Interactive effects of elevated CO2 and mineral nutrition on growth and CO2 exchange of sweet chestnut seedlings (Castanea

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1 Tree Physiology 14, Heron Publishing-Victoria, Canada Interactive effects of elevated CO2 and mineral nutrition on growth and CO2 exchange of sweet chestnut seedlings (Castanea sativa) A. EL KOHEN and M. MOUSSEAU CNRS URA 1492, Laboratoire d Ecologie ve gktale, Batiment 362, Universite Paris-Sud, Orsay Cedex, France Received October 18, 1993 Summary The effects of elevated atmospheric CO2 (700 pmol mol- ) and fertilization were investigated on 2-year-old sweet chestnut (Castanea sativa Mill.) seedlings grown outdoors in pots in constantly ventilated open-sided chambers. Plants were divided into four groups: fertilized controls (+F/-CO& unfertilized controls (-F/-CO& fertilized + COz-treated plants (+F/+CO2) and unfertilized + COztreated plants (-F/+CO$. Dry matter accumulation and allocation were measured after one growing season and CO2 exchange of whole shoots was measured throughout the growing season. Shoot growth and total leaf area of unfertilized plants were not affected by elevated CO2, whereas both parameters were enhanced by elevated CO2 in fertilized plants. Elevated CO2 increased total biomass by about 20% in both fertilized and unfertilized plants; however, biomass partitioning differed. In unfertilized plants, elevated CO2 caused an increase in root growth, whereas in fertilized plants, it stimulated aboveground growth. At the whole-shoot and leaf levels, photosynthetic activity of both fertilized and unfertilized plants increased in response to elevated C02, but the seasonal pattern of this enhancement varied with nutrient treatment. In unfertilized plants, a downward acclimation of photosynthesis was observed early in the season (June), and was related to reductions in nitrogen and chlorophyll content and to starch accumulation. The decrease in the slope of the A/Ci curve suggested a decrease in Rubisco activity. In both fertilized and unfertilized plants, shoot respiration decreased during the night in response to elevated CO2 until mid-july. The decrease was not related to changes in sugar concentration. Keywords: biomass partitioning, carbon budget, deciduous trees, dry weight, gas exchange, nitrogen partitioning, photosynthesis, respiration. Introduction Predicting forest growth response to elevated CO2 is difficult because of the interactions with other environmental factors, especially nutrients and temperature. One approach to developing a reliable method of prediction is to build mechanistic carbon budget models in which CO2 and nutrient availability are the experimental variables and seedlings are the experimental material (Saugier et al. 1993). The limitations of this approach include the need to scale up from seedlings to adult trees and the need to perform the experiments over a long period because most plants undergo a gradual inhibition of photosynthesis during acclimation to elevated CO*. Stitt (1991) argued that the inhibition was related to differences in the source-sink status of the plant. Source-sink relationships are tightly controlled by the nutrient status of the plant

