Summary For 20 weeks during the growing season, cuttingsof one birch clone (Betula pendula Roth.) were exposed in theBirmensdorf fumigation chambers to O3-free air (control) or75 nl O3 l

−1. Ozone was supplied either from 1900 until 0700 h(nighttime regime), from 0700 until 1900 h (daylight regime),or all day (24-h regime). By autumn, reductions in whole-plantbiomass production, root/shoot biomass and stemweight/length ratios were evident in all three O3 regimes. Thereductions in cuttings receiving the 24-h O3 treatment wereabout twofold larger than in cuttings receiving the daylight O3

treatment. Stomata were open at night, and stomatal conduc-tance was about 50% of its maximum daytime value. Wecalculated that the rate of O3 uptake into leaves in the darkapproached 4 nmol m−2 s−1. Whole-plant production and carb-on allocation were more sensitive to O3 during the night thanduring the day; however, O3 exposure caused similar visibleleaf injury in both of the 12-h regimes, although the leavesexposed to O3 at night exhibited delayed O3-induced shedding.Overall, changes in production and carbon allocation weredetermined by the external O3 dose rather than by the kind ofO3 exposure, indicating that, at the seasonal scale, the internaldose of ozone that was physiologically effective was a constantfraction of the external O3 dose. We conclude that nighttime O3

exposures should be included in the daily time period fordetermining critical concentrations of O3 causing injury intrees.

Keywords: biomass production, carbon allocation, foliage de-velopment, stomatal conductance.

Introduction

Ozone can limit tree development in controlled experimentswith young plants (Reich 1987, Pye 1988), but O3 effects onmature trees vary between sites and species (Miller 1973,Peterson et al. 1987, Schulze et al. 1989). Geographical infor-mation systems (GIS) are gaining importance as a means ofassessing large-scale ecological risk, e.g., for estimating theconstraint ozone poses on trees and forests (Hogsett et al.

1995). However, implementation of GIS for risk assessment ofO3 injury (see UNECE 1994) requires that critical levels of O3

exposure (CL) be established below which no adverse effectsare assumed to occur in plants (UNECE 1988). Thus, CLs needto be defined for the time periods during which trees aresensitive to ozone. It has not been determined whether ozone-sensitive periods are restricted to the daylight hours of thegrowing season for trees as proposed for agricultural cropplants (Fuhrer 1994). Forest sites that are not continuouslyaffected by ozone from local urban sources can experiencehigh O3 concentrations in the evening and early morning hours(Lefohn and Jones 1986).

To determine whether trees are sensitive to ozone during thenight, we analyzed the influence of nighttime exposure toozone on foliage development and annual biomass productionof a birch clone (Betula pendula Roth.) relative to the influenceof ozone exposures during daylight hours or throughout theday. In a previous study with this clone, continuous O3 expo-sure throughout the growing season caused declines in CO2

assimilation and biomass production that were accompaniedby marked changes in leaf differentiation and whole-plantcarbon allocation (Matyssek et al. 1991, 1992, Günthardt-Go-erg et al. 1993). Although O3 sensitivity of deciduous treespecies is restricted to the growing season, it is not knownwhether ozone is taken up at night. The capacity of stomata toopen without light stimulation (Tobiessen 1982) may be fa-vored under warm and humid conditions (Lösch 1979, Schulzeand Hall 1982), depend on leaf age (Field 1987), or be causedby sluggishness in stomatal regulation under O3 stress (Kellerand Häsler 1987). To distinguish among these possibilities, weexamined leaf gas exchange during the nighttime.

