Interactions of elevated CO2, NH3 and O3 on mycorrhizal infection,gas exchange and N metabolism in saplings of Scots pine

Four-year-old saplings of Scots pine (Pinus sylvestris) (L.) were exposed for 11 weeks in controlled-environment chambers to charcoad-filtered air, or to charcoal-filtered air supplemented with NH₃ (40 g m^–3), O₃ (110 g m^–3 during day/ 40 g m^–3 during night) or NH₃+O₃. All treatments were carried out at ambient (259 L L^–1) and at elevated CO₂ concentration (700 L L^–1). Total tree biomass, mycorrhizal infection, net CO₂ assimilation (P_n), stomatal conductance (g_s), transpiration of the shoots and NH₃ metabolization of the needles were measured. In ambient CO₂ (1) gaseous NH₃ decreased mycorrhizal infection, without significantly affecting tree biomass or N concentration and it enhanced the activity of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in one-year-old needles; (2) ozone decreased mycorrhizal infection and the acitivity of GS in the needles, while it increased the activity og GDH; (3) exposure to NH₃+O₃ lessened the effects of single exposures to NH₃ and O₃ on reduction of mycorrhizal infection and on increase in GDH activity. Similar lessing effects on mycorrhizal infection as observed in trees exposed to NH₃+O₃ at ambient CO₂, were measured in trees exposed to NH₃+O₃ at elevated CO₂. Exposure to elevated CO₂ without pollutants did not significantly affect any of the parameters studied, except for a decrease in the concentration of soluble proteins in the needles. Elevated CO₂ _NH₃ strongly decreased root branching and mycorrhizal infection and temporarily stimulated P_n and g_s. The exposure to elevated CO₂+NH₃+O₃ also transiently stimulated P_n. The possible mechanisms underlying and integrating these effects are discussed. Elevated CO₂ clearly did not alleviate the negative effects of NH₃ and O₃ mycoorhiral infection. The significant reduction of mycorrhizal infection after exposure to NH₃ or O₃, observed before significant changes in gas exchange or growth occurred, suggest the use of mycorrhizal infection as an early indicator for NH₃ and O₃ induced stress.Abbreviations DW dry weight - FA filtered air - FA_a filtered air at ambient CO₂ - FA_e filtered air at elevated CO₂ - FW fresh weight - GDH glutamate dehydrogenase - GS glutamine synthetase - g_s stomatal conductance - P_n net CO₂ assimilation - RWR root weight ratio - SRL specific root length     

Stomatal SO2 uptake and sulfate accumulation in needles of Norway spruce stands (Picea abies) in Central Europe

Monthly uptake rates and the annual deposition of gaseous SO₂ via the stomata of six Norway spruce canopies (Picea abies (L.) Karst.) in Germany (Königstein im Taunus, Witzenhausen, Grebenau, Frankenberg, Spessart, Fürth im Odenwald) were calculated (i) from statistical response functions of stomatal aperture depending on meteorological data, and (ii) from the synchronously measured SO₂ immission at these stands. The stomatal response functions had been derived on the basis of thorough stomatal water conductance measurements in the field. Calculations of the SO₂ conductance of spruce twigs and SO₂ uptake rates via stomata need continuously measured complete data sets of the (i) light intensity, (ii) air temperature, (iii) air humidity and (iv) SO₂ concentration in spruce forests from all the year. These data were recorded half hourly in different German spruce forests. The apparent needle water vapour pressure difference and transpiration rates were calculated from meteorological data. Additional use of canopy through flow data in dry years allowed the estimation of the mean stomatal conductance for H₂O and SO₂ of whole spruce canopies. The annual SO₂ uptake of a mean unit needle surface in spruce forests was 32% of the SO₂ uptake rate of exposed needles at the top of spruce crowns. There is significant SO₂ uptake all the year. The mean SO₂ dose at all sites and years received through the stomata was (0.25±0.07) mol SO₂ m^-2 (total needle surface) (nPa Pa^-1)^-1 (annual mean of SO₂ immission; 1 nPa (SO₂) Pa^-1 (air) = 1 ppb) day^-1 (vegetation period per year). Comparison of calculated SO₂ uptake rates into needles with measured SO₄ ^2- accumulation rates in needles from the mentioned sites and additionally from Würzburg, Schneeberg (Fichtelgebirge) and from three sites in the eastern Erzgebirge (Höckendorf, Kahleberg, Oberbärenburg) revealed that oxidative SO₂ detoxification (SO₄ ^2- formation) dominates only at sites with high SO₂ immission and short vegetation periods. Under these conditions 70 to 90% of the annual stomatal SO₂ uptake is detoxified via SO₄ ^2- accumulation in needles. Cations are needed for neutralization of accumulating SO₄ ^2- which are inavailable to support growth. Thus, SO₂ induces a dominant and competitive additional nutrient cation demand, cation deficiency symptoms and enhanced needle loss (spruce decline symptoms) mainly at sites, where the ratio R=(SO₂ immission): (length of the vegetation period) is higher than R=0.07 nPa Pa^-1 day^-1. Correlation analysis of the relative needle loss versus the SO₂-dependent SO₄ ^2- formation rate revealed a significant increase of needle loss at the 98% level (Student). At sites with small SO₂ immission and long vegetation periods (R<0.07 nPa Pa^-1 day^-1) reductive SO₂ detoxification via growth (and/or phloem export of SO₄ ^2-) is not kinetically overburdened. Under these conditions only 30% of the annual SO₂ uptake is detoxified via SO₄ ^2- formation and spruce decline is small or absent. On the basis of the critical value R0.07 nPa Pa^-1 day^-1 recommended SO₂ immission limits can be deduced on a mere ecophysiological basis. These deduced values are close to the proposed SO₂ immission limits of the IUFRO, WHO and the UNECE.     

