Long-Term Photosynthetic Acclimation to Increased Atmospheric CO2 Concentration in Young Birch (Betula Pendula) Trees

Long-term photosynthetic acclimation to increased atmospheric CO2 concentration in young birch (Betula pendula) trees

A. REY and P. G. JARVIS

Institute of Ecology and Resource Management, Darwin Building, Edinburgh University, Mayfield Road, Edinburgh EH9 3JU, U.K. Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021

Received September 17, 1997

Summary To study the long-term response of photosynthe- Introduction sis to elevated atmospheric CO2 concentration in silver birch (Betula pendula Roth.), 18 trees were grown in the field in Because CO2 is the substrate of photosynthesis, photosynthesis -1 is the main plant process directly affected by increases in open-top chambers supplied with 350 or 700 mmol mol CO2 for four consecutive growing seasons. Maximum photosyn- atmospheric CO2 concentration (Mott 1990). For this reason, many studies have focused on the photosynthetic response of thetic rates, stomatal conductance and CO2 response curves were measured over the fourth growing season with a portable plants to elevated CO2 concentration and, almost invariably, an photosynthesis system. The photosynthesis model developed increase in photosynthesis has been found (see reviews by Eamus and Jarvis 1989, Ceulemans and Mousseau 1994, Cur- by Farquhar et al. (1980) was fitted to the CO2 response curves. Chlorophyll, soluble proteins, total nonstructural carbohy- tis 1996). Although the mechanisms involved in the short-term drates, nitrogen and Rubisco activity were determined response of photosynthesis to elevated CO2 have been studied monthly. Elevated CO2 concentration stimulated photosynthe- in detail (Long and Drake 1992, Long et al. 1993) and are well sis by 33% on average over the fourth growing season. How- understood (Lawlor and Mitchell 1991), the response of pho- ever, comparison of maximum photosynthetic rates at the same tosynthesis to long-term exposure to elevated CO2 is less clear -1 CO2 concentration (350 or 700 mmol mol ) revealed that the (Stitt 1991, Long et al. 1993). Specifically, it is not known photosynthetic capacity of trees grown in an elevated CO2 whether the initial increase in photosynthesis will be main- concentration was reduced. Analysis of the response curves tained after several growing seasons in the field, and, if so, showed that acclimation to elevated CO2 concentration in- whether it will translate into long-term increases in growth and volved decreases in carboxylation efficiency and RuBP regen- productivity. eration capacity. No clear evidence for a redistribution of Down-regulation of photosynthesis is said to occur when nitrogen within the leaf was observed. Down-regulation of plants grown in elevated CO2 have lower photosynthetic rates photosynthesis increased as the growing season progressed and than plants grown in ambient CO2, when compared at a com- appeared to be related to the source--sink balance of the trees. mon CO2 concentration (Long 1991). Down-regulation of pho- Analysis of the main leaf components revealed that the reduc- tosynthesis has been observed in several studies after tion in photosynthetic capacity was accompanied by an accu- prolonged exposure to elevated CO2 concentration (e.g., mulation of starch in leaves (100%), which was probably DeLucia et al. 1985). It has been suggested that the reduction responsible for the reduction in Rubisco activity (27%) and to in photosynthetic capacity after long periods in elevated CO2 a lesser extent for reductions in other photosynthetic compo- is more common in plants grown in pots than in field-grown nents: chlorophyll (10%), soluble protein (9%), and N concen- plants (Arp 1991, Long and Drake 1992, Drake et al. 1997). trations (12%) expressed on an area basis. Despite a 21% This suggestion is supported by several studies showing no reduction in stomatal conductance in response to the elevated loss of photosynthetic enhancement in plants grown in the field CO2 treatment, stomatal limitation was significantly less in the over several growing seasons (Wullschleger et al. 1992, Gun- elevated, than in the ambient, CO2 treatment. Thus, after four derson et al. 1993); however, other studies have reported a growing seasons exposed to an elevated CO2 concentration in decline in photosynthetic capacity even when plants were the field, the trees maintained increased photosynthetic rates, grown under conditions that did not limit rooting volume although their photosynthetic capacity was reduced compared (Petterson and McDonald 1992, Gunderson and Wullschleger with trees grown in ambient CO . 2 1994, Sage 1994). Based on a meta-analysis of 41 woody species, Curtis (1996) highlighted the need to understand the Keywords: carbohydrates, carboxylation capacity, chloro- conditions in which down-regulation of photosynthesis occurs, phyll, electron transport capacity, elevated CO2, nitrogen, because pot size or root restriction did not always explain this Rubisco activity, soluble proteins, stomatal limitation. phenomenon. Different photosynthetic responses among spe- 442 REY AND JARVIS cies have been explained on the basis of the source--sink made in duplicate. balance of the plant (Stitt 1991, Woodrow 1994). This expla- Light-saturated photosynthesis Light-saturated rates of pho- nation implies that species with indeterminate growth and tosynthesis (A ) were measured monthly with an LI-6200, therefore large sink capacity would be less likely to exhibit max portable photosynthesis system (Li-Cor) with a 0.25-dm3 leaf down-regulation than species with determinate growth. If this chamber. The light source was a dichroic reflector halogen reasoning is