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Keywords:

  • Pinus ;
  • chlorophyll fluorescence;
  • climate change;
  • field experiment;
  • nitrogen;
  • photosynthesis;
  • Rubisco

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

In this experiment, the photosynthetic acclimation of successive needle cohorts of Scots pine were studied during 3 years of growth at elevated CO2 and temperature. Naturally regenerated Scots pine (Pinus sylvestris L.) trees were subjected to elevated CO2 concentration (+CO2, 700 p.p.m), elevated temperature (+T, ambient +2 to +6 °C) and to a combination of elevated CO2 and temperature (+CO2 + T) in closed-top chambers, starting in August 1996. Trees growing in chambers with ambient CO2 and ambient temperature served as controls (AmbC). Elevated CO2 influenced the dark reactions more than the light reactions of photosynthesis, as in the 1996 and 1997 cohorts the carboxylation capacity of Rubisco was reduced in the first and second year of exposure, but there was no consistent change in chlorophyll fluorescence. Net photosynthesis measured at growth concentration of CO2 was higher at +CO2 than at AmbC on only one measuring occasion, was generally lower at +T and was not changed at +CO2 + T. However, trees grown at +T tended to invest more nitrogen (N) in Rubisco, as Rubisco/chlorophyll and the proportion of the total needle N bound to Rubisco occasionally increased. The interaction of +CO2 and +T on Rubisco was mostly negative; consequently, in the second and third year of the experiment the carboxylation capacity decreased at +CO2 + T. In the 1996, 1997 and 1998 cohorts, the structural N concentration of needles was lower at +CO2 than at AmbC. Elevated CO2 and elevated temperature generally had a positive interaction on N concentration; consequently, N concentration in needles decreased less at +CO2 + T than at +CO2. At +CO2 + T, the acclimation response of needles varied between years and was more pronounced in the 1-year-old needles of the 1997 cohort than in those of the 1998 cohort. Thus, acclimation was not always greater in 1-year-old needles than in current-year needles. In the +CO2 + T treatment, elevated temperature had a greater effect on acclimation of needles than elevated CO2.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Increase in atmospheric CO2 concentration and mean temperature are inevitably two of the most important changes caused by man that affect plant growth and carbon balance of an ecosystem. The CO2 concentration has increased from the pre-industrial 280 µmol mol−1 to the current 370 µmol mol−1. With the present rate of increase, 1.5 µmol mol−1 per year, CO2 concentration will reach 700 µmol mol−1 by the end of this century (Schimel et al. 1996). Together with other greenhouse gases, this is predicted to result in an increase of 2.5–4.5 °C in winter temperatures in northern Europe. During summer, the range of increase in temperatures may be even larger, but the upper limit of the range is about 4.5 °C for southern and northern Europe (Kattenberg et al. 1996).

Over the long-term, even small changes in CO2 and temperature are likely to affect plant growth, since both have direct and indirect effects on carbon metabolism and plant development. Light-saturated photosynthesis is initially stimulated at elevated CO2, but during long-term growth at elevated CO2, photosynthetic rates may decline (e.g. Tissue, Thomas & Strain 1993; Kohen & Mousseau 1994, Thomas, Lewis & Strain 1994). In a meta-analysis of 15 field-based experiments on European forest tree species, photosynthesis measured at a common CO2 concentration was 10–20% lower in trees grown at elevated CO2 (Medlyn et al. 1999). This reduction was linked to decreases in nitrogen (N) concentration of leaves, in carboxylation capacity and in electron transport capacity. Despite lower photosynthetic capacity, net photosynthesis measured in growth conditions is generally higher at elevated CO2 than at ambient CO2 (Medlyn et al. 1999; Norby et al. 1999). In studies with field-grown trees over one season or several seasons at elevated CO2, photosynthesis was stimulated, on average, by 51% (Medlyn et al. 1999) or by 66% (Norby et al. 1999).

Due to the kinetic properties of ribulose bis-phosphate carboxylase-oxygenase (Rubisco), the temperature optimum for assimilation increases with increasing CO2, and the relative stimulation of assimilation by elevated CO2 should be greater at higher temperatures (Long 1991). This is supported by some (e.g. Kellomäki & Wang 1996; Wang & Kellomäki 1997b; Tjoelker, Oleksyn & Reich 1998; Lewis, Olszyk & Tingey 1999), but not all studies with trees grown at elevated CO2 and temperature (e.g. Callaway et al. 1994; Wang, Kellomäki & Laitinen 1995; Kellomäki & Wang 1997; Tjoelker et al. 1998). The optimum temperature for photosynthesis is highly dependent on previous growth temperature (Hikosaka, Murakami & Hirose 1999; Teskey & Will 1999), and shifts in the temperature optimum may reduce the positive effect of elevated temperature on the relative response of photosynthesis to elevated CO2 (Lewis et al. 2001). Acclimation to elevated temperature (Kellomäki & Wang 1997; Tjoelker, Reich & Oleksyn 1999) and to elevated CO2 (Curtis 1996; Poorter et al. 1997; Cotrufo, Ineson & Scott 1998) may lead to changes in the foliar concentrations of N and carbohydrates, which are reflected in the altered rates of photosynthesis and respiration.

Decreases in photosynthetic capacity at elevated CO2 are usually more marked when the N supply is low (Petterson, McDonald & Stadenberg 1993; Tissue et al. 1993; Kohen & Mousseau 1994; Thomas et al. 1994; Laitinen et al. 2000), and acclimation has been related to increased demand for N at elevated CO2 (Petterson et al. 1993; Paul & Driscoll 1997). Acclimation has also been explained as being due to limitations related to the experiment, like small rooting volume (Arp 1991). However, in the meta-analysis by Medlyn et al. (1999), acclimation could not be attributed to any one single experimental factor (water or nutrient availability, functional group or age of the plant, or type of experiment). Instead, there was some evidence of increasing down-regulation of photosynthesis with foliage age, as has also been found in many other studies, where acclimation has occurred only in older foliage or taken place earlier in older leaves than in younger leaves (Turnbull et al. 1998; Griffin et al. 2000; Jach & Ceulemans 2000; Laitinen et al. 2000; Tissue et al. 2001).

In Scots pine growing in northern Europe, the rate of recovery of photosynthetic capacity in 1-year-old needles is the main factor causing variation in net photosynthesis between years and is closely related to temperature (Troeng & Linder 1982). Temperature, along with day length, is also an important factor in regulating, for example, the start of the bud burst (Koski 1990), the rate of shoot elongation (Junttila 1986) and the length of the shoot elongation period (Oleksyn, Tjoelker & Reich 1998) in Scots pine. Thus, even small changes in mean temperature could be of great importance for the total biomass production of Scots pine. Provenance experiments have shown that an increase in annual mean effective temperature sum close to that which is expected in northern areas would increase wood production of Scots pine (Beuker 1994; Persson & Beuker 1997). However, because of complex interaction of CO2 and temperature on the balance between assimilate supply and sink activity via changes in photosynthesis, respiration and growth of organs (see review by Morison & Lawlor 1999), the growth response in the future climate is extremely difficult to predict.

