New Phytol. (1996), 134, 257-266

Absorption of atmospheric(Picea abies) trees

by spruce

II. Parameterization ofchamber experiments

fluxes by controlled dynamic

BY BARBARA THOENEi, HEINZ RENNENBERG^^AND PAUL WEBERi 2*

^ Fraunhofer-Institut fiir Atmosphdrische Umweltforschung, Kreuzeckbahnstrasse 19,D-82467 Garmisch-Partenkirchen, Germany

{Received 4 January 1996 ; accepted 21 May 1996)

SUMMARY

The dynamic chamber technique was applied to investigate NO., influx into Picea abies (L.) Karsten branches, andits effects on net photosynthesis and transpiration, as well as its dependency on irradiance, temperature andrelative humidity. The study aimed to quantify effects of climate on atmospheric NO2 fluxes to spruce.Experiments were performed with 3- to 4-yr-old branches of 8- to 9-yr-oId potted trees under controlledenvironmental conditions. With ambient NO., concentrations increasing from 3*5 to 50 nl T^ a linear increase inthe NO2 influx of up to c. 6-8 //mol m"^ s"" was observed. From this increase a compensation point of 1-64 nl 1~NO.2 was calculated by linear regression analysis. In the range of the NO, concentrations studied, netphotosynthesis of spruce was not affected. The responsiveness of the stomata to changes in irradiance and relativehumidity was reduced at 45 nl 1" NO2 compared with 25 nl 1" NOg. With increasing irradiance up to 1000/imolm~" s ^ PAR, increasing NOj flux to spruce branches was observed, which was attributed to a light-dependent

increase in stomatal aperture. Variation of the temperature between 14 and 35 °C did not affect the NOg fiux, inlight or in darkness. Higher temperatures, up to 45 °C, resulted in an increase in NOg influx in the light; indarkness, changes in NO. flux were not observed under these conditions. An increase in relative humidity from5 to 60 % in the light caused an increase in NOj influx, whereas in darkness NOg influx was not affected by changesin relative humidity. The increase in NOj flux in response to r.h. observed in the light could not be explained bychanges in stomatal aperture. A solution of NO, in ultra-thin water films covering the needle surface might explainthis phenomenon.

Key words: Air temperature, dynamic chamber technique, irradiance, nitrogen dioxide, relative humidity.

INTRODUCTION

In temperate forest ecosystems, nitrogen, derivedfrom the soil as nitrate and/or ammonium, is thenatural growth-limiting nutrient of trees. Owing toanthropogenic emissions (Becker et al., 1985), treesare in addition exposed to atmospheric nitrogen,especially NOg and NHg, as a supplementary sourceof nitrogen (Wellburn, 1990; Pearson & Stewart,1993). The uptake of atmospheric NOg proceedspredominantly by diffusion through the stomata intothe substomatal cavity (Wellburn, 1990). From thiscompartment, NO2 dissolves rapidly in the aqueous

* To whom correspondence should be addressed.^ Present address: Institut fiir Forstbotanik und Baum-

physiologie, Professur fur Baumphysiologie, Universitat Frei-burg, Am Flughafen 17, D-79085 Freiburg i.Br., Germany.

phase of the apoplastic space and can either undergodisproportionation reaction to nitrate and nitrite(Lee & Schwartz, 1981) and/or can react withascorbate to nitrite and dehydro-ascorbate (Ramge etal.., 1993). The additional amount of nitrogen takenup via the leaves enters the nitrogen pool derivedfrom xylem transport. It is rapidly transported intothe symplasm and is incorporated into the generalnitrogen metabolism of the leaves (Wellburn, 1990).In laboratory experiments with spruce seedlingsatmospheric NO2 was shown to contribute between20 and 40 % of total nitrogen assimilation (Mulier,Touraine & Rennenberg, 1996). In the field thisvalue might depend highly on atmospheric NOgconcentration, climate, soil properties, plant species,the stage of plant development (Imsande & Tour-aine, 1994; Nilsson & Wiklund, 1994), and factors

258 B. Thoene, H. Rennenberg and P. Weber

that modulate nitrogen uptake from the soil. Aconsequence of the uptake of atmospheric nitrogen isthe supplementary formation of amino acids in theleaves (Nussbaum et aL, 1993; Weber et al., 1995).Trees might respond to this supplementary N-nutrition either by increased growth (Kenk &Fischer, 1988; Mead & Tamm, 1988) or by reducednitrogen uptake from the soil (Muller et al., 1996).