correct, it is to be expected that the extent to which lamp (Thorn M81, 12V) providing saturating PPFD (1200 down-regulation of photosynthesis occurs will be not only mmol m-2 s-1) evenly distributed across the leaf surface. Neu- species dependent, but also influenced by the time of year, tral density filters were used to reduce the photosynthetic because sink strength varies during the growing season. photon flux density (PPFD) when required. Maximum eleva- As part of a long-term study to characterize the response of tion of leaf temperature above ambient air temperature was silver birch (Betula pendula Roth.) to increased atmospheric kept below 2 °C by means of a heat filter (Calflex-C, Balzers) Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 CO concentration, we analyzed the photosynthetic response 2 placed between the leaf chamber and light source and two small of young birch trees during the fourth year of growth in the fans that ventilated the outside of the leaf chamber. field. Our aim was to test whether: (1) increases in leaf photo- Leaf photosynthetic rates were measured at CO concentra- synthetic rates in response to elevated CO concentration can 2 2 tions of 350 and 700 mmol mol-1 in all treatments. To obtain be maintained in the field for long periods; (2) long-term the desired CO concentration inside the chamber, a tube from exposure to elevated CO leads to down-regulation of photo- 2 2 the air inlet into an elevated or ambient chamber was con- synthetic capacity, and if so, how acclimation is manifested; nected to the Li-Cor LI-6200 and a flow switch was used to (3) seasonality plays a role in the photosynthetic response to toggle the system between open and closed mode. All the elevated CO ; and (4) physiological variables other than pho- 2 leaves were first measured at the growth CO concentration. tosynthetic rate, for example stomatal conductance or transpi- 2 ration, are affected by elevated CO . The system was run as an open system for a few minutes until 2 the concentration in the chamber reached the target concentration at the beginning of each measurement, and then run as a Materials and methods closed system when the rate of change in CO2 concentration was measured. Measurements were made during the second Material and experimental design week of each month. When leaves were measured at reciprocal CO concentrations, a leak test was performed every day be- In March 1991, seeds of silver birch (Betula pendula) were 2 fore measurements were made (see Li-Cor Application Note germinated in an ambient (about 350 mmol mol-1) or elevated -1 No. 103, 1991). (ambient + 350 mmol mol ) atmospheric CO2 concentration and grown in pots in open-top chambers (OTCs) in the field at The CO2 response curves The response of photosynthesis to Bush Estate (15 km south of Edinburgh) for one year. After one internal CO2 concentration (A/Ci) was measured at PPFD satu- year in pots, 18 birch seedlings were planted in the ground and ration (1200 mmol m-2 s-1) at the end of June, the beginning of transferred to individual OTCs at Glencorse (17 km south of August and in mid-September with the Li-Cor LI-6200 oper- Edinburgh; 55°31¢ N, 3°12¢ W) for three consecutive growing ating as a closed system (McDermitt et al. 1989). Measure- seasons until November 1994 when the trees were harvested. ments were made in two stages: starting at the growth CO2 By the end of the experiment, the height of the trees exceeded concentration and increasing until the CO2 compensation con- 4 m. During 1994, trees were not fertilized but were watered centration was reached; and then from about 1300 mmol mol-1 from mid-July, because that summer was unusually dry. More down to the growth CO2 concentration. High CO2 concentra- details about the field site and the design of the chambers can tions were obtained by connecting the Li-Cor LI-6200----while be found elsewhere (Rey and Jarvis 1997). The elevated CO2 operating as an open system----to the air inlet into the elevated treatment was maintained year-round. A description of the CO2 CO2 chambers for a few minutes. Because of the large CO2 control and monitoring system has been published elsewhere gradients a leak correction was made (McDermitt et al. 1989). (Barton et al. 1993). A complete sequence of measurements took between 20 and The experiment had a completely randomized design with 40 min. three treatments. Six trees were grown in OTCs at approxi- The Farquhar et al. (1980) model of photosynthesis was mately twice the current, ambient atmospheric CO2 concentra- fitted to the data. According to the model, the rate of photosyn- -1 tion (i.e., ambient + 350 mmol mol ) (E), six trees were grown thesis is assumed to be limited either by Rubisco activity and -1 in OTCs at ambient CO2 concentration (about 350 mmol mol ) kinetics (Ac) or by RuBP regeneration driven by photosyn- (A) and six trees were grown outside (control treatment) (C). thetic electron transport (Aj). The parameters maximum carboxylation capacity (Vcmax ), electron transport (J) and day Gas exchange measurements respiration (Rd) were derived. Because the A/Ci curves were Gas exchange measurements were made over the fourth grow- measured at PPFD saturation, calculated J was effectively the ing season on young, fully expanded leaves with the same maximum electron transport capacity (Jmax ). The temperature orientation and at the same layer in the crown (middle-bot- dependencies of the Michaelis constants for carboxylation and tom). The outlines of the leaves were drawn on paper, the leaf oxygenation (Kc and Ko, respectively), and CO2 compensation shapes cut out and their areas measured with an LI-3100 concentration in the absence of mitochondrial respiration (G*)

