Response of Two Populations of Holm Oak (Quercus rotundifoliaLam.) to Sulfur Dioxide

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY

40, 42—48 (1998)

ENVIRONMENTAL RESEARCH, SECTION B ARTICLE NO.

ES981640

Response of Two Populations of Holm Oak (Quercus rotundifolia Lam.) to Sulfur Dioxide D. Garcı´ a,* J. Rodrı´ guez,* J. M. Sanz,- and J. Merino* *Departmento de Ecologı´ a, Facultad de Biologı´ a, Apdo 1095, Sevilla 41080, Spain; and -MOPTMA, Paseo de la Castellana s/n, Madrid, Spain Received July 30, 1996

et al., 1988). On the other hand, stress induced by air pollution leads to significant increases in plant maintenance cost (Amthor, 1988). Thus the capacity to withstand a determined level of air pollution can also depend on the ability to divert part of the energy available for the individual’s growth to the processes of maintenance, thereby satisfying the additional energy demands resulting from the stress (Szaniaswski, 1987; Taylor, 1989). The holm oak (Quercus rotundifolia) is the predominant evergreen tree species in the Mediterranean landscapes of the Iberian peninsula. It is found both as conserved populations, forming relatively dense woodlands, and as scattered individuals on grassland (savanna; locally known as ‘‘dehesa’’). It extends from Southern Morocco (latitude 30°40@ N) to the north of the lberian peninsula (latitude 42°30@ N), where it is substituted by deciduous formations typical of the temperate European climate areas (Quercus pyrenaica, Castanea sativa, and Fagus silvatica). The main aim of this study has been to compare the sensitivity to SO of individuals in two very sepa2 rated populations occupying distinct biogeographic locations. More precisely, the morphological and physiological responses to fumigation with SO of individuals 2 belonging to a population in northern Spain (latitude 42°22@ N), located at the northern boundary of the distribution area of this species, have been compared with those of individuals of a population in southern Spain (latitude 37°58@ N). The comparison took in leaf variables related to physiology (photosynthetic rate, respiratory rate, maintenance respiration), structure (leaf weight ratio, leaf area ratio, specific leaf weight, leaf area of the individuals, and shoot to root ratio), and relative growth rate of individuals. It is hypothesized that individuals originating from populations at the boundary of their distribution area should be more resistant to the effect of SO because, 2 in such environments, the species have been selected to resist multiple, and generally more intense, levels of stress (Parsons, 1993).

Experiments were carried out with seedlings of Quercus rotundifolia Lam., an evergreen schlerophyllous tree typical of the Spanish Mediterranean climate environments. Fruits were collected in two distant (800 km) populations located in the center (southern Spain) and northern border (northern Spain) of the area of distribution of the species. One-month-old potted plants were grown for 130 days in an enriched atmosphere of SO2 (0.23 ppm, 14 h/day) in controlled (growth chamber) conditions. Both northern and southern plants underwent a significant decrease in growth rate as a consequence of the treatment. Even so, plants appear to be quite resistant to SO2 compared with either more temperate or more productive species. The southern population was more sensitive to the treatment, as reflected by the bigger decrease in both growth and photosynthetic rates. Differences in resistance appear to be related to the biogeographic origin of the populations studied, which underlines the importance of biogeographic aspects in studies of resistance to air pollutants. ( 1998 Academic Press

INTRODUCTION

Much attention has been given to analyzing the response of species to the effect of air pollutants (Weigel et al., 1990; Whitmore and Freer-Smith, 1982). There are fewer studies on the response structure in populations of the same species originating from different habitats and, more especially, concerning their geographical location. However, knowing the resistance to an air pollutant of a species throughout its biogeographic area of distribution is mandatory if we want to predict the effects of that particular pollutant on the persistence of that species. The characteristics conferring resistance to a specific air pollutant are multiple and varied [see Mooney and Winner (1991)]. The intrinsic plasticity of individuals is an exceptional one, because it enables resources to be best allocated for each concrete situation (Hunt and Nichols, 1986). The shoot—root ratio (S/R) has received much attention in studies of resistance to stress, owing to its great sensitivity to environmental factors, including air pollutants (Mooney 42 0147-6513/98 $25.00 Copyright ( 1998 by Academic Press All rights of reproduction in any form reserved.