The influx of NOg into leaves might vary underfield conditions as a consequence of changes not onlyin atmospheric NOg gas-mixing ratios but also inclimatic factors (Weber & Rennenberg, 1996). Theaim of the present study was to quantify effects ofclimatic parameters on the flux of atmospheric NOgto Norway spruce {Picea abies), one of the mostimportant forest tree species in Europe. For thispurpose NOg influx and its effects on net photo-synthesis and stomatal aperture, as well as itsdependency on (i) irradiance, (ii) temperature, and(iii) r.h. were determined with the dynamic chambertechnique under controlled conditions. The experi-ments were performed at relatively low (25 nl 1~ )and relatively high (45 nl l~^) NOo concentrations,which are well within the range 0-25—100 nl 1~typically found in the troposphere (Crutzen, 1979).Nitrogen dioxide concentrations of 25 and 45 nl 1"were chosen because measured and predicted con-ductance of spruce twigs for NOg were found to bedifferent at 25 nl 1" NO.2, but similar at 50 nl 1"

in a previous stud}^ (Thoene et al., 1991).

MATERIALS AND METHODS

Plant material, gas handling system, andmeasurement of flux data

Experiments were performed wath 3- to 4-yr-oldbranches of 8- to 9-yr-old potted trees of Picea abies[L.] Karsten cv. Vorallgauer Fichte. Fluxes of NO2,CO2 and H2O were studied by the dynamic chambertechnique (for detail see Rennenberg, Schneider &Weber, 1996; Weber & Rennenberg, 1996) using thegas handling system and the analytical devicesdescribed by Thoene et al. (1991). Exposure oftwigs attached to the trees to atmospheric NO2 wasperformed in chambers wath glass walls (Thoene etal, 1991). Fluxes of NO2, CO2, and H2O betweenthe enclosed branches and the atmosphere werederived from the difference in concentrations be-tween the outlet ports of the chamber containing thebranch and an empty, control chamber (De Kok,Stahl & Rennenberg, 1989; Thoene et al, 1991;Weber & Rennenberg, 1996; Rennenberg et al,1996). The leaf conductance for H2O was calculatedby eqn (1),

where ^(H2O) is the transpiration rate per needlesurface area, and AH2O is the difference between the

concentrations of aqueous vapour inside the sub-stomatal space and in the fumigation chambercontaining a spruce twig. The aqueous vapourconcentration inside the stomatal cavities was cal-culated as saturated vapour concentration at ambientair temperature.

By analogy with eqn (1) the measured leafconductance for NO2 was calculated using eqn (2),

where yil>lO^ is the flux of NO2 per needle surfacearea of the spruce branches enclosed, and C(NO2) isthe ambient NO, concentration in the fumigationchamber containing the twig. As the NO2 con-centration inside the enclosed spruce needles isconsidered to be close to zero, the ambient NO2concentration represents the difference between theNO2 concentrations in the substomatal space of theenclosed needles and in the fumigation chamber.

In addition, the predicted conductance for NO2was calculated by eqn (3),

piNO.,) = ^(H^O) X

W'here

H2O and

(3)

is the leaf conductance for H2O, andand MW\TO, are the molecular weights of

respectively.

Fumigation experiments

The experiments were performed using the uppertwigs of spruce trees. Sections of 1- and 2-yr-oldtwigs were placed into the fumigation chamber andwere exposed to deflned amounts of atmosphericNO2. In each set of experiments one individualbranch was exposed per day; experiments beingrepeated three times with different trees. Eachexperiment was started with an acclimation period ofat least 2 h, during which the parameter studied wasset at its lowest level and the other parameters wereadjusted at standard level. The standard levelsapplied were an irradiance of 600 jbtmol m~'~ s" PAR,a relative air humidity of 45 + 10% r.h., and an airtemperature of 24 + 4 °C. After the acclimationperiod, the parameter studied was increased in thegiven range. Each combination of climate parameterswas maintained for 1 h.