The effect of air pollution on the mechanism of stomatal control

Summary A long-term field experiment permanently measuring gas exchange in the top of a 70-year-old spruce, continued for through the 1990 growing season. Two gas exchange chambers were run simultaneously under identical climatic conditions. One of two similar twigs was exposed to ambient air whereas the other received pure air. These experiments aimed to examine the ability of the stomata to control water balance, comparing pure and ambient air. This was done not only in natural climatic conditions but also in experimental, specifically maintained stress situations. Special care was taken to ensure that only steady state values of stomatal responses are related to the environmental stimuli. During a drought period lasting several weeks, overshooting transpiration values were documented for the ambient air. The two twigs do not merely differ in their control capacity, but the behaviour of the stomata in ambient air deviates from the norm. The increasingly uncontrolled water losses during the drought period have a negative effect on photosynthetic capacity. The influence of water deficit on stomatal response to other environmental factors (light, CO₂) is shown. Due to deficient control quality of the stomata lower stress tolerance in the face of drought is suggested in ambient air as compared with pure air. By tracing dysfunctions to structural changes in the cell walls of the stomatal apparatus, a mechanism is described explaining forest decline under the combined influence of air pollutants and drought stress.     

The effect of vapor pressure on stomatal control of gas exchange in Douglas fir (Pseudotsuga menziesii) saplings

Summary Increasing leaf to air vapor pressure deficit (VPD) caused reductions in stomatal conductance of both current year and previous season needles of Pseudotsuga menziesii saplings. The stomata of current year needles were found to be more responsive to changes in VPD than those of previous season needles. The reductions in stomatal conductance of current year needles were not associated with decreases in xylem pressure potential. In fact, the reductions in stomatal conductance of current year needles were sometimes sufficient to reduce transpiration and thus raise xylem pressure potential even though VPD was increasing. There was a decline in stomatal responsiveness to VPD in current year needles between early and late summer. Pressure-volume curves determined for different age needles at different times of the year suggested that differences and changes in stomatal responsiveness to VPD may have been caused in part by differences and changes in needle water potential components. Hexane washes of current year needles during the late summer succeeded in partially restoring their VPD sensitivity, suggesting that changes in the water permeability of the external cuticle during needle maturation may also have played a role in causing the summer decline in VPD responsiveness.In both current and previous year needles VPD-induced changes in stomatal conductance had a greater relative effect on transpiration (q _w) than on net photosynthesis (Ph_N). In maturing needles the ratio of the sensitivities of transpiration and net photosynthesis to changes in stomatal conductance, (q _w/g _s)/Ph_N/g _s), remained nearly constant as VPD was varied. This provides experimental support for a recent hypothesis that stomata respond to environmental fluctuations in such a manner as to maintain the above ratio constant, which optimizes CO₂ uptake with respect to water loss.     

Effect of temperature on net CO2 assimilation and photosystem II quantum yield of electron transfer of French bean (Phaseolus vulgaris L.) leaves during drought stress

The effect of leaf temperature on stomatal conductance and net CO₂ uptake was studied on French bean (Phaseolus vulgaris L.) using either dehydrated attached leaves (25–40% water deficit) or cut leaves supplied with 10^–4 M abscisic acid (ABA) solution to the transpiration stream. Decreasing leaf temperature caused stomatal opening and increased net CO₂ uptake (which was close to zero at around 25° C) to a level identical to that of control leaves (without water deficit) at around 15° C. (i) The ABA effect on stomatal closure was modulated by temperature and, presumably, ABA is at least partly responsible for stomatal closure of french bean submitted to a drought stress. (ii) For leaf temperatures lower than 15° C, net CO₂ uptake was no longer limited by water deficit even on very dehydrated leaves. This shows that dehydrated leaves retain a substantial part of their photosynthetic capacity which can be revealed at normal CO₂ concentrations when stomata open at low temperature. In contrast to leaves fed with ABA, decreasing the O₂ concentration from 21% to 1% O₂ did not increase either the rate of net CO₂ uptake or the thermal optimum for photosynthesis of dehydrated leaves. The quantum yield of PSII electron flow (measured by F/F_m) was lower in 1% O₂ than in 21% O₂ for each leaf pretreatment given (non-dehydrated leaves, dehydrated leaves, and leaves fed with ABA) even within a temperature range in which leaf photosynthesis at normal CO₂ concentration was the same in these two O₂ concentrations. It is concluded that this probably indicates an heterogeneity of photosynthesis, since this difference in quantum yield disappears when using high CO₂ concentrations during measurements.Abbreviations and Symbols ABA abscisic acid - F_m maximum chlorophyll fluorescence - F difference between steady-state chlorophyll fluorescence and F_m - PPFD photosynthetic photon flux density We would like to thank Dr. J.-M. Briantais (Laboratoire d'écologie végétale, Orsay, France) for help during fluorescence measurements and Ms. J. Liebert for technical assistance.     