43

RESPONSE OF Quercus rotundifolia TO SO 2

METHODS

Fruits of Q. rotundifolia were collected during October and November 1994 in the two localities selected and were sown in pots with a substrate of sand and vermiculite (1 : 1). On germination, the seedlings were kept in a greenhouse (45 days) until reaching 10 cm in height. Ninety seedlings were selected at random from each population, and 60 were put into two indoor growth and fumigation chambers. The remaining 30 were used to estimate the mean dry weight (initial weight) of the individuals of each population. The seedlings were irrigated daily with Hoagland’s solution. Experimental conditions were the following: illumination, 10 h of darkness, 14 h of light at a photosynthetic photon flux density of 312 lmol/m2/s; temperature, 24°C day and 18°C night; relative humidity of the air during the period of illumination was kept at about 30—35%. The chambers were fed with a continuous current of air taken from outside the building and filtered with active charcoal. The air in each chamber was totally renewed approximately each minute. The fumigation chamber had an injection device for fumigation from a concentrated SO 2 tank. An automatic pumping system enabled the sampling of the plant’s atmosphere inside the fumigation chamber and the almost continuous monitoring of the SO concen2 tration, using an analyzer (Model-4108, Dasibi, Glendale, CA, USA). The SO concentration in the fumigation cham2 ber atmosphere was kept at 0.23 ppm (v/v) by the injection of SO . This concentration is high compared with the aver2 age values recorded in areas potentially affected by centers of pollution in the Iberian peninsula (Mas Garc´ıa, 1993). The fumigation time was 14 h daily, coinciding with the period of illumination. The experiment lasted 130 days. Each seedling was then separated into its stem, leaf, and root fractions, oven dried at 80°C for 48 h, and weighed (final weight). The relative growth rate (RGR, mg/g/day) of each individual was calculated from the initial weight (the same for all the treatments) and from the final weight of the individual. The ratios LWR (leaf weight ratio: weight of leaves to plant weight), LAR (leaf area ratio: total surface of leaves to plant weight), SLW (specific leaf weight: leaf weight to leaf surface), and S/R (shoot to root weight) were also calculated. The values determined for each individual were used to calculate the mean values for each population studied. The leaf photosynthetic rate and the stomatal conductance were estimated by gas exchange techniques under the same conditions of temperature, relative humidity, and light intensity as those of cultivation, but in the absence of SO . 2 The determinations were made in intact individual leaves located near the middle of the stem of randomly selected plants, at the middle of the experimental period, using an open gas exchange system (Model LCA-2; ADC, Hoddes-

don, UK), calibrating the IRGA daily from synthetic air of known CO concentration. The calculations of gas ex2 change parameters were carried out in accord with Field et al. (1981), the values referring to only one side of the leaf surface. Immediately after the gas exchange analysis, each seedling was returned to the growth chamber to continue the treatment. The respiration rates (total and maintenance) were estimated in the period between days 93 and 128, counting from the beginning of the treatment. Only intact leaves in the growth phase were considered. For the estimate of total respiration rate, the CO ex2 change in darkness and at 20°C was determined for all the selected leaves. The determinations were carried out by using an open gas exchange system (Armstrong, Palo Alto, CA). The plants in which the determinations were made were kept in darkness to avoid the effect of carbohydrate level on respiration (Azcon-Bieto et al., 1983; Villar et al., 1995). After the determination, the leaf was separated from the plant, dried, and weighed. The respiration rate was expressed as milligrams of CO per gram per day. 2 The maintenance respiration of the leaves was estimated in accord with the linear model of Hesketh et al. (1971) and the approximation of Kimura et al. (1978), consisting of regression (linear model) of the individual leaf specific respiration rate (SRR, mg CO /g/day) in a set of leaves, on 2 their respective specific growth rates (SGR, mg/g/day). Extrapolation of the SRR (dependent variable) to zero SGR (independent variable) gives the respiration rate in the absence of growth; that is, the maintenance respiration [for more details, see Amthor (1989)]. The SGR of each leaf was determined from the increase in leaf surface in a period of 50 h and from SLW. The analysis CO evolution for the calculation of SRR was made in the 2 same leaves and at the same time as the determination of the growth rate for the calculation of SGR. The differences in maintenance respiration between populations and treatments were tested by means of covariance analysis (Sokal and Rohlf, 1981). In regard to the remaining physiological and morphological variables, the differences between populations and treatments were tested by means of variance analysis, considering the population and the treatment to be factors. RESULTS

Plant Structure and Growth No external signs of injury were observed in the leaves of the fumigated plants. Table 1 presents the weights reached at the end of the experiment by the different organs of the plants belonging to the southern and northern populations in the control treatment and in the treatment with 0.23 ppm of SO . 2

44

GARCI¨A ET AL.