The effect of various concentrations of atmos-pheric NO2 on the NO2 uptake rate by spruceneedles was studied in the light; therefore, theenclosed branches were exposed to ambient NO2concentrations between 3-5 and 50 nl T NO2. Inother experiments the effect of atmospheric NO2 onphotosynthesis was determined at ambient concen-trations of 10, 20 and 50 nl T^ NO2. During theseexperiments irradiance increased from darkness to1000/^mol m~ s" PAR. In addition, parameteriz-ation experiments were performed to investigate the

fluxes to needles of spruce trees 259

dependency of the NOg flux on irradiance, airtemperature and relative humidity. These experi-ments were performed at relatively low (25 nl 1 )and at relatively high (45 nl I"-") atmospheric NOgconcentrations in combination with changing con-ditions of the climatic parameters indicated above.

Needle surface area

The surface area of needles within the chamber wasobtained from the projected needle surface area(measured with an area meter (Delta-T Devices,Cambridge, UK)) multiplied by 2-65, according toOren et al. (1986).

Data recording and statistical analysis

During the experiments data were recorded at 1 minintervals. Data measured during the last 30-minperiod of each combination of parameters were usedfor calculation of means of the fluxes. Results wereobtained from three independent experiments per-formed on different trees. Significance of differences(P ^ 0-01) between means was evaluated usingDuncan's multiple range test (Zofel, 1988).

RESULTS

c) flux and its effect on photosynthesis

Exposure of branches to atmospheric NO2 resultedin a flux of NO., to the shoot (Fig. 1). With anincrease in ambient NO., concentration from 3-5 to50 nl r \ the NOg influx increased linearly from 0"12

0 10NO, concentration (nl I '')

50

Figure 1. Influence of ambient NOg concentration on theNO2 flux (JNO,) to spruce branches. Three- to 4-yr-oldbranches of 8- to 9-yr-old spruce trees were enclosed infumigation chambers and were exposed to various NOgconcentrations during the Hght (600 [imo\ m"'^ s~ PAR).Experiments were performed at air temperature of22±2°C and at relative humidity of 40 + 5%. Theexperiments were started with an acclimation period in thepresence of NOg-free air. After acclimation, the NO.concentration was increased stepwise from 3-5 up to 50 nlr^ NOg. Each NO.2 concentration was kept constant for atleast 1 h. Eor calculation of the individual data points theflux data of the second 30 min were used. The experimentswere repeated seveD times with different trees. Data of allexperiments were pooled within the upper curve.f{x) = -0-23 + 0-14X- r = 0-98.

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to 6-77 fimol m~~ h~^. A compensation point for NO.,of 1-64 + 0-30 nl 1" was calculated by linear regres-sion analysis. Application of NO2 concentrationsbelow the compensation point was not possible,because of the detection limit of the N O , analyserused, and consequently, emission fluxes of NO, outof the shoot into the atmosphere could not bedetected. In addition to the influx of NO,, the flux ofCO2 was determined at different NO2 concentrationsof 10, 20 and 50 nl 1" NO2 at irradiances between 0and 1000 /tmol m^- s" PAR (Fig. 2). Independent ofthe NO2 concentration applied, CO2 fixation show^edbiphasic kinetics with increasing irradiance. Up to300 jumol m"' s~ PAR, CO2 fixation increased at ahigh rate with increasing irradiance, whereas athigher values, up to 1000/^mol m~"- s"' PAR, a lowrate of increase in CO2 fixation was observed. CO2fluxes did not differ significantly (F^O-01), whendifferent NO2 concentrations were applied. Ap-parently, atmospheric NOg up to 50 nl 1~ did notaffect photosynthetic CO2 fixation.

Dependency of the NO^ flux on irradiance

In three individual replicates of experiments,branches were exposed to NOg concentrations of 25(Fig. 3 a, c, e) and 45 nl I'' (Fig. 3 b, d,f) at 35-50%

