Trees (1998) 12: 196 ± 200

Ó Springer-Verlag 1998

O R I G I N A L A RT I C L E

C. Nali ? L. Guidi ? F. Filippi ? G.F. Soldatini G. Lorenzini

Photosynthesis of two poplar clones contrasting in O3 sensitivity

Received: 30 June 1997 / Accepted: 3 September 1997

AbstractmTwo clones of poplar known for their phenomenological difference in response to ozone were fumigated with 150 nl l±1 of ozone for 5 h. In both clones the treatment significantly reduced the light-saturated rate of CO2 uptake of recently mature leaves and this was accompanied by a decrease in stomatal conductance. Intercellular CO2 concentration of the resistant clone increased following the fumigation. After 20 h of recovery, photosynthesis recovered completely only in the resistant clone. Electrolyte leakage of leaf disks increased in both clones to indicate damage to membranes; after the recovery time this parameter only reached values of the control in the resistant clone. The photochemical efficiency of PSII slightly decreased in the resistant clone. In the other clone, the treatment caused a decline of all chlorophyll fluorescence parameters and only some of them returned to normal values after the recovery time. The physiological response appears to be different in the two clones. In the resistant one, the most probable mechanism involved in the photosynthetic reduction was a regulatory reduction in CO2 fixation. Also data obtained by the solute leakage indicate that in the resistant clone repair mechanisms play a role. The reduction of photosynthesis observed in the sensitive clone is related both to strong stomatal closure and to an impairment in fluorescence parameters. These alterations can indicate a general disruption at the membrane level as confirmed by the solute leakage data. Key wordsmGas exchange ? Chlorophyll fluorescence Membrane leakage ? Populus C. Nali ? G. Lorenzini Dipartimento di Coltivazione e Difesa delle Specie Legnose, Sezione Patologia Vegetale, UniversitaÁ degli Studi, Via del Borghetto, 80 I-56124 Pisa, Italy L. Guidi ? F. Filippi ? G.F. Soldatini ( ) Dipartimento di Chimica e Biotecnologie Agrarie, UniversitaÁ degli Studi, Via del Borghetto, 80 I-56124 Pisa, Italy Tel.: +39-50-571557; Fax: +39-50-598614; e-mail [email protected]

Introduction

Among the gaseous air pollutants, ozone (O3) is the most prevalent during the growing season and is a potent, widespread toxicant that causes significant biological and economic damage to plants (Adams et al. 1989). Relatively little is known about O3-stress tolerance in forest trees; even though many reports are published on the effects of O3 on tree growth (see Pye 1988 for a review), the intimate mechanisms underlying such effects are not well defined. Coleman et al. (1995a) reported that the negative effects of O3 on biomass production in aspen clones were largely determined by lower photosynthetic productivity. In another work the same group (Coleman et al. 1995b) reported that the different response of these clones was related to the maintenance of high photosynthetic rate in recently mature leaves. Genetic variations in response to O3 exposures of trees have been found (Wang et al. 1986; Coleman et al. 1995a, b), but very little is known about the mechanism(s) involved. The toxicology of this pollutant is very complex, many factors such as leaf age playing important roles in determining the overall plant response (Guderian et al. 1985). In a previous study (Guidi et al. 1997a) we observed that fully mature (symptomatic) leaves of two poplar clones differing in their phenomenological response to O3 both showed a decline of photosynthetic activity following fumigation, but the recovery attitude differed, with only the resistant clone being able to fully restore the photosynthetic machinery. Since the response of leaves showing visible symptoms is complicated by contemporary phenomena of senescence in this work we examine the response of photosynthetic performance in asymptomatic leaves of these two clones exposed to a realistic pulse of O3.

197 Table 1mEffects of a pulse of O3 (150 nl l±1 for 5 h) on average solute leakage from disks of leaves of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana I-214, O3resistant). The analyses were carried out at the end of the fumigation and after 20 h of recovery in clean air. Values represent the mean of five replications. In each column, numbers flanked by the same letters are not different for P = 0.05 Solute leakage (% of total)

