At their current rate of increase, CO₂ levels are expected to double to 750 ppm by the end of this century. Elevated CO₂ significantly influences both soil nutrient availability and soil microbes that are associated plants (Janus et al. 2005). Numerous studies that have investigated the effects of elevated CO₂ on plants found that elevated CO₂ increases the growth rate of plants (Gifford 1994, Naidu et al. 1998) while significantly affecting physiological function (Melillo et al. 1990). In addition, elevated atmospheric CO₂ causes an increase in the C:N ratios of plants by reducing the N concentration (Berntson and Bazzaz 1996). Such results were due to changes in the level of Rubisco or respiratory proteins, or to the dilution of nitrogen resulting from the accumulation of non-structural carbohydrates. Sims et al. (1998) recently reported that nitrogen levels are higher in plants under elevated CO₂. However, Saxe et al. (1998) found that elevated CO₂ had no effect on the nitrogen or carbon content of oak and beech seedlings. These results suggest the level of nitrogen in plant tissue exposed to elevated CO₂ is species-dependent.

Enzymatic changes under elevated CO₂ can alter the microbial demand for N and therefore the flow of N between soil microorganisms and plant roots (Zak et al. 2000). This in turn alters the overall chemical composition of plants as well as the types of organic substrates available for microbial metabolism (Finzi et al. 2006). Elevated CO₂ can also increase microbial activity in the soil (Hungate et al. 1996) mainly by providing extra C sources for rhizospheric microorganisms (Zak et al. 2000). Soil microbial activity can be measured in terms of CO₂ production or based on the activities of major metabolic enzymes (Klose et al. 2003).

The responses of plants and microorganisms to elevated CO₂ vary according to the plant species (Cotrufo et al. 1998). While much information is available on the effect of elevated CO₂ on trees, little is known about how pine seedlings are affected. Since pine trees are a dominant tree species in eastern Asia, we believe that such information would be highly valuable.

The purpose of this study was to investigate the growth of pine seedling as well as soil microbial activity in response to elevated CO₂ in a growth chamber. We report on the impact of increased CO₂ on the root and shoot growth, biomass (dry weight) and C:N ratio of Pinus densiflora, emphasizing the interdependency of soil chemistry and microbiology, soil moisture and plant growth.

Natural soil was sampled from the pine forest on the Ewha Woman’s University campus in Seoul, Korea. Soil samples (1 kg/pot, diameter 10 cm) were used for the planting of three-year-old pine seedlings (P. densiflora), which were obtained from the Korean forest service. The plants were incubated for 12 months in growth chambers (Dasol scientific Co., Hwaseong, Korea) under 380 or 760 ppmv CO₂. The growth chamber was controlled at 25°C and 60% humidity, then subjected to a 16 h light/8 h dark cycle. The CO₂ concentrations were chosen based on Intergovernmental Panel on Climate Change (IPCC) reports (2007) that found the concentration of CO₂ in the atmosphere during the 21^st century will rise to 700 ppm, which is double the current concentration. Further, 30 mL of water and 20 mL of 1/2 nutrient solution (KNO₃ 606.6 6 mg/L, Ca(NO₃) ₂·4H₂O 944.60 mg/L, NH₄H₂PO₄ 115.02 mg/L, MgSO₄ 492.94 mg/L, FeCl₂·7H₂O 492.94 mg/L, MnCl₂·4H₂O 1.78 mg/L, H3BO3 2.84 mg/L, ZnSO4·7H2O 0.23 mg/L, CuSO₄·5H₂O 0.075 mg/L) were added to the soil one time per week. Every 4 months, all the aboveground and underground parts were harvested. The shoot was separated and the roots were collected from the soil by washing. All plant parts were oven-dried before measuring dry matter at 80°C for 12 hours. Then, the shoot, root dry weight, and C, N content were measured. After incubation for 12 months, dissolved organic carbon (DOC) and enzyme activities were measured after bulk and rhizosphere soil were divided. The bulk soil was that remaining after the roots were picked from the pot. The rhizosphere soil was that which still adhered to the roots after gentle shaking.

The pine seedlings were planted in a growth chamber at 25°C and 60% humidity, and were subjected to a 16 h light/8 h dark cycle. Shoot, root length, and biomass (dry-weight) were measured every four months. All tests were performed in triplicate. Percent dry weight of N and C content were estimated from leaf and root powder using a Flash EA 1112 Analyzer (Thermo Electron Corporation, Waltham, MA, USA).

