Photosynthesis Research 15:163-176 (1988) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Effects of elevated atmospheric carbon dioxide on gas exchange and growth of white clover I. N I J S , l I. I M P E N S 1 & T. B E H A E G H E 2 1Department of Biology, University of Antwerp, U.LA., Universiteitsplein 1, B-2610 Wilrijk, Belgium; 2Faculteit van de Landbouwwetenschappen, University of Gent, R.U.G., Coupure Links 653, B-9000 Gent, Belgium Received 21 April 1987; accepted in revised form 9 October 1987

Key words: carbon dioxide, growth analysis, photosynthesis, production, transpiration, wateruse efficiency Abstract. Effects of rising atmospheric CO2 concentrations on gas exchange, growth and productivity were investigated on an important grassland species, Trifolium repens L. cv. Blanca. Pure stands of this species were cultivated over an entire growing season in small acrylic greenhouses with an artificial atmosphere of + 367 or _ 620ppm CO2, respectively. Effects on growth and development were examined in a functional growth analysis, while consequences for gas exchange were determined by photosynthesis and transpiration measurements on canopy level. The stands were regularly clipped for production assessment. Canopies grown at high CO2 levels showed an average increase in productivity of almost 75%. Growth analysis indicated development of a larger foliage area as the major cause, particularly in the first days of regrowth after cutting. The growth advantage that began in this stage was maintained or bettered during the following weeks. The difference between gas exchange measurements expressed per unit leaf area and per unit ground area suggested that changes in net photosynthesis and respiration did not contribute to the increase in total yield. Transpiration declined under high CO2 if expressed on a leaf area basis but total canopy transpiration was at least as large as in ambient CO2 due to the larger leaf area. Water-use efficiency calculations on the summer data indicated a 35% improvement with a doubling of CO2 concentration.

Introduction

Since 1958, when a continuous monitoring program for atmospheric carbon dioxide concentration was begun at Mauna Loa, Hawai, a steady increase in atmospheric CO2 levels has been observed] Though there is still uncertainty about future CO2 levels, many CO2 emission models predict an increase in CO2 concentration between 150 and 300 ppm by 2050 (Edmonds and Reilly 1985). While research on the area of CO2-induced climatic change has only recently boomed (Luther 1985), there has been a long history of

164 experiments investigating plant and vegetation responses. Most of this research concentrated on short-term physiological effects and only in the past ten years attention has shifted towards the long-term approach. The basic reasoning in these studies is that increasing the carbon dioxide concentration, as a limiting factor for photosynthesis, will change CO2 fixation and by doing so also growth, development and yield. An increasing CO2 concentration can result in changes of different nature. Dahlman et al. (1985) give a survey of possible effects on structure (branching, leaf area, root/shoot ratio . . . . ), growth and development (vegetative and/or sexual), productivity, reaction to water and nutrient stress, biochemistry (chlorophyll concentration, enzyme activity, starch storage, carbon/ nitrogen ratio . . . . ), physiology (leaf water potential, stomatal conductance, photosynthesis, transpiration, water use efficiency. . . . ), microbial activity, competition, etc. Lemon (1983) has discussed these effects in more detail. Several changes caused by rising CO2 are expected to lead to adaptation of management practises in agriculture, e.g. in breeding programs for the selection of new varieties, in irrigation and fertilizer application, in weed control and even in genetic manipulation to incorporate positive responses to increased CO2 concentration (Kimball 1985). The purpose of this research was to determine the long-term effects of increased CO2 on an important grassland species, namely Trifolium repens. The effects studied were: (1) photosynthesis, transpiration and water-use efficiency; and (2) growth, development and productivity. The experimental procedure consisted of growing monocultures in a semi-controlled environment, formed by transparent growth-rooms placed in a larger greenhouse.