Only a few experiments, in which whole trees have been exposed over a growing season or longer, have explored the combined effect of elevated CO2 and temperature on photosynthesis of conifers (Wang et al. 1995; Kellomäki & Wang 1996, 1997; Wang & Kellomäki 1997b; Lewis et al. 1999, 2001). In the present experiment we addressed this topic by studying the effects of elevated CO2 and temperature on photosynthesis and on the composition of Scots pine needles during 3 years of continuous treatments. CO2 and temperature were successfully elevated day- and year-round in closed-top chambers that were constructed around naturally regenerated Scots pine trees growing on a typical N-poor site (Kellomäki, Wang & Lemettinen 2000). Photosynthetic capacity of current-year and 1-year-old needles was studied every growing season as gas exchange, chlorophyll fluorescence and amount and activity of Rubisco protein. Needle samples of the same shoots were analysed for biochemical characteristics. Our aim was to determine whether there is photosynthetic acclimation during 3 years of growth at elevated CO2 and elevated temperature in field-grown Scots pine. During the period covered by this study, we followed the development of two separate cohorts from current-year to 1-year-old needles. This gave us an excellent opportunity to study whether the acclimation response differs between the cohorts and whether the acclimation response is greater in 1-year-old needles than in current-year needles.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Site and chambers

The experiment was conducted in a naturally regenerated Scots pine (Pinus sylvestris L.) stand close to Mekrijärvi Research Station (University of Joensuu) in eastern Finland (62°47′ N, 30°58′ E, 145 m a.s.l). The mean stand density is 2500 trees ha−1. Trees of the stand were 25 to 30 years old and had a mean height of 3.5 m. The soil is sandy loam with water retention of 40 mm at field capacity and 20 mm at the wilting point for the top 30 cm of soil. According to the classification of Cajander (1949), the forest at this site is Vaccinium type. In 1998, the N concentrations in the uppermost mineral soil and in the B horizon at the site were low, around 0.1%.

In the summer of 1996, 16 Scots pine trees with about the same crown size and height were chosen and enclosed individually in closed-top chambers. To reduce shading by other trees, all nearby trees within 2 m from the chambers were cut down 1 year before the start of the experiment. Four trees were subjected to elevated CO2 concentration (+CO2), four to elevated temperature (+T), four to a combination of elevated CO2 and elevated temperature (+CO2 + T) and four trees were controls with ambient CO2 and ambient temperature in the chamber (AmbC). The treatments were started on 24 August 1996 and continued throughout the year.

Each chamber covers a ground area of 5.9 m2 and has an internal volume of 26.5 m3. The walls of the chambers were built in an octagonal ground structure. Four sides facing southwards were of heatable glass consisting of two elements (clear glass + anti-sun green; Eglas Oy, Imatra, Finland) and the other four of double-wall acrylic tunnel sheets (standard 16 mm polymethylmethacrylate). During the growing season, solar radiation in the chambers was reduced by 50–60% for 82% of the time (Kellomäki et al. 2000). The walls of the chambers extended 50 cm below the mean ground surface level in order to sever any root connections and to protect the soil in the chambers from freezing. The temperature of the chambers was automatically regulated by a heat exchanger linked to a cooler (CAJ-4511YHR; L’Unité Hermetique, Barentin, France) installed in the top of each chamber. Unfiltered air was blown into the chamber at the rate of 0.04–0.14 m3 s−1 from a height of 3.5 m by a fan blower. The CO2 concentration was elevated by injecting pure CO2 and mixing it with the outside air. During the growing season, the soil in the chambers was watered to correspond with the water content in the ambient soil. Technical details and the performance of the chambers are presented in Kellomäki et al. (2000).

In +CO2 treatment, CO2 concentration was approximately doubled (693 ± 30 µmol mol−1) compared with the ambient chamber (362 ± 43 µmol mol−1) all day and night throughout the year. The CO2 concentration in the ambient air was 363 ± 38 µmol mol−1. During the growing season (15 April to 15 September), CO2 concentration in the +CO2 chambers was 600–725 µmol mol−1 for 82% of the time. The temperature elevation was adjusted to follow the climatic scenario predicted for the site after doubling of the atmospheric CO2 concentration (Kellomäki & Väisänen 1997). The set temperature increase was 7.5, 6.0, 4.5 and 6.0 °C above the ambient temperature during December–February, March–May, June–August and September–November, respectively. The actual increase in temperature, however, was lower, on an average 2.8 and 6.2 °C above the ambient temperature during the warmest summer and coldest winter months, respectively.

Measurements and sampling

Photosynthesis of current-year and 1-year-old needles was measured before the beginning of the exposure in the summer of 1996 and during the next three growing seasons. In 1996, 1998 and 1999, measurements were made in late June or early July and in August. In 1997 measurements were made only in August. In June/July, when new shoots were not yet fully formed, only 1-year-old needles were studied; and in August, both current-year and 1-year-old needles were measured. After the measurement, two replicate samples of needles from the same shoots were collected for the analysis of Rubisco, soluble proteins and chlorophyll (Chl), one sample for analysis of the C/N ratio and N, and two samples for analysis of starch. Gas exchange and chlorophyll fluorescence measurements and samplings were completed during two or three consecutive days (except in August 1999, when these took 5 d).

Gas exchange measurement

Light-saturated net photosynthesis (PPDF ≥ 800 µmol m−2 s−1), stomatal conductance and transpiration were measured with a portable open gas-exchange system incorporating a conifer shoot cuvette (LCA-4; Analytical Development Co. Ltd, Hoddesdon, UK in 1996 and 1997) or a needle cuvette (LiCor-6400; Li-Cor Inc., Lincoln, NE, USA in 1998 and 1999). Gas exchange was measured at growth concentration of CO2 of the respective treatment. The results of 1996 and 1997, which are measured with the shoot cuvette, are given on a basis of silhouette area of the shoot (Laitinen et al. 2000), and the results of 1998 and 1999, which are measured with the needle cuvette, are given on a basis of projected area of needles measured with WinNeedle 3.1 (Regent Instruments Inc, Quebec City, Canada). In 1996 and 1997, gas exchange was measured at the growth temperature of the respective treatment, and in 1998 and 1999, at a common temperature of 20 °C.

Chlorophyll fluorescence

Chlorophyll fluorescence of current-year and 1-year-old needles was measured at room temperature (approximately 20 °C) using a portable, pulse-amplitude-modulated fluorometer (MINI-PAM; Heinz Walz GmbH, Effeltrich, Germany). Detached needles were mounted side by side on a piece of removable adhesive tape and put into a dark-leaf-clip (DLC-8; Heinz Walz GmbH). After 15 min dark adaptation, the minimal fluorescence level (F0) with all photosystem II (PSII) reaction centres open was determined using a low-intensity modulated measuring light (< 0.1 µmol m−2 s−1). A 1.0-s white light pulse of about 9000 µmol m−2 s−1 was used to produce transient closure of PSII reaction centres, and the maximal fluorescence level (Fm) was recorded. During all measurements, the measuring pulse frequency was set at 0.6 kHz. The maximal quantum yield of PSII photochemistry in a dark-adapted state (ΔF/Fm) was calculated as (Fm − F0)/Fm (Genty, Briantais & Baker 1989). After the saturating pulse, needles were exposed to actinic light of 500–750 µmol m−2 s−1 (in 1997–99) for 2 min, after which the steady-state fluorescence (Fs) was recorded, and a saturating pulse was applied to determine the maximal fluorescence at steady-state (Fm′). The quantum yield of PSII photochemistry in light (ΔF/Fm′) was calculated as (Fm′ − Fs)/Fm′ (Genty et al. 1989). In 1997–99, non-photochemical quenching (NPQ) was determined according to the Stern–Volmer equation as (Fm − Fm′)/Fm′ (Bilger & Björkman 1990).

