 ) Rubisco (µmol m 2 s 1 ) Elevated Ambient Elevated Ambient Elevated Ambient Skidegate a 18.33 ± 0.99 9.39 ± 0.52 *** 21.97 ± 1.24 27.28 ± 0.69 ** 29.97 ± 1.99 52.41 ± 7.48 ** Skidegate b 16.33 ± 0.72 9.27 ± 0.11 ** 19.86 ± 1.19 26.86 ± 0.95 * 28.10 ± 2.13 36.23 ± 2.77 * North Bend a 15.88 ± 0.49 11.15 ± 0.23 *** 19.50 ± 1.17 26.01 ± 0.92 * 27.51 ± 4.41 45.48 ± 6.35 * North Bend b 16.19 ± 0.67 11.29 ± 1.28 ** 19.23 ± 0.96 27.14 ± 0.57 ** 25.94 ± 2.90 40.75 ± 5.33 * Figure 1. The relationship between net CO 2 assimilation rate (A) of Sitka spruce saplings of the four clones and intercellular CO 2 concentration (C i ) in saturating PPFD (> 1700 µmol m 2 s 1 ). The measurements were made on shoots of twelve saplings per [CO 2 ] treatment. Mean A max, averaged across the four clones, was statistically different between treatments (P < 0.001). biochemical limitation to photosynthesis. These A--C i measurements showed some downward acclimation of photosynthesis in the saplings grown in elevated [CO 2 ] (Figure 1). The decrease in A max in the elevated [CO 2 ] treatment was about 25% (P < 0.001). A downward acclimation of A max was observed in all four clones grown in elevated [CO 2 ] (Table 2). There were no differences in A max among clones in either elevated or ambient [CO 2 ]. The A--C i response curves also showed that ambient [CO 2 ]- grown saplings had higher carboxylation efficiencies than elevated [CO 2 ]-grown saplings (Figure 1). Carboxylation efficiency is generally interpreted as a limitation by Rubisco activity, which was significantly reduced in vitro by 36% by elevated [CO 2 ] (Table 1). All clones in elevated [CO 2 ] showed downward acclimation of carboxylation efficiency (data not shown). In parallel, Rubisco activity in vitro significantly decreased in response to long-term growth in elevated [CO 2 ] by about 22% in Skidegate b, 36% in North Bend b, 39% in North Bend a, and 43% in Skidegate a (Table 2). However, there were no significant differences in Rubisco activity among the clones in either [CO 2 ] treatment. Total chlorophyll concentration was significantly lower at all sampling times in saplings grown in elevated [CO 2 ] compared with saplings grown in ambient [CO 2 ] in both growing seasons (Table 3). Figure 2 shows needle and root sugar concentrations per unit of dry mass of the Sitka spruce saplings harvested at the beginning and end of both the second and third growing seasons. The sugar concentration was greater in both needles (Figure 2a) and roots (Figure 2b) of plants grown in elevated [CO 2 ] compared with plants grown in ambient [CO 2 ], but the differences were significant only in needles harvested on Day 382. There was a significant increase in starch concentration at the end of both the second and third growing season (Days 551 and 972, respectively) in needles (Figure 3a) and roots (Figure 3b) of saplings grown in elevated [CO 2 ] compared with ambient [CO 2 ]-grown saplings. Figure 4 shows the relationships between leaf N concentration and mean total dry mass of the saplings in both ambient and elevated [CO 2 ]. Growth in different CO 2 concentrations affected the nitrogen concentrations when the saplings were the same size, as demonstrated by the linear relationships between mean total dry mass and nitrogen concentration of leaves (R 2 = 0.926 for the elevated [CO 2 ] saplings, and R 2 = 0.993 for the ambient [CO 2 ] saplings). Growth in elevated [CO 2 ] also affected foliar nitrogen concentration of the four Table 3. Total chlorophyll concentration (mg cm 2 ), measured at monthly intervals in the second and third growing seasons, in needles of Sitka spruce saplings grown in ambient or elevated [CO 2 ]. Data are means of 20 plants per treatment ± 1 SEM. Abbreviation: ns = not significant. Second growing season Third growing season Elevated Ambient Elevated Ambient February -- -- 91.27 ± 6.62 107.90 ± 5.51 March 77.53 ± 2.93 87.98 ± 3.83 78.66 ± 6.51 83.95 ± 4.88 April 70.53 ± 2.93 80.98 ± 3.83 105.53 ± 6.96 111.04 ± 7.16 May 52.09 ± 1.61 57.68 ± 2.10 111.90 ± 6.43 122.73 ± 7.39 June 73.28 ± 3.99 84.46 ± 4.08 33.18 ± 2.56 36.14 ± 1.72 July 66.44 ± 3.05 76.76 ± 3.53 31.15 ± 2.91 41.21 ± 2.92 August 75.70 ± 9.36 96.01 ± 7.14 40.44 ± 3.95 50.71 ± 3.73 September 80.14 ± 4.07 85.54 ± 4.52 33.45 ± 3.22 51.20 ± 4.31 October 79.75 ± 5.46 86.49 ± 3.66 37.80 ± 3.50 50.70 ± 2.97 Statistical significance: CO 2 P < 0.001 P < 0.001 Time P < 0.001 P < 0.001 Interaction ns ns TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

