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Human activities have increased the input of oxidised and reduced nitrogen compounds into forest ecosystems (Wellburn, 1990; Pearson & Stewart, 1993). As a consequence, many forests in Central Europe are exposed to excessive amounts of N (Wellburn, 1990; Fangmeier et al., 1994; Rennenberg et al., 1998; ; Geßler & Rennenberg, 1998). Reduced atmospheric nitrogen compounds (mainly NH3 and NH4+) predominantly originate from intensive agriculture (Fangmeier et al., 1994; Bundesministerium für Umwelt et al. 1996) whereas the main source of oxidised nitrogen in the atmosphere (mainly NO, NO2, NO3−) is fossil fuel combustion (Wellburn, 1990; Mosier, 2001). The oxidised N compound preferentially produced in and emitted from combustion processes is NO that reacts with ozone or peroxyl radicals to form NO2 (Fowler et al. 1998).
In many areas of Central Europe the patchiness of landscape has resulted in close vicinity of densely populated industrialised areas, agricultural land and forests (Rennenberg et al., 1998). As a consequence, forest ecosystems are exposed to both oxidised and reduced N compounds. These compounds can be removed from the atmosphere by rain- and wash-out (Wellburn, 1990; Goulding et al., 1998) thereby contributing as NH4+ and NO3−/NO2−, respectively, to the N pool of the soil and to soil acidification (Wellburn, 1990; Asman et al., 1998). In addition, both, gaseous (NH3, NO2) and dissolved (NH4+, NO3−, NO2−) reactive N compounds are taken up by plants through the stomata (Van Hove et al., 1989; Thoene et al., 1991; Brumme et al., 1992; Pearson & Stewart, 1993; Burkhardt & Eiden, 1994; Geßler & Rennenberg, 1998) and, subsequently, are dissolved in the aqueous phase of the apoplast. From this compartment, NH4+, NO3− and/or NO2− are transported into mesophyll cells, assimilated to amino compounds and contribute to the N supply of the whole plant (Wellburn, 1990; Stulen et al., 1998). Trees supplied with additional N from the atmosphere may react with additional growth and/or N storage (Stulen et al., 1998), or may adapt N uptake by the roots to their actual demand depending on their nutritional state (Imsande & Touraine, 1994; Muller et al., 1996; Geßler et al., 1998b).
NH3 and NO2 exchange between the atmosphere and plants can occur as a bi-directional flux, the direction of which is determined by the NH3 and NO2 concentration gradient between leaf interior and ambient air (Husted & Schjoerring, 1996; Thoene et al., 1996; Hereid & Monson, 2001). Hence, depending on the plant internal and the ambient concentration, plants can act as both, sources and sinks for NH3 and NO2.
In various gas exchange studies with different species under controlled conditions it was observed that JNO2 and JNH3 were affected by ambient CNO2 and CNH3 as well as by various climatic and physiological factors (Van Hove et al., 1990; Schjoerring et al., 200; Thoene et al., 1991; Thoene et al., 1996; Weber & Rennenberg, 1996; Hanstein et al., 1999; Geßler et al., 2000; Hereid & Monson, 2001). A whole set of micrometeorological experiments has shown that ecosystem fluxes of NH3 and NO2 mainly depended on climatic parameters as temperature and air or leaf surface humidity and on processes related to soil or litter (e.g. Walton et al., 1997; Sutton et al., 2000; Nemitz et al., 2000).
With the present study we intended to assess – isolated from other ecosystem fluxes – the mechanisms of NH3 and NO2 exchange between twigs of adult spruce subjected to chronically high atmospheric N input and the ambient atmosphere using the dynamic chamber technique. We wanted to test the hypothesis, that mainly the concentration of trace gas and stomatal conductance determines NH3 and NO2 exchange of adult trees in the field as observed in previous studies with spruce (Kesselmeier et al., 1993; Thoene et al., 1996) and other species (e.g. NH3: Populus euamericana (Van Hove et al., 1989); Hordeum vulgare; Husted et al. (1996)); NO2: Triticum aestivum (Weber & Rennenberg, 1996); sunflower (Latus et al., 1990)) under controlled conditions. However, due to the chronically high N-input into the ecosystem examined (Rennenberg et al., 1998) and the colonisation of spruce needles with chemolithoautotrophic nitrifiers at this site, which are able to consume NH3, NH4+ and NO2−(Papen et al., 2002), compensation point, internal (mesophyll) resistances and/or cuticular adsorption may differ significantly from those determined under laboratory conditions and with other species in the field. Hence, it is important to understand the magnitude of canopy effects and the primary environmental and physiological controls over NH3 and NO2 exchange in the field, in order to accurately quantify and parameterise regional NO2 and NH3 exchange inventories (Sparks et al., 2001).