Biochemical determinations

For biochemical determinations, two replicates of needles were collected under saturating light, frozen immediately in liquid N and stored at −80 °C. The frozen needles were ground in liquid-N and were further homogenized in ice-cold extraction buffer containing 50 mm 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.8, 20 mm MgCl2, 50 mmβ-mercaptoethanol and 1% TWEEN® 80 (Vapaavuori, Rikala & Ryyppö 1992). Aliquots of the crude extract were analysed for chlorophyll (Chl) content by the method of Arnon (1949) in 1996–98 and by the method of Porra, Thompson & Kriedemann (1989) in 1999. The method of Arnon (1949) overestimates Chl concentration and underestimates Chl a/b; consequently, Chl-levels were lower and Chl a/b -levels higher in 1999 than in 1996–98. To compare values measured in 1999 with the values of three previous years, 14 needle samples collected at the same site were analysed for Chl by both methods, and conversion coefficients were calculated. According to these, Chl concentration [mg Chl g−1 fresh weight (FW)] measured by the method of Arnon (1949) is 1.0514x + 0.0271 (r2 = 0.997), where x is the Chl concentration (mg Chl g−1 FW) measured by the method of Porra et al. (1989), and Chl a/b measured by the method of Arnon (1949) is 0.7579x + 0.3854 (r2 = 0.88), where x is Chl a/b measured by the method of Porra et al. (1989). For Fig. 1, the 1999 values are converted for corresponding values measured by the method of Arnon (1949). After centrifugation, initial and total activity of Rubisco were determined as incorporation of 14C into acid-stable products as in Laitinen et al. (2000). The activation state of Rubisco is expressed as the ratio of initial activity to total activity. The amount of Rubisco protein was determined by polyacrylamide gel electrophoresis (PAGE) (Ruuska, Vapaavuori & Laisk 1994) and the soluble protein content by the method of Bradford (1976). Twenty needles of the same shoots were collected and kept on ice in the dark until measurement of chlorophyll fluorescence, which was completed within 1 h of collection. Fresh weight, projected needle area (LI-3050A leaf area meter; Li-Cor Inc.) and dry weight (DW; dried at 60 °C for 48 h) of the same needles were measured. The concentrations of C and N in the dried needles were determined by a CHN Elementar Analyzer (Model 1106; Carlo Erba Strumentazione, Milan, Italy). The proportion of N bound to Rubisco was calculated as a percentage of total needle N content, assuming that Rubisco protein contains 16.67% N (Evans 1989). In 1996 and 1997, starch was measured by an enzymatic method (Steen & Larsson 1986). The method measures non-structural polysaccharides which are not extracted in the 0.05 m acetate buffer at 60 °C and are hydrolysed to glucose by a-amylase and amyloglucosidase. In 1998 and 1999, starch was measured colorimetrically (Hansen & Møller 1975).

image

Figure 1. Light-saturated net photosynthesis (µmol CO2 m−2 s−1) and chlorophyll concentration (mg g−1 FW) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two- to four- replicate trees (SEM indicated by an error bar), except in the 1998 and 1999 cohorts, when in some cases only one tree was studied.

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Statistical analysis

The effects of treatments and time as well as their interactions were analysed statistically using a mixed model program MLwiN 1.1 (Multilevel Models Project, Institute of Education, University of London, UK) which takes the time aspect into account in the analysis. CO2 and T treatments, time effects and their interactions were regarded as fixed treatment effects, and replicate samples and chambers as sources of random variation (random effects). A maximum likelihood method was used to estimate the linear functions of the fixed effects and the variances associated with the random effects. The statistical significance of the differences between treatments was determined by a likelihood ratio test with a chi-square distribution. The effects of +CO2 and +T were tested using AmbC as the basic level. Thus, the chamber effect was excluded from the effects of the treatments. In order to distinguish whether the effects of +CO2 and +T are purely additive or not, the response at +CO2 + T was tested as the interaction of +CO2 and +T. There is an interaction between +CO2 and +T, if the response at +CO2 + T differs from the additive response of +CO2 and +T. A positive interaction means that the level of the variable at +CO2 + T is greater than the summative effects of +CO2 and +T; and a negative interaction means that the level of the variable is lower. The data for different cohorts were tested separately (see Table 6). For example, the needles formed in 1997 (the 1997 cohort), which were studied as current-year needles in August 1997 and as 1-year-old needles in June 1998 and in August 1998, were analysed separately from other data. This approach shows how the treatments affect needles developed in a specific year, at different needle ages.

Table 6.  Statistical analysis of parameters studied in four consecutive cohorts of Scots pine at elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T) in Mekrijärvi site. Significant increases at P ≤ 0.10, P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 are shown as ([UPWARDS ARROW]), [UPWARDS ARROW], [UPWARDS ARROW][UPWARDS ARROW], [UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW], respectively. Significant decreases at P ≤ 0.10, P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 are shown as ([DOWNWARDS ARROW]), [DOWNWARDS ARROW], [DOWNWARDS ARROW][DOWNWARDS ARROW], [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]. Non-significant differences are shown as n.s. The responses at +CO2 + T were tested as interaction of CO2 and T. Positive interactions at P ≤ 0.10, P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 are shown as (+), + , + + , + + +, respectively, and negative interactions at P ≤ 0.10, P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 are shown as (–), –, – –, – – –, respectively. Hence, the signs + and – do not tell directly how the response at +CO2 + T differs from that at AmbC, but rather in which direction the response differs from the additive response of +CO2 and +T
Physiological parameter1996 Cohort1997 Cohort1998 Cohort1999 Cohort
+CO2+TCO2 × T+CO2+TCO2 × T+CO2+TCO2 × T+CO2+TCO2 × T
Pn (µmol CO2 m−2 s−1)
 Current-year needles, Aug.   n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW]++n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]++n.s.([DOWNWARDS ARROW])+
 1-year-old, June   n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW]++n.s.n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.s.[UPWARDS ARROW]n.s.– –n.s.n.s.n.s.   
gs (mmol m−2 s−1)
 Current-year needles, Aug.   n.s.n.s.n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]++
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.[UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.n.s.+   
Trans (mmol H2O m−2 s−1)
 Current-year needles, Aug.   n.s.n.s.n.s.n.s.n.s.n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]++
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.[UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.n.s.n.s.   
ΔF/Fm
 Current-year needles, Aug.   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++n.s.n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   [UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]– – –n.s.n.s.n.s.   
 1-year-old, Aug.[UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.n.s.n.s.   
ΔF/Fm
 Current-year needles, Aug.   n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.n.s.[UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.n.s.n.s.   
NPQ
 Current-year needles, Aug.   n.s.n.s.n.s.[DOWNWARDS ARROW]n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.sn.s.n.s.n.s.n.s.([UPWARDS ARROW])(–)   
SLW (g m−2)
 Current-year needles, Aug.   n.s.n.s.n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.   
C/N ratio
 Current-year needles, Aug.   [UPWARDS ARROW][UPWARDS ARROW]n.s.– – –([UPWARDS ARROW])([UPWARDS ARROW])n.s.n.s.n.s.
 1-year-old, June   [UPWARDS ARROW][UPWARDS ARROW]n.s.– – –([UPWARDS ARROW])n.s.n.s.   
 1-year-old, Aug.[UPWARDS ARROW]n.s.n.s.[UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]– – –([UPWARDS ARROW])n.s.n.s.   
N (% DW)
 Current-year needles, Aug.   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++[DOWNWARDS ARROW]([DOWNWARDS ARROW])+n.s.[UPWARDS ARROW][UPWARDS ARROW]n.s.
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++[DOWNWARDS ARROW]n.s.   
 1-year-old, Aug.[DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.++[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]+++[DOWNWARDS ARROW]n.s.n.s.   
Structural N (%)
 Current-year needles, Aug.   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++[DOWNWARDS ARROW][DOWNWARDS ARROW]+ +n.s.n.s.n.s.
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++[DOWNWARDS ARROW]n.s.n.s.   
 1-year-old, Aug.[DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.+++[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]+++[DOWNWARDS ARROW]n.s.n.s.   
N (g m−2)
 Current-year needles, Aug.   [DOWNWARDS ARROW]n.s.++[DOWNWARDS ARROW][DOWNWARDS ARROW]n.sn.s.n.s.n.s.n.s.
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW]++[DOWNWARDS ARROW]n.s.+   
 1-year-old, Aug.n.s.n.s.n.s.[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]++[DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.n.s.   
Starch (% DW)
 Current-year needles, Aug.   [UPWARDS ARROW]n.s.n.s.[UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   n.s.n.s.+n.s.n.s.+   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.+ +   
Soluble protein (mg g−1 FW)
 Current-year needles, Aug.   n.s.n.s.n.s.n.s.n.s.n.s.[DOWNWARDS ARROW]n.s.n.s.
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.n.s.n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.   
Chl (mg g−1 FW)
 Current-year needles, Aug.   n.s.([UPWARDS ARROW])++n.s.[DOWNWARDS ARROW]n.s.n.s.n.s.n.s.
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.n.s.n.s.[DOWNWARDS ARROW]– – –   
 1-year-old, Aug.n.s.n.s.++([DOWNWARDS ARROW])n.s.– – –[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.   
Chl a/b
 Current-year needles, Aug.   n.s.n.s.(–)n.s.n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   n.s.[DOWNWARDS ARROW]n.s.n.s.n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.n.sn.s.n.s.+   
Rubisco (mg g−1 FW)
 Current-year needles, Aug.   ([DOWNWARDS ARROW])n.s.n.s.n.s.n.s.n.s.n.s.[UPWARDS ARROW]n.s.
 1-year-old, June   ([DOWNWARDS ARROW])n.s.[UPWARDS ARROW]n.s.– – –   
 1-year-old, Aug.[DOWNWARDS ARROW][DOWNWARDS ARROW][DOWNWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]n.s.([DOWNWARDS ARROW])n.s.n.s.n.s.n.s.   
Tot. act. (µmol min−1 g−1 FW)
 Current-year needles, Aug.   ([DOWNWARDS ARROW])[UPWARDS ARROW][UPWARDS ARROW]n.s.[DOWNWARDS ARROW]n.s.n.s.n.s.[UPWARDS ARROW][UPWARDS ARROW]
 1-year-old, June   [DOWNWARDS ARROW][DOWNWARDS ARROW]n.s.n.s.n.s.n.s.– – –   
 1-year-old, Aug.[DOWNWARDS ARROW][UPWARDS ARROW]n.s.[DOWNWARDS ARROW]n.s.n.s.n.s.n.s.n.s.   
Activation state (%)
 Current-year needles, Aug.   n.s.n.s.+n.s.n.s.– – –n.s.n.s.
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.– – –   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.– – –   
Spec. act. (µmol CO2 s−1 g−1)
 Current-year needles, Aug.   n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.
 1-year-old, June   [DOWNWARDS ARROW]n.s.n.s.([DOWNWARDS ARROW])n.s.n.s.   
 1-year-old, Aug.n.s.n.s.n.s.n.s.n.s.++n.s.([UPWARDS ARROW])   
Rbc/Chl (mg mg−1)
 Current-year needles, Aug.   [DOWNWARDS ARROW]n.s.n.s.n.s.n.s.n.s.[DOWNWARDS ARROW][UPWARDS ARROW]
 1-year-old, June   n.s.n.s.n.s.n.s.n.s.   
 1-year-old, Aug.[DOWNWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.n.s.n.s.(+)   
Rubisco-N (% total N)
 Current-year needles, Aug.   n.s.n.s.n.s.n.s.n.s.n.s.n.s.[UPWARDS ARROW]n.s.
 1-year-old, June   n.s.([UPWARDS ARROW])– –[UPWARDS ARROW][UPWARDS ARROW]n.sn.s.   
 1-year-old, Aug.[DOWNWARDS ARROW][UPWARDS ARROW][UPWARDS ARROW]n.s.n.s.n.s.– –n.s.([DOWNWARDS ARROW])n.s.   