TABLE 1 Average Weight (g) 6 One Standard Deviation of the Mean of the Different Plant Fractions (Leaves, Stems, and Roots) and Results in RGR (mg/g/day) in the Two Populations Considered Leaves Northern population Control Fumigated Effect of treatment

Stems

Roots

RGR

n

1.25$0.15 0.61$0.11 0.67$0.12 17.3$2.8 17 1.16$0.13 0.52$0.07 0.68$0.09 16.0$2.0 12 * * ns *

Southern population Control 1.79$0.17 0.93$0.11 1.37$0.14 27.6$3.0 24 Fumigated 1.33$0.17 0.69$0.08 0.88$0.10 18.5$2.5 18 Effect of treatment * * * * Interpopulation differences * * * * Note. n, sample size. * Statistical significant difference at P(0.05; ns, no significant difference.

As can be seen, the weight of the different organs of the control plants is always higher (P(0.05) in the southern population. Southern individuals were bigger, with greater leaf biomass and stem biomass and with a greater root system, resulting in plants with a higher growth rate (17.3 mg/g/day for the northern population and 27.6 mg/ g/day for the southern one). The effect of the SO treatment was general for the two 2 populations and resulted in a significant (P(0.05) decrease in the final weight of the different organs studied. The effect was stronger in the southern population, with decreases on average between 22 and 36% with respect to the control. In the northern population, the decreases were about 10%, except for root weight, where the effect of the treatment was zero (Table 1). Eventhough not significant, the leaf area per individual (LA) in the southern population paralleled the decrease undergone by the rest of the plant organs. In contrast, LA of the fumigated individuals in the northern

population remained the same as that of the control plants (Table 2). The decrease in the weight of the organs as an effect of the treatment a significant decrease in the growth rate. In the northern population, growth rate fell from 17.3 mg/ g/day to 16.0 mg/g/day (a decrease of 8%) and, in the southern population, from 27.6 mg/g/day to 18.5 mg/g/day (a decrease of 33%). It is important to note that, although the relative decrease in weight of the different organs was always greater in the southern population, the absolute weight of each fraction in this population never fell below that of the northern population. Thus the growth rates of the fumigated plants of the two populations were very similar (16.0 and 18.5 mg/g/day for the northern and southern populations, respectively).

Biomass Allocation The individuals of the two populations have a different morphology, because all the indices considered in the study were significantly different (P(0.05) (Table 2). The northern population allocates more biomass to aerial structures (greater LWR, S/R, LAR) than does the southern one. No significant differences were detected between populations in respect to SLW, with values of 169.9 g/m2 and 164.0 g/m2 in the northern and southern populations, respectively. The SO treatment did not significantly modify the values 2 of most of the indices studied (LWR, LAR, S/R), suggesting that in each population the morphology of the plants is tightly controlled and, consequently, not altered by the effect of the SO concentration considered in the present 2 study. In contrast, SLW underwent a significant change in the northern population individuals.

TABLE 2 Average Values 6 One Standard Deviation of the Mean for the Biomass Allocation Indexes in the Two Populations Considered LWR

LAR

S/R

LA

SLW

n

Northern population Control Fumigated Effect of treatment

0.51$0.02 0.50$0.02 ns

30.2$1.0 33.5$1.3 ns

3.3$0.4 2.7$0.3 ns

81.7$14.5 83.1$12.3 ns

169.9$2.5 147.6$3.0 *

17 12

Southern population Control Fumigated Effect of treatment Interpopulation differences

0.44$0.01 0.45$0.02 ns *

27.0$0.8 27.4$1.3 ns *

2.1$0.1 2.4$0.2 ns *

117.2$14.7 85.9$12.6 ns ns

164.0$5.3 164.0$4.1 ns ns

24 18

Note. LWR, weight of leaves to plant weight (g/g); S/R, shoot weight to root weight (g/g); LAR, leaf area to plant weight (cm2/g); SLW, leaf weight to leaf area (g/m2); LA, total leaf area of the individuals (cm2); n, sample size. * Statistical significant difference at P(0.05; ns, no significant difference.