Impairment of gas exchange and structure in birch leaves (Betula pendula) caused by low ozone concentrations

Summary Injury caused by low O₃ concentrations (0, 0.05, 0.075, 0.1 l 1^-1) was analyzed in the epidermis and mesophyll of fully developed birch leaves by gas exchange experiments and low-temperature SEM: (I) after leaf formation in O₃-free and ozonated air, and (II) after transferring control plants into ozonated air. In control leaves, autumnal senescence also was studied in O₃-free air (III). As O₃ concentration increased, leaves of (I) stayed reduced in size, but showed increased specific weight and stomatal density. The declining photosynthetic capacity, quantum yield and carboxylation efficiency lowered the light saturation of CO₂ uptake and the water-use efficiency (WUE). Carbon gain was less limited by the reduced stomatal conductance than by the declining ability of CO₂ fixation in the mesophyll. The changes in gas exchange were related to the O₃ dose and were mediated by narrowed stomatal pores (overriding the increase in stomatal density) and by progressive collapse of mesophyll cells. The air space in the mesophyll increased, preceded by exudate formation on cell walls. Ozonated leaves, which had developed in O₃-free air (II), displayed a similar but more rapid decline than the leaves from (I). In senescent leaves (III), CO₂ uptake showed a similar decrease as in leaves with O₃ injury but no changes in mesophyll structure and WUE. The nitrogen concentration declined only in senescent leaves in parallel with the rate of CO₂ uptake. A thorough understanding of O₃ injury and natural senescence requires combined structural and functional analyses of leaves.     

Histological examination of spruce needles from a long-term gas exchange experiment in pure and polluted air in the field

Summary At the end of a 4-year period of gas exchange measurements in a natural stand in the Lower Bavarian Forest, needles of an adult spruce [Picea abies (L.) Karst.] were harvested from two chambers, one with pure air and the other with ambient air. The needles were examined as to their histological properties in the stomatal apparatus and in the bundle sheath. In needles from the polluted air UV absorbance at 280 nm was decreased in the walls of the stomatal apparatus. Simultaneously, the deposition of compounds with an absorption maximum at 310 nm increased within the encrusted plate-like thickenings of the subsidiary cells. The contents of the lumina of hypodermal cells and of the bundle sheath exhibited a greater degree of autofluorescence in ambient-air material than in pure-air leaf organs. Differences between needles exposed to pure and polluted air are gradual. The damaged condition is rare in pure air, common in polluted air. The needles from outside the chambers occupied an intermediate position between pure-air and ambient-air needles. This fact is traced to an unnaturally high pollutant load in the liquid phase of the needle surfaces within the ambient-air chamber because in order to compensate pollutant losses within the system, SO₂ and O₃ were added even during periods of irrigation. The reduction of absorption capacity at 280 nm in the walls of the stomatal apparatus is attributed to destruction of lignin due to the high reactivity of the pollutants in the liquid phase on the damp needle surface. The importance of delignification with regard to hydroregulation is discussed.     

Free Radical Scavengers and Photosynthetic Pigments in Pinus Cembra L. Needles as Affected by Ozone Exposure

The goal of this study was the characterization of the antioxidative protection system of current and 1-year-old needles of a cembran pine (Pinus cembra L.) and its possible responses to elevated concentrations of atmospheric O₃. Twigs of a mature cembran pine at the alpine timberline (1950 m a.s.l.) were exposed in climate-controlled twig chambers for 91 d to charcoal-filtered air (CF), ambient air O₃ concentration (A), and two-fold ambient air O₃ concentration (2A). Additionally, a chamberless control group (AA) was used to examine chamber effects. At the end of the fumigation period the contents of free radical scavengers and photosynthetic pigments were measured in the needles. Independent from O₃ exposure, total ascorbate and -tocopherol contents were higher in 1-year-old needles compared to the current flush while the opposite was found for glutathione. The amounts of pigments and antioxidants in P. cembra needles were comparable to those in other conifers growing at high-elevation sites. The only hint toward O₃ induced changes in the composition of antioxidants was an increase in the glutathione redox state toward more oxidation in 1-year-old needles upon exposure to A or AA conditions, but not upon 2A exposure. Chlorophyll and carotenoid contents were not affected by O₃ neither in current- nor in previous-year needles. The de-epoxidation state of the xanthophyll cycle pigments, however, was significantly increased in 1-year-old needles under A and AA compared to the CF control, but not under 2A. Hence, Pinus cembra, which is well adapted to the extreme environment of the timberline ecotone, exhibited only marginal biochemical changes in response to elevated O₃.     

