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- Materials and Methods
Nitrous oxide (N2O) is a strong greenhouse gas contributing approx. 6% to the anticipated global warming (IPCC, 2001). Microbial production by denitrification and nitrification processes in soils is considered to be the main source of N2O into the atmosphere (Kroeze et al., 1999). Currently, the emission estimates from natural ecosystems are based solely on soil emission measurements using soil enclosures. In the enclosure technique, the soil is covered with a shallow chamber, and the emission from the soil is calculated from the increase in N2O concentration inside the chamber (e.g. Butterbach-Bahl et al., 1997). Such enclosures exclude tall plants and trees and may therefore underestimate the whole soil–plant N2O emissions.
Agricultural and wetland plants have been found to contribute significantly to the total N2O emissions from soil–plant systems (Chang et al., 1998; Rusch & Rennenberg, 1998; Yan et al., 2000; Smart & Bloom, 2001; Chen et al., 2002; Müller, 2003). Rusch & Rennenberg (1998) found that black alder (Alnus glutinosa), a wetland tree species, emitted N2O through the bark of the tree when the gas concentration in the soil solution was above the ambient concentration. They suggested that the gases diffuse through the aerenchyma of the bark. Yan et al. (2000) found that 87% of the N2O emissions from a rice field occurred through rice plants when the soil was flooded and 18% when the soil was unsaturated, whereas Chen et al. (2002) and Zou et al. (2005) found that N2O emissions from maize, soybean and wheat plants accounted for up to 11, 16 and 62% of the total soil–plant N2O emissions. All of these field studies, however, failed to identify the mechanisms responsible for plant-mediated N2O emissions.
In a laboratory study, Chang et al. (1998) found that barley (Hordeum vulgare) and canola (Brassica napus) plants can serve as a conduit for dissolved N2O from the root zone to the atmosphere. They suggested that this transpiration-mediated N2O emission may be a common phenomenon with several upland plants. Smart & Bloom (2002) and Hakata et al. (2003) found, also in the laboratory, that N2O can be formed enzymatically inside wheat leaves during nitrite (NO2−) photoassimilation. According to Smart & Bloom (2002), this enzymatic production of N2O in the leaves could account for 5–6% of the total N2O emissions from agricultural soil–plant systems.
In summary, the studies reporting plant-mediated N2O emissions have all been conducted with agricultural or wetland plants, whereas studies with upland forest trees are lacking. We hypothesize that tree leaves can be a significant source of N2O into the atmosphere and that N2O produced in the soil can be transported to the atmosphere via the transpiration stream.
To test our hypothesis, we conducted two laboratory experiments with potted beech (Fagus sylvatica) seedlings to examine whether beech leaves emit N2O under controlled conditions and whether N2O can be transported from the soil solution to the atmosphere via transpiration stream.
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The increased N2O and 15N2O concentrations inside the shoot chamber during the enclosure period demonstrated that soil fertilization with 15NH415NO3 and glucose-induced N2O emissions from the beech foliage. The addition of mineral nitrogen and organic carbon substrates activates soil denitrifying bacteria to produce N2O in the soil (Azam et al., 2002). Hence, we assumed that denitrifying bacteria in the soil produced N2O and 15N2O using the glucose and 15NH415NO3 as substrates. Part of this N2O and 15N2O was then taken up by beech roots from the soil water and transported to the leaves in the transpiration stream. The 15NH415NO3 added to the soil was approximately 5% enriched with 15N, whereas the emitted N2O into the shoot chamber was approx. 6% enriched with 15N for S2 and 0.4% enriched for S1. The results from S2 indicate that the emitted N2O was derived solely from the added inorganic nitrogen, whereas the results from S1 indicate that an additional unlabelled inorganic N pool already present in the pot–root environment was available for N2O production.
In the fertilization experiment, the shoot emissions are a sum of all processes that could lead to N2O emissions from the leaves and shoot, such as transpiration, N2O formation in the leaves and N2O diffusion through the bark (Rusch & Rennenberg, 1998; Smart & Bloom, 2001; Hakata et al., 2003). We considered gaseous N2O diffusion through the bark of the beech seedlings as a minor factor contributing to the total N2O emissions, because beech trees lack aerenchyma structure in the stem to facilitate gas diffusion from the stem to the atmosphere. However, part of the N2O emitted from the leaves may have been produced inside the leaves during NO2− assimilation, as reported by Smart & Bloom (2001) and Hakata et al. (2003).
To test whether transpiration could account for the N2O emissions from beech leaves, we conducted another experiment. In this experiment, the beech roots were exposed to elevated concentration of N2O in the root solution. The solution in the root compartment was made with deionized water, which is depleted with NO3−. This way, we were able to eliminate the possible NO3− assimilation and consequent production of N2O in the leaves. As a water-soluble gas, N2O can theoretically be taken up by roots of the trees and transported to the leaves via transpiration stream. We discovered that N2O was taken up by beech roots and transported in the transpiration stream to the leaves and further to the atmosphere.
High N2O concentrations in the leaves of the laboratory seedlings confirmed that the beech leaves were sources of N2O to the atmosphere. Our finding that the leaf N2O concentrations were much higher in the laboratory seedlings than in the forest trees indicates that the natural N2O emissions are much smaller than those measured in the laboratory. However, the leaf N2O concentrations in the beech forest were higher than the ambient atmospheric N2O concentration, indicating that beech leaves may also be sources of N2O in the forest.
Using the leaf area index (LAI) value of 5 measured at the Lille Bøgeskov beech forest (P.T. Sørensen, Risø National Laboratory, pers. comm.), the observed leaf-based N2O emissions from the fertilization and the root chamber experiments correspond to area-based emissions of 5.2 and 10.0 µg N2O-N m−2 h−1, respectively. These rough estimates of the canopy emissions in a forest ecosystem are of the same order of magnitude as soil-derived N2O emissions from north and central European forest ecosystems (Ambus et al., 2001; Beier et al., 2001; Butterbach-Bahl et al., 2002). In the root chamber experiment, the beech seedlings were exposed to a root solution containing 2300–10 400 µg of N2O l−1 of solution. These N2O concentrations were similar to what Chan et al. (1998) used in their studies with canola and barley and also similar to the concentrations measured from agricultural soils (Heincke & Kaupenjohann, 1999). However, the concentrations were 1–3 orders of magnitude higher than those measured from natural forest ecosystems (Heincke & Kaupenjohann, 1999; Papen & Butterbach-Bahl, 1999). Hence, our results from the root chamber experiment more likely represent a potential for transpiration-mediated N2O emissions in forest ecosystems. The emission estimate from the 15N fertilization experiment represents a situation of N-affected forest ecosystem and, hence, may apply to forests exposed to high atmospheric N-deposition or fertilization management, such as poplar plantations.
Current emission estimates from forest ecosystems are based on soil enclosure measurements and the effect of forest canopies on N2O emissions has been ignored. Our results indicate that the reported N2O emissions from forest ecosystems are an underestimate of the total N2O emissions from forest ecosystems. Field measurements are needed to quantify the potential N2O emissions from forest canopies. Measurements of N2O concentration in the soil solution and inside tree leaves may help to estimate the magnitude of canopy exchange of N2O. Nevertheless, our experiments demonstrate the existence of a previously unknown pathway of N2O to the atmosphere in forest ecosystems, and bring about a need to investigate the magnitude of this phenomenon in larger scales.