260 B. Thoene, H. Rennenberg and P. Weber

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Figure 3. Influence of light intensity on NOg flux ( O , ^ N O )> transpiration ( • , J ^ Q), measured leaf conductancefor HgO (D, gs^o)' measured ( # , g^^o), and predicted (O, P^o ) conductance for NOj. Three- to 4-yr-oldbranches of 8- to 9-yr-old spruce trees were enclosed into fumigation chambers and were exposed to NOgconcentrations of 25 {a,c,e) or 45 (b, d,f) n\lSlO^\~^, respectively. Experiments were performed at airtemperature of 24 + 4 °C and at relative humidity of 35-50%. The experiments were started with anacclimation period in NO2-free air in darkness. After acclimation, the irradiance was increased stepwise up to1000 /imol m"^ s~ PAR. For details of calculation of the fluxes see legend to Figure 1. Curves represent meansof three independent experiments performed with different trees. Maximal standard deviation (SD) : J^^ :SD + 0-05/^mol m"^ h" ; J^HO- SD + 0-15 mol m~^ h"^; gH,o' SD + 0-15 mm s"-*; §'1,-0,: SD + 0-03mms~^SD + 0-13 mm s~^

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r.h. and 24 + 4 °C. During the experiments ir-radiance was raised from 0 to 1000 /tmol mf^ s"^PAR. At 25 nl 1~ NO2, a small flux was observedthat increased from 0-33 /^mol m^^ h"^ to 0-75 fimolm" ^ h~ within the range of irradiances applied (Fig.3 a). Application of 45 nl 1~ NO2 caused a sig-nificantly (P ^ 0-01) higher flux of NO2 that showedoptimum kinetics with increasing irradiance. Maxi-mum influx of 1-51 /^mol NO2 m"^ h~ was calculatedat 530/^molm-''s"'PAR (Fig. 3^). The rate oftranspiration and leaf conductance for H2O, mea-sured at 25 nl NOg r \ showed optimum kineticswith increasing irradiance (Fig. 3{c)). Maximum

transpiration of 2-60 mol H2O m ^ h ^ was observedat 700 jLtmol rrT'^ s"^ PAR; maximum leaf conduc-tance for H2O of 1-64 mm s~ at 670//mol m~ s ^PAR. Exposure of spruce branches to 45 nl NO21"^also showed saturation kinetics of transpiration andleaf conductance for H^O with increasing irradi-ances, but reached a lower maximum transpirationof 1-82 mol HgOm-'h-^ at 700/xmol m" 's" 'PARand a lower maximum leaf conductance for HgO of072mms~i at 600/imol m'^ s" PAR (Fig. 3d).Apparently, the responsiveness of stomatal apertureto light was reduced in the presence of 45 comparedwith 25 nl T^ NO2. During exposure to 25 and 45 nl

fluxes to needles of spruce trees 261

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measured conductance for NOg varied radiance of 600 //mol m " s PAR (Fig. 4) and in0-08 and 0-35 mm s ^ and showed no

significant difference (P ^ 0-01) between both treat-ments (Fig. 3e,/) . At 25nir^NO2 the predictedconductance for NO2 was up to fivefold higher thanthe measured conductance, whereas at 45 nl T^ NO2predicted and measured conductance were notstatistically different (P ^ 0-01).

Dependency of the NO^^ flux on temperature

During exposure of spruce branches to 25 and 45 nlr^ NO2, temperature was varied between 14 and45 °C in three individual experiments at an ir-

darkness (Fig. 5), respectively. At the beginning ofexposure to NO2, i.e. at the lowest temperatureapplied, relative humidity was adjusted to c. 45 % inlight and to 35 % in darkness. With increasingtemperature, absolute humidit}^ inside the chambersof 5-8 ± 0-4 g m= during light and of 4-5 ± 0-4 g m ^during darkness remained constant.

During experiments performed at 25 nl 1~ NO2 inthe light, an increase in temperature from 18 to 33 °Cresulted in a small increase in the NO2 flux fromc. 1-00 to 1-18 /imol NO2 m"^ h"^ (Fig. 4a). A furtherincrease in temperature from 35 to 43 °C caused amuch higher increase in NO2 flux, to c. 1 -40 /imol

262 B. Thoene, H. Rennenberg and P. Weber

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m ^ h . Exposure of spruce branches to 45 nl 1 ^NO.2 showed a similar pattern at a significantly(P^O-01) higher flux level (Fig. 4b). At tempera-tures between 20 and 35 °C the NOg flux remainedconstant at c. 2-5 jLtmol NOg m^ h~ , whereas a fur-ther increase in temperature up to 45 °C caused anincrease in the NO2 flux up to 3-22 jiimol m"^ h~ .The rate of transpiration increased almost linearlywithin the range of temperatures applied. Significantdifferences (P O'Ol) between the exposure torelatively low (Fig. 4c) and relatively high (Fig. 4d)NO2 concentrations were not observed. The leafconductance for H2O increased from 0"10 to l'O6 mms" during exposure to 25 nl 1~ (Fig. 4c) and from0-40 to 1-10 mm s~ during exposure to 45 nl 1" (Fig.4d), respectively. Up to 21 °C at 25 nl T^ NO2 andup to 22-5 °C at 45 nl T^ NOg the predicted con-ductance for NO9 was below the measured con-

ductance. At higher temperatures the predictedconductance for NO2 was higher than the measuredconductance for NOg.