2 680 EL KOHEN AND MOUSSEAU (Ingestadt and Agren 1991). For example, in sugar maple, the soil nitrogen availability influences the seasonal carbon allocation pattern (Burke et al. 1992). Because elevated CO;? plays a major role in plant nutrition (Conroy 1992), most studies of the effects of elevated CO2 on plant processes have been done under optimal nutritional conditions (Eamus and Jarvis 1989). Although several studies have investigated the interaction between nutrients and elevated CO2 (Wong 1979, Hocking and Meyer 1985, Wong 1990, Sinclair 1992), few studies have documented these interactions in trees (Brown and Higginbotham 1986, Shipley et al. 1991, Silvola and Ahlholm 1992). It has been shown that growth is enhanced by elevated CO2 even when the nutrient supply is restricted (Norby et al. 1986a), and that CO;! x nitrogen effects are species dependent (Wong et al. 1992). Elevated CO;? might also affect respiration, thus changing the carbon loss component of the carbon budget (Amthor 1991, Bunce 1992) In this paper, we investigated the combined effects of elevated CO2 and nutrient availability on growth and CO2 exchange to obtain information on variations in the carbon budget of young tree seedlings in response to climate change. Sweet chestnut, Castanea sativa Mill., was chosen because it is a fast-growing deciduous tree with a relatively high photosynthetic activity (Ceulemans and Saugier 1992). In addition, changes in dry weight and nitrogen partitioning in sweet chestnut in response to elevated CO2 have already been shown to be dependent largely on nutrient availability (El Kohen et al. 1992). Material and methods At the beginning of March, 2-year-old sweet chestnut seedlings were divided into four treatment groups (24 seedlings per treatment): +F and-f refer to plants growing with or without addition of fertilizers, respectively, +COz and -CO2 refer to plants growing in elevated CO2 or ambient air, respectively. Each seedling was potted in 12 1 of forest soil (El Kohen et al. 1992) consisting of the upper 15 cm organic layer of a chestnut forest soil, yielding about 0.65 g N per year per pot. Each plant in the +F treatments was fertilized monthly with 40 fertilizer granules that were spread over the pot surface to provide 0.82 g N, 0.78 g P and 0.4 g K. These quantities were three times as high as the final mineral content of a tree at the end of one year of growth (El Kohen et al. 1992). All plants were grown outdoors in constantly ventilated open-sided chambers (2 m long, 1 m wide, 1.25 m high) in natural light and watered daily to compensate for evapotranspiration. Chambers providing the elevated CO2 treatments were enriched to 700 pmol mol- CO2 with pure industrial CO2 (a detailed description of the chambers and CO2 enrichment procedure is given in Mousseau and Enoch 1989). Plants were harvested at the end of the growing season to determine dry weight. Leaf area (S) was computed from nondestructive measurements of length (L) and width (I+) of all leaves, based on the relationship S = 0.65LW, previously established on a population of similar plants. During the growing season, light-saturated photosynthetic activity and night res-

3 CO2 AND MINERAL NUTRITION ON GROWTH AND GAS EXCHANGE 681 piration rate were measured outdoors twice a week on four different plants of each treatment. Measurements of the rate of decrease or increase in CO2 concentration inside an acrylic chamber placed over a whole shoot for a few minutes were made with a portable CO2 analyzer (LCA2, Analytical Development Company, Hoddesdon, Herts, UK) (El Kohen et al. 1993). Measurements were performed at the CO2 concentration in which the plants were grown. Dark respiration rate was measured at the end of the night period with the same experimental system. A dark cloth was placed over the acrylic chamber to insure total darkness during the measurement. When the climatic conditions did not allow the measurements to be made outdoors, measurements were performed under an artificial light source in the laboratory. For one plant per treatment, an A/Ci curve of an attached leaf in controlled conditions was performed every month. The trees were transported in their pots to the laboratory and the 5th leaf was enclosed in an assimilation cuvette. Measurements were performed at saturating light and 25 C in an open gas-exchange circuit. Leaf nitrogen concentration was determined by automatic element analysis on leaf samples taken from the 5th leaf of five different trees. Chlorophyll content was measured in acetone extracts (McKinney 1941) of four leaf discs (0.6 cm diameter) sampled randomly from four different plants. Starch and total soluble sugars were extracted in 80% ethanol from 2 g of fresh leaf tissue collected from five plants. The anthrone calorimetric method (Ashwell 1957) was used to determine total soluble sugar concentration. The starch concentration was determined by enzymatic hydrolysis (Thivend et al. 1965). The glucose molecules liberated by hydrolysis were measured with an industrial sugar analyzer (ISY 2700, Biochemistry Analyzer). Student t-tests were used for comparison of means. Results Biomass accumulation and partitioning The increase in total dry matter in response to elevated CO2 did not differ significantly between fertilized and unfertilized plants (20 and 25% for -F/+COz and +F/+COz plants, respectively). Fertilization altered the allocation pattern of the increase in dry matter induced by elevated CO*. In unfertilized plants, the total increase in dry weight was allocated to roots (Table l), suggesting that unfertilized plants were nutrient limited, whereas in unfertilized plants, it was allocated to aboveground parts (Table 1). In unfertilized plants, elevated CO2 did not have any significant effect on total leaf area, whereas in fertilized plants elevated CO2 caused a significant enhancement of foliage production by increasing both the number of leaves and the mean leaf area (El Kohen 1993). Net photosynthesis Seasonal changes in shoot net photosynthetic activity are presented on a shoot basis