Therefore, we determined the effects of (a) CNH3 and CNO2, respectively, and (b) different climatic (T, PPFR, RH) and physiological (JH2O, JCO2 and gH2O) parameters on JNH3 and JNO2 between twigs of mature spruce trees (Picea abies) – the most common coniferous tree species in Central Europe – and the atmosphere. Twigs of spruce trees were exposed to different CNH3 or CNO2 representative for the field site. JNH3 (in the NH3 exposure experiments) and JNO2 (in the NO2 exposure experiments) were determined in the field under varying natural climatic conditions and, as a consequence, varying physiological parameters. Additional 15N-NH3 fumigation experiments were performed in order to trace the fate of NH3 taken up and validate the NH3 exchange measurements.
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- Materials and Methods
The present study was aimed at assessing the effects of different climatic (T, RH, PPFR) and physiological parameters (JH2O, gH2O, JCO2) as well as the effects of different NO2 and NH3 concentrations on the JNO2 and JNH3 between the needles of spruce trees and the atmosphere under field conditions – isolated from other ecosystem fluxes of NH3 and NO2. A whole set of studies on NH3 and NO2 exchange have been performed with gas exchange chambers in the laboratory (e.g. Van Hove et al., 1990; Husted et al., 1996; Thoene et al., 1996; Hanstein et al., 1999; Hereid & Monson, 2001) with the aim of parameterising the effect of climatic patterns on trace gas exchange and understanding the physiological mechanisms that drive NH3 and NO2 emission and deposition. In addition, intensive micrometeorological research is performed with the aim of characterising ecosystem NO2 and NH3 fluxes between forests and the atmosphere and underlying mechanisms (e.g. Walton et al., 1997; Andersen et al., 1999; Sutton et al., 2000, 2001; Pryor et al., 2001). However, the assessment of NH3 and NO2 fluxes to and from an isolated compartment – the twigs/needles of adult spruce – is of particular interest in the forest ecosystem examined, since spruce needles have been found to be colonised by chemolithoautotrophic nitrifying bacteria (Papen et al., 2002). Since these organisms are able to consume NH3 as well as NH4+ and NO2− (Bock et al., 1989), that is produced when NH3 and NO2, respectively, dissolve in water, compensation points, internal resistances and surface deposition may be significantly different from plants grown and fumigated under controlled conditions or field grown plants without colonialisation by nitrifiers. From previous studies (Papen et al., 2002) it is known that NH3 deposition fluxes to spruce needles are increased significantly as a consequence of metabolic activity of nitrifying bacteria in and/or on the needles. However, until now no information has been available on how different climatic and physiological parameters influence NH3 and NO2 fluxes to nitrifier-colonised needles.
In the fumigation experiments twigs of spruce trees were exposed to defined concentrations starting from the lower to the higher concentration consecutively. In order to avoid time-dependent chamber effects, and, as a consequence changes in physiological performance of the incubated twigs, near-natural conditions were maintained in the chambers by adapting air temperatures within the chambers to ambient air temperature and using chamber material with > 90% PAR transmission. Experiments with beech (Geßler et al., 2000), where a comparable experimental design was applied, showed that over a period of up to 3 d transpiration, photosynthesis and JNH3 and JNO2 remained constant at a given trace gas concentration.