(Li-Cor Inc., Lincoln, NE) area meter. All measurements were are given by Lloyd et al. (1995) and Jmax and Vcmax by Harley

TREE PHYSIOLOGY VOLUME 18, 1998 PHOTOSYNTHETIC ACCLIMATION TO ELEVATED CO2 IN BIRCH 443 and Baldocchi (1995). An example of a measured and fitted to express all of the other chemical results on an area basis. CO2 response curve is given in Figure 1. Samples were immediately frozen in liquid nitrogen. Chloro- phyll was extracted from intact tissue by direct immersion in Stomatal limitation Stomatal conductance (gs) and photosynthesis were measured at the same time. However, even N,N-dimethylformamide (DMF) and determined colorimetri- though g was calculated every time measurements were made cally with a spectrophotometer (CE 303, Cambridge Instru- s ments, Cambridge, U.K.). at ambient and elevated CO2 concentrations, the stomata did not have time to adjust over such a short period of time, so that Rubisco activity and soluble proteins For the determination only the measurements made in the growth CO2 concentrations of total Rubisco activity at 25 °C, three discs, 1 cm in diameter, are reported. Leaf transpiration rate (E) was also simultane- were taken from the central portion of three leaves. Samples ously measured. Short-term, instantaneous transpiration effi- were frozen in liquid nitrogen and stored at -80 °C until ciency (ITE) was calculated from measurements of analyzed. The extraction and analysis procedure was as de- Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 photosynthesis and transpiration as the molar ratio of H2O scribed by Besford (1984) and Paulillo et al. (1994). Soluble transpired to CO2 assimilated. protein was extracted from ground freeze-dried material with Stomatal limitation to photosynthesis (L) was calculated 0.1 N NaOH and determined colorimetrically by the Bradford from the A/Ci response curves by comparing the rate of CO2 Protein Assay (Bradford 1976) as modified by Jones et al. assimilation at a given Ca (A) (growth concentration) with that (1989). which would be obtained (Ao) if there was no stomatal limitation, so that Ci = Ca (Farquhar and Sharkey 1982), i.e.: Carbohydrates Starch was extracted from finely ground freeze-dried material for 30 min at room temperature with

L = (Ao - A)/Ao. HClO4 (32% v/v) and determined colorimetrically by a modification of the iodine method described by Allen (1989). Sol- Biochemical measurements uble carbohydrates were extracted three times with Samples of leaves for phytochemical determinations were double-distilled water at 30 °C in a water bath for 15 min and taken throughout the 1994 growing season. The trees grown then measured by anion-exchange chromatography with pulse outside the OTCs (control treatment) had not yet flushed in amperometric detection (Ion Chromatography System HPLC, May 1994, so no control leaves could be sampled in May. From DX500, Carbopak, PA; carbohydrate column, Dionex Corpo- eight to 10 of the most recent fully expanded leaves from the ration, Sunnyvale, CA). A set of 10 pure polyols and sugars middle crown and from the same azimuthal orientation (west) (inositol, manitol, fucose, rhamnose, arabinose, galactose, glu- were collected. Leaves were taken on the same day every cose, xylose, fructose and sucrose) was run as a standard with month and always between 1100 and 1300 h to minimize any each set of samples. The concentration of total nonstructural possible diurnal fluctuations. carbohydrates (TNC) was calculated as the sum of starch and soluble sugars and expressed as percentage of dry mass. Chlorophyll Chlorophyll a, chlorophyll b and total chlorophyll were determined on fresh material according to the Nitrogen Leaf samples for nitrogen analysis were oven-dried method described by Porra et al. (1989). From three leaves, at 70 °C for 48 h, ground and kept in a desiccator until analyzed. three discs 1 cm in diameter were taken with a circular hole Nitrogen concentration was determined by a gas diffusion punch from the central portion of the leaf, avoiding the central method using a flow injection analyzer (Fiastar, Tecator Ltd., vein. Similar discs were taken for dry mass and leaf area Wilsonville, OR). determinations so that measured properties could be expressed both on an area and on a dry mass basis. These data were used Statistical analysis For the overall mean comparison between treatments, a one- way analysis of variance (ANOVA) model I was used (Sokal and Rohlf 1995). When comparing only elevated and ambient