Performance of two Picea abies (L.) Karst. stands at different stages of decline

Summary Photosynthetic rates and nutrient contents of spruce needles were measured in a region with high levels of air pollution in NE Bavaria, Germany (FRG), and compared to spruce grown under clean air conditions at Craigieburn, in the South Island of New Zealand (NZ). The absolute rates of CO₂ uptake, the slope of the CO₂ response curve at 240 l l^–1 internal CO₂ concentration, and the change of photosynthetic rates with needle age at ambient and saturated CO₂ concentrations were virtually identical at both measuring sites. These results confirm an earlier conclusion, that there is no long-term effect of atmospheric pollutants directly on photosynthetic CO₂ uptake rates with persistent exposure at the FRG site to high levels of anthropogenic air pollution. Photosynthetic capacity at saturating CO₂ concentration was three times higher in the NZ spruce. Needles with high photosynthetic capacity in NZ had lower nitrogen and higher calcium concentrations per unit dry weight but higher concentrations of nitrogen, phosphorus, potassium, magnesium and calcium per unit leaf area, and twice the specific leaf weight.     

Ameliorating effect of UV-B radiation on the response of Norway spruce and Scots pine to ambient ozone concentrations

Elevated levels of both ozone and UV-B radiation are typical for high-altitude sites. Few studies have investigated their possible interaction on plants. This study reports interactive effects of O₃ and UV-B radiation in four-year-old Norway spruce and Scots pine trees. The trees were cultivated in controlled environmental facilities under simulated climatic conditions recorded on Mt Wank, an Alpine mountain in Bavaria, and were exposed for one growing season to simulated ambient or twice-ambient ozone regimes at either near ambient or near zero UV-B radiation levels. Chlorotic mottling and yellowing of current year needles became obvious under twice-ambient O₃ in both species at the onset of a high ozone episode in July. Development of chlorotic mottling in relation to accumulated ozone concentrations over a threshold of 40 nL L^–1 was more pronounced with near zero rather than ambient UV-B radiation levels. In Norway spruce, photosynthetic parameters at ambient CO₂ concentration, measured at the end of the experiment, were reduced in trees cultivated under twice-ambient O₃, irrespective of the UV-B treatment. Effects on photosynthetic capacity and carboxylation efficiency were restricted to trees exposed to near zero levels of UV-B radiation, and twice-ambient O₃. The data indicate that UV-B radiation, applied together with O₃, ameliorates the detrimental effects of O₃. The data also demonstrate that foliar symptoms develop more rapidly in Scots pine than in Norway spruce at higher accumulated ozone concentrations. Symbols and abbreviations: LSD, least significant difference; PAS300, UV-B irradiance weighted according to the plant action spectrum of Green et al. (1974) normalized at 300 (nm); AOT40, (AOT = accumulated over threshold) reflects the sum of hourly ozone concentrations above 40 nL L^–1 during daylight hours (> 50 Wm^–2) ( Kärenlampi & Skärby 1996 ); A₃₅₀, net photosynthesis at ambient CO₂; G₃₅₀, stomatal conductance for water vapour at ambient CO₂; A₂₅₀₀, net photosynthesis at saturating CO₂ (maximal potential photosynthetic activity); CE, carboxylation efficiency; ROS, reactive oxygen species; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; GLM, general linear model.     

Rapid stomatal closure triggered by a short ozone pulse is followed by reopening to overshooting values