As observed in the light, exposure of sprucebranches to atmospheric NO2 in darkness caused aNO2 flux to the branches. The observed increase inthe NO2 fluxes with increasing temperature wassignificantly (P 0-01) lower in darkness than in thelight. During darkness, application of 25 nl 1"' NO2caused an increase in NO2 flux from 0-25 to 0-42 ptmolm~^ h" at temperatures from 13 to 43 °C (Fig. 5 a),whereas an increase in temperature from 15 to 45 °Cat 45 nl 1~ NO2 caused an increase in NO2 flux from0-45 to 0-71 fimol m"' h" (Fig. 5b). With increasingtemperature the rate of transpiration increased from^.0-05 to l-43molH2Om"'s-^ (Fig. 5 c, d). Sig-nificant differences (P ^0-01) between the two NO2concentrations applied were not observed. In the

NO 2 fluxes to needles of spruce trees 263

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range of temperatures applied, neither leaf con-ductance for H.,O (Fig. Sc,d) nor measured norpredicted conductance for NOg (Fig. 5e,f) showedany variation.

Dependency of the NO^ flux on relative humidity

During exposure of branches to NO2 in the light(600 ju,mo\ m~ s ^ PAR), temperature was adjustedto 26 + 2 °C (Fig. 6), during exposure in darkness to22 + 2 °C (Fig. 7). Relative humidity varied between5 and 60 %. In the light, increasing relative humidityresulted in changes in the NO2 flux to sprucebranches. During exposure of spruce branches to25 nl r^ NO2, the NO2 flux was slightly increasedfrom M to l-4//,mol m"^ h~ (Fig. 6 a); whereasexposure to 45 nl T^ NO2 caused an increase from

1-2 to 2-2 jumol m " h ^ (Fig. 6^). Independent of theNO2 concentration applied, the rate of transpirationdecreased from c. 2-20 mol m^^ h~ at 5 % r.h. toc. 1-20 mol m"^h~^ at 60% r.h. (Fig. 6c, d). In thesame range of relative humidities, leaf conductancefor H2O increased from 0*77 to 1-61 mm s"^ at 25 nl1"- NO2 (Fig. 6 c) and from 0-42 to 0-92 mm s""- at45 nl 1~ NO2 (Fig. 6d). Apparently, responsivenessof the stomatal aperture to changes in relativehumidity was reduced in the presence of 45 nl 1"NO2 compared with 25 nl l"^ NO2. In all experi-ments the measured conductance for NO2 variedbetween 0*17 and 0*3 8 mm s ^ and showed nosigniflcant differences (P^O-01) between the NO2exposures (Fig. 6e,f). i\t 25 nl 1" NO2, the pre-dicted conductance for NO2 was twofold or threefoldhigher than the measured conductance, whereas at

264 B. Thoene., H. Rennenberg and P. Weber

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45 nl 1 ^ NO2 the differences between predicted andmeasured conductance were much smaller.

In contrast to experiments performed in hght, theNO2 influx was not affected by changes in relativehumidity during darkness (Fig. 7a, b). In all ex-periments a decrease in the rate of transpiration fromc. 1-60 mol H2O m-^ hT^ at 5 % r.h. to about 0-60 molH2O m~ h"^ at 60% r.h. was found, similar to thatobserved during experiments performed in light. Atboth NO, concentrations, 25 and 45 nl 1" NO2, leafconductance for H2O was only slightly affected byvariation in relative humidity (Fig. 7 c, d). Indarkness the predicted conductance for NOg washigher than the measured conductance for NOg (Fig.1 e,f), with small differences at 20 to 40% r.h., andsomewhat greater differences below 20 and above40% r.h.