CO2 treatments, a t-test was used. Data on Amax , gs, E and ITE were analyzed as a mixed model nested analysis of variance because several leaves were measured from the same tree (Sokal and Rohlf 1995). To take account of seasonal variation, results from this analysis and all biochemical data were analyzed by a two-way ANOVA with repeated measures on CO2 as a factor. Fisher’s least-significance difference (LSD) test was used to test for treatment differences when the previous analysis was significant at the 0.05 level of probability (Mil- liken and Johnson 1992). All statistical analyses were carried out with SAS software (SAS Institute Inc., Cary, NC). All data were checked for normality (Shapiro-Wilk test) and homoge- Figure 1. Examples of CO2 response curves (A/Ci) measured with a neity of variances (Hartley’s test) and met these assumptions, portable photosynthesis system (Li-Cor LI-6200) (· · ·) and modeled except for the starch data which were log-transformed. The using the Farquhar et al. (1980) model (------). CO2 response curves of individual leaves were modeled with a

TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com 444 REY AND JARVIS nonlinear equation (Farquhar et al. 1980) by nonlinear regres- masked any differences between CO2 treatments when meas- -1 sion techniques. Differences among CO2 treatments were ex- ured at 700 mmol mol (P > 0.15). amined by comparison of the curves fitted to each treatment following Mead and Curnow (1983) as recommended by The CO2 response curves Potvin et al. (1990). The advantage of using this statistical approach is that the mechanistic model used to fit the curves Table 1 lists the estimated values of the parameters obtained has a biological meaning, is distribution free and does not from fitting the A/Ci response curves at different times during assume any correlation among parameters. The percentage the growing season. Temperatures at which the curves were difference in a given variable between the elevated CO2 treat- measured on each occasion were not significantly different in ment (E) and the ambient CO2 treatment (A) was calculated as any case (P > 0.05). Both Vcmax and J were lower in the E [(E - A)/A]100. treatment than in the A treatment, in agreement with the Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 monthly photosynthetic measurements. Although the trend over the growing season was the same in both CO treatments, Results 2 the decline in the slope of the curve was more pronounced in Light-saturated photosynthesis the E treatment than in the A treatment. Pair-wise comparisons (cf. Mead and Curnow 1983) revealed statistically significant Trees grown in elevated CO2 (E treatment) had consistently differences between the curves in August (P < 0.05) and higher A than trees grown in ambient CO (A treatment) max 2 September (P < 0.01). The E treatment decreased V by 9% when measured at the growth CO concentration, but treatment cmax 2 at the beginning of the growing season (third week of June) and differences decreased over the growing season (Figure 2). In by 23% at the end of the growing season (second week of June the difference between CO treatments was large (60%) 2 September). The E treatment also decreased J to a similar and significant (P < 0.05), whereas in September the difference max degree, so that the Jmax :Vcmax ratio (at 25 °C) was similar in was only 10% (P > 0.05). In all treatments, Amax declined at the end of the season as a result of leaf senescence and consequent both CO2 treatments: 2.17 and 1.96 in June, 2.33 and 2.39 in N remobilization. August and 2.32 and 2.04 in September for the E and A treatments, respectively. When Amax was compared at the same CO2 concentration, trees grown in the E treatment always had lower photosynthetic rates than trees grown in the A treatment, indicating Stomatal response down-regulation of photosynthesis (Figure 2). The overall Comparison of g measured at the growth CO concentrations treatment effect was more significant when measured at 350 s 2 (Table 2) showed a mean decrease of 21% in trees in the E mmol mol-1 (P < 0.03) than at 700 mmol mol-1 (P < 0.08). In treatment compared to trees in the A treatment; however, June, differences in A between CO treatments measured at max 2 treatment differences were statistically significant only in Sep- the same CO2 concentration were not significant at either 350 or 700 mmol mol-1 (P > 0.05 in both cases); however, differ- tember (P < 0.01). These results are consistent with the Ci/Ca ences became significant in July (P < 0.02) and increased until ratios for the different treatments and months (Table 2). De- the end of the growing season. Large variability between trees spite lower gs, stomatal limitation to photosynthesis was sig- within the same treatment at the end of the growing season nificantly reduced in trees in the E treatment (Table 2). Although instantaneous transpiration rates (E) were always lower in trees in the E treatment (Table 2), differences between CO2 treatments were generally not significant (except in Sep- tember, when P < 0.05). The E treatment increased ITE by 55% on average.

Table 1. Least-squares estimates of model parameter values: Vcmax and J were obtained by fitting the A/Ci response curves using the Farquhar et al. (1980) model over the growing season (n = 6 except for August where n = 4). The mean temperature (T) at which curves were measured is given.

Parameter Month T (°C) CO2 Treatment Elevated Ambient -2 -1 Vcmax (mmol m s ) June 29 54.3 ± 0.91 59.4 ± 0.95 August 26 40.2 ± 0.97 45.5 ± 1.32 -2 -1 Figure 2. Light-saturated photosynthesis (Amax , mmol m s ) of Sept. 21 17.1 ± 1.03 21.8 ± 0.77 young fully expanded leaves throughout the growing season measured J (mmol m-2 s-1) June 29 107.9 ± 1.06 117.7 ± 1.44 at CO concentrations of (a) 700 mmol mol-1 and (b) 350 mmol mol-1. 2 August 26 86.1 ± 1.80 99.0 ± 2.32 Mean leaf temperatures (T) and vapor pressure deficit (VPD) (kPa) are Sept. 21 43.1 ± 2.03 60.5 ± 1.83 given. Symbols represent means ± 1 SE (n = 5 or 6).