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Effect of elevated CO2

During the three first years of this experiment, +CO2 significantly increased the rate of net photosynthesis (Pn) measured at growth concentration of CO2 only in 1-year-old needles of the 1997 cohort (Fig. 1). Stomatal conductance (gs) and transpiration (E) were higher at +CO2 than at AmbC in the 1996 cohort, but lower in the 1999 cohort (Table 1). For all treatments, gs and E measured in 1996–97 were much greater than those measured in 1998–99. This is due to the change in the equipment of gas exchange measurement. In 1996–97 gas exchange was measured on shoots and was calculated on the basis of silhouette area whereas in 1998–99 gas exchange was measured on needles and data were calculated on the basis of projected area of the needles. The maximal photochemical efficiency of PSII (ΔF/Fm) of Scots pine needles was close to the average values measured for 44 vascular species at 77 K (0.832, Björkman & Demmig 1987). There was no consistent effect of +CO2 on ΔF/Fm(Table 2). The apparent quantum yield of PSII photochemistry after light-adaptation (ΔF/Fm′) and non-photochemical quenching (NPQ) are dependent on actinic light, which varied from one measuring date to another. Within a given measurement date, however, there were no significant differences in actinic light for the treatments (data not shown), which ensures that the treatments are comparable. ΔF/Fm′ was not changed at +CO2 in any of the cohorts, but NPQ was lower in the current-year needles of the 1998 cohort (Table 2).

Table 1.  Stomatal conductance (mmol m−2 s−1) and transpiration (mmol H2O m−2 s−1) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two to four replicate trees (± SEM), except in the 1998 and 1999 cohorts, when in some cases only one tree was studied.
 Stomatal conductance (mmol m−2 s−1)Transpiration (mmol H2O m−2 s−1)
Current-year August1-year-old June1-year-old AugustCurrent-year August1-year-old June1-year-old August
1996 Cohort
 AmbC  0.338 ± 0.043  4.54 ± 0.31
 +CO2  0.585 ± 0.165  6.48 ± 1.03
 +T  0.553 ± 0.082  6.94 ± 0.59
 +CO2 + T  0.945 ± 0.122  7.59 ± 1.39
1997 Cohort
 AmbC0.781 ± 0.0310.125 ± 0.0750.023 ± 0.0015.65 ± 1.251.65 ± 0.181.13 ± 0.15
 +CO20.803 ± 0.1390.063 ± 0.0140.025 ± 0.0086.75 ± 0.851.08 ± 0.381.46 ± 0.44
 +T0.667 ± 0.1560.037 ± 0.0140.020 ± 0.0105.73 ± 0.880.79 ± 0.301.77 ± 1.10
 +CO2 + T0.951 ± 0.0940.056 ± 0.0100.011 ± 0.0035.88 ± 2.121.16 ± 0.231.15 ± 0.28
1998 Cohort
 AmbC0.081 ± 0.0260.035 ± 0.0080.0272.42 ± 0.120.38 ± 0.140.37
 +CO20.052 ± 0.0110.048 ± 0.0150.0332.47 ± 0.780.37 ± 0.030.34
 +T0.0320.016 ± 0.0030.0121.450.29 ± 0.010.15
 +CO2 + T0.037 ± 0.0140.019 ± 0.0080.1272.18 ± 0.860.32 ± 0.061.39
1999 Cohort
 AmbC0.138 ± 0.036  1.33 ± 0.29  
 +CO20.039 ± 0.027  0.44 ± 0.35  
 +T0.041  0.49  
 +CO2 + T0.104 ± 0.023  0.85 ± 0.08  
Table 2.  The maximal quantum yield of PSII photochemistry in a dark-adapted state (ΔF/Fm), the apparent quantum yield of PSII photochemistry in light (ΔF/Fm′) and non-photochemical quenching (NPQ) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two to four replicate trees (± SEM)
 ΔF/FmΔF/FmNPQ
Current-year August1-year-old June1-year-old AugustCurrent-year August1-year-old June1-year-old AugustCurrent- year August1-year-old June1-year-old August
1996 Cohort
 AmbC  0.788 ± 0.012  0.292 ± 0.02  4.58 ± 0.50
 +CO2  0.812 ± 0.008  0.274 ± 0.06  4.02 ± 0.94
 +T  0.833 ± 0.003  0.424 ± 0.02  3.38 ± 0.03
 +CO2 + T  0.838 ± 0.005  0.320 ± 0.01  3.23 ± 0.47
1997 Cohort
 AmbC0.837 ± 0.0040.778 ± 0.0210.859 ± 0.0020.358 ± 0.0240.365 ± 0.0260.165 ± 0.0404.19 ± 0.702.49 ± 0.415.03 ± 0.30
 +CO20.796 ± 0.0160.819 ± 0.0050.850 ± 0.0040.350 ± 0.0640.332 ± 0.0270.161 ± 0.0203.81 ± 1.052.63 ± 0.233.95 ± 0.16
 +T0.834 ± 0.0020.827 ± 0.0070.845 ± 0.0030.333 ± 0.0410.328 ± 0.0140.168 ± 0.0274.14 ± 1.123.04 ± 0.254.56 ± 0.28
 +CO2 + T0.836 ± 0.0080.822 ± 0.0050.835 ± 0.0090.324 ± 0.0090.276 ± 0.0360.158 ± 0.0363.83 ± 1.023.07 ± 0.194.65 ± 0.21
1998 Cohort
 AmbC0.857 ± 0.0040.824 ± 0.0130.845 ± 0.0030.196 ± 0.0280.286 ± 0.0140.213 ± 0.0165.35 ± 0.444.29 ± 0.214.42 ± 0.48
 +CO20.854 ± 0.0020.821 ± 0.0080.837 ± 0.0020.190 ± 0.0160.271 ± 0.0060.218 ± 0.0424.21 ± 0.693.92 ± 0.064.45 ± 0.14
 +T0.852 ± 0.0040.823 ± 0.0050.838 ± 0.0050.182 ± 0.0140.245 ± 0.0410.204 ± 0.0274.89 ± 0.654.37 ± 0.105.10 ± 0.30
 +CO2 + T0.848 ± 0.0110.829 ± 0.0040.841 ± 0.0110.176 ± 0.0440.247 ± 0.0220.216 ± 0.0174.43 ± 0.294.28 ± 0.234.23 ± 0.34
1999 Cohort
 AmbC0.845 ± 0.003  0.223 ± 0.013  5.53 ± 0.50  
 +CO20.842 ± 0.005  0.229 ± 0.016  6.02 ± 0.41  
 +T0.852 ± 0.006  0.268 ± 0.047  4.92 ± 0.68  
 +CO2 + T0.843 ± 0.006  0.275 ± 0.025  4.68 ± 0.36  