Filtered air Ozonated Recovery

Eridano (O3-sensitive) 5.8 a 9.5 b 12.3 c

I-214 (O3-resistant) 8.5 ab 10.3 b 7.0 a

Materials and methods Plant material Rooted cuttings of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana clone I-214, O3-resistant) were grown for 2 months in plastic pots containing a steam sterilized soil:peat:perlite (1:1:1 volume) mix in a greenhouse and watered every other day. Uniform plants were selected when ten leaves were fully expanded and when they were 25±30 cm tall. Exposure chambers and fumigation techniques O3 treatments were performed in a Perspex fumigation apparatus placed inside a walk-in growth chamber. Temperature was maintained at 20+1 °C, RH at 85+5%; a photon flux density at plant height of 530 mmol photons m±2s±1 was provided by incandescent lamps for a 12-h photoperiod. O3 was generated by a silent electrical discharge in pure oxygen. Details of the fumigation facilities are reported elsewhere (Lorenzini et al. 1994). Plants were pre-adapted to the chamber conditions for 48 h and exposed to a single pulse of 150 nl l±1 of O3 for 5 h from 0900 to 1400 h. Another group of plants for each clone, which were exposed to charcoal-filtered air, was used as control. Gas exchange measurements These were carried out using an open system (Heinz Walz, Effeltrich, Germany). The apical portions of leaves were enclosed in a gas exchange chamber with a volume of approximately 500 ml. The cuvette was equipped with a light source which provided maximal photon flux densities greater than 1000 mmol m±2s±1 photosynthetically active radiation. Cuvette temperature was controlled by a Peltier battery and standardized at 25 °C. Incoming air humidity was controlled at a dew point of 16 °C, thereby producing an air RH of about 60%. The CO2 uptake and release as well as the water loss of the plant material in the chamber were determined by differential measurement techniques, using an infra-red gas analyser (Binos, Leybold-Heraeus, Germany). The readings were performed at the end of the treatment and after a 20-h recovery in filtered air. Data obtained for differentials in CO2 and water vapour, light, temperature and humidity were displayed and processed on-line by means of a specific data acquisition system. Measurements of gas exchange in the leaf were performed at 345 ml l±1 CO2, 25+0.2 °C and at 60+10.3% RH. The maximum photosynthetic rate under light saturation was determined at light saturation levels (800±900 mmol m±2s±1) and each value was recorded when photosynthesis had reached a steady-state level. Gas exchange parameters were computed according to Von Caemmerer and Farquhar (1981).

Fig. 1mEffects of O3 (150 nl l±1 for 5 h) on the photosynthetic activity at saturating light (A), stomatal conductance (B) and intercellular CO2 concentration (C) in the recently mature leaves of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana I-214, O3-resistant). The analyses were carried out at the end of the fumigation and after 20 h of recovery in clean air. Each bar represents the mean of three replications. In each sub-graph, bars marked with the same letters are not different for P = 0.05 Chlorophyll fluorescence analysis A pulse amplitude modulation fluorometer (PAM-2000, Heinz Walz, Effeltrich, Germany) was used to monitor chlorophyll fluorescence and to measure the status of the electron transport of PSII. Details of procedures are given elsewhere (Guidi et al. 1997b). Chlorophyll fluorescence was determined in vivo on the same leaves utilized for gas exchange, after 30 min of dark adaptation. The quenching components qP and qNP were calculated as defined by Schreiber et al. (1986), while the actual quantum yield of PSII was computed as reported by Genty et al. (1989).

Solute leakage Disks (10 mm) were excised between 2nd-order veins in leaves homologous for age to those used for gas exchange and washed in distilled water for 10 min in a cheesecloth bag, in order to eluate the exogenous material and endogenous electrolytes released by the cut surface. Twenty disks taken from three plants were placed in 10 ml

198 Table 2mEffects of a pulse of O3 (150 nl l±1 for 5 h) on transpiration rate (E) and water use efficiency (WUE) in leaves of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana I-214, O3-resistant). The analyses were carried out at the end of the fumigation and after 20 h of recovery in clean air. Values represent the mean of three replications. In each column, numbers flanked by the same letters are not different for P = 0.05

Eridano (O3-sensitive) Filtered air Ozonated Recovery I-214 (O3-resistant) Filtered air Ozonated Recovery

E (mmol H2O m±2 s±1)

WUE (mmolCO2/ mmol H2O)

2.96 a 1.33 c 2.05 b

2.07 a 2.22 a 2.09 a

2.49 b 1.66 c 3.15 a

2.60 a 1.87 b 2.05 b

Table 3mQuenching analysis coefficients determined on leaves of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana I-214, O3-resistant) exposed to a pulse of O3 (150 nl l±1 for 5 h). The analyses were carried out at the end of the fumigation and after 20 h of recovery in clean air. Each value represents the mean of five replications. In each sub-column, numbers flanked by the same letters are not different for P = 0.05