Stomatal pores, surrounded by the pairs of guard cells, regulate plant gas exchange. Correct stomatal regulation is crucial for plant survival under various stress conditions. We have recently utilized the air pollutant ozone (O₃) to study stomatal signaling and showed that application of O₃ induces rapid decrease in stomatal conductance. Here we have addressed the recovery of stomatal conductance and show that after exposures of plants to high O₃ pulses stomatal conductance recovered faster, reaching higher, “overshooting” values than were the pre-exposure values. We propose the hypothetical mechanism for this phenomenon and discuss it in the frames of current stomatal signaling models.Key words: ozone, stomata, signaling, Arabidopsis, overshooting, guard cells, stressRapid progress in understanding structural and molecular mechanisms of the core abscisic acid (ABA) signaling pathway and subsequent stomatal closure (reviewed in ref. ¹) has been achieved by using a variety of mostly in vitro technologies and approaches. Data on early induction of stomatal response by a brief ABA pulse in vivo is almost absent, largely due to difficulties in rapid removal of ABA from intact guard cells. Application of O₃, an air pollutant efficiently utilized to study stomatal signaling,^2–4 lacks this disadvantage and allows monitoring stomatal responses to brief, clean-cut, strictly dosed pulses of this powerful oxidant in planta. Application of O₃ for 1 min to intact Arabidopsis rosette triggered a Rapid Transient Decrease (RTD) in stomatal conductance which, after lasting for 8–10 min, was followed by a 3–4 times slower recovery.³ The entire RTD, lasting for up to 40–50 min, is a conserved response in plants; to date it is found to be present in about 90 Arabidopsis ecotypes/mutants³ and also in tobacco and birch (unpublished results). Absence of RTD in protein phosphatase ABI1 and ABI2 mutants (abi1-1 and abi2-1) which are unable to form complex with PYR/PYL ABA receptors, in protein kinase OST1 and in guard cell plasma membrane anion channel SLAC1 mutants, indicates that O₃-triggered signal propagates through the same phosphatase/kinase pair as does the signal triggered by ABA.³ Results of mostly proteomic, pharmacological and electrophysiological studies allow to suggest that the most likely reason for the rapid stomatal closure during RTD is the ABI1, ABI2 and OST1 mediated alterations in a battery of plasma membrane ion channels, including the outward-rectifying anion channel SLAC1 and the depolarization-activated K⁺ channel GORK1 which after their sequential activation result in efflux of osmotica, turgor loss and stomatal closure.Physiological background of the recovery during RTD which takes place also under continuous exposure to ozone² is less understood. To study this process further we exposed whole rosettes of intact 22–25 day old Arabidopsis plants to different O₃ concentrations for 3 min as described earlier³ and observed that after exposures to high concentration O₃ pulses stomatal conductance recovered faster and reached higher values than were the preexposure values. We term this phenomenon “overshooting”.Ozone concentration of 70 nl l⁻¹ did not induce RTD (Fig. 1A). At higher concentrations O₃ induced intense decrease in stomatal conductance within 4–6 min after application. This was followed by rapid stoppage of the closure, a brief transition period and a sluggish, almost linear recovery where the pre-exposure value of stomatal conductance was reached about 30 min after the onset of O₃ (Fig. 1A). The rates and extents of the O₃-induced stomatal closure, as well as rates of reopening were concentration dependent. Continuation of the linear increase in stomatal conductance after reaching the pre-exposure value resulted in almost two-fold higher values at 50 min after the onset of 385 nl l⁻¹ of O₃. Overshootings were dependent on ozone concentration (Fig. 1B) and on the extent of the initial decrease in stomatal conductance (Fig. 1C). Both dependencies were exponential indicating a presence of threshold at 150–200 nl l⁻¹ of O₃ and at 20% of initial O₃-induced decrease in stomatal conductance, respectively.Open in a separate windowFigure 1Ozone-triggered rapid decrease in stomatal conductance is followed by recovery to higher “overshooting” values. (A) Typical asymmetric time patterns of stomatal conductance after exposure of 22–25 day old Arabidopsis plant leaf rosettes to different concentrations of ozone as described in Kollist et al.² In (B and C) O₃-induced “overshooting” is plotted against O₃ concentration and O₃-induced decrease in stomatal conductance, respectively.What could be the reason and mechanistic explanation for described O₃-induced “overshooting” in stomatal conductance? The protein kinase OST1 is required for induction of rapid closure phase of the O₃-triggered RTD.³ Besides phosphorylating SLAC1,^3,5 OST1 has been shown to phosphorylate also the inward-rectifying K⁺ channel KAT1 resulting in its inhibition.⁶ Inhibition of K⁺ uptake, which allows faster membrane depolarization and stomatal closure, has been shown to occur under various stresses.⁷ Presumably, H⁺-ATPase activity and proton pumping, tightly coupled to K⁺ uptake via channel energization⁸ are also suppressed by O₃. It has been shown that in depolarized guard cell, plasma membrane proton pumping may precede volume and turgor increase.⁹ We speculate that in the O₃-triggered, SLAC1- and GORK-mediated stomatal closure, when ion efflux and turgor loss proceed at high rates, reactivation of H⁺-ATPase and proton pumping and associated recovery of K⁺ uptake are induced to avoid guard cell plasmolysis.¹⁰ Guard cells begin to regain turgor and stomata reopen. At the same time outward-rectifying ion channels are transiently locked (inactivated) as stomata become completely insensitive to repeated O₃-pulses during recovery phase.³ This interpretation is supported by our observation that the recovery in stomatal opening is heavily suppressed in kincless mutant³ where the inward rectifying K⁺ current is abolished.¹¹ In addition, peak densities of inward K⁺ currents (2–4 µA/cm² membrane⁹) are shown to be much lower than those for outward anion and K⁺ currents (17–20 µA/cm).^2,8,12,13 This could be a reason why stomatal reopening is much slower than the initial O₃-induced closure. Our findings (Fig. 1) suggest that the faster and deeper the O₃-triggered turgor loss, the faster and extensive is its recovery. The “overshootings” suggest plasma membrane hyperpolarization and predict a viable oscillation-like stomatal behavior where the system tends to restore the initial equilibrium. Longer experiments are needed to address whether such an oscillating response exists in Arabidopsis elicited by O₃.Taken together, our data suggest the presence of a “security” mechanism in plant guard cells which avoids the excessive dehydration and precipitous turgor loss by reswitching the reaccumulation of osmotica ultimately leading to stomatal opening. Molecular mechanism(s) linking feedback from low turgor to activation of plasma membrane proton pumping and subsequent ion uptake are obscure. Irrespective of mechanism(s), our data indicate that stomata tend to recover from stress the faster the stronger has been the perturbation at its onset. Undoubtedly, rapid O₃-induced transient decrease in stomatal conductance is one of countless expressions of the Le Chatelier''s principle having numerous wordings like: “any change in status quo prompts an opposing reaction in the responding system,” or paraphrased on the basis of our results—the stronger the stimulus (O₃ concentration) the stronger the response (“overshooting”).     

