Responses of citrus leaf photosynthesis, chlorophyll fluorescence, macronutrient and carbohydrate contents to elevated CO2

J. Plant Physiol. 158. 1307 – 1316 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Responses of citrus leaf photosynthesis, chlorophyll fluorescence, macronutrient and carbohydrate contents to elevated CO2 Norbert Keutgen*, Kai Chen Institut für Obstbau und Gemüsebau, Universität Bonn, D-53121 Bonn, Germany

Received March 2, 2001 · Accepted June 7, 2001

Summary Ninety days exposure to elevated atmospheric CO2 concentrations at and above 600 ppm resulted in ‹down-regulation› of leaf net photosynthesis rate (A) in completely expanded and old leaves of Citrus madurensis Loureiro (Calamondin). The decrease of A at high CO2 levels could be explained by a lower activity and/or concentration of Calvin cycle enzymes, which was accompanied by a reduced leaf N content. Nevertheless, C. madurensis did not suffer from nutrient deficiency. Instead, N was redistributed in the plant according to the sink capacity for this macronutrient, which changed at elevated CO2 levels. Mutual shading of older leaves, which occurred especially at elevated atmospheric CO2 concentrations due to the accelerated tree growth, and as a consequence, adaptation to a slightly darker environment may also have played a role for the ‹down-regulation› of A. A decrease of the chlorophyll fluorescence parameter Fv/Fm in completely expanded and old leaves of C. madurensis at and above 750 ppm CO2 is interpreted to indicate photoinhibition at high CO2 levels. Especially in young leaves of C. madurensis, but also in older ones, the increase of photosynthetic water use efficiency was an important effect of elevated atmospheric CO2 concentrations. Key words: calamondin – chlorophyll fluorescence – elevated CO2 – leaf gas exchange Abbreviations: A net photosynthesis rate. – Chl chlorophyll. – Fm maximum fluorescence of darkadapted leaves. – Fm′ maximum fluorescence of light-adapted leaves. – Fo dark fluorescence of dark-adapted leaves. – Fo′ quenched Fo. – Fs′ actual fluorescence of light-adapted leaves. – Fv/Fm maximum quantum yield of PS II. – ∆F/Fm′ actual quantum yield of PS II. – PS photosystem. – qN so-called non-photochemical quenching. – qP photochemical quenching. – Rubisco ribulosebisphosphate-carboxylase/oxygenase. – WUE photosynthetic water use efficiency

Introduction Citrus is an evergreen fruit tree that accumulates a considerable amount of carbohydrates in the leaves, which remain * E-mail corresponding author: [email protected]_

on the tree for two to three years (Sanz et al. 1987, Magel et al. 2000). As a consequence, an increase of the atmospheric CO2 level to 710 ppm resulted in a decrease of the root/shoot ratio e.g. in Citrus aurantium L. (Syvertsen and Graham 1999). By comparison, the dry weight ratio of leaves and roots in apple trees increased when plants were transferred to 700 ppm 0176-1617/01/158/10-1307 $ 15.00/0

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Figure 1. About two years old, representative trees of Citrus madurensis Loureiro after 90 days of growth in different atmospheric CO2 concentrations. From left to right: 300 ppm, 450 ppm, 600 ppm, 750 ppm and 900 ppm CO2.

atmospheric CO2, but the root/shoot ratio did not change significantly (Chen, unpublished). Apple trees accumulate photoassimilates in the trunk and root (Wibbe and Blanke 1995), and the different storage capacity of roots in citrus and apple may explain their different growth responses to elevated atmospheric CO2 concentrations. With respect to storage preferences, strawberry is more similar to apple than citrus. The main storage organs of strawberry plants are crown and root. In strawberry, CO2 concentrations above 750 ppm resulted in an increase in leaf and root weight ratio, but a decrease in crown weight ratio, indicating a limitation of this plant organ for further carbohydrate storage. The root/shoot ratio increased considerably (Chen et al. 1997a). From the principle differences in carbohydrate storage and growth response at elevated atmospheric CO2 concentrations between citrus, apple and strawberry, the hypothesis is deduced that photosynthetic acclimation of citrus to elevated CO2 levels may differ considerably from that observed earlier in strawberry. Strawberry leaves growing for two months at an elevated atmospheric CO2 concentration of or above 750 ppm responded with a ‹down-regulation› of net photosynthesis rate that could be attributed to end product inhibition by elevated carbohydrate concentration in the leaf, combined N and P deficiency, and photoinhibition, possibly due to the damage of PS core complexes (Keutgen et al. 1997). Citrus, however, should respond in a different way to a long-term CO2 enrichment because the storage capacity of citrus leaves for carbohydrates is larger than that of strawberry leaves. In addition, the acclimation response of tree species to elevated CO2 has been mentioned to be less distinct when compared with herbaceous plants; effects on leaf photosynthesis have also been reported (Norby et al. 1999). The objective of the present study was to investigate the long-term response of gas exchange, macronutrient, and carbohydrate contents of citrus leaves to sub-optimal to optimal

(supra-optimal) CO2 concentrations ranging from 300 to 900 ppm. This broad range was selected to include a CO2 level close to the one before the Industrial Revolution, and a level that could be expected to induce a strong ‹down-regulation› of photosynthesis. As the same CO2 concentrations and a comparable experimental set-up were used with strawberry cv. ‹Elsanta› (Keutgen et al. 1997), the results of the citrus experiment could be compared more easily with the latter.