Materials and methods Trifolium repens L. cv. Blanca seeds were planted in steamed loamy soil in plastic containers on 4 September 1985 at a density of 60 kg/ha. The containers were 13 cm of depth and had a 121cm 2 surface area. During the following winter, the plants were grown in a greenhouse with a minimum temperature of 10 °C. When the stands reached a height of 30 cm, they were cut and removed and the remaining five cm high stubble was refertilized with a standard N P K mixture. Container border effects were minimized by hemming them with gauze. On 12 March 1986 the containers were moved to transparent acrylic growth-chambers within the greenhouse with a ground area of one m 2 and a 60 cm clearance above the soil. The containers were arranged without interspace so the small vegetations in them were assembled to a closed

165 canopy. One of the chambers was ventilated with outside air ( C O 2 conc e n t r a t i o n approximately 367 _+ 23 ppm), while the other was aerated with CO2-enriched air (CO2 concentration approximately 620 _ 35 ppm), both at a rate of 1 m3/min. To avoid contamination from exhaust fumes the ventilation system's inlet was placed at 5 m altitude. The CO2 concentration was continuously measured with a differential infra red gas analyser (UNOR-SN, Maihak) and registered with a HewletPackard 7155A strip-chart recorder. There was no additional illumination so the stands were cultivated under normal daily and seasonal light fluctuations. Photosynthetic active radiation (P.A.R.), relative humidity and air temperature in the growth-rooms were measured with quantum sensors (LI-190S, LICOR), capacitive moisture sensors (Vaisala, type 1514) and copper-constantan thermocouples, respectively. While P.A.R. and relative humidity were somewhat less than natural values outside the greenhouse (25% and 5% respectively), the air temperature inside the growth-chambers was 5 to l0 °C higher than in the open field on warm bright days. The average increase over air temperature was 6.6 °C. In the course of the growing season, the mean values for each month of the minimum air temperature in the chambers rised from 7.5°C to 13.6°C (22.7°C and 33.1 °C for maximum air temperature). The monthly averages for the maximum values of photosynthetic active radiation ranged from 966 to 1592/~mol m -2 s- ~. Gas exchange measurements were conducted on entire stands by taking them out of the growth-chamber and placing them in a cuvette, incorporated in a classical open gas exchange system, described by Koch et al. (1968). A perspex cuvette with temperature (Peltier element) and windspeed control was used. In the cuvette, the canopy boundary layer conductance, calculated from the average windspeed above the canopy and from the length of the vegetation surface, was approximately 7 m m s -1 . Gas exchange measurements included net canopy photosynthesis, canopy respiration (the sum of root and root nodule respiration, soil respiration and shoot respiration) and evapotranspiration. Since there was little difference between the stands' evapotranspiration and their transpiration, the process will from now on be referred to as transpiration. CO2 exchange rate was calculated as P =

(C*J)/A

with P net photosynthetic rate, J flow rate of air through the cuvette, C difference in CO2 concentration between sample and reference air and A ground or leaf area. Transpiration rate was calculated analogously. A rectangular hyperbola

166 Table 1. Maximum photosynthesis (Pmax), dark respiration (RD), relative quantum efficiency (~) and light compensation point (Io) of the net photosynthesis vs. P.A.R. response curves for Trifolium repens in the spring of 1986 (April and May). Values are averages and standard deviations of three stands and are expressed on ground area basis (a) and on leaf area basis (b). Climate control in the cuvette included temperature and dewpoint regulation (set at 25 °C and 6 . 8 g m 3, respectively) as well as CO2 concentration adjustment at 350 or 600ppm, depending on the experiment. The leaf area indices (LAI) of the stands grown at high and at low CO2 were 7.3 and 5.7, respectively. Growth and measuring concentration of CO2 (ppm)

Pmax ( 1 0 - 6 g m - 2 s -I)

RD ( 1 0 - 6 g m 2s-I)

ct (gmol i)

Io (10-6molm-2s-~)

350 a)