Materials and methods

Plants and treatments

From April 17 until September 27, 1990, cuttings of one birchclone (Betula pendula) were each grown in a 10-l pot filledwith sand and a basal layer of inert synthetic clay beads. The

Nighttime exposure to ozone reduces whole-plant production in Betulapendula

RAINER MATYSSEK,1,2 MADELEINE S. GÜNTHARDT-GOERG,1 STEFAN MAURER1 andTHEODOR KELLER1

1 Swiss Federal Institute for Forest, Snow and Landscape, Research, Zürcherstrasse 111, CH-8903 Birmensdorf ZH, Switzerland2 New address: Department of Forest Botany, University of Munich, Hohenbachernstr. 22, D-85354 Freising, Germany

Received April 4, 1994

Tree Physiology 15, 159--165© 1995 Heron Publishing----Victoria, Canada

cuttings were fertilized and well watered. On May 17, 1990,when the shoot length of the plants was about 3 cm, they weretransferred to the Birmensdorf field fumigation chambers andsubjected to one of four O3 treatments (five plants per treat-ment, one plant per chamber). The O3 concentrations were< 0.003 µl l−1 (control, charcoal-filtered air, regarded as O3-free, 24 h day−1) and 0.075 µl l−1, the latter being applied dailyeither from 0700 until 1900 h (daylight regime), from 1900until 0700 h (nighttime regime, including dawn and dusk), orthroughout the day (24-h regime). Ozone was generated frompure oxygen (Fischer, model 502) and added to charcoal-fil-tered air. All treatments were continuously monitored with aMonitor Labs 8810 instrument. The birch clone, cultural prac-tices and fumigation chambers have been described byMatyssek et al. (1991, 1992, see also Landolt et al. 1989). Onclear sunny days, a shade roof limited the photon flux densityto a maximum of about 600 µmol m−2 s−1 to prevent over-heat-ing in the open-top chambers. The roof was not used underovercast and cloudy conditions, or at dawn and dusk.

Macroscopic leaf injury

Visible symptoms of O3-induced leaf injury were classified onthe basis of leaf color, which progressed from dark-green to theoccurrence of light-green dots, brownish-bronze discoloration,necroses and premature leaf loss. The early symptoms (i.e.,light-green dots spread over the leaf lamina) were assigned toClass 1 and premature leaf loss was assigned to Class 4,following Günthardt-Goerg et al. (1993). The trees were exam-ined for symptoms of visible injury in early and late June, earlyAugust and mid-September.

Biomass analysis

On September 27, 1990, the five trees in each treatment wereharvested. Whole-plant foliage area was determined with a leafarea meter (MK2, Delta-T Devices, U.K.) by summing theone-sided area of all leaves. Attached and prematurely shedleaves were counted. All tree organs (leaves, branches, stem,root and initially planted cutting) were dried at 65 °C toconstant weight. The ratio of root/shoot biomass comprisedthe plant organs that had developed from the initial cutting(about 13 cm long, 0.6 g in April), otherwise the cuttingincrement was part of the whole-plant dry weight.

Measurements of leaf gas exchange

Measurements are not available from the 1990 experiment, butmeasurements were conducted in 1992 with the same birchclone. In 1992, the daily O3 regime was 0.09 µl l−1 from 0700until 2100 h and 0.04 µl l−1 during the rest of the day (thecontrol treatment and all other practices were the same as in1990). Attached complete leaves were measured with a ther-moelectrically climate-controlled cuvette system (Walz, Ger-many) as described by Matyssek et al. (1991). Diurnal coursesof leaf gas exchange were recorded from the plants exposed inthe fumigation chambers. Temperature and air humidity werereproduced inside the gas exchange cuvette at any instant oftime, and the photon flux density was reduced by about 8% bythe cuvette lid (CO2 concentration of the ambient air was about

340 µl l−1). Gas exchange rates were based on the one-sidedleaf area. The rate of O3 uptake into the intercellular spaces ofthe leaf mesophyll was calculated according to the water vaporsurrogate method (Laisk et al. 1989). That is, by convertingstomatal conductance for water vapor (gH2O) to stomatal con-ductance for ozone (gO3

), based on the ratio of the diffusioncoefficients (DH2O /DO3

= 1.68), and assuming that the O3

concentration in the intercellular spaces of the mesophyll waszero. The expression ‘‘external O3 dose’’ denotes the O3 con-centration of the ambient air multiplied by the duration ofexposure.