Needle N concentrations (Table 3) were low but typical for Scots pine grown at this N-deficient site (Kellomäki & Wang 1997, 1998a; Laitinen et al. 2000), ranging, on average, from 9 to 12 mg g−1 DW at AmbC. +CO2 increased the C/N ratio (Fig. 2) and decreased N concentration both on a mass basis and on an area basis (Table 3). In the 1996 cohort, the decline in N concentration was already very clear, and in the 1997 cohort the reduction was even stronger. In the 1998 cohort, the decline was smaller, and finally, in the 1999 cohort the change was not significant. When calculated on an area-basis, relative decreases in N concentration (on average, 11, 24, 14 and 7% in the 1996, 1997, 1998 and 1999 cohorts, respectively) (Table 3) were comparable with reductions in mass-based N concentration (16, 24, 11 and 9%, respectively). Decreases in N concentration were, in general, not caused by accumulation of starch, because concentration of structural N (Fig. 2), which takes into account the dilution effect caused by starch, also declined. Starch concentration (Table 4) was significantly higher at +CO2 only in the current-year needles of the 1997 and 1998 cohorts. Specific leaf weight (SLW, Table 3) was, in general, not changed at +CO2.

Table 3.  SLW (specific leaf weight, g m−2), N concentration on a mass (% DW) and on an area (g m−2) basis in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two to four replicate trees (± SEM)
 SLW (g m−2)N (% DW)N (g m−2)
Current-year August1-year-old June1-year-old AugustCurrent-year August1-year-old June1-year-old AugustCurrent- year August1-year-old June1-year-old August
1996 Cohort
 AmbC  562 ± 16   9.84 ± 0.77  2.47 ± 0.20
 +CO2  569 ± 13   8.26 ± 0.21  2.20 ± 0.08
 +T  556 ± 21  10.24 ± 0.69  2.61 ± 0.16
 +CO2 + T  533 ± 12  10.68 ± 0.38  2.64 ± 0.05
1997 Cohort
 AmbC676 ± 31613 ± 46630 ± 2111.93 ± 0.6910.80 ± 0.4410.70 ± 0.633.04 ± 0.193.17 ± 0.232.90 ± 0.22
 +CO2646 ± 34623 ± 24605 ± 11 9.71 ± 0.75 8.31 ± 0.44 7.46 ± 0.522.60 ± 0.152.40 ± 0.201.98 ± 0.13
 +T625 ± 18602 ± 35633 ± 1412.40 ± 0.9110.07 ± 0.44 8.49 ± 0.533.15 ± 0.202.72 ± 0.222.36 ± 0.14
 +CO2 + T627 ± 22574 ± 25633 ± 2212.76 ± 0.56 9.52 ± 0.25 7.78 ± 0.223.13 ± 0.122.49 ± 0.112.04 ± 0.17
1998 Cohort
 AmbC660 ± 27538 ± 28573 ± 2710.84 ± 0.65 9.66 ± 0.34 9.04 ± 0.402.57 ± 0.212.25 ± 0.012.32 ± 0.19
 +CO2576 ± 27546 ± 21562 ± 26 9.27 ± 0.35 9.00 ± 0.87 7.95 ± 0.461.97 ± 0.102.12 ± 0.211.98 ± 0.20
 +T605 ± 4529 ± 38519 ± 28 9.69 ± 0.8610.13 ± 0.13 9.72 ± 0.362.31 ± 0.212.35 ± 0.152.25 ± 0.21
 +CO2 + T561 ± 27485 ± 30492 ± 510.34 ± 0.63 8.53 ± 0.24 8.77 ± 0.932.22 ± 0.191.85 ± 0.141.87 ± 0.07
1999 Cohort
 AmbC513 ± 18  11.16 ± 0.89  2.34 ± 0.10  
 +CO2543 ± 19  10.13 ± 0.42  2.18 ± 0.16  
 +T518 ± 38  12.24 ± 0.87  2.50 ± 0.40  
 +CO2 + T542 ± 38  11.31 ± 0.11  2.47 ± 0.21  
image

Figure 2. C/N ratio and structural N concentration (mg g−1 DW) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August.Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two- to four-replicate trees (SEM indicated by an error bar).

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Table 4.  Starch (% DW) and soluble protein (mg g−1 FW) concentrations and Chl a/b in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two to four replicate trees (± SEM)
 Starch (% DW)Soluble protein (mg g−1 FW)Chl a/b
Current-year August1-year-old June1-year-old AugustCurrent-year August1-year-old June1-year-old AugustCurrent-year August1-year-old June1-year-old August
1996 Cohort
 AmbC   7.30 ± 0.90  23.4 ± 1.6  3.02 ± 0.04
 +CO2   8.89 ± 0.85  22.0 ± 1.6  3.11 ± 0.09
 +T   7.10 ± 0.78  22.8 ± 1.1  3.04 ± 0.05
 +CO2 + T   9.99 ± 0.83  22.2 ± 0.7  3.01 ± 0.05
1997 Cohort
 AmbC 7.43 ± 1.07 9.54 ± 2.00 7.52 ± 0.4720.6 ± 1.428.4 ± 0.622.8 ± 2.33.11 ± 0.062.91 ± 0.092.85 ± 0.11
 +CO2 9.81 ± 1.67 8.12 ± 0.55 8.29 ± 0.3618.7 ± 0.721.8 ± 1.118.8 ± 2.63.20 ± 0.132.98 ± 0.102.92 ± 0.03
 +T 8.08 ± 0.98 7.34 ± 0.60 7.84 ± 0.2023.1 ± 2.025.3 ± 0.721.4 ± 1.03.15 ± 0.032.71 ± 0.112.83 ± 0.02
 +CO2 + T 9.32 ± 1.3010.10 ± 0.9610.21 ± 1.0822.5 ± 1.021.2 ± 1.315.8 ± 1.83.02 ± 0.042.72 ± 0.093.03 ± 0.03
1998 Cohort
 AmbC 9.80 ± 0.41 5.73 ± 0.90 6.11 ± 0.6520.4 ± 1.214.5 ± 2.715.0 ± 1.93.08 ± 0.053.04 ± 0.103.06 ± 0.11
 +CO212.53 ± 0.91 5.62 ± 0.31 6.38 ± 0.2317.8 ± 0.714.8 ± 2.311.4 ± 2.73.01 ± 0.043.15 ± 0.073.11 ± 0.05
 +T 9.82 ± 0.91 5.57 ± 0.64 6.99 ± 0.6918.8 ± 0.316.0 ± 2.216.6 ± 0.73.01 ± 0.053.02 ± 0.062.90 ± 0.07
 +CO2 + T11.04 ± 1.74 7.36 ± 0.3811.52 ± 0.1918.6 ± 1.116.7 ± 2.114.6 ± 0.82.96 ± 0.053.10 ± 0.123.27 ± 0.14
1999 Cohort
 AmbC 9.84 ± 1.01  19.2 ± 1.5  3.01 ± 0.09  
 +CO210.70 ± 1.09  16.4 ± 1.0  3.32 ± 0.09  
 +T 8.45 ± 0.89  19.8 ± 2.2  3.26 ± 0.05  
 +CO2 + T10.44 ± 0.41  20.4 ± 1.9  3.31 ± 0.09  