2202 ___196 387.4 +29.8 2557 +328 352.7 +40.9

- 127.4 +20.8 - 25.7 +8.5 - 182.1 +38.3 - 22.4 ___12.1

4.797 +0.624 0.866 +0.094 6.281 +0.819 0.844 +0.077

16.77 __+14.25 16.77 ___14.25 27.77 +20.73 27.77 ___20.73

b) 600 a) b)

Y =

A + B/(X + C)

(X is the independent variable, Y is the dependent variable) was used to fit observed values on photosynthetic response diagrams. Using the constants A, B and C, maximum net photosynthetic rate Pmax, dark respiration rate RD, relative quantum efficiency ~ (slope of the fitted curve for X = 0) and light compensation point Io were calculated. ~ compounds both differences in light absorption in the canopy and in the true quantum efficiency. Ceulemans et al (1980) used this equation successfully for light response curves though in this study it was also used for CO2 response curves without causing any special problems. During the growth period from 13 May 1986 to 2 June 1986 canopy dry weight, fresh weight and leaf area (measured with a portable leaf area meter Licor, LI-3000) were determined every three or four days for all treatments on four randomly selected containers and these data were used for a functional growth-analysis. This type of analysis is preferred because no rigid harvesting schedule is required and random fluctuations in plant parameters are smoothed so that small deviations from general trends are damped. Polynomial regression was used to fit the data since no specific growthmodel was known for this species under the experimental conditions used. A computer program by Hunt and Parsons (1974) for stepwise polynominal regression selected the polynomial's degree based on statistical considera-

167 Table 2. Transpiration rate ( 1 0 - 4 g m 2s-I) at three selected photosynthetic photon flux densities (PPFD) for Trifolium repens in the spring of 1986. Values are averages and standard deviations of three stands and are expressed on ground area basis (a) and on leave area basis (b). Climate control in the cuvette included temperature and dewpoint regulation (set at 25 °C and 6 . 8 g m -3, respectively) as well as CO2 concentration adjustment at 350 or 600ppm, depending on the experiment. The leaf area indices (LAI) of the stands grown at high and low CO2 were 7.3 and 5.7, respectively. G r o w t h and measuring concentration of C O 2 (ppm)

P P F D (10 -6 mol m -2 s-~) 654 198

1510

350 a)

1376 _ 131 242 ___28 1496 +81 206 + 16

1949 __+196 343 4- 17 2055 +208 281 +44

b) 600 a) b)

1661 __+177 292 +28 1859 +161 258 +39

tions and thus avoids overfitting. The calculated parameters relative growth rate (RGR), leaf area ratio (LAR) and net assimilation rate (NAR) slightly deviate from their general definition, since dry weight is determined on small vegetations instead of on individual plants, and since only above-ground material was used.

Results

A. Canopy gas exchange measurements Expressed on a ground area basis there was a 16% increase in maximum net photosynthesis when the stands were grown and measured in a high CO2 instead of in a low CO2 atmosphere during spring (Table 1). Dark respiration and relative quantum efficiency increased 41 and 31% respectively, while the light compensation point was 65% higher compared to its value in ambient air. Due to the high variability in the data and the small samples used, none of these differences was statistically significant. Expressed on a leaf area basis net photosynthesis decreased slightly though not significantly under high CO2 conditions. Dark respiration and relative quantum efficiency showed a similar response. Transpiration behaved somewhat differently than net photosynthesis during the same measuring period. It showed a 15% decrease (on a leaf area

168 Table 3. Maximum photosynthesis (Pmax) and CO 2 compensation point (Co) for Trifolium repens in the spring of 1986. Values are averages and standard deviations of three stands and are expressed on ground area basis (a) and on leaf area basis (b). Climate control in the cuvette included temperature and dewpoint regulation (set at 25 °C and 6.8 g m -3, respectively) as well as CO2 concentration adjustment at 350 or 600 ppm, depending on the experiment. The leaf area indices (LAI) of the stands grown at high and low CO2 were 7.3 and 5.7, respectively. Growth and measuring concentration of CO 2 (ppm)

Pmax (10 6 g m - 2 s- ' )

Co (ppm)

350 a)