4 682 EL KOHEN AND MOUSSEAU Table 1. Biomass (gow & SE) of different plant parts and total leaf area (dm2) of young sweet chesmut seedlings after one year of growth in ambient (-CO2) or elevated CO2 (+COz) on fertilized (+F) or unfertilized soil (-F). R/S = root/shoot ratio, n = 24, * = differing from the control (ambient CO2) at P < 0. 05, ** = no SE could be attributed to these numbers because the litter was collected as a whole. Initial DW Unfertilized (-F) Fertilized (+F) Ambient (-CO2) Elevated (+CO2) Ambient (-CO2) Elevated (+COz) Litter 11.5** ** 40.9** Shoot 4.1 f f k * 21.5* Root 8.6 k k f 10.2* Total 12.7 k xi f 15.2* * 40.7* Leaf area A 8* 37.3 f 8 R/s * * in Figure la and on a leaf area basis in Figure lb (whole-shoot photosynthetic activity divided by total leaf area) for the sweet chestnut plants in the four treatments. During April and May, young leaves in the elevated CO2 treatment had a higher net photosynthetic activity than young leaves in the ambient CO2 control treatment. The low photosynthetic rates of control plants in early spring suggest that, functionally, leaves were still not fully developed. From July, the elevated-coz-induced increase in photosynthetic rate of unfertilized plants was not statistically significant (as shown by SE bars in Figure la), although photosynthesis of COZ-enriched plants remained slightly higher than that of control plants. A second increase in carbon fixation occurred at the end of the summer, and was correlated with an unusual regrowth of the terminal bud, probably caused by a warm period (which enhanced the CO2 effect) prevailing at that time. Unfertilized Fertilized 7 20 E f ~...~ ,~ P - 9 I I 1 APT May Jun JUI Aug Sep APT MOY JUll Jul Aug SSP Figure 1. Effects of elevated CO2 in two contrasting nutrient treatments on the light saturated net assimilation rate of young chestnut seedlings. The measurements were made in situ on whole shoots twice a week during the growing season. Each point represents the mean of at least four measurements (*SE). Solid symbols = COz-enriched trees; open symbols = ambient CO:! (control) trees. (a) Assimilation on a whole shoot basis; (b) assimilation on a leaf area basis.

5 CO2 AND MINERAL NUTRITION ON GROWTH AND GAS EXCHANGE 683 Because elevated CO2 had no effect on total leaf area of unfertilized plants, the unit leaf assimilation rate of COz-enriched plants was strongly enhanced during April and May (Figure lb). After May, the stimulation decreased and became nonsignificant. A downward acclimation of photosynthesis was observed after May, suggesting nutrient deficiency. The effect of elevated CO2 on fertilized plants differed from the effect on unfertilized plants. As a result of the combined effects of CO2 on leaf area and leaf photosynthetic activity, the total carbon fixation of fertilized plants in elevated CO2 remained higher than that of +F/-CO2 plants until the end of the season (Figure la). Total carbon fixation of fertilized plants decreased when leaf abscision began. On a leaf area basis, no limitation in photosynthetic rate was observed before mid-august (Figure lb). Evidence for a down regulation of photosynthesis in unfertilized plants compared to fertilized plants was indicated by the A/Ci curves (Table 2). In the ambient CO2 treatments, fertilization caused an increase in the COzsaturated photosynthetic rates. In May, the rates doubled in response to CO2 enrichment in both nutrition treatments. In unfertilized plants, there was little effect of CO2 enrichment on the COz-saturated photosynthetic rate in July, but a slight decrease was observed in August (Table 2). In unfertilized plants, the slope of the A/Ci curve increased in response to elevated CO2 only in May and then decreased from July to the end of the season. Because the slopes of the A/Ci curves are thought to represent plants carboxylation efficiency, it may be inferred that, in unfertilized plants, there was a decrease in carboxylation efficiency in response to elevated CO*. In contrast, in the fertilized plants, the slope values increased in response to elevated CO* until the end of the season. Starch and sugar concentrations The down regulation of photosynthesis could be due to a negative feedback resulting from the accumulation of starch in the chloroplasts (Yelle et al. 1989). Figure 2 shows the seasonal variation in starch and soluble sugar concentrations of leaves in the Table 2. Characteristics of the A/Ci curves of the 5th leaf of 2-year-old sweet chestnut trees grown in ambient (-CO2) or elevated CO2 (+CO2) on fertilized (+F) or unfertilized soil (-F). Each curve was made from measurements on the same leaf each month. Month -F/-CO2 -F/+CO2 -F/+CO2 p- tf/-co2 +F/tCO2 -F/-CO2 +F/+CO2 +FJ-CO2 A at saturating C, (~01 mm2 SF ) May July August Slope ofaici curve (mmol mm2 s-l) May July August