Materials and Methods Plant material and treatments In autumn 1994, 25 cuttings of Citrus madurensis Loureiro (Calamondin) were planted individually in 5 L plastic containers filled with 0.7– 1.2 mm ∅ quartz sand. The surface was covered with a layer of pebbles (1– 2 cm ∅) to prevent incrustation of the sand. The plants received modified Hoagland nutrient solution (Sruamsiri and Lenz 1985) twice a week and tap water when needed. After a pre-culture of one year in the glasshouse, the trees were transferred into five growth chambers with CO2-levels in the air of 300 ± 20, 450 ± 20, 600 ± 20, 750 ± 20, or 900 ± 20 ppm. The other environmental conditions were: 16/8 hours day/night rhythm, 25 ± 2/20 ± 2 ˚C day/night air temperature, and 70 % ± 5 %/80 % ± 5 % day/night relative air humidity. Light was supplied with Osram Power star HQI-T 400W/D lamps (about 550 µmol m – 2 s –1 photosynthetically active radiation at the level of the upper leaves). Within plants, self-shading of leaves occurred so that the light intensity available, especially for old leaves, was reduced to a certain extent. The shading effect was larger in plants grown at high atmospheric CO2 concentrations due to differences in shoot growth and leaf development under the five CO2 levels (cf. Fig. 1). The citrus plants remained in the growth chambers for 90 days. During the last two weeks of the experiment, leaf gas exchange was measured under growth chamber conditions, but light intensity of the measured leaves was kept between 500 and 550 µmol m – 2 s –1. Leaves were grouped into three age-types: young ( < 4 weeks), fully expanded (5–12 weeks), and old leaves ( >13 weeks). Chlorophyll fluorescence measurements

Elevated CO2 effects on citrus were conducted parallel to the gas exchange measurements. At the end of the experiment, the leaves were harvested. Chlorophyll contents were determined from 6 leaves of each age-type. The remaining leaves of each plant were lumped with respect to age-type. From the five samples per variant, three were randomly selected to determine macronutrient and carbohydrate contents.

Leaf gas exchange and chlorophyll fluorescence measurements Net photosynthesis rate (A), transpiration rate (E), stomatal conductance (gs ), and leaf internal CO2 concentration (Ci ) were measured between 8:00 and 12:00 a.m. with the portable CIRAS-1 CO2/H2O infrared gas analyser connected to the PLC broad Parkinson leaf cuvette (PP Systems, Hitchin, Herts., England). The photosynthetic water use efficiency (WUE) was calculated as the ratio A/E. Chlorophyll fluorescence measurements of Fm and Fo were conducted on dark-adapted leaves, as described in Keutgen et al. (1997), with the pulse amplitude modulation fluorometer PAM 2000 (Heinz Walz GmbH, Effeltrich, Germany). Maximum quantum yield was expressed as the ratio Fv/Fm = (Fm – Fo)/Fm

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leaf dry mass. Freeze-dried leaf samples were extracted with methanol and the methanol extracts were used for the identification of glucose, fructose, and sucrose, the residues for starch analyses. Chl in the methanol extracts was minimised with charcoal. The extracts were purified by centrifugation. For the sequential enzymatic degradation procedure to determine the sucrose contents, β-fructosidase and hexokinase/glucose-6-phosphatedehydrogenase were used; for the determination of glucose and fructose, hexokinase/glucose-6-phosphatedehydrogenase was applied first, and then phosphoglucoseisomerase. The absorption of the carbohydrate solutions was detected at 340 nm using a UV detector before and after enzyme applications. For the starch analyses 4 mL H2O bidest. were added to the residue of the methanol extraction and boiled in a water bath for 140 min. Then, 4 mL acetate buffer and 50 µL amyloglucosidase were added to the starch solution and kept for 20 min at 60 ˚C. The solutions were allowed to cool down to room temperature and then were centrifuged. The supernatant was exposed to hexokinase/glucose-6phosphatedehydrogenase and the absorption was measured at 340 nm before and after enzyme application. The sum of the glucose, fructose, and sucrose contents was taken as soluble carbohydrate, and the sum of soluble carbohydrates and starch as the total carbohydrate content.

(1)

Data analyses Thirty seconds after the saturation pulse to determine Fm, the leaves were exposed to actinic light (about 100 µmol m – 2 s –1). For a period of 300 s saturation pulses were triggered every 20 s to determine Fs′ and Fm′ until a quasi steady-state was reached. Finally, Fo′ was recorded on the again darkened leaves exposed to a weak modulated measuring beam. ∆F/Fm′, qP and qN were calculated according to Schreiber et al. (1995): ∆F/Fm′ = (Fm′ – Fs′)/Fm′

(2)

qP = (Fm′ – Fs′)/(Fm′ – Fo′)

(3)

qN = 1-(Fm′ – Fo′)/(Fm – Fo)

(4)

Leaf chlorophyll, macronutrient and carbohydrate analyses Total [Chl] was measured in methanol extracts of the freeze-dried leaf samples. Chl concentrations were determined spectrophotometrically according to Šesták (1971). Total [N] was analysed by a modified Kjeldahl method. The organic and inorganic N were reduced to ammonium by acid hydrolysis with H2SO4 together with catalysts at 400 ˚C. Total [N] was estimated by colorimetry in a SFAS-5100 Skalar auto-analyser (Skalar GmbH, Erkelenz, Germany) after adding salicylic acid and hypochlorite in relation to the N concentration. P, K, Ca, and Mg were determined after digestion at 200 ˚C with H2O2 and HNO3 in an MLS-1200 digestion/drying module microwave laboratory system (MLS GmbH, Leutkirch im Allgäu, Germany). [P] was measured by colorimetry with the Skalar auto-analyser. The concentrations of K, Ca, and Mg were analysed separately by a PerkinElmer-373 atomic absorption and emission spectrophotometer (Perkin-Elmer Corp., Analytical Instruments, Norwalk, CT, USA) (cf. Chen et al. 1997b). Starch, glucose, fructose, and sucrose contents were measured enzymatically according to Boehringer (1995), and expressed in % of

The experimental data was analysed with the SPSS statistical package (SPSS INC., Chicago, USA). A 5 % probability level was accepted to indicate significant differences. The data was tested for normal distribution and variance homogeneity and was compared by Tukey HSD or Wilcoxon-tests. For the latter, the probability level was corrected according to Bonferroni.