In the 1997 cohort, chlorophyll (Fig. 1) and soluble protein (Table 4) concentrations declined at +CO2 more in 1-year-old than in current-year needles. In 1-year-old needles of the 1998 cohort measured in August, chlorophyll concentration was also reduced. In the 1996 cohort, +CO2 reduced the carboxylation capacity of Rubisco, as the amount (Fig. 3) and total activity of Rubisco (Table 5), proportion of Rubisco relative to Chl (Rbc/Chl, Table 5) and proportion of Rubisco N of total needle N (Rubisco-N, Fig. 3) decreased. In the 1997 cohort, the amount and total activity of Rubisco were also lower, whereas in the 1998 and 1999 cohorts, reductions in the parameters related to Rubisco were infrequent and inconsistent.

image

Figure 3. Amount of Rubisco (mg g−1 FW) and Rubisco-N (% of total needle-N) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two- to four-replicate trees (SEM indicated by an error bar).

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Table 5.  Total activity (µmol CO2 min−1 g−1 FW) and activation state (%) of Rubisco and Rubisco/Chl (mg mg−1) in four consecutive cohorts of Scots pine, measured as current-year needles in August and as 1-year-old needles in June and August. Trees were grown at ambient control chamber (AmbC), elevated CO2 (+CO2), elevated temperature (+T), or at elevated CO2 and elevated temperature (+CO2 + T). Treatments were started on 24 August 1996. Values are means of two to four replicate trees (± SEM)
 Total activity of Rubisco (µmol CO2 min−1 g−1 FW)Activation state of Rubisco (%)Rubisco/Chl (mg mg−1)
Current-year Aug1-year-old June1-year-old AugCurrent-year Aug1-year-old June1-year-old AugCurrent- year Aug1-year-old June1-year-old Aug
1996 Cohort
 AmbC  4.52 ± 0.67  33.9 ± 3.0  3.52 ± 0.60
 +CO2  3.31 ± 0.48  32.7 ± 0.3  2.26 ± 0.31
 +T  6.55 ± 1.30  32.0 ± 1.6  5.30 ± 0.26
 +CO2 + T  5.97 ± 0.62  32.3 ± 2.7  2.67 ± 0.45
1997 Cohort
 AmbC3.83 ± 0.3510.30 ± 0.457.07 ± 1.2429.2 ± 1.839.6 ± 2.834.7 ± 4.13.49 ± 1.003.37 ± 0.503.13 ± 0.24
 +CO22.77 ± 0.80 6.47 ± 0.924.18 ± 0.8326.2 ± 4.137.7 ± 1.838.1 ± 2.92.23 ± 0.353.34 ± 0.212.49 ± 0.32
 +T6.87 ± 2.0411.12 ± 0.827.52 ± 1.1929.8 ± 1.044.4 ± 2.237.1 ± 4.13.47 ± 0.344.03 ± 0.822.46 ± 0.79
 +CO2 + T5.09 ± 0.72 6.41 ± 0.793.00 ± 0.0334.3 ± 2.339.7 ± 3.340.2 ± 0.63.09 ± 0.612.76 ± 0.331.39 ± 0.30
1998 Cohort
 AmbC5.32 ± 1.03 5.06 ± 0.344.87 ± 0.7039.6 ± 3.238.1 ± 4.843.6 ± 2.32.87 ± 0.313.79 ± 0.603.96 ± 0.83
 +CO24.22 ± 0.44 6.13 ± 0.593.53 ± 0.3238.8 ± 0.941.8 ± 6.043.7 ± 1.42.63 ± 0.154.11 ± 0.193.84 ± 0.60
 +T4.83 ± 0.72 5.99 ± 0.455.39 ± 0.1238.3 ± 5.842.7 ± 3.048.4 ± 2.93.40 ± 0.484.27 ± 1.343.27 ± 0.13
 +CO2 + T4.15 ± 1.12 1.86 ± 0.892.76 ± 0.3329.1 ± 6.236.8 ± 6.236.3 ± 3.52.29 ± 0.533.87 ± 2.754.07 ± 0.03
1999 Cohort
 AmbC4.68 ± 0.21  55.3 ± 2.3  4.86 ± 0.11  
 +CO24.11 ± 0.75  51.4 ± 5.9  3.44 ± 0.15  
 +T6.77 ± 0.72  49.8 ± 1.3  6.11 ± 0.74  
 +CO2 + T4.55 ± 0.87  40.1 ± 2.5  2.64 ± 0.12  

Effect of elevated temperature

In the 1997, 1998 and 1999 cohorts, net photosynthesis was lower at +T than at AmbC (Fig. 1). The reduction was significant in the current-year needles of these cohorts and in the 1-year-old needles of the 1997 cohort in June 1998. In these cases, reduction in Pn was not caused by loss of the carboxylation capacity of Rubisco (Fig. 3, Table 5). In the 1998 cohort, however, Chl concentration decreased at +T (Fig. 1). In the 1996 cohort, stomatal conductance and transpiration were higher at +T than at AmbC, but in the 1999 cohort gs and E were lower (Table 1).

The carboxylation capacity of Rubisco was occasionally improved at +T. The response was clear in the first treatment year in the 1996 cohort and in the third year in the 1999 cohort, when the amount (Fig. 3) and total activity of Rubisco (Table 5), Rbc/Chl (Table 5) and Rubisco-N (Fig. 3) increased. In the 1996 cohort, higher carboxylation capacity coincided with higher maximal and apparent quantum yield of PSII (Table 2). In contrast, in 1-year-old needles of the 1998 cohort, +T slightly reduced the amount (Fig. 3) and total activity of Rubisco (Table 5) and Rubisco-N (Fig. 3). In the second year of the treatments in the 1997 and 1998 cohorts, +T increased the C/N ratio (Fig. 2) and decreased N concentration (Fig. 2, Table 3). In the third year of the experiment in the 1999 cohort, contradictory, +T increased mass-based N concentration.

Interaction of elevated CO2 and elevated temperature

In the 1997, 1998 and 1999 cohorts, +CO2 and +T had a positive or, in one case, a negative interaction on net photosynthesis (Table 6). Positive interactions were observed when +T alone decreased Pn, and a negative interaction was found when +CO2 alone increased Pn. Consequently, Pn at +CO2 + T was not changed compared with that at AmbC (Fig. 1). In the 1996 cohort, in which +CO2 and +T increased gs and E, there was no interaction. Consequently, gs and E increased also at +CO2 + T (Table 1). In the 1999 cohort, in which +CO2 and +T decreased gs and E, there was a positive interaction. Therefore, gs and E were not changed at +CO2 + T compared with that at AmbC. On chlorophyll fluorescence parameters +CO2 and +T had either a negative, positive or no interaction (Table 6).