810 CENTRITTO AND JARVIS Figure 4. Linear relationships between the combined mean foliar nitrogen concentration and total dry mass of the Sitka spruce saplings grown in ambient or elevated [CO 2 ]. Data are means of 20 to 40 plants per treatment ± 1 SEM. clones when the saplings were the same size (Figure 5). There was a trend to produce more biomass with a lower foliar N concentration in the more southerly clones when they were younger. Foliar N concentration decreased to similar values in all four clones as they grew larger, although the North Bend clones maintained a greater total dry mass than the Skidegate clones. There was a positive linear relationship between best-fit estimates of J max, V cmax, and A max and leaf nitrogen concentration when expressed on a leaf area basis in both elevated and ambient [CO2] (Figure 6). However, saplings grown in elevated [CO 2 ] had lower values of all three parameters per unit of foliar nitrogen on a leaf area basis than saplings grown in ambient [CO 2 ]. The values of V cmax and J max were similar in magnitude to those reported by Walcroft et al. (1997) for Pinus radiata D. Don, when based on projected leaf area. A strong positive, linear correlation was also observed between the best-fit estimates of J max and V cmax (Figure 7); i.e., J max = 2.39V cmax. Discussion Figure 2. Needle (a) and root (b) sugar concentrations per unit of dry mass in the Sitka spruce saplings grown in ambient or elevated [CO 2 ], plotted against time since the beginning of the experiment. Data are means of 20 to 40 plants per treatment ± 1 SEM. The significance level (** = P < 0.01) shows the difference in sugar concentration in response to the [CO 2 ] treatments. Figure 3. Needle (a) and root (b) starch concentrations per unit of dry mass in the Sitka spruce saplings grown in ambient or elevated [CO 2 ], plotted against time since the beginning of the experiment. Data are means of 20 to 40 plants per treatment ± 1 SEM. The significance levels (* = P < 0.05, ** = P < 0.01, *** = P < 0.001) show the difference in starch concentration in response to the [CO 2 ] treatments. Figure 5. Relationships between foliar nitrogen concentration and total dry mass of the four clones of Sitka spruce grown in elevated [CO 2 ]. Data are means of five to 10 plants per treatment ± 1 SEM. The four clones of Sitka spruce were grown in stress-free conditions (adequate nutrition and water) to assess the effect of elevated [CO 2 ] on tree physiology, thus ruling out any TREE PHYSIOLOGY VOLUME 19, 1999

EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY 811 Figure 6. Linear relationships between best-fit estimates of the photosynthetic parameters derived from individual A--C i curves (using the model of Farquhar et al. 1980) and foliar nitrogen concentration on a leaf area basis; (a) J max (R 2 = 0.533 for the elevated [CO 2 ] plants, R 2 = 0.553 for the ambient [CO 2 ] plants), (b) V cmax (R 2 = 0.523 for the elevated [CO 2 ] plants, R 2 = 0.510 for the ambient [CO 2 ] plants), and (c) A max (R 2 = 0.569 for the elevated [CO 2 ] plants, R 2 = 0.502 for the ambient [CO 2 ] plants). Figure 7. Linear relationship (R 2 = 0.934) between the maximum rate of electron transport (J max ) and the maximum rate of carboxylation (V cmax ), derived as best-fit estimates from individual A--C i curves. effects that insufficient water or nutrient supply might have caused. Recently, Drake et al. (1997) have stressed the importance of available nutrients in determining the extent of the stimulation of A in elevated [CO 2 ]; in a review of eight experiments the average stimulation dropped from 57% when nitrogen availability was high to 23% when nitrogen availability was low. Long-term growth in elevated [CO 2 ] increased instantaneous photosynthetic rates of the clonal Sitka spruce saplings by about 62% (Table 1). Similar increases in assimilation rate were found in juvenile foliage of nine-year-old Pinus taeda L. trees exposed for the second year to a doubling of CO 2 concentration (Murthy et al. 1997). In two-year-old Sitka spruce clones, Townend (1993) found a significant effect of growth in elevated [CO 2 ] (600 µmol mol 1 ) on the response of A to PPFD, but did not quantify this increase. Lucas (1998) found that A of 3-year-old Sitka spruce seedlings was 35% higher in elevated [CO 2 ] (700 µmol mol 