6 684 EL KOHEN AND MOUSSEAU Unfertilized Fertilized May Jun JUI Aug S.=P act MOY Jun Jul Aug SeP act Figure 2. Seasonal changes in starch and total soluble sugar concentrations of young chestnut leaves grown in elevated CO2 (closed symbols) or ambient CO2 (open symbols) in two nutritional treatments. No error bars are indicated because each analyzed sample (2 g of fresh material) represents the mean value of several leaf disks punched on 5-8 different leaves and pooled together. elevated and ambient CO2 treatments. The fertilization treatment lowered leaf starch concentrations of plants in the ambient CO2 treatment (Figure 2). Elevated CO:! enhanced starch accumulation by a factor of four to live in both fertilized and unfertilized plants during the middle of the vegetative season. However, starch accumulation occurred earlier in the unfertilized plants than in the fertilized plants. The accumulation of soluble sugars was slightly greater in plants in the elevated CO2 treatment than in plants in the ambient CO2 treatment. Soluble sugars accumulated early in spring in unfertilized plants, whereas they accumulated only at the end of the season in fertilized plants. In response to elevated CO2, the fertilized plants seemed to transform all their starch to soluble sugars, so that their soluble sugar concentration increased greatly before leaf fall. Norby et al. (19866) also noted that abscised leaves from seedlings of Quercus alba in elevated CO2 contained higher concentrations of soluble sugars than abscised leaves from seedlings in ambient conditions. Nitrogen and chlorophyll concentrations Figure 3 shows that, although the fertilization treatment increased the overall nitrogen content of the leaves, N concentrations were reduced by elevated CO2 in both nutrient treatments during the entire vegetative season. In fertilized plants, elevated CO2 caused a 20% reduction in nitrogen compared to plants in ambient CO2, whereas in unfertilized plants, it caused a progressive reduction from 20% in the spring to 40% in the fall (Figure 3). In unfertilized plants, elevated CO2 caused the amount of chlorophyll per unit leaf area to decline (Table 3). The reduction was even greater when chlorophyll concentration was expressed on a dry weight basis (Table 3) because the leaf mass per unit

7 CO2 AND MINERAL NUTRITION ON GROWTH AND GAS EXCHANGE 685 Unfertilizad Fertilized * i 01 I I I May Jun Jul Aug Sap Ott Nov May Jun Jul Aug Ssp Ott Figure 3. Seasonal change in leaf nitrogen concentration of young chestnut leaves grown in elevated CO2 (closed symbols) or ambient CO2 (open symbols) in two nutritional treatments. For each date, values represent the nitrogen concentration of pooled leaf disc samples of at least five plants. Table 3. Leaf chlorophyll concentrations (* SE, n = 4) of sweet chestnut trees grown in four CO:! x nutrient treatments: +COz = COS-enriched plants, -CO2 = ambient air, -F = plants growing on sandy forest soil, and +F = plants growing on fertilized forest soil. Ratio refers to the ratio of -F/-CO2 to +F/+COz. Date of measurements was July 7 for -F/+CO2 and -F/-CO2 plants and July 17 for +F/-CO2 and +F/+COa plants. An asterisk (*) indicates a value statistically different (P < 0.05) from that of the control (ambient CO2 ). Treatment Chl a Chl b Chl a/h Chl total Chl a Chl b Chl a/b Chl total (mg mm2) (mg me2) (mg mm2) (mg gow- ) (mg gow- ) (mg DW- ) -F/-CO2 190 f 22 60f F/+CO ll* 56f k 19* Ratio F/-CO2 344?r f f FJ+CO2 375 k 6 135f f Ratio area increased in response to elevated CO2 (Mousseau and Enoch 1989). The elevated CO2 treatment had no effect on leaf chlorophyll concentrations of fertilized trees (Table 3). This result confirms findings already documented for other tree species (Wullschleger et al. 1992). The elevated CO2 treatment caused a substantial shift in the ratio of chlorophyll a to chlorophyll b (Chl a/b) in the unfertilized plants but had no effect on the ratio in fertilized plants (Table 3). Night respiration Figure 4 shows a typical set of curves of hourly changes in dark respiration (Rd) of leaves in the spring. The Rd rate of attached leaves at constant temperature (16 C) decreased during the night period. The decrease was always significantly less in leaves in the elevated CO2 treatment than leaves in the ambient CO2 treatment. At the beginning of the night, the difference due to CO2 enrichment was greater in fertilized plants than in unfertilized plants. Because Rd rates were steady during the final hours of the night in all treatments, we chose this time to compare Rd of plants