In the 1996 and 1997 cohorts, +CO2 and +T had mostly a negative interaction on the C/N ratio and a positive on N concentration (Table 6). This trend was not as clear in the 1998 and 1999 cohorts. Positive interaction caused that at +CO2 + T, N concentration declined less and less frequently than at +CO2 (Fig. 2, Table 3). In the 1997 cohort, the response of N concentration at +CO2 + T followed that at +T, and the decrease in mass-based N concentration in 1-year-old needles was 12% in June and 27% in July (Table 3). In the 1998 cohort, the reductions in N concentration were not as large as in the previous cohort, and were not enhanced in 1-year-old needles. +CO2 and +T had in some cases a positive interaction on starch concentration, which was higher at +CO2 + T than at AmbC in all cohorts, and most distinctly in 1-year-old needles (Table 4).

In the 1996 cohort, +CO2 and +T did not have an interaction on any of the parameters associated with Rubisco (Table 6). Because of the opposite effects of +CO2 and +T on the amount, total activity, Rbc/Chl and Rubisco-N in this cohort, the carboxylation capacity of Rubisco was not changed at +CO2 + T compared to AmbC (Fig. 3, Table 5). In the 1997, 1998 and 1999 cohorts, +CO2 and +T had mostly a negative interaction on the parameters related to Rubisco (Table 6). In the 1-year-old needles of the 1997 cohort, the amount (Fig. 3) and total activity of Rubisco (Table 5), Rbc/Chl (Table 5) and Rubisco-N (Fig. 3), as well as concentrations of Chl (Fig. 1) and soluble protein (Table 4), were lower at +CO2 + T than at AmbC. Again, the reduction relative to AmbC was enhanced from June to August 1998. In the 1998 cohort, the amount, activation state, total and specific activity of Rubisco, Rubisco-N and Chl concentration decreased more in 1-year-old needles than in current-year needles, but the decrease was not enhanced from July to August 1999. In the 1999 cohort, the activation state of Rubisco (Table 5), Rbc/Chl and Rubisco-N were also lower at +CO2 + T than at AmbC.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Effect of elevated CO2

During 3 years of growth at elevated CO2, the most consistent response in Scots pine was an increase in the C/N ratio and a decrease in N concentration of the needles. Reduced N concentration was not caused by accumulation of starch, but rather by a decline in concentration of structural N. Reductions in N concentration of needles or leaves are a general response in plants grown at elevated CO2 (Curtis 1996; Poorter et al. 1997; Cotrufo et al. 1998; Medlyn et al. 1999) and have frequently been interpreted as improved efficiency of N use because of higher rates of photosynthesis or greater growth per unit of N. However, it is not easy to distinguish whether a lower N concentration at elevated CO2 is a direct consequence of elevated CO2, or whether it is related to accessibility of N (see Stitt & Krapp 1999). A decrease in the N concentration of a plant may simply be caused by lower availability of N in the soil or by size-dependent dilution of N resulting from accelerated plant growth at elevated CO2 (Stitt & Krapp 1999). In this study, the low N availability of the soil is a very probable reason for reduction of foliar N concentration at +CO2. In the 1998 cohort, N concentration at AmbC was always lower than in the 1997 cohort at the respective age; and probably because of initially lower N concentration, +CO2 did not lead to as large reductions in N in the 1998 cohort as in the 1997 cohort. It is possible that content of N in the 1998 cohort was already so low that it could not be reduced by further redistribution of N at +CO2. This kind of situation has been proposed by Arp et al. (1998).

Starch concentrations are commonly higher in non-fertilized than in fertilized plants (Ericsson 1979; Paul & Driscoll 1997); and in N-limited plants exposed to elevated CO2, there is further accumulation of nonstructural carbohydrates (see Stitt & Krapp 1999). In our study, in current-year needles of the 1996 and 1997 cohorts starch concentration was higher at +CO2 than at AmbC. However, the starch concentration of 1-year-old needles was not changed at +CO2 in any of the 3 years, possibly because of increasing mobilization of starch reserves from mature needles when the shoot starts to grow (Ericsson 1979). At the time of our measurements in June/July, the sink size of the growing tissues has probably been large enough to ensure remobilization of carbohydrates from 1-year-old needles.

Acclimation of photosynthesis at elevated CO2 may allow the optimization of N use by re-allocation of N from Rubisco to other constituents of photosynthesis or to growing sinks of the plant (see Bowes 1991; Moore et al. 1999). However, the relative allocation of protein N to different functions of photosynthesis is affected by the N concentration of the leaf, as the proportion of Rubisco N decreases with decreasing N concentration (Evans & Seeman 1989). Thus growth at elevated CO2 may reduce the investment of N in Rubisco as a consequence of lower concentration of N in the leaves and not as a direct consequence of elevated CO2 (Theobald et al. 1998; Harmens et al. 2000). In this experiment, in the 1996 and 1997 cohorts lower N concentration at +CO2 was also associated with decreased amount and activity of Rubisco. However, the proportion of Rubisco N of the total N declined only in the 1996 cohort, and Rbc/Chl in the 1996 and 1997 cohorts in the first year of the treatments. Therefore, the reduction in needle N was caused not only by a selective decrease in Rubisco, but also by a general decline in leaf proteins or other N-containing compounds.

Reductions in the carboxylation capacity of Rubisco and in Chl concentration at +CO2 led to lower photosynthetic capacity. Net photosynthesis measured at growth concentration of CO2 was stimulated significantly at + CO2 on only one measuring occasion. Elevated CO2 did not consistently change gs or E, which at +CO2 increased, decreased or were not changed. Other long-term experiments with field-grown trees have also shown variable responses of gs to elevated CO2 and, on average, no significant reductions in gs (Curtis 1996; Curtis & Wang 1998). The stomata of trees may be less responsive to elevated CO2 than the stomata of herbaceous species (Saxe, Ellsworth & Heath 1998). However, Kellomäki & Wang (2000) observed decreased cumulative sap flow at +CO2 in the second year of this experiment. Their model computations showed that smaller gs was mainly responsible for a decrease in sap flow, and changes in needle area or absorption of total radiation had less effect (Kellomäki & Wang 2000).

Elevated CO2 had no effect on ΔF/Fm′, either increased or decreased ΔF/Fm and decreased NPQ on only one occasion. Contrasting effects of elevated CO2 on photochemical yield have also been observed earlier in trees (Saxe et al. 1998). In Scots pine exposed to elevated CO2 in branch-bags (Wang & Kellomäki 1997a) or in open-top chambers for 3 years (Gielen, Jach & Ceulemans 2000) there was no change in chlorophyll fluorescence at elevated CO2. According to our results, elevated CO2 influenced the dark reactions more than the light reactions of photosynthesis. The carboxylation capacity of Rubisco reduced, especially in the 1996 and 1997 cohorts; but as concluded on the basis of fluorescence parameters, there was no consistent change in the function of PSII.