1 ) than in ambient [CO 2 ]. Barton (1997) found that A of both current-year and 1-year-old shoots of mature Sitka spruce doubled in response to elevated [CO 2 ] (700 µmol mol 1 ) in a branch bag experiment. The more southerly clones (North Bend a and b) showed an increase in assimilation rate of about 43%, which is close to the average increase for conifers reported in the survey by Gunderson and Wullschleger (1994), whereas the increases in photosynthetic rates of the more northerly clones (Skidegate a and b) were well above the average (Table 2). However, the Skidegate a and b clones produced significantly less total dry mass than the North Bend clones in elevated [CO 2 ] at the end of the third growing season (Centritto et al. 1999). Either the increases in instantaneous assimilation rates of each clone, measured at the beginning of August in the third growing season, were not representative of the average increase over the entire growth period studied, or there were large losses of carbon in respiration, volatilization, root exudation and fine root turnover in the more northerly clones. These processes were not studied in this work, but they can account for a large proportion of assimilates lost (Wang et al. 1998). Furthermore, other important factors, including mutual leaf shading, leaf area per plant and respiratory costs, affect growth responses. Oleksyn et al. (1992) found that, in Pinus sylvestris L., root respiration accounted for about two-thirds of total respiratory cost. At the beginning of the growth period, the Skidegate clones allocated proportionally more dry mass to the roots compared with the North Bend clones (data not shown), which might have increased whole-plant respiratory losses and led to an initially smaller growth stimulation by [CO 2 ] than in the North Bend clones. Long-term growth in elevated [CO 2 ] may result in reduced amounts of photosynthetic pigments and enzymes (Eamus and Jarvis 1989). However, Arp (1991) showed that downward acclimation of photosynthetic capacity and size of pot were highly correlated, and that constrained rooting caused by inadequate pot volume may cause downward acclimation of photosynthetic capacity in elevated [CO 2 ]. In contrast, plants rooted in the ground show little acclimation of photosynthetic capability (Liu and Teskey 1995, Curtis and Wang 1998). A three-year exposure to elevated [CO 2 ] of branches of mature Sitka spruce in a stand did not cause acclimation of A in current-year needles, but caused some downward acclimation in one-year-old needles (Barton 1997). There were no significant changes in the A--C i relationship measured in branches exposed to elevated [CO 2 ] of 22-year-old Pinus taeda trees in all the three years of growth, indicating that elevated [CO 2 ] (ambient + 330 µmol mol 1 ) did not alter the photosynthetic TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

812 CENTRITTO AND JARVIS capacity of the foliage when adequate sinks were available (Teskey 1997). There were no significant differences in A--C i curves between Betula pendula Roth plants grown in elevated (700 µmol mol 1 ) and ambient [CO 2 ] in Ingestad units with steady-state nutrition (Pettersson and McDonald 1992). In the present experiment, the Sitka spruce saplings were supplied with free access to nutrients, and dry mass allocation was identical when the plants were the same size suggesting that they were not pot limited (Centritto et al. 1999). Yet, downward acclimation of photosynthetic capacity occurred (Figure 1) in each clone (Table 3), as was also found by Lucas (1998) in three Sitka spruce clones and Barton (1997) in 2-year-old seedlings of Sitka spruce. The A--C i response curves showed both a decrease in A max, which is a function of photosynthetic electron transport and regeneration of RuBP, and in carboxylation efficiency, which is limited by Rubisco activity. The latter observation is consistent with the in vitro Rubisco activities, which decreased in elevated [CO 2 ]-grown saplings (Table 1). This decline of photosynthesis was in agreement with the downward acclimation of Rubisco (Van Oosten et al. 1995), which may result from both lowered enzyme activation and enzyme amount (Tissue et al. 1993, Vu et al. 1997). Nitrogen concentration was affected by growth in elevated [CO 2 ] (Centritto et al. 1999). Reduction in leaf N concentration in elevated [CO 2 ] was only partially caused by starch dilution, because