Fig. 3 shows that although absolute values of g H2O – which are known to vary with water availability, water pressure deficit of the air, nutrition, and needle age ( Wieser et al., 2000 ; Phillips et al., 2001 ) – and J NH3 may have been different between the two times of measurement (May 95; September 95), the regression between J NH3 and g H2O at a given concentration (135 nmol mol −1 ) remained constant as previously also observed for beech ( Geßler et al., 2000 ). Independent of the time of measurement, partial correlation procedures ( Table 2 ) that were used to differentiate between the influence of stomatal function and climatic parameters, produced comparably high (NO 2 : R between –0.657 and –0.941; NH 3 : R between –0.622 and –0.901) and highly significant correlations between J NH3 or J NO2 and g H2O at concentrations above c . 5 nmol mol −1 where significant fluxes of NH 3 or NO 2 could be observed ( Figs 3 and 4 ). Hence, it is concluded that – for the analysis of the factors determining J NH2 and J NO2 – measurements performed at different times can be compared.
JNH3 and JNO2 are related linearly to trace gas concentration
In both, NH3 and NO2 fumigation experiments, trace gas fluxes (JNH3 and JNO2) depended linearly on CNH3 and CNO2 in the concentration range found at the field site studied. Apparently, the difference in NH3 and NO2 concentrations between the gaseous phase of the leaf interior and the atmosphere is the driving force for NH3 and NO2 exchange. In addition, it must be concluded that the capacity of spruce for NH3 and NO2 acquisition was not exceeded even at high NH3 and NO2 concentrations (Hanstein et al., 1999) different from observation of Sparks et al. (2001) for tropical tree species. The consecutive increase in NO2 or NH3 concentration applied in the present study may result in transient changes of apoplastic conditions and/or expression/activity of nitrate and ammonium transporters, hence, either increasing or decreasing fluxes of both trace gases. A decrease in NO2 or NH3 flux with increasing incubation time and, thus, increasing concentration can be ruled out, since no saturation was observed in the relation between JNH3 or JNO2 and trace gas concentration. However, the possibility that the observed linearity of this relation in a higher concentration range was due to a consecutive adaptation of the exposed twigs to higher NO2 or NH3 concentrations can not be ruled out. Nevertheless, in accordance with the present results linear dependencies between JNH3 and CNH3 have already been observed for Phaseolus vulgaris (Farquhar et al., 1980; Van Hove et al., 1987), Populus euamericana (Van Hove et al., 1989), Picea abies (Kesselmeier et al., 1993) and Hordeum vulgare (Husted et al., 1996) under controlled environmental conditions and for Fagus sylvatica (Geßler et al., 2000) and Picea abies (Peters & Bruckner-Schatt, 1995; Andersen et al., 1999) in the field. Due to the activity of chemolithoautotrophic nitrifiers, colonizing the needles of spruce at the field site ‘Höglwald’ the sink strength of the adult spruce trees for NH3 is increased by 24–37% compared with needles without bacterial activity (Papen et al., 2002). The bacterial activity can explain the c. 1.4-fold higher NH3 deposition fluxes at a given gH2O and CNH3 determined for spruce in the present study compared with beech (Geßler et al., 2000) from the same forest stand, but without colonisation of leaves with nitrifying bacteria (H. Papen, pers. comm.). However, JNH3 at a given CNH3 was about one order magnitude lower as observed for herbaceous species (Van Hove et al., 1990; Husted & Schjoerring, 1995; Husted et al., 1996, Husted & Schjoerring, 1996).
A linear correlation between JNO2 and CNO2 has also been determined in a number of studies (Johansson, 1987; Latus et al., 1990; Thoene et al., 1991; Rondón et al., 1993; Thoene et al., 1996, Hereid & Monson, 2001, Sparks et al., 2001). At a given NO2 concentration between 5 and 25 nmol mol−1 and under comparable environmental conditions, NO2 deposition flux for adult spruce was in the same range as adult tropical tree species (Sparks et al., 2001) but about 2-fold higher – at comparable gH2O– as for young spruce trees under controlled conditions (Thoene et al., 1996). The observed difference can be attributed to the activity of chemolithoautotrophic nitrifiers (nitrite oxidisers) consuming nitrite and living on the surface or within the apoplast of needles (Papen et al., 2002) of the adult trees. However, JNO2 observed with adult spruce was still about 5-fold smaller as the NO2 fluxes observed between the atmosphere and herbaceous plants (Weber & Rennenberg, 1996; Hereid & Monson, 2001).