Results Gas exchange Net photosynthesis rate (A) of citrus leaves was largest at atmospheric CO2 concentrations between 450 and 600 ppm (Table 1, Fig. 2). Nevertheless, the maximum of A was more distinct in completely developed than in young and old leaves. In tendency, the optimum CO2 concentration for the maximum A decreased from young to old leaves from 600 to 450 ppm. A similar relationship and a corresponding shift of the optimum CO2 concentration were also observed for the stomatal conductance (gs ) and transpiration rate (E). Moreover, there was a linear relationship between gs and E, which was independent from the atmospheric CO2 concentration and leaf age (Fig. 3). Photosynthetic water use efficiency (WUE) increased in tendency from 300 to 600 ppm CO2, and remained stable between 600 and 900 ppm CO2 in completely expanded and old leaves. Only in young leaves did WUE seem to increase along the entire range of investigated CO2 concentrations.

Chlorophyll fluorescence The chlorophyll fluorescence parameters Fv/Fm and Fm were characterised by a maximum at an atmospheric CO2 concen-

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Figure 2. Carpet plot for the leaf gas exchange and chlorophyll fluorescence data of young, completely expanded, and old leaves at different atmospheric CO2 concentrations. Data is represented in % deviation of the results of the 300 ppm CO2 treatment.

tration of 450 ppm (Table 2, Fig. 2). However, the results for Fm were unusual because an effect of the CO2 level in the growth chambers was only detected at 450 ppm. With regard to the dark fluorescence Fo, the data indicated an increase at 750 and 900 ppm CO2, with the exception of young leaves. This increase caused the decrease of Fv/Fm at the two highest CO2 concentrations because Fv/Fm is defined as the ratio (Fm – Fo)/Fm. The actual quantum yield (∆F/Fm′) was also well in line with the low A at 750 and 900 ppm CO2 (Table 2, Fig. 2). The so-called non-photochemical quenching (qN)

increased up to 600 ppm CO2 and then remained at an elevated level. The photochemical quenching (qP) did not depend on the atmospheric CO2 concentration, but decreased with leaf age.

Leaf chlorophyll and macronutrient contents Total [Chl] of leaves was independent of the CO2 concentration in the growth chambers, at least after a growth period of

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90 days. However, [Chl] was affected by leaf age. Young leaves were characterised by significantly lower Chl contents than old ones, and completely expanded leaves were intermediate (Table 3). The macronutrient content of leaves was only slightly affected by the atmospheric CO2 level. Whereas young leaves did not develop a significant CO2 effect, [N] decreased distinctly at atmospheric CO2 concentrations above 600 ppm in completely expanded and old leaves (Table 3). The other macronutrient concentrations were not significantly reduced at elevated CO2 levels. However, K decreased in tendency in completely developed and old leaves and also N in young leaves.

Leaf carbohydrate contents In young citrus leaves, the concentrations of starch as well as of the soluble carbohydrates glucose, fructose, and sucrose were not affected by the development under different atmospheric CO2 concentrations (Table 4). Nevertheless, in completely expanded and old leaves, total carbohydrate contents rose significantly at 750 and 900 ppm CO2. The increase was mainly due to elevated starch levels. In tendency, a comparable effect was also detected for glucose and fructose, but not for sucrose.

Table 1. Gas exchange parameters of C. madurensis leaves grown at different atmospheric CO2 concentrations (CO2 in ppm, A in µmol CO2 m – 2 s –1; E and gs in mmol H2O m – 2 s –1; WUE in µmol CO2 m – 2 s –1/mmol H2O m – 2 s –1). CO2

A

E young leaves 0.7 ± 0.3 a 1.0 ± 0.5 a 1.2 ± 0.5 a 0.6 ± 0.5 a 0.4 ± 0.3 a

300 450 600 750 900

1.0 ± 0.3 a 2.6 ± 1.9 a 3.3 ± 1.0 a 1.9 ± 1.3 a 1.8 ± 0.9 a

300 450 600 750 900

1.5 ± 0.4 b 6.3 ± 2.3 a 6.1 ± 2.1 a 2.4 ± 1.5 b 2.4 ± 1.1 b

300 450 600 750 900

1.1 ± 0.7 c 2.7 ± 0.9 a 2.1 ± 1.0 ab 1.4 ± 0.6 bc 1.7 ± 0.7 abc

gs

36 ± 16 a 40 ± 26 a 47 ± 23 a 27 ± 22 a 18 ± 14 a

completely expanded leaves 1.0 ± 0.1 bc 43 ± 10 b 2.0 ± 0.7 a 72 ± 19 a 1.6 ± 0.9 ab 59 ± 25 ab 1.0 ± 0.4 bc 41 ± 25 bc 0.5 ± 0.3 c 19 ± 12 c old leaves 1.0 ± 0.4 ab 1.1 ± 0.5 a 0.5 ± 0.2 bc 0.5 ± 0.4 c 0.5 ± 0.2 c

42 ± 10 a 44 ± 20 a 24 ± 11 ab 22 ± 18 b 19 ± 6 b

WUE

1.8 ± 0.9 a 2.5 ± 0.9 a 3.2 ± 1.3 a 3.7 ± 2.3 a 5.6 ± 4.1 a 1.5 ± 0.3 b 3.2 ± 0.3 a 4.3 ± 1.2 a 2.4 ± 1.3 ab 5.9 ± 2.3 a 1.2 ± 0.6 a 2.7 ± 1.0 a 4.4 ± 1.1 a 3.3 ± 2.0 a 4.4 ± 2.3 a

Values represent means ± standard deviation. Within a column, different letters indicate significant differences.