TREE PHYSIOLOGY VOLUME 18, 1998 PHOTOSYNTHETIC ACCLIMATION TO ELEVATED CO2 IN BIRCH 445

Table 2. Stomatal conductance (gs), leaf transpiration (E) and instantaneous transpiration efficiency (ITE) measured over the growing season at the growth CO2 concentration, Ci/Ca calculated from monthly photosynthesis measurements and stomatal limitation to photosynthesis (L) calculated from A/Ci response curves measured throughout the growing season, using the Farquhar and Sharkey (1982) model. Values are the means ± 1 SE (n = 6), and significance (P) (t-test, P < 0.05) is shown by the symbols: ns = not significant, * = 0.05, ** = 0.01, *** = 0.001.

Parameter Month CO2 Treatment P

Elevated Ambient Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 -2 -1 gs (mol m s ) June 0.19 ± 0.06 0.29 ± 0.09 ns July 0.32 ± 0.10 0.27 ± 0.04 ns August 0.17 ± 0.03 0.22 ± 0.02 ns Sept. 0.10 ± 0.01 0.18 ± 0.01 ** E (mmol m-2 s-1) June 2.13 ± 0.49 2.40 ± 0.49 ns July 2.45 ± 0.39 2.55 ± 0.31 ns August 2.29 ± 0.36 2.80 ± 0.36 ns Sept. 1.20 ± 0.12 1.70 ± 0.20 * Figure 3. Concentrations of (a) total chlorophyll, (b) soluble proteins, (c) starch and (d) nitrogen of young fully expanded leaves expressed ITE June 108.80 ± 18.67 191.41 ± 28.30 *** on a dry mass basis (%) over the growing season. Each bar represents July 154.69 ± 16.27 208.87 ± 34.13 ns the mean ± 1 SE (n = 5 or 6). August 136.42 ± 10.75 215.29 ± 23.08 *** Sept. 158.77 ± 26.18 241.01 ± 47.79 ns

Ci/Ca June 0.50 ± 0.07 0.53 ± 0.06 ns less (27% on average; P < 0.01) in trees in the E treatment than July 0.69 ± 0.02 0.59 ± 0.02 ** in trees in the A treatment and it decreased over the growing August 0.69 ± 0.02 0.63 ± 0.02 ns season in all treatments. The time and treatment interaction Sept. 0.77 ± 0.02 0.74 ± 0.03 ns was significant (P < 0.002), as well as the treatment effect L June 0.14 ± 0.02 0.27 ± 0.03 *** (P < 0.008). Although Rubisco activities expressed on a nitro- August 0.05 ± 0.01 0.23 ± 0.03 *** gen basis were lower in the E treatment than in the A treatment Sept. 0.01 ± 0.003 0.18 ± 0.001 *** (Table 3), these differences were smaller than the differences observed when Rubisco activities were expressed on an leaf area basis. Chlorophyll Soluble carbohydrates Total chlorophyll, both on a dry mass and on an area basis, did not differ significantly between CO2 treatments (P > 0.05 in all Growth in the E treatment resulted in a significant (100% on cases), although total chlorophyll concentration of leaves was average; P < 0.001) accumulation of starch in leaves at all consistently lower (12% on average) in the E treatment than in measurement times (Figure 3), despite great variability within the A treatment (Figure 3). The chlorophyll a/b ratio was trees in the same treatment. Consequently, leaves in the E unchanged by elevated CO2 concentration. Total chlorophyll concentration showed the same seasonal pattern in all treatments with a maximum in July (Figure 3). Total chlorophyll per unit nitrogen concentration (Table 3) did not differ between treatments (P > 0.05).

Soluble proteins and Rubisco activity Throughout the growing season, trees grown in the E treatment had lower leaf soluble protein concentrations on a dry mass basis (9% on average) than trees grown in the A and C treatments (Figure 3). However, this difference was only statistically significant (P < 0.05) in the middle of the growing season (July and August), when differences between the A and E treatments were maximal (15%). The differences in soluble protein between CO2 treatments were consistent with the dif- Figure 4. Rubisco activity expressed on an area basis (mmol m-2 s-1) ferences in foliar N concentrations. of young fully expanded leaves over the growing season. Bars repre- We observed down-regulation of Rubisco activity from June sent the means ± 1 SE (n = 6). Bars with different letters are signifi- to September (Figure 4). Rubisco activity was significantly cantly different (LSD, P < 0.05).

TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com 446 REY AND JARVIS

Table 3. Total chlorophyll (mg g-1 N) and Rubisco activity (mmol g-1 s-1) expressed on a nitrogen basis over the growing season. Values are the means ± 1 SE (n = 6), and significance (ANOVA, P < 0.05) is shown by the symbols: ns = not significant, * = 0.05. Means with different letters are significantly different (LSD, P < 0.05).