Stomatal apertures were investigated by low-temperaturescanning electron microscopy (Scheidegger et al. 1991).

Results

The tolerance of leaves to O3 stress varied with both season andthe daily period of O3 exposure. Leaves developing in summergenerally tolerated higher external O3 doses before exhibitingvisible injury than leaves that were formed in the spring (Fig-ure 1A). The O3 tolerance of leaves in the daylight and night-time O3 regimes increased similarly during the growing

Figure 1. Range of external O3 dose (five plants/treatment, see Meth-ods) to induce early macroscopic leaf injury (A) and premature leafloss (B) under nighttime (NIGHT), daylight (DAY) or 24-h O3 expo-sure; determination on leaves, the development of which was followeduntil the time periods shown (the birch cuttings display indeterminateshoot growth until early September). In the 24-h regime, no leaves hadbeen formed in September to develop early leaf injury (see A); notethe different scales of the O3 dose in A and B.

160 MATYSSEK ET AL.

season, but O3 tolerance was lower than in leaves in the 24-hO3 regime. In the daylight O3 regime, no leaves were shed byearly June, and in the nighttime O3 regime, no leaves were lostby mid-September (Figure 1B). The external O3 dose thatcaused premature abscission was higher in leaves that haddeveloped during the second half of the growing season thanin leaves formed in the spring (Figure 1B). The dose range thatinduced leaf shedding was small.

The total number of leaves formed per plant throughout thegrowing season did not differ among treatments (Figure 2A).In the nighttime regime, premature leaf loss did not occur untilthe end of September, and the number of leaves shed was lessthan in the other two O3 regimes (Figure 2A). In both thedaylight and nighttime regimes, leaf loss was too small tocause a perceptible reduction in crown foliage area relative tothat of the control trees (Figure 2B). Although leaf loss wassimilar in trees in the daylight and 24-h O3 regimes, only treesin the 24-h O3 regime displayed an apparent reduction in

whole-plant foliage area as the result of a decrease in stemrather than branch foliage area (Figure 2B). A reduction in thesize of the stem leaves, but not of the branch leaves, in responseto 24-h O3 exposure (Figure 2C) resulted in the decreasedcrown foliage area.

Ozone had a greater effect on whole-plant biomass produc-tion than on foliage area during the growing season (Fig-ure 3A). The decline in biomass production was accompaniedby marked changes in whole-plant carbon allocation. Withincreasing external O3 dose, annual stem production, based onthe increment in stem length, was reduced (Figure 3B, stemweight/stem length) as was the root/shoot biomass ratio (Fig-ure 3C). The ozone sensitivity of biomass production andcarbon allocation was higher in trees in the nighttime than inthe daylight O3 regime, but the combined effects of the treat-ments were similar to those of the 24-h treatment (Figures3A--C).

We compared two 37-h sequences of leaf gas exchange thatdiffered in weather conditions, leaf age and O3 exposure (Fig-ure 4). Gas exchange of the 4-week-old leaf, which had beencontinuously exposed to O3-free air, is shown for a period ofhot midsummer weather (Figure 4, solid line). Day tempera-

Figure 2. Whole-plant leaf number and prematurely shed leaves (A),whole-plant foliage area on stem and branches (B), and mean area ofthe stem and branch leaves (C) at the end of the growing season asrelated to the external O3 dose applied during the growing season. Datarepresent means of five plants per treatment ± SD; for graphicalreasons, the symbols of the NIGHT and DAY regimes were groupedaround the O3 dose of 120 µl l−1 h in both treatments. Dashed linesillustrate the overall trend in the response to the ozone dose.

Figure 3. Whole-plant biomass (A), stem weight/stem length (B) androot/shoot biomass ratio (C) at the end of the growing season asrelated to the external O3 dose applied during the growing season(means ± SD, arrangement of the data points and dashed lines as inFigure 2).