DISCUSSION

In the present study, the Bux of NO2 to branches, itseffects on net photosynthesis and stomatal aperture,and its dependency on irradiance, temperature andrelative humidity were studied in order to quantifythe effects of climate on atmospheric NO2 flux tospruce. As observed in other studies, exposure ofspruce branches to increasing atmospheric NO2concentrations caused a linear increase in the NO2flux to the branches (Johansson, 1987; Thoene et al.,1991; Rondon, Johansson & Granat, 1993). Thislinear increase in the NO2 flux indicates that thedriving force of the NO2 gas exchange betweenplants and the atmosphere is the difference betweenNO2 concentrations of leaf interior and of theatmosphere. Therefore, it is likely that spruce trees

NO2 fiuxes to needles of spruce trees 265

emit NO2 into the atmosphere when exposed toNO2-free air, provided that NO2 is produced in theneedles. From the present laboratory experiments acompensation point for NO2 of 1-64 nll~^ wascalculated for spruce by linear regression analysis.This compensation point is considerably higher thanthat determined for spruce outdoors within a foreststand (0-6 nl r \ Rondon & Granat, 1994), but is stillin the same order of magnitude. In comparison,Weber & Rennenberg (1996) calculated a NOgcompensation point of 1-15 nl T^ for wheat leaves.

During exposure to atmospheric NO2 in concen-trations up to 50 nl 1~\ the rate of net CO2 fixation(photosynthesis) was not affected. This flnding isconsistent with previous experiments performedwith wheat plants (Weber & Rennenberg, 1996) andwith several cultivars of potted plants (Saxe, 1986).Inhibition of photosynthesis has been reported,however, during long-term exposure to NO2(^ 20 h) or exposure to much higher NO2 concen-trations, of up to 1000 nl r ' (Hill & Bennett, 1970;Saxe, 1986; Darrall, 1989).

Increasing temperature from 18 to 35 °C did notaffect the NO2 flux to spruce branches in light or indarkness. When temperature was raised to 45 °C, theNO2 flux increased only in the light, especiallyduring exposure to 45 nl l~^ NO2. In contrast to theNO2 flux, transpiration and leaf conductance forHgO increased continuously with increasing tem-perature as reported by other authors (Brunner &Eller, 1974; Jarvis, 1976). Apparently, the tempera-ture-dependent changes in NO2 flux to sprucebranches are not a consequence of changes instomatal aperture. Calculations of NO2 fluxes inrecent modelling approaches (Ramge et al., 1993)were based on the assumption that solubility ofgaseous NO2 in the aqueous phase of the apoplasticspace decreases with increasing temperature, andsolubility of atmospheric NO2 will become the mainresistance to NO2 flux into leaves at higher tempera-tures. The finding that NO2 fluxes decrease withincreasing temperature in experiments with wheatleaves is consistent with this assumption (Weber &Rennenberg, 1996). Such a dependency on tem-perature of NO2 flux was not found in the presentstudy. It may therefore be concluded that tempera-ture-dependent changes in the NOo fluxes to sprucebranches are not determined by the solubility of NO2in the aqueous phase of the apoplastic space. Thereasons for this difference between spruce and wheatremain to be elucidated.

In temperate forests air temperature does notusually increase above 35 °C. Therefore, the spon-taneous increase of the NO2 flux observed in bothNO2 exposure treatments is likely to have beencaused by damage to the needles. Similar effects onplants exposed to relatively high air temperatureshave been reported from other fumigation experi-ments (Weber & Rennenberg, 1996).

Variation in relative humidity in the light causedan increase in NO2 flux to spruce branches, but indarkness the NO2 flux was not affected. The samewas observed for leaf conductance for H2O. Thepattern of the increase of the NO2 flux showedsaturation kinetics, whereas the increase of the leafconductance for H2O with increasing relative hu-midity was exponential. As the kinetics of the NO2flux pattern are different from those of leaf con-ductance for H2O, changes in the NO2 flux to sprucebranches with changing relative humidity cannot beattributed to alterations in stomatal aperture alone.This result is surprising, since changes in stomatalaperture as a consequence of changing relativehumidity are frequently observed (e.g. Lange et al.,1971). From the results of the present experiments itis more likely that high relative humidities cause theformation of water films on the needle surfaces, asobserved by Burkhard & Eiden (1994), that mightact as sinks for atmospheric NO2. Similar conclu-sions were recently drawn from experiments withwheat (Weber & Rennenberg, 1996).