starch concentration was not always affected (Figure 3a). There are several processes by which elevated [CO 2 ] can lead to decreased leaf N concentration. These include inhibition of photorespiration and reduced amounts of photosynthetic pigments and enzymes. Rubisco is the largest pool of nitrogen in leaves (Drake et al. 1997), and its content can be reduced by about 35% in elevated [CO 2 ] before there is co-limitation of A (Long and Drake 1992). Carbon assimilation rate of the four clones was increased by about 51% in elevated [CO 2 ] (Table 2) despite a reduction in V cmax per unit of leaf nitrogen (Figure 6b). Thus, because less Rubisco was required in elevated [CO 2 ] (Table 1), increased carbon assimilation per unit of leaf N led to increased nitrogen use efficiency (NUE). The chlorophyll--protein complexes also constitute major pools of N in leaves (Evans 1997), and are usually decreased by elevated [CO 2 ] (Drake et al. 1997), as found here (Table 3). Wullschleger (1993) found that, in a retrospective analysis of the A--C i curves from a large number of C 3 species, the carboxylation and light-harvesting capabilities were closely coupled. Coordinated regulation of the biochemical limitations to photosynthesis has also been shown in elevated [CO 2 ]. Van Oosten et al. (1995) reported that downward acclimation of the light-harvesting complexes in tomato plants grown in elevated [CO 2 ] followed the decline in carboxylation capacity. In our study, a strong positive linear relationship between the best-fit estimates of J max and V cmax was also found (Figure 7). This may indicate that the decline in chlorophyll concentration (on a leaf area basis) and the downward acclimation of J max per unit of leaf N (also expressed on a leaf area basis) in elevated [CO 2 ] (Figure 6a), resulted from the tight coordination of the activities of thylakoid proteins and soluble proteins to match each other. The ability to maintain a constant ratio between the carboxylation and light-harvesting activities across a wide range of environmental conditions (Wullschleger 1993) originates from a functional balance in the allocation of N between thylakoid and soluble proteins. This allows optimization of resource allocation (Evans 1997) and leads to a more efficient use of N (Von Caemmerer and Farquhar 1981). Furthermore, inhibition of photorespiration in plants grown in elevated [CO 2 ] may also reduce the amount of nitrogen required per unit of dry mass produced (Conroy and Hocking 1993). Thus, changes in the biochemistry of photosynthesis and photorespiration, which usually occur when N uptake does not keep pace with carbon uptake (Jarvis 1995), may be regarded as an optimization process that involves reallocation of nitrogen away from non-limiting components to more limiting processes or organs (i.e., additional or larger sinks for the extra carbon assimilated), and consequently leading to increased NUE. The lower N concentration per unit of leaf area in elevated [CO 2 ] may account for the acclimation of the photosynthetic capability of the four clones, despite free access to nitrogen and the large rooting volume (10 dm 3 ), although, as Drake et al. (1997) pointed out, downward acclimation of A is the exception rather than the rule when the rooting volume exceeds 10 dm 3. However, a recent study on the interactive effects of elevated [CO 2 ] (700 µmol mol 1 ) and N supply in field-grown rice has shown that reduced foliage concentration of N (even if calculated as a percentage of structural dry mass) was always associated with increased [CO 2 ] (Ziska et al. 1996). This is a common observation in many species and is hard to explain. In the Sitka spruce saplings, leaf N concentration was clearly lower when the plants were the same size in the two [CO 2 ] treatments (Figure 4), indicating that growth in elevated [CO 2 ] increased the dry mass produced per unit of nitrogen taken up. Increased growth per unit of plant nitrogen and phosphorus was also noted with seedlings of Pinus ponderosa Dougl. ex Laws. grown in 700 µmol mol 1 of CO 2 (DeLucia et al. 1997). Van Oosten and Besford (1996) have described a molecular model for photosynthetic acclimation. The model invokes metabolite regulation of gene expression, which probably occurs when the production of new assimilates is larger than the capacity to handle them. This can involve a source--sink imbalance leading to feedback