Figure 3. Relationship between stomatal conductance (gs ) and transpiration rate (E) of young (䊉), completely expanded (䉬), and old leaves (䊏) at different atmospheric CO2 concentrations in C. madurensis. The regression line is y = 0.029 x – 0.146 and the correlation coefficient r2 equals 0.97. For comparison, the same relationship is given for strawberry cv. ‹Elsanta› (䊊, 䉫, 䊐; y = 0.006 x + 0.608; r2 = 0.79; cf. Keutgen et al. 1997).

Discussion Investigations of Idso et al. (1991), Martin et al. (1995), and Syvertsen and Graham (1999) on different citrus species indicated an increase of citrus growth by about 20 % to 90 % at elevated atmospheric CO2 concentrations of 660 to 720 ppm. The increase was correlated with net photosynthesis rates (A) of single leaves, which were about 20 % to 120 % larger when compared with A at 360 ppm CO2. The present experiments with C. madurensis indicated an optimum CO2 concentration for A between 450 to 600 ppm, which was in accordance with measurements of the efficiency of electron transport across the thylakoid membrane (∆F/Fm′). Higher atmospheric CO2 concentrations resulted in a ‹down-regulation› of photosynthesis, i.e., a reduction of A and ∆F/Fm′. This result corresponds well with an earlier experiment, where strawberry plants were grown at comparable atmospheric CO2 levels for two months (Keutgen et al. 1997). Strawberry showed a maximum of A at 600 ppm atmospheric CO2 at all leaf age stages. Photoinhibition of photosynthesis had originally been defined as a decrease in the rate of photosynthesis, particularly in the efficiency of photosynthetic energy conversion. In chlorophyll fluorescence studies, the term photoinhibition was used almost synonymously with a damage of PS II or a photoprotective response of the light reactions of photosynthesis (Demmig-Adams and Adams 1992), usually indicated by a decrease of the photochemical conversion efficiency of PS II, Fv/Fm (Schulzová 1996). The decrease of Fv/Fm at atmospheric CO2 concentrations above 750 ppm in fully expanded and old citrus leaves can thus be interpreted as a strong ar-

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Table 2. Chlorophyll fluorescence data of C. madurensis leaves in relative units at different atmospheric CO2 concentrations (CO2 in ppm). CO2

Fo

Fm

300 450 600 750 900

0.32 ± 0.04 a 0.31 ± 0.02 a 0.32 ± 0.02 a 0.34 ± 0.02 a 0.32 ± 0.04 a

1.00 ± 0.20 b 1.42 ± 0.20 a 0.97 ± 0.17 b 0.96 ± 0.12 b 0.90 ± 0.13 b

300 450 600 750 900

0.30 ± 0.02 b 0.30 ± 0.02 b 0.30 ± 0.04 b 0.38 ± 0.04 a 0.39 ± 0.04 a

1.01 ± 0.16 b 1.44 ± 0.13 a 1.09 ± 0.19 b 1.08 ± 0.12 b 1.12 ± 0.15 b

300 450 600 750 900

0.30 ± 0.02 c 0.31 ± 0.02 bc 0.32 ± 0.03 bc 0.38 ± 0.04 a 0.34 ± 0.04 b

1.19 ± 0.17 b 1.42 ± 0.06 a 1.24 ± 0.14 b 1.17 ± 0.08 b 1.20 ± 0.14 b

Fv/Fm

yield

young leaves 0.67 ± 0.08 b 0.78 ± 0.02 a 0.67 ± 0.06 b 0.64 ± 0.04 b 0.65 ± 0.05 b

0.52 ± 0.13 a 0.49 ± 0.07 a 0.49 ± 0.04 a 0.41 ± 0.07 ab 0.35 ± 0.11 b

completely expanded leaves 0.70 ± 0.05 b 0.42 ± 0.10 ab 0.79 ± 0.01 a 0.49 ± 0.06 a 0.72 ± 0.04 b 0.49 ± 0.06 a 0.65 ± 0.05 c 0.34 ± 0.10 b 0.65 ± 0.04 c 0.32 ± 0.12 b old leaves 0.74 ± 0.04 ab 0.78 ± 0.01 a 0.74 ± 0.04 b 0.67 ± 0.02 c 0.71 ± 0.04 bc

0.35 ± 0.03 a 0.36 ± 0.07 a 0.35 ± 0.06 a 0.26 ± 0.04 b 0.27 ± 0.05 b

qN

qP

0.16 ± 0.07 a 0.22 ± 0.08 a 0.38 ± 0.12 a 0.33 ± 0.13 a 0.29 ± 0.20 a

0.80 ± 0.22 a 0.67 ± 0.09 a 0.85 ± 0.07 a 0.75 ± 0.13 a 0.63 ± 0.16 a

0.14 ± 0.08 c 0.18 ± 0.08 bc 0.34 ± 0.17 a 0.28 ± 0.12 ab 0.14 ± 0.03 c

0.63 ± 0.20 a 0.65 ± 0.10 a 0.79 ± 0.08 a 0.60 ± 0.18 a 0.52 ± 0.17 a

0.14 ± 0.07 a 0.18 ± 0.03 a 0.30 ± 0.21 a 0.27 ± 0.11 a 0.20 ± 0.07 a

0.50 ± 0.05 a 0.48 ± 0.09 a 0.54 ± 0.11 a 0.44 ± 0.09 a 0.41 ± 0.08 a

Values represent means ± standard deviation. Within a column, different letters indicate significant differences.