Parameter Month CO2 Treatment P-Value Elevated Ambient Control Total chlorophyll June 0.19 ± 0.01a 0.21 ± 0.02a 0.16 ± 0.01a ns July 0.31 ± 0.03a 0.29 ± 0.02a 0.27 ± 0.02a ns August 0.30 ± 0.02a 0.32 ± 0.02a 0.24 ± 0.01b * September 0.29 ± 0.03a 0.28 ± 0.03a 0.22 ± 0.02a ns

Rubisco activity June 10.35 ± 0.81a 13.94 ± 1.08b 10.55 ± 0.97a * Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 July 11.05 ± 1.88a 17.23 ± 1.32a 12.23 ± 0.94a ns August 8.99 ± 0.60a 12.64 ± 1.02b 12.50 ± 1.03b * September 8.01 ± 0.96a 12.48 ± 2.45a 10.60 ± 1.37a ns

treatment had significantly more TNC than leaves in the A and photosynthetic rates of trees grown and measured in ambient C treatments (P < 0.05). No significant differences were found CO2 concentration. A persistent increase in photosynthesis for any sugars at any time (P > 0.05 in all cases). after long periods in elevated CO2 concentration has also been found for other deciduous tree species (see Ceulemans and Nitrogen Mousseau 1994, Curtis 1996) and for trees grown in the field Leaves in the E treatment contained on average 13% less under conditions that did not limit rooting volume (e.g., Gun- nitrogen per unit dry mass than leaves in the A treatment derson et al. 1993, Tissue et al. 1993, Lewis et al. 1996, Heath (Figure 3). A similar decrease, although not significant in this and Kerstiens 1997). case, was observed in soluble protein concentrations. Leaves Comparison of photosynthetic rates measured at the same grown in the E treatment also had consistently lower N con- CO2 concentration, revealed a decline in the photosynthetic centration on an area basis, and even on a starch-free mass capacity of trees grown in elevated CO2 concentration. Down- basis, than leaves grown in the A treatment (Table 4), but regulation of photosynthesis increased as the growing season differences between CO2 treatments were smaller than when N progressed; that is, differences between CO2 treatments that was expressed on a mass basis. Leaves in the C treatment had were not significant at the beginning of the growing season, significantly higher N concentrations than leaves in the other when the trees were actively growing, became larger and treatments. significant later in the season. The reduction in photosynthetic capacity in the E treatment was accompanied by a reduction in all photosynthetic components: total chlorophyll, soluble pro- Discussion teins and nitrogen concentrations showed similar reductions. The E treatment also caused highly significant decreases in Photosynthetic response Rubisco activity and Vcmax . Because the assay failed to activate Enhancement of leaf photosynthesis in birch trees grown in the the enzyme fully----Rubisco activities were about half of maxi- E treatment was maintained during the fourth year of growth mum carboxylation rates----it is also possible that storage at in the field: leaf photosynthetic rates of trees grown and meas- -80 °C caused enzyme deactivation. However, this should not ured in elevated CO2 concentration were always higher than invalidate comparisons among CO2 treatments, because there

Table 4. Concentration of nitrogen on an area basis (g m-2) and on a starch-free basis (%) of young fully expanded leaves over the growing season. Values are the means ± 1 SE (n = 6) and significance (ANOVA, P < 0.05) is shown by the symbols: ns = not significant, * = 0.05, ** = 0.01, *** = 0.001. Values with different letters within the same row are significantly different (LSD, P < 0.05).

Parameter Month CO2 Treatment P-Value Elevated Ambient Control N (g m-2) June 1.86 ± 0.11a 1.80 ± 0.15a 2.46 ± 0.30a ns July 1.10 ± 0.04a 1.38 ± 0.06b 1.73 ± 0.07c *** August 1.05 ± 0.04a 1.30 ± 0.07b 1.62 ± 0.08c *** September 1.01 ± 0.13a 1.19 ± 0.13a 1.58 ± 0.13b * N (%) June 2.86 ± 0.20a 2.85 ± 0.12a 3.07 ± 0.09b ** July 2.18 ± 0.11a 2.53 ± 0.11a 3.12 ± 0.16b *** August 2.20 ± 0.01a 2.42 ± 0.05a 2.69 ± 0.07b ** September 2.13 ± 0.17a 2.22 ± 0.22a 2.80 ± 0.15b *