Soil pH was determined by adding soil to water at a ratio of 1:5 (w:v). Soil moisture was determined gravimetrically by drying at 105°C for 24 hours, and organic matter content was determined by loss on ignition at 700°C (MAS 7000 oven; CEM, Mattews, NC, USA). Soil cation-exchange capacity was determined according to EPA 9081 methods (US Environmental Protection Agency 1986). Soil nitrate (NO₃-) content was determined by extracting soil with deionized water and then measuring NO₃- content in the liquid phase using an NO₃- electrode (Gelderman and Beegle 1998).

To measure the concentration of DOC, soil was added to water at a ratio of 1:10 (w/v) after which the DOC content was measured using a TOC 5000 (Shimadzu Co., Kyoto, Japan) meter. Specific UV absorbance (SUVA) reveals the nature or quality of DOC in a given sample and is used as a surrogate measurement of DOC aromaticity (Chin et al. 1994). SUVA was measured at 254 nm (SUVA₂₅₄) due to the strong absorption of natural organic matter at this wavelength. This value correlates strongly with the aromatic carbon content of organic matter (Chin et al. 1994). Phenolic compound content was assayed using Folin-Ciocalteau phenol reagent (Box 1983). One milliliter of sample was added to 1.5 mL of Na₂CO₃ solution (50 g/L). Then, 0.5 mL of Folin-Ciocalteau solution (diluted 1/4 with deionized water; 0.5 N) was added, followed by incubation of the mixture for 2 hours at room temperature. A standard curve was prepared by applying the same chemicals to a series of 0 to 2 mg/L phenol solutions.The color change of reactants was measured spectrophotometricallyat 750 nm. Once out of range of the standard curve, the samples were diluted with distilled water and the procedure was repeated. Phenol oxidase activity was determined using 10 mM L-dihydroxyphenylalaninesolution as a substrate, according to Pind et al. (1994).

The activities of four extracellular enzymes (β-glucosid ase, N-acetylglucosaminidase, phosphatase, and arylsulfatase) were measured by the MUF-substrate method (Freeman et al. 1996). The concentrations of the MUF-β-glucoside, MUF-N-acetylglucosamine, and MUF-arylsulfate substrate solutions were 400 μM (Sigma, St. Louis, MO, USA) while the concentration of the MUF-phosphate substrate solution was 800 μM (Sigma). Enzyme activities in a slurry containing soil and substrate solution (1:5 w/v) were measured using a fluorimeter. Dehydrogenase activity was measured by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) assay (Tabatabai 1982). Mixtures of soil (3 g fresh soil) and substrate solution were incubated for 24 hours at 37℃ after which the reaction products were detected using a spectrophotometer (DR/3000 Spectrophotometer; HACH, Mount Holly, NJ, USA) at 485 nm.

Data were analyzed by one-way ANOVA using SPSS ver. 9.0 (SPSS Inc., Chicago, IL, USA). Tukey’s test after one-way ANOVA was used to determine significance differences in soil parameters and soil enzyme activities in each sample. Growth, biomass, DOC, SUVA, phenolic compounds and phenol oxidase activity were tested for significance by a t-test between ambient and elevated CO₂ (P < 0.05).

[Table 1.] Carbon concentration, Nitrogen concentration, and the C:N ratio of Pinus densiflora grown under ambient or elevated atmospheric CO2

Carbon concentration, Nitrogen concentration, and the C:N ratio of Pinus densiflora grown under ambient or elevated atmospheric CO2

Although root and shoot elongation were not significantly affected by elevated CO₂, the root dry weight of pine under elevated CO₂ was increased (P < 0.05) (Fig. 1). C:N ratios of pine needles were increased under elevated CO₂ (Table 1) as well. However, no difference in C:N ratios were observed in roots.

The physicochemical parameters of the soils are listed in Table 2. Under elevated CO₂, both NO₃^- and water content were decreased in soil. The soil pH was found to be mildly acidic (6.3-7.1) while the organic matter content ranged from 2.3 g/kg to 3.5 g/kg.

Elevated CO₂ had no effect on DOC concentration in soil (Fig. 2a). However, the composition of DOC did appear to be influenced. Under elevated CO₂, the proportion of aromatic material in DOC (estimated by SUVA₂₅₄) was increased in bulk soil but decreased in rhizosphere soil (Fig. 2b). In contrast, phenolic compound content was decreased in both rhizosphere and bulk soil (Fig. 2c). Interestingly, phenol oxidase activity was decreased only in rhizosphere soil (Fig. 2d).