Table 3. Macronutrient and total chlorophyll contents of C. madurensis leaves in mg/g dry matter at different atmospheric CO2 concentrations (CO2 in ppm). CO2

N

P

300 450 600 750 900

41.7 ± 4.3 a 43.6 ± 2.6 a 39.9 ± 5.1 a 39.2 ± 2.5 a 37.9 ± 2.3 a

3.8 ± 0.3 a 3.6 ± 0.4 a 3.3 ± 0.4 a 3.4 ± 0.3 a 3.5 ± 0.2 a

300 450 600 750 900

37.8 ± 2.0 a 35.6 ± 1.4 ab 31.9 ± 2.2 bc 30.1 ± 1.0 c 30.6 ± 1.5 c

2.4 ± 0.2 a 2.6 ± 0.2 a 2.1 ± 0.4 a 2.4 ± 0.1 a 2.4 ± 0.4 a

300 450 600 750 900

30.7 ± 2.5 a 30.9 ± 2.6 a 25.2 ± 1.7 ab 25.1 ± 1.9 ab 24.8 ± 2.0 b

1.6 ± 0.3 a 1.7 ± 0.2 a 1.7 ± 0.3 a 1.7 ± 0.1 a 1.7 ± 0.2 a

K

Ca

young leaves 28.2 ± 1.7 a 28.0 ± 1.1 a 25.7 ± 3.3 a 26.3 ± 2.3 a 25.4 ± 4.5 a completely expanded leaves 27.9 ± 1.9 a 28.0 ± 1.2 a 24.7 ± 6.0 a 22.0 ± 3.2 a 22.4 ± 4.1 a old leaves 17.4 ± 5.1 a 16.4 ± 5.5 a 13.7 ± 1.6 a 16.4 ± 6.0 a 13.5 ± 1.6 a

Mg

Chl

13.3 ± 0.5 a 13.9 ± 1.5 a 13.3 ± 1.4 a 13.4 ± 1.3 a 12.7 ± 0.6 a

3.0 ± 0.3 a 3.1 ± 0.2 a 3.0 ± 0.3 a 3.0 ± 0.2 a 3.1 ± 0.1 a

2.0 ± 0.9 a 1.9 ± 1.0 a 2.2 ± 0.4 a 2.0 ± 1.1 a 2.0 ± 0.5 a

27.0 ± 0.3 a 28.7 ± 3.1 a 26.4 ± 0.5 a 25.7 ± 3.0 a 26.8 ± 0.4 a

4.0 ± 0.1 a 3.2 ± 0.1 b 3.8 ± 0.4 ab 3.6 ± 0.1 ab 3.7 ± 0.4 ab

2.4 ± 0.7 a 2.4 ± 0.8 a 2.6 ± 0.7 a 2.3 ± 0.9 a 2.4 ± 0.5 a

43.4 ± 5.6 a 41.0 ± 1.2 a 42.2 ± 1.0 a 44.0 ± 4.5 a 44.4 ± 3.5 a

2.8 ± 0.6 a 2.9 ± 0.6 a 3.0 ± 0.3 a 2.9 ± 0.4 a 2.8 ± 0.5 a

2.6 ± 0.6 a 2.6 ± 0.4 a 2.8 ± 0.6 a 2.6 ± 0.3 a 2.6 ± 0.4 a

Values represent means ± standard deviation. Within a column, different letters indicate significant differences.

gument for photoinhibition caused by high CO2 levels. According to the model of photosynthetic energy dissipation proposed by Butler and co-workers (Butler and Kitajima 1975, Butler 1978), fluorescence yield is a function of the rate constants for fluorescence (KF), non-radiative energy dissipation

in the pigment bed (KD), exciton transfer from PS II to PS I (KT), and photochemistry of PS II (KP). In the case of greater nonradiative dissipation (higher KD), Fo would theoretically decrease, as would Fm. On the other hand, damage to PS II (lower KP) would lead to a higher Fo and lower Fm (Franklin et

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Table 4. Carbohydrate contents of C. madurensis leaves in % of dry matter at different atmospheric CO2 concentrations (CO2 in ppm; tot. C. = total carbohydrates). CO2

starch

glucose

fructose

sucrose

tot. C.

300 450 600 750 900

0.98 ± 0.50 a 0.75 ± 0.52 a 0.96 ± 0.65 a 1.05 ± 0.52 a 0.98 ± 0.66 a

0.83 ± 0.02 a 0.82 ± 0.04 a 1.91 ± 0.12 a 1.56 ± 0.31 a 1.82 ± 0.45 a

young leaves 0.23 ± 0.01 a 0.23 ± 0.01 a 0.45 ± 0.05 a 0.32 ± 0.18 a 0.42 ± 0.14 a

1.09 ± 0.03 a 1.05 ± 0.04 a 1.11 ± 0.08 a 0.98 ± 0.22 a 1.14 ± 0.18 a

3.12 ± 0.52 a 2.84 ± 0.52 a 4.43 ± 0.79 a 3.91 ± 1.22 a 4.36 ± 0.97 a

300 450 600 750 900

0.96 ± 0.21 c 1.53 ± 1.47 bc 1.33 ± 0.81 bc 6.86 ± 1.63 a 4.57 ± 1.57 ab

completely expanded leaves 0.25 ± 0.11 a 0.13 ± 0.01 a 0.20 ± 0.12 a 0.15 ± 0.09 a 0.20 ± 0.03 a 0.11 ± 0.01 a 0.26 ± 0.01 a 0.22 ± 0.02 a 0.35 ± 0.09 a 0.26 ± 0.08 a

0.77 ± 0.20 b 1.30 ± 0.12 a 0.88 ± 0.09 b 1.38 ± 0.06 a 1.38 ± 0.07 a

2.12 ± 0.11 b 3.18 ± 1.31 b 2.52 ± 0.83 b 8.72 ± 1.56 a 6.55 ± 1.47 a

300 450 600 750 900

1.83 ± 0.98 a 1.28 ± 0.97 a 2.10 ± 0.17 a 3.85 ± 0.29 a 3.69 ± 0.33 a

0.33 ± 0.14 a 0.42 ± 0.15 a 0.31 ± 0.01 a 0.59 ± 0.18 a 0.69 ± 0.17 a

old leaves 0.27 ± 0.15 a 0.37 ± 0.15 a 0.26 ± 0.01 a 0.54 ± 0.17 a 0.64 ± 0.16 a

1.02 ± 0.43 a 1.29 ± 0.44 a 0.80 ± 0.01 a 1.13 ± 0.13 a 1.05 ± 0.14 a

3.45 ± 0.28 b 3.36 ± 0.23 b 3.47 ± 0.16 b 6.11 ± 0.08 a 6.08 ± 0.16 a

Values represent means ± standard deviation. Within a column, different letters indicate significant differences.