2462 ___342 433.2 _+38.5 2954 + 379 406.7 + 27.3

44.79 _ 13.40 44.79 4- 13.40 50.52 __+18.60 50.52 + 18.60

b) 600 a) b)

basis) under high CO2 concentration but a 9% increase on a ground area basis (Table 2). Carbon dioxide response curves showed hardly any difference in pmax and Co (CO2 compensation point) between stands grown and measured in 350 ppm and stands grown and measured in 600 ppm (Table 3). In the summer of 1986 net photosynthesis and transpiration measurements were repeated and from these data water-use efficiency (W.U.E.) was calculated as the ratio of net canopy photosynthetic r a t e ( 1 0 - 6 g m -2 S-1) to canopy transpiration r a t e ( 1 0 - 6 g m - 2 s - l ) . Averaged over three selected Table 4. Water use efficiency (* 10- 5mg CO2/mg H2 O) at three selected photosynthetic photon flux densities (PPFD) for Trifolium repens in the summer of 1986. Values are averages and standard deviations of four stands. Climate control in the cuvette included temperature and dewpoint regulation (set at 25 °C and 6.8 g m -3, respectively) as well as CO2 concentration adjustment at 350 or 600 ppm, depending on the experiment. Growth and measuring concentration of CO 2 (ppm)

P P F D (10 -6 mol m-2 s- i ) 198 654

1510

350 600

163 256

553 697

740 878

57

26

19

Relative increase in W.U.E.

(%)

169 2.25 2.00 600 PPM

1.75 1.50 1.25 1.O0

350 PPM

0.75 0.50 0.25 0.00

I

I

I

3.

I

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6.

I

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9.

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I-

12.

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TIME (DRYS AFTER CUTTIMG) Fig. 1. Above-ground dry matter changes with time for Trifoliurn repens grown in a 350 and in a 600 ppm atmosphere. Dry weight is expressed on container basis (with a 121 cm 2 area).

E

450.

!

6oo :

400. 350. 300. 250. 200.

(IS .._1

1.

350 PPM

150. 100. 50. O.

I

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TIME (DRYS RFTER CUTTIIY5) Fig. 2. Changes in foliage area and leaf area index (LAI) with time for Trifolium repens grown in a 350 and in a 600ppm atmosphere. Foliage area is expressed on container basis (with a 121 cm 2 area).

170 0.600

"T

0.500 O. 400

!

0.300 0.200 0.I00

600

t

0.000

600 PPM

-.I00

35o PPM

-.200

-.300

I

3.

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1

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TIME (2AYSAFTERCUTTIMG)

Fig. 3. Relativegrowth rate (RGR) during regrowth of Trifoliumrepens stands grown in a 350 and in a 600ppm atmosphere. Vertical bars are 95% confidence limits.

light intensities the high CO2 treatment showed a 34% increase in W.U.E. compared to the ambient CO: treatment (Table 4). The difference was the largest at the lowest light intensities.

Canopy dry matter production High CO 2 caused a significant canopy dry matter production increase of

75% (average of four growing periods from March to July). There were no significant changes in dry matter content (dry weight/fresh weight).

Functional growth analysis During one growing period (from 13 May 1986 to 2 June 1986) data were collected for the analysis of the stands' growth pattern. The polynomials fitted through these data are represented in figs. 1 and 2. Above-ground dry weight and foliage area were always higher in the high CO: environment and the major difference between treatments was established in the first days after cutting. Derived growth parameters (RGR, L A R and N A R ) were subject to little change (figs. 3, 4 and 5).

171 500. 400. 300. 350 PPM ..-.I

200.

600 PPM

I00. O.

I

3.

I

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6.

I

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9.

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12.

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1

15.

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TIME (DAYSAFTER CUTTIM6) Fig. 4. Leaf area ratio (LAR) during regrowth of Trifolium repens stands grown in a 350 and in a 600ppm atmosphere. Vertical bars are 95% confidence limits.