However, a response of NO2 flux to increasingrelative humidity was not observed in darkness. It islikely that light-dependent processes are involved inNO2 uptake by spruce. Experiments with tomatoplants unable to regulate their stomata showed thatunknown physiological processes can be regulatedby changes in irradiance; apparently, these physio-logical processes, located in the leaf's interior, alsoaffect NO2 deposition (Murray, 1984). An increasein irradiance up to 1000 jumol PAR m~" s~ caused anincrease in NO2 flux to spruce branches. Thesimultaneous increase in transpiration indicates thatthis is a result of changes in stomatal aperture. Stillother factors, such as internal resistances, seem tocontribute to the NO2 exchange between sprucebranches and the atmosphere.

In all experiments the predicted conductance forNO2 was higher than the measured conductance,which indicates the existence of internal resistancesfor the NO2 fluxes. Internal resistances for NO2fluxes have been observed in previous experimentswith Picea abies (Thoene et al., 1991) and Pinussylvestris (Johansson, 1987), and were also found forthe fluxes of other trace gases, e.g. of H2S toSpinacea oleracea (De Kok, Rennenberg & Kuiper,1991). Internal resistances for NO2 were not ob-served in experiments with Pinus taeda (Hanson etal., 1989) and Triticum aestivum (Weber & Ren-nenberg, 1996). Apparently, species-speciflc dif-ferences in internal resistance have to be consideredin calculations of NO2 fluxes to vegetation. Exposureof spruce branches to 45 nl l"^ NO2 reduced theresponsiveness of the stomata as compared with anexposure to 25 nl T^ NO2. It was surprising that thisreduction was observed only in response to changesin irradiance and relative humidity, but not inresponse to changes in temperature; it might be a

266 B. Thoene, H. Rennenberg and P. Weber

consequence of the exposure of spruce branches to25 nl r^ NO2 at air temperature of 25-28 °C, and to45 nl r^ at air temperatures of 20-23 °C. Figure 4shows that the leaf conductance for H^O as well asthe rates of transpiration are twice as high at 27 °C asat 22 °C. Therefore, the reduction in responsivenessof stomata which was observed in the exposureexperiments of spruce branches to 45 nl 1~ NOg wasmainly caused by the difference in air temperatureand was not, therefore, an effect of increasedatmospheric NO., concentration.

ACKNOWLEDGEMENTS

The authors thank Drs B. Huber, H. Papen, P. Schroderand K. Stahl (IFU, Garmisch-Partenkirchen) for helpfuldiscussions on the experiments.

REFERENCES

Becker KH, Fricke W, Lobel J, Schurath U. 1985. Formation,transport, and control of photochemical oxidants. In: GuderianR. ed. Air Pollution by Photochemical Oxidants. Berlin:Springer-Verlag, 3-111.

Brunner U, EUer BM. 1974. Offnen der Stomata bei hohenTemperaturen im Dunkeln. Planta 121: 293-302.

Burkhard J, Eiden R. 1994. Thin water films on coniferousneedles. Atmospheric Ettvironment 28: 2001-2017.

Crutzen PJ. 1979. The role of NO and NO^ in the chemistry ofthe troposphere and stratosphere. Annual Reviezv of EarthPlanetation Science 7: ^AZ-All.

Darrall NM. 1989. The effect of air pollutants on physiologicalprocesses in plants. Plant, Cell and Environment 12: 1-30.

De Kok LJ, Rennenberg H, Kuiper PJC. 1991. The internalresistance in spinach leaves to atmospheric H. S deposition isdetermined by metabolic processes. Plant Physiology andBiochemistry 29: 463^70.

De Kok LJ, Stahl K, Rennenberg H. 1989. Fluxes ofatmospheric hydrogen sulphide to plant shoots. New Phytologist111. 533-542.'

Hanson PJ, Rott K, Taylor GE Jr, Gunderson CA, LindbergE, Ross-Todd BM. 1989. NOj deposition to elements rep-resentative of a forest landscape. Atmospheric Environment 23:1783-1794.

Hill AC, Bennett JH. 1970. Inhibition of apparent photosynthesisby nitrogen oxides. Atmospheric Environment 4: 341-348.

Imsande J, Touraine B. 1994. N demand and regulation ofnitrate uptake. Plant Physiology 105: 3-7.