8 686 EL KOHEN AND MOUSSEAU 1.2,,,,,,,,,,,,,,, v-v-7-. Fertilized ;cl d-v., p : E v. o : cr.y r I -. v-v-v -14 i Unfertilized II Obo- : Q-O-0-Q.. E a o- o-q~o~ a :* a 0.5 I I Time of day (h) Figure 4. Typical curve of the hourly change in dark respiration rate (& = CO2 output) of attached chestnut leaves during the night. Measurements were made at growth CO2 concentration and constant temperature (16 C) in an open gas analysis system in the laboratory. Closed symbols = COa-enriched trees; open symbols = ambient CO2 (control) trees. from different treatments. For each CO:! and nutrition treatment, whole-shoot Rd was measured in situ at the end of the night during the entire leafy period. Figure 5 shows the results plotted on a whole shoot basis (Figure 5a) and on a leaf area basis (Figure 5b). Respiration rates were high in spring, which was especially warm in the study year. At the beginning of the growing season, COz-enriched plants had a significantly lower Rd rate than plants in the ambient CO2 treatment in both nutrition treatments (Figure 5). This difference decreased with time and was negligible by June in unfertilized plants, but persisted until mid-july in fertilized plants. Discussion Biomass accumulation increased in response to elevated CO2 regardless of nutrient treatment. The response (20 to 25% total biomass increase per tree) was of similar magnitude to that reported for other woody species (Eamus and Jarvis 1989). In both fertilized and unfertilized plants, the rate of carbon fixation approximately doubled in response to elevated CO2. The relative increase in photosynthesis due to elevated CO2 was greater than the corresponding increase in biomass, whereas fertilization had a greater effect on biomass (Table 1) than on photosynthetic rate. This response to elevated CO2 and fertilization is identical to that of willow (Silvola and Ahlholm 1992), which is probably because the photosynthetic measurements were performed at light saturation and, therefore, do not represent the real carbon

9 COz AND MINERAL NUTRITION ON GROWTH AND GAS EXCHANGE 687 Unfertilized Fertilized 1.0 i i n 0.6 z x 'ij & :, 6.lY 6 (4 6 ( ; a 0.0 r 20 May Jun JUI Aug May JlJn JUI Aug Figure 5. Seasonal variation in the end-of-night dark respiration rate (& = CO2 output) expressed per shoot (a) or per unit leaf area (b) of sweet chestnut seedlings under two CO2 x nutrition treatments. Closed symbols = C02-enriched trees; open symbols = ambient CO2 (control) trees. Each point represents the mean of at least four measurements (& SE). fixation in fluctuating environmental conditions. Down regulation of photosynthesis was observed in unfertilized plants in the elevated CO2 treatment. This reduced photosynthetic activity cannot be attributed to a restriction of root growth, i.e., a sink limitation, because the pot volume was about 12 1; however, increases in the length of fine roots, which can be enhanced severalfold by elevated CO;! (Berntson et al. 1993, Pettersson et al. 1993), could have been restricted by the pots. The decline in photosynthetic rate was correlated with a decrease in leaf nitrogen concentration (Figure 3), and corresponded to declines in both the slope and plateau of the A/Ci curves (Table 2), indicating possible limitation by Rubisco (Von Caemmerer and Farquhar 1981) and limitation as a result of a reduction in end product synthesis, respectively. The accumulation of starch in COz-enriched leaves has been found in many species (Koch et al. 1986, Downton et al. 1987, Wong 1990). Our finding that the extent of starch accumulation in the fertilized and unfertilized plants was similar could indicate that the seedlings were unable to utilize all of the carbohydrates that they had assimilated. The stimulation of net carbon assimilation was not paralleled by a stimulation of other metabolic pathways. For example, respiratory activity decreased in response to elevated CO*. In the unfertilized plants, starch accumulation may indicate a lack of new sinks to incorporate the carbohydrate surplus, i.e., the -F/+CO;! plants did not increase their leaf area in response to elevated CO2, whereas the +F/+COz plants increased their leaf area. The lowering of the respiration rate in COZ-enriched plants is an intriguing phenomenon. It has been found in many woody species (Bunce 1992, Idso and Kimball 1992, Wullschleger et al. 1992) and some herbaceous species (Amthor et al. (b) +