[Fig. 1.] Root, shoot length and biomass of pine seedling under ambient and elevated CO2. (a) Root length, (b) shoot length, and (c) biomass. C, CO2 380 ppmv; E, CO2 760 ppmv; 4M, after 4 months; 8M, after 8 months; 12M, after 12 months.

[Fig. 2.] (a) Dissolved organic carbon concentration, (b) specific UV absorbance (SUVA254), (c) phenolic compounds, and (d) phenol oxidase activity after 12 months. C, 380 ppmv, bulk soil; CR, 380 ppmv, rhizosphere soil; E, 760 ppmv, bulk soil; ER, 760 ppmv, rhizosphere soil.

Elevated CO₂ increased dehydrogenase activity (a measure of microbial intracellular activity) in bulk soil only (Table 3). β-glucosidase, N-acetylglucosaminidase, and phosphatase activities under elevated CO₂ were increased in soils, especially rhizosphere soil (Table 3).

Elevated CO₂ affected the C:N ratio of pine seedling as well as soil enzyme activity and N content. However, plant growth and soil DOC content were unaffected. Interestingly, elevated CO₂ increased the C:N ratio of plants as well as microbial activities. It has been suggested that increased CO₂ decreases the level of nitrate in soil. Elevated CO₂ could likely decrease N into the soil, both of which are necessary for plant and microbial decomposition function. These decreases in soil nitrate levels are extremely important because net primary productivity is nitrogen dependent (Janus et al. 2005). Thus, as the amount of available N for plant uptake is decreased, the C:N ratios in pine needles are increased. Gifford et al. (2000) also similarly reported that the C:N ratio of pine seedling is increased 15% due to a 21% decrease in the N content of needles under elevated CO₂.

[Table 2.] Soil physical and chemical parameters

Soil physical and chemical parameters

[Table 3.] Enzyme activities in soil under ambient and elevated CO2

Enzyme activities in soil under ambient and elevated CO2

It was observed that the root dry weight of pine seedling was increased under elevated CO₂, although root and shoot elongation were unaffected (Fig. 1). Many studies show that the overall biomass is more affected than either root or shoot growth under elevated CO₂. Pushnik et al. (1999) reported that the root biomass of Pinus ponderosa is significantly increased at 500 and 700 ppmv CO₂. Further, King et al (2001) reported that the root biomass of pine is increased by 96% under elevated CO₂. It has been hypothesized that elevated CO₂ will increase biomass partitioning to fine roots (Curtis et al. 1994), thereby increasing the total root surface area. The increase in root biomass would be expected to favor the growth of microbial fungi in soil due to altered soil chemical composition.

The microbial activities of β-glucosidase, N-acetylglucosaminidase, and phosphatase were increased under elevated CO₂ (Table 2), particularly in rhizosphere soil (P < 0.05). These increases in enzyme activities may be related to an increase in root exudates and rhizosphere microbe activity (Fig. 1). Several rhizospheric bacterial species are known to produce compounds such as phytohormones, antifungal molecules or siderophores that assist the plant through atmospheric nitrogen fixation (Rillig et al. 1997). Increases in the activities of N-acetylglucosaminidase and phosphatase often occur in response to nitrogen (Gifford et al. 2000). It has been shown that under elevated CO₂, β-glucosidase releases more C from organic matter into soil (Larson et al. 2002, Henry et al. 2005) and that both C and N in general, which are important for microbial metabolism, are released.

Elevated CO₂ did not influence the concentration of dissolved organic matter in soil, but the concentrations of phenolic compounds in soil and aromatic compounds in rhizosphere soil were decreased (Fig. 2). Phenolic compounds are resistant to the nitrification of microbial decomposition activity. Therefore, elevated CO₂ could promote their use as carbon sources by microbes (Rouhier and Read 1998). This notion is supported by our finding that phenol oxidase activity in soil was reduced under elevated CO₂.

In this study, changes in the level of nitrogen in soil could explain the effects of elevated CO₂ on microbial activities and pine seedling growth. However, as our work is limited to the growth chamber, further investigation is needed study.

The results of this study demonstrated that elevated CO₂ had significant effects on the growth of pine seedling as well as soil microbial activity. These findings suggest that rising levels of atmospheric CO₂ cause a reduction in pine seedling biomass as well as distinct changes in soil chemistry and microbiology.