NIGHTTIME EXPOSURE TO OZONE 161

tures reached 33 °C, nighttime temperatures did not dropbelow 20 °C, and the air was moderately dry during the night(leaf/air mole fraction difference of water vapor, ∆w > 10mmol mol−1; Figures 4B and 4C). During the day, stomatalconductance (gH2O) allowed CO2 uptake to follow the timecourse of irradiance; however, at night gH2O still reached about50% of the daily maximum value (Figures 4A, 4D and 4E),indicating that stomata remained open in the dark. Gas ex-change of the 7-week-old leaf (Figure 4, dotted line), whichhad been continuously exposed to ozone since bud break, isshown for a period during late summer when night tempera-tures fell to 7 °C at ∆w = 4 mmol mol−1 (Figures 4B and C).Stomatal conductance remained high at night (Figure 4E),even though stomata may narrow in the light in aging leaves or

under prolonged O3 exposure (cf. Matyssek et al. 1991). Basedon the assumption that the O3 concentration of the ambient airremained constant at 0.075 µl l−1 (corresponding to the 24-hO3 regime in 1990), the calculated rate of O3 uptake, whichthen parallels the time course of gH2O, could have reached 3--4nmol m−2 s−1 at night in both the 4- and 7-week-old leaves(Figure 4F), indicating that O3 uptake at night was substantialrelative to that during the day. The stomatal behavior shown inFigure 4E was representative of the daily leaf gas exchange ofthe birch clone investigated (Maurer and Matyssek, unpub-lished data), and was confirmed by microscopic analysis (Fig-ure 5). Stomatal pores were found to be open in the dark, butless so than during the day, regardless of the O3 regime.

Figure 4. Thirty-seven-hour sequence of a4-week-old leaf in August 1992 (exposedto O3-free air since bud break; solid line)and a 7-week-old leaf in September 1992(exposed to 0.09/0.04 µl l−1 during day/night since bud break; dotted line; leaf ofinjury class 2, i.e., light-green or bronze-green discoloration according to Günthardt-Goerg et al. 1993); time course of photonflux density (A), leaf temperature (B), dif-ference in leaf/air vapor mole fraction (C),CO2 assimilation rate (D), stomatal conduc-tance for water vapor (E), and rate of O3uptake into the leaf (F). The O3 uptake ratewas calculated according to the water va-por surrogate method (Laisk et al. 1989)based on an assumed constant O3 concen-tration = 0.075 µl l−1 of the ambient air(corresponding to the 24-h O3 regime in1990).

162 MATYSSEK ET AL.

Discussion

Nighttime exposure to O3 caused leaf injury, limited biomassproduction and decreased the root/shoot biomass ratio (R/S)as a result of changed carbon allocation. Reduced R/S hasoften been observed in response to the growth-limiting in-fluence of O3 or SO2 (Freer-Smith 1985, Mahoney et al. 1985,Tsukahara et al. 1986, Chappelka et al. 1988, Schier et al.1990, Matyssek et al. 1992, 1993b). Low R/S ratios may re-sult from impeded assimilate translocation caused by eithercell collapse in the mesophyll or impaired phloem structureand function (Michin and Gould 1986, Günthardt-Goerg etal. 1993); however, it may also indicate acclimation toozone. During the acclimation process, assimilates can beused in the leaves for repair and detoxification processes(enhanced demand for energy and antioxidants; Reich1983, Polle 1994) instead of being allocated to roots. Suchbehavior tends to maintain set-points between the plant’sinternal fluxes of carbon and nutrients during O3 stress(Mooney and Winner 1991), and may explain the insensitivityof leaf formation to the ozone treatments (cf. Figure 2A, Mooi1980, Matyssek et al. 1993a, 1993b). Leaf formation in birchinherently relates to the length increment of stem and branchaxes, which precedes radial growth (i.e., biomass increment).Radial growth becomes limited (cf. Figure 3B) as CO2 assimi-lation declines with O3 dose (i.e., exposure time), and maycease before latewood formation is initiated (Matyssek et al1991, 1993a).