al. 1992). In C. madurensis, however, only Fo increased, whereas Fm remained constant (Table 2). Noteworthy, in strawberry a similar photoinhibitory response was observed (Keutgen et al. 1997): The decrease of Fv/Fm at CO2 concentrations above 750 ppm was due to an increase of Fo, but Fm also rose. According to the model of Butler and co-workers the response in strawberry should point to a reduction of nonradiative dissipation (lower KD). To what extent this interpretation is also valid for citrus is questionable due to the different responses of Fm in citrus and strawberry. A decrease in [Chl] may also result in an increase of Fo because less Chl may cause a lower rate of re-absorption of the emitted fluorescence light and thus result in an increased total fluorescence emission. Nevertheless, the leaf [Chl] of C. madurensis was independent from the applied CO2 level. This result is in line with an earlier observation of Martin et al. (1995) on C. limon f. ‹Eureka›, where elevated CO2 concentrations (680 ppm CO2) had no effect on the [Chl a] when temperature was moderate (29/21 ˚C day/night air temperature). In consequence, changes in [Chl] may not have affected Fo in citrus. In strawberry, contrary to citrus, total [Chl] decreased with increasing atmospheric CO2 concentrations, even when CO2 was risen from 300 to 450 ppm (Keutgen et al. 1997). Worth noting is that the experiments not only revealed an immediate effect of increasing atmospheric CO2 levels on leaf photosynthesis of C. madurensis, but also an indirect. As can be seen in Figure 1, citrus growth was accelerated when CO2 concentrations rose. This resulted in self-shading, especially

of older leaves, and caused an adaptation to this light environment, e.g. an increase of [Chl] (cf. Table 3). Although it has frequently been reported that [Chl] decreases with leaf age (Hikosaka 1996), it rose in citrus. A high [Chl], however, is required to optimise light harvesting especially at low light intensities. As a consequence, at low growth irradiance, N investment in Chl-protein complexes should be greater relative to that in Calvin cycle enzymes and electron carriers. Thus, the ratio [Chl]/[N], which increases if N is preferably incorporated into Chl-protein complexes, but also if [N] decreases, is an appropriate measure. In young leaves of C. madurensis, the ratio [Chl]/[N] was 0.05 and did not depend on the atmospheric CO2 concentration. Completely expanded leaves growing under 300 and 450 ppm CO2 were characterised by a [Chl]/[N] ratio of 0.06 and 0.07, but for leaves growing under 600 to 900 ppm CO2, by a ratio of 0.08. For older leaves, the difference in the ratio was most distinct: 0.08 at 300 and 450 ppm CO2, but 0.10 to 0.11 at 600 to 900 ppm CO2. This result corresponds well with the hypothesis that older citrus leaves have adapted to the more shaded environment within the canopy, and that this adaptation is more obvious in citrus grown at elevated CO2 levels, where the accelerated tree growth enhanced mutual shading. In strawberry (cf. Keutgen et al. 1997), the ratio [Chl]/[N] changed in a slightly different way when compared with citrus. It was about 0.21 at atmospheric CO2 levels of 300 and 450 ppm, 0.24 at 600 ppm, and 0.19 at 750 and 900 ppm CO2. It may be concluded that mutual shading did not occur in strawberry at 300 and 450 ppm CO2, but it might have af-

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fected leaf [Chl] at 600 ppm. Between 600 and 750 ppm CO2, the [Chl] dropped distinctly by about 50 %. This and the significant decrease of the Chl a/b ratio from 2.7 to 2.4 indicate a severe impairment of light reaction of photosynthesis in strawberry. In C. madurensis, a comparably distinct response was not observed. Total carbohydrate content of completely expanded and old leaves rose in C. madurensis, which was mainly due to an increase in starch content. Two distinct groups could be separated: 300 to 600 ppm and 750 to 900 ppm CO2 (Table 4). In strawberry cv. ‹Elsanta›, for comparison, total carbohydrate and starch contents of leaves grew in parallel to the increase of the atmospheric CO2 level, but also in strawberry the largest difference, i.e., a 74 % increase in starch content, appeared between 600 and 750 ppm CO2 (unpublished results). The starch content in completely expanded strawberry leaves at elevated CO2 levels (750 and 900 ppm) was more than twofold larger when compared with citrus. This may indicate a more pronounced source-limitation in strawberry than in citrus. Growth of the herbaceous strawberry was more limited by its genetic potential, which limits meristem production much more than in citrus trees, and thus curtailed further growth (Morison and Lawlor 1999). In addition, strawberry growth at elevated atmospheric CO2 levels was limited by nutrient deficiency (Keutgen et al. 1997). As a result, the accumulation of carbohydrates in the leaves was more distinct in strawberry than in citrus. It has been argued (Graham 1996, Koch 1996, Jang and Sheen 1997) that elevated concentrations of soluble carbohydrates in the leaf suppress the expression of several photosynthetic genes. Among them is LHCII (or cab), which encodes the major light harvesting proteins. Thus, soluble carbohydrate repression, as it occurs in the leaves, should cause a decrease of [Chl]. This response was detected in strawberry, but not in citrus. Because Chl b is located in the light-harvesting complexes of PS I and II, and Chl a is a component of these complexes as well as of PS I and II core complexes, a repression of the LHCII or other photosynthetic genes may result in a change of the Chl a/b ratio (Von Willert et al. 1995). In strawberry, the a/b ratio decreased significantly at 750 and 900 ppm CO2, which was interpreted by Keutgen et al. (1997), in accordance with literature (Van Oosten and Besford 1995, Wilkins et al. 1994) as a damage of the core complexes of the photosystem. Nevertheless, other scientists argue that the accumulation of soluble carbohydrates is less important to induce ‹downregulation› of A because starch synthesis is an effective buffer for photoassimilates (Heineke et al. 1999). In C. madurensis, the soluble carbohydrate content ranged between 1.2 % and 3.5 % of dry matter and was thus indeed buffered by starch (Table 4). Changes in [Chl] due to the CO2 enrichment were not observed (cf. also Martin et al. 1995), which is in line with the almost constant and, in comparison to strawberry, low soluble carbohydrate content. In strawberry, however, soluble carbohydrate content rose from 7.1 % at 300 ppm to