Photosynthesis of cotton plants exposed to elevated levels of carbon dioxide in the field

The cotton (Gossypium hirsutum L.) plant responds to a doubling of atmospheric CO₂ with almost doubled yield. Gas exchange of leaves was monitored to discover the photosynthetic basis of this large response. Plants were grown in the field in open-top chambers with ambient (nominally 350 l/l) or enriched (nominally either 500 or 650 l/l) concentrations of atmospheric CO₂. During most of the season, in fully-irrigated plants the relationship between assimilation (A) and intercellular CO₂ concentration (c_i) was almost linear over an extremely wide range of c_i. CO₂ enrichment did not alter this relationship or diminish photosynthetic capacity (despite accumulation of starch to very high levels) until very late in the season, when temperature was somewhat lower than at midseason. Stomatal conductance at midseason was very high and insensitive to CO₂, leading to estimates of c_i above 85% of atmospheric CO₂ concentration in both ambient and enriched chambers. Water stress caused A to show a saturation response with respect to c_i, and it increased stomatal closure in response to CO₂ enrichment. In fully-irrigated plants CO₂ enrichment to 650 l/l increased A more than 70%, but in water-stressed plants enrichment increased A only about 52%. The non-saturating response of A to c_i, the failure of CO₂ enrichment to decrease photosynthetic capacity for most of the season, and the ability of the leaves to maintain very high c_i, form in part the basis for the very large response to CO₂ enrichment.Abbreviations c_a- atmospheric CO₂ concentration - c_i- intercellular CO₂ concentration - A- rate of assimilation of CO₂ - g_s- stomatal conductance to water vapor - g_b- boundary layer conductance to water vapor - g_m- mesophyll conductance to CO₂ - VPD- vapor pressure deficit - _w leaf water potential - L- stomatal limitation to CO₂ uptake     

Estimates of potential ozone stomatal uptake in mature trees of Quercus ilex in a Mediterranean climate

This paper is focused on evaluation of the potential ozone stomatal fluxes (FO₃) under seasonally varying microclimatic conditions at two levels of the canopy of an evergreen Mediterranean plant species, Holm oak (Quercus ilex L.), and on comparing them with ozone hourly averages, in order to assess what are the main environmental/physiological variables that most affect the definition of critical levels for Mediterranean vegetation. Microclimatic factors such as radiation, temperature and wind velocity greatly affected gas exchange rates and stomatal conductance to water vapour measured at different heights. O₃ concentration values highlight a daily cycle with higher nocturnal O₃ concentrations above the canopy than below it. Similar O₃ trends have been observed by using passive diffusive samplers. As a consequence, potential O₃ stomatal fluxes calculated for the upper layer of the canopy are higher than those below the canopy. Moreover, O₃ concentration values show an opposite seasonal trend compared to FO₃. These opposite trends are clearly due to the stomatal closure in drier months, as a protection against excessive water losses that yield low FO₃ values during high O₃ concentration. This paper highlights the different contribution of different Holm oak canopy portions to overall O₃ uptake impact, attributing important roles to microclimatic conditions and to physiological activity related to stomata opening, which in turn is affected by internal and external effectors (hormones, water availability, hydraulic conductance, etc.). We cannot exclude an O₃ effect on stomatal cell guards and on the carbon assimilation process working in the mesophyll cells. Further research needs to be considered to evaluate more clearly the risk of O₃ on Mediterranean vegetation taking into consideration microclimatic conditions, plant physiology and possible plant canopy defensive reactions to O₃, so as to define an air quality standard to protect the vegetation.     

Influence of elevated ozone and limited nitrogen availability on conifer seedlings in an open-air fumigation system: effects on growth, nutrient content, mycorrhiza, needle ultrastructure, starch and secondary compounds

Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies Karst.) seedlings were exposed to realistically elevated O₃ levels in open‐air experiments over three growing seasons. The total O₃ exposure doses were 1.2 × (1991), 1.5 × (1992) and 1.7 × (1993) ambient levels. During the 1992 and 1993 growing seasons pine and spruce seedlings received two different levels of nitrogen supply. Effects on growth, mycorrhiza formation, needle ultrastructure, primary and secondary compounds were studied. Ozone exposure had only slight effects on biomass production, growth height and nutrient content of studied conifers. Higher nitrogen availability improved growth of the seedlings and resulted in higher concentration of nitrogen in needles. In Scots pine O₃ exposure did not have effects on quantity of total mycorrhizas and short roots, while higher nitrogen availability decreased quantity of mycorrhizas and short roots. In both tree species O₃ exposure induced O₃‐related ultrastructural symptoms, e.g. granulation and dark staining of the chloroplast stroma in the needle mesophyll cells, at both nitrogen availability levels. Ozone exposure and nitrogen availability did not have significant effects on starch concentrations in either tree species. Concentrations of some individual terpenes were higher in O₃‐exposed needles, while concentrations of individual and total resin acids, total phenolics and catechins were not affected by O₃ exposure. Nitrogen availability did not have substantial effects on concentrations of monoterpenes. By contrast, concentrations of some individual and total resin acids were lower in pine needles and higher in spruce needles with higher nitrogen availability, while phenolic concentration in spruce needles decreased at higher nitrogen availability. The results suggest that realistically elevated levels of O₃ in the field can have some negative effects on the mesophyll ultrastructure of conifer needles, but carbon allocation to root and shoot growth and secondary metabolites are not affected substantially.     