effects on photosynthesis by endproduct accumulation (Stitt 1991). The cytoplasmatic pool of glucose may provide a regulatory signal for coarse control, which determines the amount of photosynthetic systems by repressing photosynthetic gene expression (rbcs, cab-7, cab- 3C, rca), thereby triggering a cascade of reactions that lead to acclimation of the photosynthetic apparatus. However, sugar concentration in needles of the Sitka spruce saplings was not increased significantly at the beginning or end of the third growing season (Figure 2), when photosynthetic acclimation in elevated [CO 2 ] was detected (Figure 1). This finding seems to conflict with the model put forward by Van Oosten and Besford (1996). However, Paul and Driscoll (1997) have recently shown that loss of photosynthetic activity is better correlated with an increase in the C/N ratio than with carbohy- TREE PHYSIOLOGY VOLUME 19, 1999

EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY 813 drate status. Our results accord with this view. The higher leaf C/N ratio in needles at the end of both the second and third growing seasons (Days 719 and 972), as a result of similar sugar concentrations (Figure 2a) but lower nitrogen concentrations (Centritto et al. 1999), may explain photosynthetic downward acclimation in elevated [CO 2 ]. This result is consistent with findings in several other studies. For instance, A of two full-sib families of Pinus ponderosa seedlings was lower in elevated [CO 2 ] than in ambient [CO 2 ] after about 39 days from germination, but the C/N ratio of the needles had already increased in elevated [CO 2 ] (Grulke et al. 1993). Reductions in Rubisco activity and chlorophyll concentration led to improved nitrogen use efficiency in elevated [CO 2 ], because A max, J max, and V cmax scaled linearly with leaf N concentration (Figure 6), and the ratio between J max and V cmax was not changed by growth in elevated [CO 2 ] (Figure 7). Wullschleger (1993) has shown that these two parameters describing photosynthetic capacity are species-specific. Furthermore, despite their importance in mechanistic models predicting the effects of global change on growth processes, there are few values of these parameters for different species in the literature (Walcroft et al. 1997). As far as we know, there are no published values of J max and V cmax, determined from A--C i curves, in relation to leaf N concentration for Picea sitchensis (Bong.) Carr. grown in elevated [CO 2 ]. In conclusion, the amount of N needed for growth was less when Sitka spruce saplings growing in elevated [CO 2 ] were the same size as saplings in ambient [CO 2 ] (Figure 4), and this is likely to result in an advantage for plants growing in a nitrogen-limited environment. Provenance did not significantly influence either photosynthetic capacity or A measured at the growth CO 2 concentrations in the clonal Sitka spruce saplings (Table 2), although, the more northerly clones were significantly less responsive to elevated [CO 2 ] than the southerly clones (Centritto et al. 1999). Genetic differences in growth response to elevated [CO 2 ] were already evident after the first year of growth, and they were magnified over time becoming significant after three full growing seasons. The more southerly clones had higher initial NUE than the more northerly clones (Figure 5), showing that they had more N available for those processes or organs that were most limiting to growth. Clonal provenance affected growth in elevated [CO 2 ] and plant nitrogen use efficiency may have played an important role. This finding is particularly important for northern countries where N is the most limiting resource and will, therefore, affect Sitka spruce growth as the atmospheric CO 2 concentration rises, unless compensated by wet and dry N deposition. Acknowledgments This research was done within the EU Project ECOCRAFT (Contract No. ENV4-CT95-0077) The likely impact of rising CO 2 and temperature on European forests. Mauro Centritto was supported by an agreement between Consiglio Nazionale delle Ricerche and British Council. We would like to thank Dr R. Besford (then at Horticulture Research International Institute, Littlehampton, U.K.) for the analyses of Rubisco activity. References Allen, S.E. 1989. Chemical analysis of ecological materials. 2nd Edn. Blackwell Science, Oxford, 384 p. Amthor, J.S. 1995. 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