Birch stomata remained open at night thus allowing pollut-ant uptake. Open stomata at night have also been observed inspruce (Wieser and Havranek 1993) and herbaceous plants(Goknur and Tibbitts 1984, Aben et al. 1989). Also, in turnipplants, nighttime exposure to ozone reduced production (Win-ner et al. 1989); however, the O3 concentration must remainenhanced at night to limit plant growth (cf. Kress et al. 1989).These observations suggest that a 24-h period may provide themost satisfactory daily basis for defining critical ozone expo-

sure for both agricultural crop plants (cf. Fuhrer 1994) andtrees.

Although the stomata of birch were open in the dark, gH O

2

and thus O3 uptake were lower than during the day (cf. Figure4); however, whole-plant production and carbon allocationtended to be more sensitive to ozone at night than during theday (cf. Figure 3). Reactive oxygen species, which are re-leased in the leaves by ozone as well as by photosynthetic pro-cesses, may be detoxified more efficiently in the light (Menser1964, Gillham and Dodge 1987, Schupp and Rennenberg1988). Both ascorbate and glutathione require NADPH, re-generated by photosynthetic electron transport, to stay re-duced and scavenge oxidants (Foyer et al. 1991). Ascorbatecan be exchanged between chloroplasts and cytosol and be de-livered to the plasmalemma and apoplast, where ozone, whenoccurring in low concentrations as applied in this study,mainly attacks the cell (Laisk et al. 1989, Urbach et al. 1989,Luwe et al. 1993). Consequently, the absence of light may ren-der plants susceptible to O3 stress. However, the increasednighttime sensitivity to ozone was not reflected in a reductionin foliage area or in substantially lowered tolerance to macro-scopic leaf injury, indicating that the amount and appearanceof foliage were not closely coupled with a decline inphotosynthetic capacity. Limited photosynthesis, however,must have impeded the overall carbon flux in the plant result-ing in O3-induced changes in whole-plant production and car-bon allocation (cf. Figure 3, Mooney and Winner 1991).

Overall, the proportional decline in biomass production,R/S and stem weight/length relative to plants in O3-free airwas determined by the external O3 dose rather than by the kindof O3 exposure (daily time interval, O3 concentration, differentgrowing seasons; Figure 6). The relationships suggest that, atthe seasonal scale, the internal dose of ozone that is physiolog-ically effective was a constant fraction of the external O3 dosein each O3 regime.

Although the nonlimiting water supply of the experimental

NIGHTTIME EXPOSURE TO OZONE 163

Figure 5. Stomatal pores in 6- to7-week-old leaves in O3-free air(A, B) or exposed to O3 (C, D) ei-ther in the dark (A, C) or in day-light (B, D).

plants may have favored stomatal opening at night, the plantsshowed a capacity for substantial O3 uptake at night and, as aconsequence, a reduction in biomass production. TroposphericO3 concentration is often highest in the afternoon and canremain enhanced during the night or may be permanentlyelevated at high altitudes (Lefohn and Jones 1986, Krupa andManning 1988, NAPAP 1991). We conclude that for definingcritical exposure for ozone in trees, a daily time basis of 24 his justified.

Acknowledgments

We gratefully acknowledge the technical assistance of Mr. U. Bühl-mann, Mr. P. Bleuler, Mr. R. Gall and Mr. A. Burkart in tending theplants and operating the O3 fumigation chambers. We also thank Dr.C. Scheidegger for his support in scanning electron microscopy. Thehelpful discussions with Dr. W. Landolt, Dr. J.B. Bucher and Dr. A.Polle and the stylistic editing of the English text by Mrs. M. Sieber are

highly appreciated. Part of the study was financed through theEUREKA 447 EUROSILVA program by the Swiss Bundesamt fürBildung und Wissenschaft.

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