Needles of spruce show compensation points for NH3 and NO2
Spruce trees exhibited compensation points for both trace gases, NH3 and NO2, indicating the release/production of NH3 and NO2 inside the needles. Hence, the adult spruce trees are not only sinks, but also sources for NH3 and NO2 depending on atmospheric concentrations. The NH3 compensation point determined amounted to 2.5 nmol mol−1 and, thus, was somewhat lower than for beech trees at the same field site (up to 3.5 nmol mol−1; Geßler et al., 2000). In literature NH3 compensation points for trees and canopies of forest ecosystem are described to range between 0.8 nmol mol−1 (montane forest with low N supply; Langford & Fehsenfeld, 1992) and 48 nmol mol−1 (N-polluted forests; Wyers & Erisman, 1998) indicating the relevance of N supply and N status for this parameter (Sutton et al., 1995; Geßler & Rennenberg, 1998; Herrman et al., 2001). The dependence of NH3 compensation point on N supply is supposed to be the reason for a higher NH3 compensation point in spruce from the N oversaturated field site ‘Höglwald’ (Rennenberg et al., 1998) compared with individuals of the same species growing at another site in SE-Germany (Meixner et al., 1997). The NO2 compensation point observed for adult spruce in the present study amounted to 1.7 nmol mol−1 and therefore was in a range comparable to that found for young trees of the same species under controlled conditions (1.6 nmol mol−1; Thoene et al., 1996), for adult beech trees at the same field site (1.8–1.9; Geßler et al., 2000) and for different tropical trees (Sparks et al., 2001), but somewhat higher than reported for corn (c. 0.9 nmol mol−1Hereid & Monson, 2001) and for Scots pine and Norway spruce at less N exposed sites in Sweden (c. 0.1–0.7 nmol mol−1; Rondón et al., 1993; Rondón & Granat, 1994). This difference between individuals of the same species growing at different sites can be due to the excess N supply of the spruce at the field site studied, as indicated by high leaf N contents (Geßler et al., 1998a), which are known to increase NO2 compensation point (Sparks et al., 2001). At the field studied, NO2 emission from spruce is supposed to play only a minor role, since atmospheric NO2 concentrations in the canopy at the field site ‘Höglwald’ are usually higher than the NO2 compensation point (Papen & Gasche, pers. comm.).
NH3 and NO2 fluxes correlate with stomatal conductance at a given NH3 and NO2 concentration
NH3 and NO2 fluxes were not only determined by CNH3 and CNO2 and, consequently, by the concentration gradients between leaf interior and exterior, but were also under control of stomatal conductance (Figs 3 and 4). These findings are consistent with observations made in a whole set of previous studies (NH3, e.g. Hutchinson et al., 1972; Rogers & Aneja, 1980; Harper et al., 1989; Van Hove et al., 1990; Geßler et al., 2000; NO2: Weber, 1993, Neubert et al., 1993; Weber & Rennenberg, 1996; Hereid & Monson, 2001; Sparks et al., 2001). In contrast to the observations by Sparks et al. (2001) for different tree species that stomatal conductance only controlled deposition and not emission, also emission from spruce needles was found to be slightly dependent on gH2O (Fig. 4).
Several authors have observed internal (mesophyll) resistances limiting NO2 deposition in different tree species (Johannson, 1987; Thoene et al., 1991; Sparks et al., 2001). Internal resistances (q < 1 cp pg. 14) were also observed under all environmental conditions in NO2 fumigation experiments with young spruce trees (Thoene et al., 1996). In the present study, however, indications of measured NO2 conductance to be smaller than NO2 conductance predicted from water vapour conductance were not found (Fig. 6E–H). Since the disproportionation reaction of NO2 in water resulting in equal amounts of nitrate and nitrite is slow at typical atmospheric NO2 concentrations, it is supposed that the reaction of ascorbate with NO2 to nitrite and dehydroascorbate is responsible for maintaining high fluxes of NO2 into the leaf and may prevent internal resistances (Ramge et al., 1993). Since the apoplastic ascorbate concentration does not only vary with species but also with environmental conditions (Polle et al., 1995, Schwanz et al., 1996) and stage of development (Luwe, 1996), the observed differences may be due to different apoplastic ascorbate concentrations. In addition, the removal of nitrite, formed in the reaction of NO2 with ascorbate, by chemolithoautotrophic nitrite oxidisers present and physiologically active in the needle apoplast of the adult trees can also contribute to the reduction of mesophyll resistance.