Discussion

The most striking response to higher carbon dioxide levels is a significant increase in this one-species ecosystem's productivity. The average rise in above-ground yield over four three-week growing periods was much larger than the 33% figure Kimball (1982) calculated as an average for 430 species under various growth conditions but always with a doubled CO2 concentration. In the open field the growth stimulation could be smaller because of nutrient limitations but on the other hand it could be larger due to higher light levels. In addition the response to rising CO2 in the field also depends on the presence of other competitive species: Overdieck et al (1984) found similar yield increases in a mixed sward of Trifolium repens and Lolium perenne. Growth analysis clearly shows that the difference between CO2 treatments originates in the first days after cutting - - during which no sampling occurred - - and that this initial advantage in dry matter production and leaf area development is at least maintained if not increased further on. Changes in derived growth parameters (RGR, LAR and NAR) are similar for both treatments and are consistent with the course of primary vegetation growth parameters. Since the initial difference between treatments is preserved throughout the growth period it is unlikely to be produced by experimental

172 0.0030 0.002S !

0.0020 350 PPM

O.O01S 0.0010 O.O00S

600 PPM

0.0000

350 PPM

-.O00S -.0010 -.O01S

I

3.

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1

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9.

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TIME (~AYS AFTER CUTTIIY6) Fig. 5. Net assimilation rate (NAR) during regrowth of Trifoliurn repens stands grown in a 350 and in a 600ppm atmosphere. Vertical bars are 95% confidence limits.

error. A possible cause may be a more extensive root system in the high CO2 treatment, though this hypothesis was not tested in this investigation. The fact that stands grown in a high CO2 environment build up a larger foliage area particularly in the first days after cutting suggests they will reach the optimal leaf area index for photosynthesis sooner than stands grown in ambient CO2. Increasing the mowing frequency to shorten the less productive phase afterwards may optimize seasonal dry matter production in such a system, though simultaneous changes in fertilizer application may be required. Since final biomass production is the integral of gas exchange during a growing period, production data and gas exchange measurements should be in line, so a higher CO2 fixation is expected in this experiment. This reasoning may be valid for single leaves, it appears not to be on the canopy level, because net photosynthesis decreased if expressed on a leaf area basis. The explanation for this apparent anomaly lies in the fact that under higher CO2 levels more leaf area is developed so more shading on the bottom leaves brings down the illumination and thus the net photosynthesis of the average leaf in the canopy. Summed over the entire canopy however still more CO2 is fixed due to the higher foliage area, supposing leaf thickness is not altered (Table 1). Another explanation that could be valid at the same time is that by reduction of dark respiration under high CO2 less of the amount of CO2

173 fixed during the day is released back to the atmosphere, as observed by Reuveni and Gale (1985) with Medicago sativum. In this case however the hypothesis is invalid since dark respiration is larger in the high CO2 environment if expressed on a ground area basis. This is again caused by a larger foliage area. Differences in photosynthetic rate between treatments being non-significant does not necessarily mean that they are not worth discussing, since there are indications for real difference. The highly significant increase in dry matter under increased COs concentration is one of them. In view of the high variability of the data, differences would probably have been significant if more repetitions had been possible and if the plant material had been more uniform. No attempt was made here to estimate yield increase under high CO2, using changes in net photosynthesis and dark respiration: gas exchange was after all measured under constant and optimal conditions, while the stands were grown under varying conditions of light, temperature and humidity. Continuous monitoring of carbon dioxide exchange rate for deriving complete mass balances of CO2 (cf. Jones, Jones and Allen 1985) was not possible here, so seasonal dependence of photosynthesis could not be fully investigated. Zelitch (1982) already pointed out that estimates of crop yield from net photosynthetic CO2 assimilation can be hazardous under these circumstances. Little evidence was found to sustain the hypothesis that photosynthetic capacity declines with increasing COs growth concentration (cf. Delucia et al. 1985): expressed per unit leaf area there was little or no decrease in Pmax on COs response curves when the stands were grown in high instead of low COs. Similar results were found in a switching experiment on soybean: photosynthetic response to short-term exposure to different COs levels adequately estimated the long-term canopy response to COs level (Jones, Allen et al. 1985). An important determinant of CO2 exchange rate is stomatal behaviour. It is not clear from our measurements whether the internal CO2 concentration changes and how it changes, though it is certain that stomatal closure occurs under high CO2 since transpiration rate per unit leaf area decreased (Table 2). This cannot be caused by a higher water vapour concentration in the cuvette, since the amount of water vapour released to the cuvette air was almost the same for both treatments, so the water vapour concentration should have been fairly similar and without gradients due to the effective stirring of cuvette air. The advantage of a lower transpiration on the leaf level is offset by a larger leaf area for the whole canopy so slightly more water is used in this highly productive system. This is not a disadvantage in