Eridano (O3-sensitive) Filtered air Ozonated Recovery I-214 (O3-resistant) Filtered air Ozonated Recovery

qP

qNP

DF/Fm,

0.607 a 0.584 a 0.632 a

0.471 a 0.544 ab 0.608 b

0.391 a 0.341 a 0.374 a

0.681 a 0.635 a 0.693 a

0.390 a 0.494 b 0.492 b

0.473 a 0.400 a 0.434 a

ultrapure water (distilled water purified using a Milli-Q 50 system) and shaken at room temperature. Conductivity of bathing solutions was measured periodically for 4 h using a 4010 Conductivity Meter (Hanna Instruments, Padova, Italy). After measurements, the vials were maintained at ±20 °C overnight, in order to completely kill the leaf tissue. After thawing, the contents were shaken for 2 h, and the conductivity was read again. The measurement on frozen and thawed tissue represented conductivity after complete membrane destruction. Conductance measured at different time intervals is expressed as percent of total electrolyte leakage (Dijak and Ormrod 1982). Statistics All experiments were repeated twice and a minimum of ten plants per treatment were used in each experiment. Analyses were carried out at the end of fumigation and after 20 h of recovery in filtered air. For comparison of the means, one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test was used. Where appropriate, angular transformation of raw data was performed.

Results

Within 24±48 h after the fumigation, Eridano, the O3sensitive clone, developed severe minute roundish dark-

Fig. 2mEffects of O3 (150 nl l±1 for 5 h) on the ground fluorescence (A), maximum fluorescence (B) and Fv/Fm ratio (C) in the recently mature leaves of two poplar clones (Populus deltoides x maximowiczii Eridano, O3-sensitive, and P. x euramericana I-214, O3-resistant). The analyses were carried out at the end of the fumigation and after 20 h of recovery in clean air. Each bar represents the mean of five replications. In each sub-graph, bars marked with the same letters are not different for P = 0.05

blackish necrosis, localized in the interveinal area of the adaxial surface of mature leaves. On the resistant clone symptoms were absent or very scarce. Solute leakage is a good marker of membrane integrity. As described in Table 1, the exposure to O3 had a different effect on solute leakage from foliar disks of the two clones. In the sensitive one, a severe and irreversible increase in leakage was observed, whilst in the resistant clone the values of ozonated material increased but were not statistically different from those of controls. It is noteworthy that a 20-h recovery in clean air was associated with a relevant reduction of leakage in treated leaves of the resistant clone, and this can be evaluated in terms of repair mechanisms. The photosynthetic response of the two clones to the treatment was similar in the short-term (i.e. a dramatic ± more than 50% ± fall in CO2 assimilation), but only in the

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resistant clone was the effect completely reversed after a 20-h recovery in clean air (Fig. 1A). Stomatal conductance showed a similar trend (Fig. 1B): a 52% decline in Eridano was only partially reversed, while in I-214 a 33% reduction observed at the end of the treatment was followed by values over the controls after the recovery time. Intercellular CO2 concentration was not affected in the sensitive clone and increased in the resistant one (Fig. 1C). The values of transpiration rate (E) and water use efficiency (WUE) are reported in Table 2. As a consequence of the treatment, E decreased in both the clones, and WUE was only reduced in I-214. After recovery, the sensitive clone showed transpiration values still lower than the controls, whereas the other clone had fully recovered this parameter, which was even higher in treated plants than in unfumigated individuals. No recovery was observed for WUE in the resistant clone following the post-fumigation period in clean air. Figure 2 reports the chlorophyll a fluorescence parameters. In the sensitive clone, ground fluorescence (F0), maximal fluorescence (Fm) and Fv/Fm ratio were depressed by ozonization. This last ratio indicates the PSII efficiency and constitutes the most reliable marker for photoinhibition (Krause 1988). After recovery, only F0 remained lower than the controls. In I-214, these parameters were unaffected at the end of the fumigation, with the exception of Fv/Fm ratio. F0 and Fm did not change after the recovery period and the ratio remained lower, even if not statistically different, than the controls. Photochemical quenching did not show differences in the two clones following O3 exposure, whilst qNP slightly increased (Table 3). The !F/Fm' ratio was not affected by O3. After 20 h of recovery qNP remained higher than the controls in both the clones.