TREE PHYSIOLOGY VOLUME 18, 1998 PHOTOSYNTHETIC ACCLIMATION TO ELEVATED CO2 IN BIRCH 447

was a close correlation between Rubisco activity and Vcmax we assessed N allocation to different photosynthetic compo- (Wang et al. 1998). Our results contrast with several studies nents by expressing total chlorophyll and proteins on a nitro- showing no effect of elevated CO2 concentration on photosyn- gen basis, we found no differences between CO2 treatments. thetic capacity (Idso and Kimball 1991, Gunderson et al. 1993, There is little evidence that the decrease in Rubisco is accom- Scarascia-Mugnozza et al. 1996, Goodfellow et al. 1997). We panied by an increase in capacity for regeneration of RuBP or conclude that down-regulation can occur when trees are grown any other enzymes of the Calvin cycle, or in the thylakoid in the field, with no apparent root limitation (cf. Webber et al. proteins (Mott 1990, Medlyn 1996), although some cases have 1994, Jacob and Drake 1995, Drake et al. 1996, Lewis et al. been reported (Woodrow 1994). 1996). Sink limitation has been proposed to explain the decrease in Analysis of the A/Ci response revealed decreases in the photosynthetic capacity at elevated CO2 concentrations (Stitt initial slope (carboxylation capacity) and the plateau of the 1991, Long and Drake 1992). High photosynthetic rates can be Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 curve (RuBP regeneration capacity). The Vcmax was lower in the maintained while the demand for carbohydrates is high. If E treatment than in the A treatment and treatment differences photosynthesis exceeds the demand for photosynthates, sugars increased toward the end of the season indicating that seasonal accumulate and can trigger a feedback inhibition of the tran- effects influence the response of photosynthesis to elevated scription of photosynthetic proteins (Krapp et al. 1993, Van CO2. The occurrence of down-regulation of photosynthesis as Oosten and Besford 1994, 1995). The result is a decrease in the the growing season progressed has also been observed in other rate of triose phosphate production and utilization (Paul and broad-leaved species such as Castanea sativa Mill. (El Kohen Driscoll 1997). We found a large increase in starch concentra- and Mousseau 1994). Maximum down-regulation at the end of tion in leaves of trees in the E treatment, indicating that the the season may be the consequence of remobilization of nutri- demand for photosynthates did not keep pace with assimila- ents in the E treatment; however, there was no evidence of an tion. There was a negative relationship between leaf starch earlier senescence in this treatment (Rey 1997). Studies on concentration and soluble proteins and Rubisco activity at the Pinus taeda L. have also demonstrated a strong dependence of end of the growing season (Figure 5) when down-regulation the photosynthetic response on time of year (Lewis et al. 1996, was most evident. Similarly, leaf photosynthesis was nega- Tissue et al. 1997). In Pinus taeda, enhancement of photosyn- tively correlated with starch concentration over the growing thesis was maximal in the summer and at a minimum during season in the E treatment (Figure 6). This response would the winter. Reduced sink strength coinciding with minimum allow a more balanced limitation with other photosynthetic growth may have contributed to this response. Changes in processes (Krapp and Stitt 1995), and would facilitate the environmental variables, such as temperature, during the year mobilization of N to parts of the plant where it is limiting (Paul may also contribute to the seasonal response of photosynthesis and Stitt 1993). Although we attribute down-regulation to sink to elevated CO2, because photosynthesis is likely to be more limitation because silver birch has indeterminate growth and responsive to elevated CO2 at high temperatures than at low therefore a capacity to increase sink-strength, no apparent temperatures (Long 1991, Woodrow 1994). reason for sink-limitation in mid-season was detected. It is Down-regulation is not fully understood but two major ex- possible that other processes were limiting the export and planations have been proposed: nutrient and source--sink limi- transport of sugars to other parts of the plant. However, several tations. As a result of accelerated growth and subsequent long-term elevated CO2 studies have found a similar decrease increased demand for nutrients, plants grown in elevated CO2 in Rubisco activity or carboxylation capacity that was unre- concentration may become nutrient limited, and particularly N lated to an increase in TNC (Lewis et al. 1996). A more recent limited. It has been suggested that plants grown in elevated hypothesis for the differential photosynthetic response of spe- CO2 concentration utilize resources more efficiently, particu- cies to increases in atmospheric CO2 concentration is based on larly nutrients, than plants grown in ambient CO2 concentra- phloem loading, but the mechanisms for this is still unclear tion (Norby et al. 1986, Drake et al. 1997). In this experiment, despite an acceleration of growth in response to the E treatment, nutrient concentrations in all non-leaf plant tissues were unchanged (Rey and Jarvis 1997) and the reduction in leaf N concentration was partly explained by an accumulation of starch. It has been suggested that the reduction in Rubisco activity or carboxylation efficiency is most likely to occur under nitrogen limiting conditions (Long and Drake 1992), but no evidence of N limitation was observed in this study. Within the photosynthetic system, an optimal acclimatory response would involve a reduction in Rubisco with increasing RuBP regeneration capacity (Sage 1994, Webber et al. 1994). Our results do not support the theory of a redistribution of N within the photosynthetic system, because Jmax was also de- Figure 5. Relationship between (a) leaf starch concentration and Ru- creased in the E treatment, and no clear differences were found bisco activity (R2 > 0.65) and (b) leaf starch concentration and soluble 2 in the Jmax :Vcmax ratio to indicate a reallocation of N away from protein (R > 0.83) at the end of the growing season (September) in the Rubisco toward proteins involved in electron transport. When ambient (᭺) and elevated (᭹) CO2 treatments (n = 6).