Cuticular deposition increases with increasing concentrations of NH3 and NO2
Cuticular NO2 and NH3 deposition onto needles of spruce trees was observed as indicated by the intercepts of the regression lines in Figs 3 and 4, respectively, and by q > 1 given in Fig. 6 and increased with increasing NH3 concentration even at the low RH < 40% applied in the fumigation experiments. At low ambient CNH3 and CNO2 flux of NH3 and NO2 could be explained almost by stomatal uptake but at high concentrations measured conductance of both trace gases exceeded the one predicted from gH2O by a factor of up to 3. This finding is in contradiction to the calculations of Wellburn (1990) for Picea abies, who concluded that even at high NO2 concentrations (140 nmol/mol) stomatal flux exceeded deposition on the cuticles by one or two orders of magnitude. NO2 deposition rates onto cuticles were also minute for sun flower and tobacco (Neubert et al., 1993), for adult beech grown at the same field site examined in the present study (Geßler et al., 2000) and for spruce trees in a forest in Sweden (Rondon et al., 1993). It is known that NO2 is able to penetrate the cuticles within a certain range (Wellburn, 1990) and can be adsorbed to and/or react with components of the cuticle (Lendzian & Kerstiens, 1988). Differences in chemical composition between different species and, as a key factor, the metabolic activity of autotrophic nitrite oxidising bacteria colonising the needles at the site examined (Papen et al., 2002) may be responsible for the different concentration-dependent patterns of cuticular NO2 deposition. Especially the tendency of cuticular deposition to decrease with increasing PPFR indicates that nitrifying bacteria – known to be inhibited by light (Vanzella et al., 1989) – are involved in cuticular deposition.
For oilseed rape, no significant NH3 deposition on the cuticles even at a relative humidity of 80% could be observed (Husted & Schjoerring, 1995). However, significant cuticular deposition to a spruce forest was assumed by Andersen et al. (1993; 1999) and onto beech leaves by Geßler et al. (2000). NH3 deposition on the cuticles may be explained by the wetness of leaf cuticles observed even in the absence of free water at low air humidity (Van Hove & Adema, 1996) that may act as additional sink for highly water soluble NH3.
If it is supposed that at least a part of the NO2 and NH3 deposited on the cuticles can be re-emitted when ambient NO2 and NH3 concentration decreases, future research will have to achieve discrimination between net emission via stomata and cuticular re-emission.
NH3 taken up by needles of adult spruce contributes to the N demand of the whole trees
The 15N[NH3] fumigation experiments demonstrated that the deposition rates calculated from the gas exchange measurement were reliable, since daily deposition rates determined from both approaches were similar. In addition, it could be directly traced for the first time in adult trees grown in the field, that NH3 taken up by adult spruce trees is subjected to long distance transport, since the 15N tracer was found in significant amounts in bark of sections inside and outside the fumigation chambers. This is a prerequisite for atmospheric NH3 to serve as additional N supply for adult forest trees. The present finding of the field experiment is consistent with the results gained from NH3 exposition of Fagus sylvatica seedlings (Geßler et al., 1998b; Geßler & Rennenberg, 1998) and Pinus sylvestris (Pérez-Soba et al., 1994; Van der Eerden et al., 1990) under controlled conditions. A 3-d fumigation of beech with c. 55 nmol mol−1 NH3 resulted in a significant enrichment of soluble amino compounds in the leaves (mostly Arg) and in the phloem (Glu, Gln, Asp, Asn) (Geßler et al., 1998b). Exposure of Scots pine to NH3 stimulated glutamine synthetase (GS) and lead to a subsequent increase in amino compounds in the tissues exposed (Pérez-Soba et al., 1994). Hence, it must be concluded that NH3 similar to NO2 (Nussbaum et al., 1993, Muller et al., 1996) contributes to the pool of soluble nitrogen compounds circulating between shoots and roots that first provides different tissues and organs of the plant with nitrogen and second is involved in the adaptation of pedospheric N uptake to the plant's demand (Imsande & Touraine, 1994; Muller et al., 1996) in adult spruce.