Jarvis PG. 1976. The interpretation of the variations in leaf waterpotential and stomatal conductance found in canopies in thefield. Philosophical Transactions of the Royal Society of London273: 593-610.

Johansson C. 1987. Pine forest: a negligible sink for atmosphericNO^ in rural Sweden. Tellus 39B: 426-438.

Kenk G, Fischer H. 1988. Evidence from nitrogen fertilisation inthe forests of Germany. Environmental Pollution 54: 199-218.

Lange OL, Losch R, Schulze ED, Kappen L. 1971. Responsesof stomata to changes in humidity. Planta 100: 76-86.

Lee YN, Schwartz SE. 1981. Evaluation of the rate of uptake ofnitrogen dioxide by atmospheric and surface liquid water.Journal of Geophysical Research 86: 11971-11983.

Mead DJ, Tamm CO. 1988. Growth and stem form changes inPicea abies as affected by stand nutrition. Scandinavian Journalof Eorest Research 3: 505-513.

MuUer B, Touraine B, Rennenberg H. 1996. Interactionbetween atmospheric and pedospheric nitrogen nutrition inspruce {Picea abies L. Karst) seedlings. Plant, Cell andEnvironment 19: 345-355.

Murray AJS. 1984. Light affects the deposition of NOg to theflacca mutant of tomato without affecting the rate of transpira-tion. New Phytologist 98: 447-450.

Nilsson LO, Wiklund K. 1994. Nitrogen uptake in a Norwayspruce stand following ammonium sulphate application, fertig-ation, irrigation, drought and nitrogen-free-fertilisation. Plantand Soil 164: 221-229.

Nussbaum S, von Ballmoos P, Gfeller H, Schlunegger UP,Fuhrer J, Rhodes D, Brunold C. 1993. Incorporation ofatmospheric ^^NOg-nitrogen into free amino acids by Norwayspruce Picea abies (L.) Karst. Oecologia 94: 408-414.

Oren R, Schulze ED, Matyssek R, Zimmermann R. 1986.Estimating photosynthetic rate and annual carbon gain inconifers from specific rate and leaf bioniass. Oecologia 70:187-193.

Pearson J, Stewart GR. 1993. The deposition of atmosphericammonia and its effects on plants. New Phytologist 125:283-305.

Ramge P, Badeck FW, Plochl M, Kohlmaier GH. 1993.Apoplastic antioxidants as decisive elimination factors withinthe uptake process of nitrogen dioxide into leaf tissues. NewPhytologist 125: 771-785.

Rennenberg H, Schneider S, Weber P. 1996. Analysis ofuptake and allocation of nitrogen and sulphur compounds bytrees in the field. Journal of Experimental Botany (in press).

Rondon A, Granat L. 1994. Studies on the dry deposition ofNO., to coniferous species at low NO., concentrations. Tellus46B: 339-352.

Rondon A, Johansson C, Granat L. 1993. Dry deposition ofnitrogen dioxide and ozone to coniferous forests. Journal ofGeophysical Research 98: 5159-5172.

Saxe H. 1986. Stomatal-dependent and stomatal-independentuptake of NO^. New Phytologist 103: 199-205.

Thoene B, Schroder P, Papen H, Egger A, Rennenberg H.1991. Absorption of atmospheric NO., by spruce {Picea abies L.Karst.) trees. I. NO, influx and its correlation with nitratereduction. New Phytologist 117: 575-585.

Weber P, Rennenberg H. 1996. Dependency of nitrogen dioxide(NOj) fluxes to wheat {Triticum aestivum L.) leaves from NOgconcentration, light intensity, temperature and relative hu-midity determined from controlled dynamic chamber experi-ments. Atmospheric Environment 30: 3001-3009.

Weber P, Nussbaum S, Fuhrer J, Gfeller H, SchluneggerUP, Brunold C, Rennenberg H. 1995. Uptake of atmospheric^^NOg and its incorporation into free amino acids in wheat{Triticum aestivum L.). Physiologia Plantarum 94: 11-11.

Wellburn AR. 1990. Why are atmospheric oxides of nitrogenusually phytotoxic and not alternative fertilizers ? New Phv-

Zofel P. 1988. Statistik in der Praxis. Stuttgart, Germany:G. Fischer Verlag, 136-141.