Elevated temperature and its interaction with elevated CO2

Previous studies have shown that trees grown at high temperature may invest more N in Rubisco than do trees grown at low temperature (Hikosaka et al. 1999). In this study, Scots pine grown at +T also showed a tendency to invest more N in Rubisco, as Rbc/Chl and Rubisco-N occasionally increased at +T, most strongly in the 1996 and 1999 cohorts. In the 1999 cohort, increased Rubisco-N was associated with higher N concentration, and may be normal reallocation of N, which would be observed at higher concentration of needle N regardless of growth temperature. Improved N status of needles in the 1999 cohort at +T may be a sign of faster N mineralization and enhanced availability of N in the soil. Increased N concentrations have previously been observed in Scots pine (Kellomäki & Wang 1997) and in Douglas-fir (Hobbie et al. 2001) after 4 years of growth at elevated temperature. In the experiment with Douglas-fir, the hypothesis about faster N mineralization at elevated temperature was supported by stimulated rhizosphere respiration, litter decomposition and oxidation of organic matter in the soil (Lin et al. 1999, 2001). In the 1997 and 1998 cohorts in the second year of our experiment, however, N concentration was lower at +T. Soil processes may have responded more slowly to elevated temperature than growth did, leading to decreased availability of N from the soil in the second year. In the third year, the N supply in the soil may have improved enough to meet the demands of faster growth. Elevated CO2 and elevated temperature generally had a positive interaction on N concentration, most strongly in the 1996 and 1997 cohorts, in which the reductions of N concentration at +CO2 were greatest. Consequently, N concentration decreased less at +CO2 + T than at +CO2. This suggests that in the combined treatment, +T alleviated the depletion of N caused by +CO2, possibly via faster mineralization. Naturally, the capacity for nutrient uptake may also be changed at elevated CO2 (see review by BassiriRad, Gutschick & Lussenhop 2001) and at elevated temperature, and is also a possible explanation for changes in the N concentration of needles.

Despite increases in the carboxylation capacity of Rubisco, net photosynthesis was usually lower at +T than at AmbC. The interaction of +CO2 and +T on the carboxylation capacity of Rubisco was generally negative; consequently, carboxylation capacity decreased at +CO2 + T in the 1997, 1998 and 1999 cohorts. Net photosynthesis was, however, regularly at the same level as at AmbC. At elevated temperature, photorespiration was evidently decreased by elevated CO2, and as predicted on the basis of mechanistic models of photosynthesis (Long 1991), elevated CO2 was more beneficial for photosynthesis at +CO2 + T than separately. Net photosynthesis at elevated temperature cannot, however, be calculated on the basis of short-term temperature responses, as during acclimation the optimum temperature of photosynthesis shifts to near the growth temperature (Berry & Björkman 1980; Hikosaka et al. 1999; Teskey & Will 1999). Temperature acclimation of photosynthesis probably involves changes in several components of photosynthetic apparatus, such as chloroplast membrane lipids and Rubisco (see Berry & Björkman 1980). Changes in the amount of Rubisco protein were also observed in this study as well as in that of Hikosaka et al. (1999). Recent data by Bunce (2000) showed that photosynthesis of Taraxacum officinale was stimulated to the same extent by elevated CO2 on the cool and warm days of the season. He suggested that changes in the apparent specificity of Rubisco for CO2 and O2 accounted for the acclimation of the temperature dependence of photosynthesis (Bunce 2000).

During the 3 years of the study no change was observed in the starch concentration at +T. In all cohorts, however, starch concentration was generally higher at +CO2 + T than at AmbC. This is surprising, as it might be expected that elevated temperature would enhance the use of carbohydrates and prevent accumulation of starch by increasing the rates of metabolic processes and transport of carbohydrates (Farrar & Williams 1991).

There was no consistent effect of elevated temperature on gs or E. Stomata may acclimate to growth temperature, as plants grown in cooler conditions had lower gs and Ci, independent of measurement temperature (Hikosaka et al. 1999). There is uncertainty regarding the direction and magnitude of stomatal acclimation in the future climate: Heath (1998) suggested that the sensitivity of stomata to changes in water vapour pressure deficit may decrease at elevated CO2, whereas Kellomäki & Wang (1998b) observed in the first year of this experiment that stomatal sensitivity to water vapour pressure deficit was increased at elevated CO2.

Is photosynthetic acclimation at elevated CO2 and temperature dependent on the age of the needles?

In many studies, photosynthetic acclimation has been attributed to the age of the foliage, as acclimation has occurred only in older foliage, or earlier in older than in younger leaves (Turnbull et al. 1998; Griffin et al. 2000; Jach & Ceulemans 2000; Laitinen et al. 2000; Tissue et al. 2001). The measurements have usually been made at the same time for different treatments, and thus fail to take into account the role of ontogenetic drift in interpreting the results (Tjoelker et al. 1998). In our study, the timing of bud burst and the rate of needle expansion were either not studied, and thus we can not conclude whether the changes in photosynthetic capacity and N concentration are due to ontogenetic drift or whether they indicate a real acclimation response at elevated CO2 and temperature.

During the 3 years of this experiment, foliar concentrations of N in Scots pine were lower at +CO2 than at AmbC. The reductions in N concentration at +CO2 were independent of needle age, and were not significantly larger in 1-year-old needles than in current-year needles. Neither were the decreases observed in the amount and activity of Rubisco or in the Rbc/Chl at +CO2 larger in 1-year-old needles than in current-year needles. On this basis, it appears that +CO2 did not accelerate needle ontogeny or enhance the remobilization of nutrients from 1-year-old needles. In 1-year-old needles of the 1997 and 1998 cohorts, however, faster decline of chlorophyll concentration at +CO2 than at AmbC may indicate ontogenetic drift.

Elevated temperature accelerated the reduction in N concentration in the 1997 cohort. In the +CO2 + T treatment, the +T factor dominated, and N concentration followed that at +T. Lower N concentration in 1-year-old needles at +CO2 + T was accompanied by a faster decline in chlorophyll, soluble protein, amount and total activity of Rubisco, Rbc/Chl and Rubisco-N than at AmbC or at +CO2. This could be interpreted as earlier onset of senescence, or faster remobilization of nutrients from 1-year-old needles. The amount of Rubisco, Rubisco-N, Rubisco/Chl and soluble protein concentration decreased at +CO2 + T as steeply as at +T. It seems that in the combined treatment, elevated temperature had a greater effect on needle ageing than elevated CO2 did. In the 1998 cohort, the carboxylation capacity of Rubisco and Chl concentration were also decreased more in 1-year-old needles than in current-year needles, but the reduction was not enhanced from July to August 1999. On the basis of these results, acclimation or ontogenetic drift at +CO2 + T was more obvious in the 1997 cohort than in the 1998 cohort. As discussed above, N concentration in needles at AmbC was always lower in the 1998 cohort than in the 1997 cohort at the respective age. In addition, concentrations of chlorophyll and soluble proteins in 1-year-old needles were lower in the 1998 cohort than in the 1997 cohort. Decreased content of N-containing compounds in the 1998 cohort may have restricted the flexibility of needle composition to acclimate to changing conditions. It seems that acclimation of needle biochemistry was not directly dependent on the age of the needles, but was probably regulated more by the general physiological state of the cohort.

CONCLUSIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

During 3 years of growth at elevated CO2 and temperature, Scots pine showed decreased carboxylation capacity of Rubisco. The acclimation response was not solely regulated by the age of the needles, and varied between cohorts. Our results underline the importance of studying the effects of elevated CO2 and temperature in a combined long-term exposure, as elevated CO2 and elevated temperature had interactions that led to a response that differed from what would be expected on the basis of separate treatments.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

We thank Mss Marja-Leena Jalkanen, Mervi Ahonpää, Eeva Vehviläinen, Anna-Maija Väänänen and Merja Essel for invaluable technical assistance and Mr Alpo Hassinen, Matti Lemettinen and the staff at the Mekrijärvi Research Station for setting up and maintaining the experiment. We thank Ms Sini Niinistö for providing us the information of soil N concentration. We are grateful to Dr Juha Lappi for statistical guidance and to Dr Joann von Weissenberg for helping with the English language. We also thank Dr Pedro J. Aphalo and Dr Jarmo K. Holopainen for useful suggestions and comments on a earlier version of the manuscript. This work was supported by funding provided by the Graduate School of Forest Sciences, Maj and Tor Nessling Foundation and The Finnish Cultural Foundation to Eeva-Maria Luomala. Funding by the Academy of Finland to the Mekrijärvi site (Project No. 64308), co-ordinated by Professor Seppo Kellomäki, is gratefully acknowledged.

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  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
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Received 10 June 2002; received in revised form 3 September 2002; accepted for publication 29 October 2002