Effects of elevated O3 and CO2 concentrations on photosynthesis and stomatal conductance in Scots pine

Naturally regenerated Scots pines (Pinus sylvestris L.), aged 28–30 years old, were grown in open-top chambers and subjected in situ to three ozone (O₃) regimes, two concentrations of CO₂, and a combination of O₃ and CO₂ treatments From 15 April to 15 September for two growing seasons (1994 and 1995). The gas exchanges of current-year and 1-year-old shoots were measured, along with the nitrogen content of needles. In order to investigate the factors underlying modifications in photosynthesis, five parameters linked to photosynthetic performance and three to stomatal conductance were determined. Elevated O₃ concentrations led to a significant decline in the CO₂ compensation point (Г^*), maximum RuP₂-saturated rate of carboxylation (V_cmax), maximum rate of electron transport (J_max), maximum stomatal conductance (g_smax), and sensitivity of stomatal conductance to changes in leaf-to-air vapour pressure difference (?g_s/?D_v) in both shoot-age classes. However, the effect of elevated O₃ concentrations on the respiration rate in light (R_d) was dependent on shoot age. Elevated CO₂(700 μmol mol^?1) significantly decreased J_max and g_smax but increased R_d in 1-year-old shoots and the ?g_s/?D_v in both shoot-age classes. The interactive effects of O₃ and CO₂ on some key parameters (e.g. V_cmax and J_max) were significant. This may be closely related to regulation of the maximum stomatal conductance and stomatal sensitivity induced by elevated CO₂. As a consequence, the injury induced by O₃ was reduced through decreased ozone uptake in 1-year-old shoots, but not in the current-year shoots. Compared to ambient O₃ concentration, reduced O₃ concentrations (charcoal-filtered air) did not lead to significant changes in any of the measured parameters. Compared to the control treatment, calculations showed that elevated O₃ concentrations decreased the apparent quantum yield by 15% and by 18%, and the maximum rate of photosynthesis by 21% and by 29% in the current-year and 1-year-old shoots, respectively. Changes in the nitrogen content of needles resulting from the various treatments were associated with modifications in photosynthetic components.     

Leaf Conductance as Related to Xylem Water Potential and Carbon Dioxide Concentration in Sitka Spruce

Current year shoots of Sitka spruce [Picea sitchensis Bong. (Carr.)] from the forest canopy were equilibrated in a leaf chamber. The shoots were excised in air, and removed at differing times in order to establish a relationship between stomatal conductance and xylem water potential. The experiment was repeated at five ambient CO₂ concentrations. A second set of excised forest shoots, and shoots excised from 2-year- old nursery seedlings were allowed to evaporate freely in a controlled environment wind tunnel until a constant rate of transpiration was measured, to establish a relationship between cuticular conductance and xylem water potential. Cuticular conductance was estimated to be 0.012 cm s^-1 at high water potential and declined linearly to 0.007 cm s^-1 at ?3.5 MPa. The implication of this decline in the subsequent calculation of stomatal and mesophyll conductance is considered. Stomatal conductance remained constant at water potentials above ?1.4 MPa and was not affected by ambient carbon dioxide concentrations between 20 and 600 cm^-3. At lower water potentials, stomatal conductance declined and approached zero at ?2.5 to ?2.6 MPa. The results suggest that stomatal aperture is not controlled by either ambient or intercellular space carbon dioxide concentration, and that stomatal closure at low water potential is unlikely to be mediated by carbon dioxide.     

Changes in gas exchange characteristics and water use efficiency of mangroves in response to salinity and vapour pressure deficit

Summary Measurements were made of the photosynthetic gas exchange properties and water use efficiency of 19 species of mangrove in 9 estuaries with different salinity and climatic regimes in north eastern Australia and Papua New Guinea. Stomatal conductance and CO₂ assimilation rates differed significantly between species at the same locality, with the salt-secreting species, Avicennia marina, consistently having the highest CO₂ assimilation rates and stomatal conductances. Proportional changes in stomatal conductance and CO₂ assimilation rate resulted in constant and similar intercellular CO₂ concentrations for leaves exposed to photon flux densities above 800 mol·m^-2·s^-1 in all species at a particular locality. In consequence, all species at the same locality had similar water use efficiencies. There were, however, significant differences in gas exchange properties between different localities. Stomatal conductance and CO₂ assimilation rate both decreased with increasing salinity and with increasing leaf to air vapour pressure deficit (VPD). Furthermore, the slope of the relationship between assimilation rate and stomatal conductance increased, while intercellular CO₂ concentration decreased, with increasing salinity and with decreasing ambient relative humidity. It is concluded from these results that the water use efficiency of mangroves increases with increasing environmental stress, in this case aridity, thereby maximising photosynthetic carbon fixation while minimising water loss.Contribution No. 459 from the Australian Institute of Marine Science     