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tration (e.g., Thomas et al. 1994, Curtis 1996), gs has also been found to increase or remain unchanged (e.g., Barton et al. 1993, Ellsworth et al. 1995). Interactions with other environmental variables, such as temperature, water stress or light, may account for these discrepancies (Eamus and Jarvis 1989). For a range of C3 species, stomatal conductance appears to interact with CO2 uptake to maintain Ci as a constant propor- tion of Ca (Wong et al. 1979, Farquhar and Sharkey 1982). Typically, this ratio is about 0.7 for C3 species at a water vapor pressure deficit (VPD) of 2 kPa (Wong et al. 1979). Although the E treatment reduced gs, the Ci/Ca ratio was similar in the E Downloaded from https://academic.oup.com/treephys/article/18/7/441/1687122 by guest on 29 September 2021 and A treatments, suggesting that there was little or no stomatal acclimation to elevated CO2 concentration. However, Ci/Ca tended to increase over the growing season in all treatments, Figure 6. Relationship between light-saturated photosynthesis (Amax ) especially toward the end, when temperatures and hence VPD -1 measured at an atmospheric CO2 concentration of 700 mmol mol and declined. The absence of any effect of elevated CO2 concentra- ᭺ leaf starch concentration (% of dry mass) for the ambient ( ) and tion on this ratio has also been observed in Pinus taeda (Liu elevated (᭹) CO2 treatments. Each point represents the values for one tree at a given time during the growing season (R2 > 0.56). and Teskey 1995). Thus, despite the observed reduction in gs in response to elevated CO2 concentration, stomata appear to be less limiting to photosynthesis in elevated CO2 concentration than in ambient CO concentration (Woodrow 1994, (see Körner et al. 1995). 2 Drake et al. 1997). It has been suggested that the positive effect of down-regulation of photosynthesis on resource use efficiency, N balance and growth is greater than its negative effect on photosynthesis Conclusions (Sage 1994, Drake et al. 1997). The suggestion is supported by a further analysis of the data obtained by Wang et al. (1998), In agreement with other long-term studies on deciduous tree who established a carbon balance for the same trees using the species, leaf photosynthetic enhancement was maintained in simulation model MAESTRO (Wang and Jarvis 1990). That silver birch trees grown in elevated CO2 concentration for analysis revealed that down-regulation of photosynthesis at the several growing seasons in the field. In contrast with other field leaf scale had little effect on net assimilation at the canopy studies, we found that the photosynthetic capacity of trees scale. High leaf photosynthetic rates over the growing season, grown in elevated CO2 concentration was reduced compared to together with large leaf areas, resulted in a doubling of net trees grown at an ambient CO2 concentration under conditions assimilation rates of trees in the E treatment compared to trees of no apparent growth limitation. This reduction in photosyn- in the A treatment over the 1994 growing season. Other studies thetic capacity showed a marked seasonal effect, which was at in natural systems have shown an increase in carbon uptake a maximum toward the end of the growing season. We con- despite a reduction in photosynthetic capacity in response to clude that seasonal changes in the response of photosynthesis elevated CO2 concentration (Drake et al. 1996). to elevated CO2 should be considered when predicting whole- Although the presence of OTCs did not affect photosynthe- tree response over the year. It is possible that down-regulation sis, there were chamber effects on several leaf components. is a positive response to maximize resources, allowing the tree Leaves of trees grown outside the OTCs did not accumulate to use nutrients for more limiting processes without large starch but had higher concentrations of nutrients than leaves of effects on the net assimilation of the whole tree; however, the trees inside the OTCs. Trees grown outside an OTC had a mechanism underlying this response remains unclear. significantly smaller biomass and leaf area than trees grown inside (Rey and Jarvis 1997). This may explain the higher leaf Acknowledgments N content, which in turn, may account for their lack of starch accumulation (e.g., McDonald et al. 1986). This work is part of a European Union funded project, ECOCRAFT (Contract No. EV5V-CT92-0127). We thank Dr. Robert Besford for Rubisco activity determinations, Dr. Craig Barton for technical assis- Stomatal response tance over the whole experiment, Dr. Ying-Ping Wang and Dr. Bart Kruijt for their help with curve fitting and Andy Gray for nitrogen Although gs measured at the growth CO2 concentrations was analysis. The research area was kindly made available on land man- not significantly affected by treatment, gs was on average 21% lower in the E treatment than in the A treatment. The lower g aged by the Institute of Terrestrial Ecology, Penicuik. This work s contributes to the Global Change and Terrestrial Ecosystems (GCTE) in the E treatment was partly the result of a lower stomatal core project of the International Biosphere Program (IGBP). density (Rey and Jarvis 1997). Field et al. (1995) reviewed the response of gs to elevated CO2 concentration in 23 tree species and found a mean decrease of 23%, which corresponds closely References to the decrease of 21% found in our experiment. Although a Allen, S.E. 1989. Chemical analysis of ecological materials, 2nd Edn. reduction in gs is a common response to elevated CO2 concen- Blackwell Scientific Publications, Oxford, 368 p.

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