10 688 EL KOHEN AND MOUSSEAU 1992, Bunce and Caulfield 1991), and has been shown to be an instantaneous and reversible response (El Kohen et al. 1991, Amthor et al. 1992). Our results show that the percentage reduction in respiration rate followed a diurnal pattern, with maximum values at the beginning of the night and minimum values at the end. The plateau in the nighttime respiration rate in the elevated-co2 treatment is probably associated with the elevated-co*-induced decreases in sucrose export and metabolism at night (Wullschleger et al. 1992). The elevated-coz-induced decrease in Rd also showed a seasonal pattern; the reduction was important when the plant was growing vigorously in May and June, and much less so at the end of the season. The decrease occurred earlier in the season for unfertilized plants than for fertilized plants and did not seem to be related to the seasonal change in leaf sugar concentration (Figure 2). The mechanism underlying this decrease in Rd is not known. Wullschleger and Norby (1992) concluded that~both growth and maintenance components of respiration should be affected. In many cases, maintenance respiration is related to the nitrogen content of the tissue (Ryan 1991). However, in Castanea, the decrease in Rd in response to elevated CO2 was evident even when the results were expressed on a nitrogen basis (El Kohen et al. 1993). To build a whole-plant carbon balance response to elevated COZ, it will be necessary to consider mycorrhizal growth and maintenance. Rouhier et al. (1994) found increased rhizospheric activity in elevated CO2. This could be due to increased fine root activity and turnover (Khmer and Amone 1992), or stimulation of microbial activity due to an increase in carbohydrate root exudates (Norby et al. 1987), or both. A plant-soil system model will be needed to reconcile all of these observations. Acknowledgments This study was supported by an EC research program (EPOCH COTU 13). It represents part of the Ph.D. research of A. El Kohen. The authors acknowledge Bernard Saugier for stimulating discussions, Jean-Yves Pontailler for technical assistance, and Bernard Legay and Jacqueline Liebert for their help in the field References and in the laboratory. Amthor, J.S Respiration in a future, higher-co2 world. Plant Cell Environ. 14: Amthor, J.S., G.W. Koch and A.J. Bloom CO2 inhibits respiration in leaves of Rumex cripus L. Plant Physiol Ashwell, G Calorimetric analysis of sugars. In Methods in Enzymology. Vol. III. Eds. S.P Colowick and N.O. Kaplan. Academic Press, New York, pp 73. Berntson, G.M., K.D.M. McConnaughay and EA. Bazzaz Elevated CO2 alters the deployment of roots in small growth containers. Oecologia 94: Brown, K. and K.O. Higginbotham Effects of carbon dioxide enrichment and nitrogen supply on growth of boreal tree seedlings. Tree Physiol. 2: Bunce, J.A Stomata1 conductance, photosynthesis and respiration of temperate deciduous tree seedlings grown outdoors at elevated concentrations of carbon dioxide. Plant Cell Environ. 15: Bunce, J.A. and F. Caulfield Reduced respiratory carbon dioxide efflux during growth at elevated carbon dioxide in three herbaceous perennial species. Ann. Bot. 67: Burke, M.K., D.J. Raynal and M.J. Mitchell Soil nitrogen availability influences seasonal carbon allocation patterns in sugar maple (Acer saccharum). Can. J. For. Res. 22:

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