174 conditions of drought stress when only a limited amount of water can be transpired, because the high CO2 adapted vegetation - - which has a better water-use e f f i c i e n c y - will fix more carbon. Though a 35% increase in W.U.E. may seem considerable, it is far below the mean values that Carlson and Bazzaz (1980) calculated for various crop and weed species under a doubled CO2 concentration (52% and 76 to 128%, respectively). Future changes in W.U.E., yield and productivity are particularly interesting knowing that the yields of many crops all over the world are limited to only 20 to 35% of their potential yields in optimal conditions: water and nutrient limitations, maladjustment to climate and radiation regime, pests and weeds are primarily responsible for this (Gifford et al. 1984). Focusing on the conditions that allow improvement on these fields in a higher CO2 world and prediction of plant and vegetation responses to future CO2 levels can therefore be helpful with long-term planning in agriculture.

Acknowledgements This study was supported by the "Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" (Brussels, Belgium). We are grateful to Dr L.H. Allen, Jr. for his comments and criticism of the manuscript.

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175 Jones P, Allen LH Jr, Jones JW and Valle R (1985) Photosynthesis and transpiration responses of soybean canopies to short- and long-term CO2 treatments. Agr. J. 77:119-126 Jones P, Jones JW and Allen LH Jr (1985) Seasonal carbon and water balances of soybeans grown under stress treatments in sunlit chambers. Transactions of the ASAE, Vol 28, No 6: 2021-2028. Published by the American Society of Agricultural Engineers. St. Joseph, Michigan Kimball BA (1982) Carbon dioxide and agricultural yield: An assemblage and analysis of 430 prior observations. Agron. J. 75:779-788 Kimball BA (1985) Adaptation of vegetation and management practises to a higher carbon dioxide world. In Strain BR and Cure JD, eds. Direct effects of increasing carbon dioxide on vegetation (DOE/ER-239), U.S. Department of Energy, Washington D.C. Springfield, Virginia: NTIS Koch W, Klein E and Walz H (1968) Elektronische gaswechselmessanlage fiir pflanzen in laboratorium und freiland (In German). Siemenz Z. 42:392-404 Lemon ER (1983) CO2 and plants: the response of plants to rising levels of atmospheric carbon dioxide, AAS Selected Symposium 84. Westview, Boulder, Colorado Luther FM (1985) Projecting the climatic effects of increasing carbon dioxide: volume summary. In McCracken MC and Luther FM, eds. The potential climatic effects of increasing carbon dioxide (DOE/ER-239), U.S. Department of Energy, Washington D.C., pp. 215246. Springfield, Virginia: NTIS Overdieck D, Bossemeyer D and Lieth H (1984) Long-term effects of an increased CO2 concentration level on terrestrial plants in model-ecosystems. I. Phytomass production and competition of Trifolium repens L. and Loliurn perenne L. Progr. Biometeorol. 3:344-352 Reuveni J and Gale J (1985) The effect of high levels of carbon dioxide on dark respiration and growth of plants. Plant, Cell Environ. 8:623-628 Zelitch I (1982) The close relationship between photosynthesis and crop yield. BioScience 32: 796-802