Discussion

From the present experiments it appears that the two poplar clones showed a different response to O3 as indicated by the effects on membrane permeability. Prior to the onset of any visible injury, in the sensitive clone there was an increase in electrical conductivity in the leachates from the disks obtained from leaves exposed to O3 compared with the controls. O3 and its oxidative derivatives probably affected the plasmalemma, which resulted in a leaky membrane (Coulson and Heath 1974) or a disruption of the plasmalemma (Rufner et al. 1975). This causes leakage of cellular liquids into the intercellular spaces. Coulson and Heath (1974) determined that O3 does not penetrate beyond the plasmalemma and that alterations in the chloroplast structure can be due to ionic or osmotic imbalances due to a leaky plasmalemma. Several hypotheses on alterations in chloroplast structure are reported by Heath (1994). Although the literature concerning other tree species presents a wide range of photosynthetic responses to O3 exposure, the negative effects of O3 seem to prevail (Pye 1988). Wallin et al. (1992) showed that in Picea abies

plants exposed to O3 photosynthetic efficiency at high light conditions were more influenced than at low light conditions. Also Tjoelker et al. (1995) reported that in Acer saccharum a reduction in net photosynthesis and an impaired stomatal function were evidenced when branches were exposed to O3. The different clonal responses to O3 in terms of maximum photosynthetic rate under light saturation can provide further information about the mechanisms of differential sensitivities. After the end of the treatment, both clones decreased their CO2 fixation ability in recently mature leaves, but the resistant clone appeared to make adjustments soon and 20 h after the end of the fumigation it completely recovered the photosynthetic activity. The resistant clone showed a peculiar response: after the end of the fumigation stomatal conductance decreased whereas the intercellular CO2 concentration increased. This could indicate that the limitation to photosynthetic processes can be due to non-stomatal components. When the stress was removed, photosynthesis and stomatal conductance completely recovered. Data of chlorophyll fluorescence suggest that the light reactions are virtually not altered in treated plants as demonstrated by the rather small reduction of the Fv/Fm ratio. The changes observed in the electron transport efficiency of O3-treated leaves of the resistant clone are consistent with the pollutant gas having a negative effect on carbon metabolism and there was no evidence for photoinhibition as reported by other authors (Heath 1994). In the sensitive clone strong negative effects of O3 on photosynthetic ability and stomatal conductance were observed, but stomatal limitations should not influence photosynthesis since the intercellular CO2 concentration did not change significantly. Also in this clone the reduction of CO2 assimilation capacity appears to be due mainly to mesophyllic limitations. The reduction of stomatal conductance seems to comply with the idea that stomatal conductance is changed in response to changes in the rate of assimilation (Farquhar and Sharkey 1982). After recovery, the photosynthetic rate partially recovered but did not reach the values of the controls. The mesophyll capacity of the leaves (i.e. CO2 fixation) can be damaged directly (e.g. via oxidative stress in photoactive compartments) or indirectly, by the disorganisation of the biomembranes as evidenced by changes in permeability due to leakage of organic and inorganic solutes. Chlorophyll fluorescence measurements of sensitive clone leaves indicate that at the end of the O3 exposure there was a decrease in F0 and Fm, which persisted during the recovery time. On the other hand, Fv/Fm ratio completely recovered 20 h after removing the O3 stress, to indicate that the efficiency in the energy conversion of the PSII was not altered. Also the yield did not change, indicating that the rate of non-cyclic electron flux through PSII was unaffected. However, the CO2 assimilation rate decreased in the sensitive clone. In these O3-treated leaves there must be a decrease in the allocation of reducing equivalents produced by electron transport to carbon assimilation relative to other electron sinks. O3 damage of the mesophyll, as detected by the gas exchange measurements, was prob-

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ably initiated indirectly by O3-induced alterations of cellular structure, as seen by the increase in electrolyte leakage, finally inhibiting photosynthetic reactions. The complex of data presented in this paper are consistent with the hypothesis that the primary target of O3 is the plasmalemma, and that inhibition of photosynthesis is of a secondary nature (Heath 1980, 1994). AcknowledgementsmThis research was supported by the National Research Council of Italy (Special Project INGA) and by MURST (40% funds), Rome.

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