22.4 % of dry matter at 900 ppm CO2. The strawberry data is well in line with the hypothesis that elevated concentrations of soluble carbohydrates are involved in the regulation of the expression of photosynthetic genes because the [Chl] decreased and the a/b ratio changed. It is concluded that the accumulation of soluble carbohydrates is at least partly responsible for the ‹down-regulation› of A in strawberry, but not in citrus. The macronutrient content of C. madurensis leaves depended only to a limited extent on the CO2 level in the growth chambers. In young leaves, only [N] decreased in tendency. In completely expanded and old leaves, however, a significant reduction of [N] was observed, especially at concentrations at and above 600 ppm CO2. The lower leaf [N] corresponded well with the maxima of A in fully expanded and old leaves at 460 ppm CO2. A lack of N affects the metabolism of the entire plant, but A is especially impaired since 50 % to 80 % of leaf N is allocated to photosynthetic proteins (Hikosaka and Terashima 1995), especially Rubisco (Mengel 1991). An impaired Rubisco activity should result in a reduced Calvin Cycle activity. If lightharvesting of PS II remained similar, the impairment of the dark reactions of photosynthesis should lead to an increasing proton gradient across the thylakoid membrane, and consequently, to an increase of qN (cf. Von Willert et al. 1995). This increase of qN was indeed detected in all citrus leaf growth stages. However, the values of qN also showed a maximum at 600 ppm CO2. Since [N] did not differ between 600, 750, and 900 ppm CO2, but qN decreased in tendency or even significantly for fully expanded leaves from 600 to 900 ppm CO2, electron transport across the thylakoid membrane at 750 and 900 ppm CO2 was reduced, possibly due to the above mentioned reduced photochemical conversion efficiency of PS II (Fv/Fm) caused by the significant increase of dark fluorescence (Fo). In this case, the increase of Fo may represent a photoprotective response of the photosystem (cf. DemmigAdams and Adams 1992). In C. madurensis, the reduction of Rubisco activity is also reflected in changes of the ratio [Chl]/[N] because [Chl] did not depend on the atmospheric CO2 concentration, whereas [N] decreased when the CO2 level rose. The question may be asked, whether the low [N] in completely developed and old citrus leaves at elevated atmospheric CO2 concentrations is due to N deficiency, to N dilution caused by accelerated tree growth, or is an adaptive response to higher CO2 levels? In the experiment with strawberry cv. ‹Elsanta›, all of the studied macronutrients (N, P, K, Ca, Mg) decreased significantly at elevated CO2 concentrations (cf. Keutgen et al. 1997), whereas in C. madurensis only [N] was significantly lower. In strawberry, high CO2 concentrations resulted in a general nutrient deficiency, but this was definitely not the case for C. madurensis. A dilution of N due to enhanced tree growth at elevated CO2 levels can also be excluded for citrus because dilution effects should be detectable for the other macronutrients, for example P. The present results favour the hypothesis that N was re-

Elevated CO2 effects on citrus distributed as an adaptive response to growth at elevated CO2 from leaves to other plant organs in C. madurensis. This is in line with the observation of Idso and Kimball (1997), that in C. aurantium trees, growing for several years at an elevated atmospheric CO2 concentration, fine root development to enhance nutrient uptake was not stimulated. For comparison, Syvertsen and Graham (1999) reported that doubling the present atmospheric CO2 concentration did not result in an increase of root weight ratio in C. aurantium and C. sinensis. However, the root weight ratio rose in strawberry cv. ‹Elsanta›, possibly to enhance nutrient uptake (Chen et al. 1997a). The responses of C. madurensis and strawberry cv. ‹Elsanta› to an increase of atmospheric CO2 concentrations can be summarised as follows: A rose up to a maximum of 450 to 600 ppm CO2 in both species; at higher CO2 levels, A was ‹down-regulated›. In strawberry, the limited growth capacity of the plants and the general nutrient deficiency resulted in a sink-limitation for photoassimilates, leading to a distinct accumulation of starch and soluble carbohydrates in the leaves. Expression of photosynthetic genes was impaired, and as a consequence, [Chl] was reduced and the photosystem damaged. Dark reactions of photosynthesis were also affected. Photoassimilate allocation in C. madurensis, on the contrary, was not sink-limited. A slight increase in leaf carbohydrate content at elevated atmospheric CO2 levels was buffered by starch, so that the soluble carbohydrate content did not rise significantly. Consequently, expression of photosynthetic genes was not impaired and [Chl] remained unchanged. In C. madurensis, the ‹down-regulation› of A can partly be explained by a reduction in leaf [N], resulting in a reduced Rubisco activity. This response can either be interpreted as an immediate CO2 effect, but may also be regarded as an indirect effect. Excessive tree growth at elevated CO2 concentrations resulted in enhanced mutual shading of leaves. The adaptation of completely expanded and old citrus leaves to a lower light level might have contributed to the reduction of A to a certain extent. The chlorophyll fluorescence parameter Fv/Fm indicated photoinhibition in strawberry and also in citrus at 750 and 900 ppm CO2. The cause for the increase of Fo in C. madurensis, which is responsible for the decrease of Fv/Fm, is still unclear and has to be investigated in further studies. Acknowledgements. The authors are grateful to Prof. em. F. Lenz for the research opportunity.