Stomatal Response and Leaf Injury of Pisum sativum L. with SO(2) and O(3) Exposures : I. INFLUENCE OF POLLUTANT LEVEL AND LEAF MATURITY

Plants of Pisum sativum L. `Alsweet' were grown under a controlled environment and exposed to SO₂ and O₃ to determine whether changes in stomatal aperture during exposure were related to subsequent leaf injury. Stomata consistently closed with injurious levels of SO₂ and O₃. Measurements with diffusion porometers demonstrated 75 and 25% lower conductance with SO₂ and O₃ exposures, respectively, compared to the conductance of control plants. Stomata also showed a closing response with noninjurious levels of SO₂ but an opening response with noninjurious levels of O₃. Stomata closed to the same degree with combinations of SO₂ plus O₃ as with SO₂ alone. Stomata of expanding leaves closed more during pollutant exposures than stomata of expanded leaves. The abaxial and adaxial stomata both exhibited closure with SO₂ and combinations of SO₂ plus O₃, but abaxial stomata tended to close and adaxial stomata tended to open with exposure to O₃ alone.     

基于树干液流技术的北京市刺槐冠层吸收臭氧特征研究

全球范围内加速的城市化导致空气质量严重退化。随着北京市建设范围不断扩大和机动汽车数量迅猛增长,空气污染日益严重。浓度不断增加的近地层臭氧作为影响全球气候变化的重要因素和危害人类健康、动植物生长的二次污染物,受到广泛关注。城市树木能够有效地去除大气污染物,进而提高空气质量。目前已有很多研究关于区域尺度上城市树木吸收臭氧,然而,冠层尺度上城市树木吸收臭氧特征少有研究。因此,本文基于树干液流技术,结合天气变化和大气臭氧浓度分析,研究夏秋季节北京市典型绿化树种刺槐（Robinia pseudoacacia）整树冠层吸收臭氧特征及环境影响因素。结果表明,在日尺度上,刺槐吸收臭氧速率变化呈单峰曲线,于下午15:00左右达到峰值;夏季峰值范围较宽,秋季峰值范围较窄;中午前后累积吸收臭氧量增加最明显。在季节尺度上,夏季刺槐吸收臭氧速率高于秋季;夏季累积吸收臭氧量显著增加,秋季略有增加。刺槐吸收臭氧的时间变化规律取决于大气臭氧浓度和冠层对臭氧的导度。臭氧浓度日变化和季节变化明显,导致刺槐吸收臭氧速率时间变化格局与之接近。在一定的臭氧浓度下,刺槐吸收臭氧速率的变化主要由冠层对臭氧的导度调控,进而受水汽压亏缺和总辐射的影响。随着水汽压亏缺降低,刺槐冠层对臭氧的导度明显下降;总辐射大于600 W/m2,冠层对臭氧的导度迅速下降。研究树种刺槐单位冠层投影面积上年吸收臭氧量约为0.16 g/m2,明显低于基于模型得到的结果,表明评估森林受臭氧危害的风险应考虑树种冠层臭氧通量。     

Thermoregulation in a large bird,the emu (Dromaius novaehollandiae)

The emu is a large, flightless bird native to Australia. Its habitats range from the high snow country to the arid interior of the continent. Our experiments show that the emu maintains a constant body temperature within the ambient temperature range-5 to 45°C. The males regulate their body temperature about 0.5°C lower than the females. With falling ambient temperature the emu regulates its body temperature initially by reducing conductance and then by increasing heat production. At-5°C the cost of maintaining thermal balance is 2.6 times basal metabolic rate. By sitting down and reducing heat loss from the legs the cost of homeothermy at-5°C is reduced to 1.5 times basal metabolic rate. At high ambient temperatures the emu utilises cutaneous evaporative water loss in addition to panting. At 45°C evaporation is equal to 160% of heat production. Panting accounts for 70% of total evaporation at 45°C. The cost of utilising cutaneous evaporation for the other 30% appears to be an increase in dry conductance.Abbreviations A _r Effective radiating surface area - BMR basal metabolic rate - C _dry dry conductance - CEWL cutaneous evaporative water loss - EHL evaporative heat loss - EWL evaporative water loss - FECO₂ fractional concentration of CO₂ in excurrent air - FF_H2O water content of chamber excurrent air - FEO₂ fractional concentration of O₂ in chamber excurrent air - FICO₂ fractional concentration of CO₂ in incurrent air - FIO₂ fractional concentration of O₂ in chamber incurrent air - MHP metabolic heat production - MR metabolic rate - REWL respiratory evaporative water loss - RH relative humidity - RQ respiratory quotient ; - SA surface area - SEM standard error of the mean - SNK Student-Newman-Keuls multiple range test - STPD standard temperature and pressure dry - T _a ambient temperature(s) - T _b body temperature(s) - T _e surface temperature(s) - flow rate of air into the chamber - carbon dioxide production - oxygen consumption - vapour pressure of water