References Boehringer GmbH (1995) Methoden der enzymatischen BioAnalytik und Lebensmittelanalytik mit Test-Kombinationen. Mannheim, Boehringer Mannheim Biochemica, BRD Butler WL (1978) Energy distribution in the photochemical apparatus of photosynthesis. Annu Rev Plant Physiol 29: 345 – 378 Butler WL, Kitajima M (1975) Fluorescence quenching in photosystem II of chloroplasts. Biochim Biophys Acta 376: 116–125

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Chen K, Hu G, Keutgen N, Lenz F (1997 a) Effects of CO2 concentration on strawberry. I. Plant growth analysis. Angew Bot 71: 168–172 Chen K, Hu G, Lenz F (1997b) Effects of CO2 concentration on strawberry. V. Macronutrient uptake and utilization. Angew Bot 71: 168 – 172 Demmig-Adams B, Adams WW (1992) Photoprotection and other responses of plants to high light stress. Annu Rev Plant Physiol Plant Mol Biol 43: 599 – 626 Franklin LA, Levavasseur G, Osmond CB, Henley WJ, Ramus J (1992) Two components of onset and recovery during photoinhibition of Ulva rotundata. Planta 186: 399 – 408 Graham IA (1996) Carbohydrate control of gene expression in higher plants. Research in Microbiology 147: 572 – 580 Heineke D, Kauder F, Frommer W, Kühn C, Gillissen B, Ludewig F, Sonnewald U (1999) Application of transgenic plants in understanding responses to atmospheric change. Plant Cell Environ 22: 623 – 628 Hikosaka K (1996) Effects of leaf age, nitrogen nutrition and photon flux density on the organisation of the photosynthetic apparatus in leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves. Planta 198: 144–150 Hikosaka K, Terashima I (1995) A model of the acclimation of photosynthesis in the leaves of C3 plants to sun and shade with respect to nitrogen use. Plant Cell Environ 18: 605 – 618 Idso KE, Kimball BA, Allen SG (1991) Net photosynthesis of sour orange trees maintained in atmospheres of ambient and elevated CO2 concentrations. Agricultural and Forest Meteorology 54: 95 – 101 Idso SB, Kimball BA (1997) Effects of long-term atmospheric CO2 enrichment on the growth and fruit production of sour orange trees. Global Change Biol 3: 89 – 96 Jang J-C, Sheen J (1997) Sugar sensing in higher plants. Trends Sci 2: 208 – 214 Keutgen N, Chen K, Lenz F (1997) Responses of strawberry leaf photosynthesis, chlorophyll fluorescence and macronutrient contents to elevated CO2. J Plant Physiol 150: 395 – 400 Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 509 – 540 Magel E, Einig W, Hampp R (2000) Carbohydrates in trees. In: Gupta AK, Kaur N (eds) Carbohydrate reserves in plants. Synthesis and regulation. Elsevier, Amsterdam, The Netherlands, 317– 336 Martin CA, Stutz JC, Kimball BA, Idso SB, Akey DH (1995) Growth and topological changes of Citrus limon (L.) Burm. f. ‹Eureka› in response to high temperatures and elevated atmospheric carbon dioxide. J Amer Soc Hort Sci 120: 1025–1031 Mengel K (1991) Ernährung und Stoffwechsel der Pflanze. 7. überarbeitete Auflage. Gustav Fischer Verlag Jena, BRD Morison JIL, Lawlor DW (1999) Interactions between increasing CO2 concentration and temperature on plant growth. Plant Cell Environ 22: 659 – 682 Norby RJ, Wullschleger SD, Gunderson CA, Johnson DW, Ceulemans R (1999) Tree responses to rising CO2 in field experiments: implications for the future forest. Plant Cell Environ 22: 683–714 Sanz A, Monerri C, Gonzalez Ferrer J, Guardiola JL (1987) Changes in the carbohydrates and mineral elements in Citrus leaves during flowering and fruit set. Physiol Plant 69: 93 – 98 Schreiber U, Bilger W, Neubauer C (1995) Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schulze E-D, Caldwell MM (eds) Ecophysiology of photosynthesis. Springer Verlag, Berlin, pp 49–70

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Norbert Keutgen, Kai Chen

Schulzová T (1996) Photoinhibition in situ in Norway spruce. J Plant Physiol 148: 129–134 Šesták Z (1971) Determination of chlorophyll a and b. In: Šesták Z, Catsk´y KJ, Jarvis PG (eds) Plant photosynthetic production. Manual of methods. Dr W Junk NV Publishers, The Hague, The Netherlands, 672–701 Sruamsiri P, Lenz F (1985) Photosynthese und stomatäres Verhalten bei Erdbeeren. I. Einfluß von Licht. Gartenbauwissenschaft 50: 78 – 83 Syvertsen JP, Graham JH (1999) Phosphorus supply and arbuscular mycorrhizas increase growth and net gas exchange responses of two Citrus spp. grown at elevated [CO2]. Plant Soil 208: 209 – 219

Van Oosten J-J, Besford RT (1995) Some relationships between the gas exchange,biochemistryandmolecularbiologyofphotosynthesisduring leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant Cell Environ 18: 1253–1266 Von Willert DJ, Matyssek R, Herppich W (1995) Experimentelle Pflanzenökologie. Grundlagen und Anwendungen. Georg Thieme Verlag Stuttgart New York Wibbe ML, Blanke MM (1995) Effects of defruiting on source-sink relationship, carbon budget, leaf carbohydrate content and water use efficiency of apple trees. Physiol Plant 94: 529 – 533 Wilkins D, Van Oosten J-J, Besford RT (1994) Effects of elevated CO2 on chloroplast proteins in Prunus avium L. Stella. Tree Physiol 14: 769–779