45

RESPONSE OF Quercus rotundifolia TO SO 2

TABLE 3 Average Values 6 One Standard Deviation of the Mean for Variables Related to the Physiology of the Seedlings of the Two Populations Considered

Northern population Control Fumigated Effect of treatment Southern population Control Fumigated Effect of treatment Interpopulation differences

An

gst

Rt

Rm

Ci

Slopea

n

10.8$0.9 7.6$0.6 *

81.9$7.7 53.2$5.4 *

77.0$5.7 58.0$4.8 *

44.7$9.2 34.3$7.8 *

291.5$7.6 281.5$7.8 ns

0.212$0.011 0.237$0.012 ns

24 24

10.5$0.8 3.9$0.4 * ns

92.8$7.6 28.5$3.1 * ns

65.8$4.6 56.8$3.7 * *

32.7$9.2 24.7$11.4 * *

309.0$5.8 293.7$9.2 ns ns

0.186$0.008 0.225$0.014 ns ns

24 24

Note. An, leaf photosynthetic rate (lmol CO /m2/s); gst, stomatal conductance to water vapor (mmol/m2/s); Rt, total leaf respiration rate (mg 2 CO /g/day), Rm, leaf maintenance respiration coefficient (mg CO /g/day); Ci, CO internal concentration (ppm). n, sample size. 2 2 2 a Slope of the An/gst curves (mmol/mol). * Statistical significant difference at P(0.05; ns, no significant difference.

Plant Physiology The two populations had an identical (P'0.05) photosynthetic rate (10.8 lmol CO /m2/s and 10.5 lmol 2 CO /m2/s for the northern and southern populations, 2 respectively; Table 3). The two populations also had virtually identical values (P'0.05) of stomatal conductance for water vapor (81.9 mmol/m2/s and 92.8 mmol/m2/s). These values are at the lower extreme of the range reported for individuals of Q. rotundifolia growing under natural conditions (Tretriach, 1993), and are lower than those published for other non-Mediterranean woody species (Yoshie, 1986). As a result of the treatment, both the stomatal conductance and the photosynthetic rate decreased significantly in both populations (P(0.05), although the decrease was less marked in the seedlings of the northern population (a decrease of about 30% with respect to the control) than in those of the southern one (a decrease of about 65%). It should be noted that, as a consequence of the treatment, the average for conductance of the northern population resulted in about double (53.2 mmol/m2/s) that estimated for the southern one (28.5 mmol/m2/s). If it is accepted that the difference between the mean values of stomatal conductance for the two populations under the experimental conditions is representative of the whole growth period, the real dose of pollutant penetrating the leaves of the northern seedlings should also be about double. There were no significant differences between populations either in the slopes of the photosynthesis-conductance curves or in the internal CO concentration. In addition, 2 these variables did not differ in the control plants and those subjected to fumigation in either of the populations studied (Table 3).

The total respiration rate of the control plants was about 15% higher in the seedling leaves of the northern population than in those of the southern one (P(0.05). Additionally, the maintenance respiration of the leaves of the northern population was significantly greater (27%, Table 3). The treatment resulted in a significant (P(0.05) decrease in total respiration rate in both populations. In the northern population, it fell from 77.0 mg CO /g/day (control plants) 2 to 58.0 mg CO /g/day (a decrease of 25%), whereas, in the 2 southern population, the decrease was less marked, falling from 65.8 mg CO /g/day to 56.8 mg CO /g/day (approxi2 2 mately 14%). The maintenance respiration also decreased significantly (P(0.05) in the two populations by a similar amount (23—25%), although its value remained higher (of the order of 28%) in the northern population. DISCUSSION

Growth Rate The results indicate that the seedlings of the southern population have a higher growth rate than those of the north. Considering that the photosynthetic rate is virtually identical in the two populations, this may be surprising, because this population invests more energy in aerial parts; that is, in the energy-gathering fraction of biomass. The higher maintenance respiration rates of the leaves of individuals in the northern population may explain their lower growth rate. Maintenance respiration represents the energy cost associated with maintenance of structures and gradients. Despite its instantaneous value, it can be considered small; integrated throughout the life of an organ, it can equal, or

46

GARCI¨A ET AL.

even surpass, the other leaf costs (Merino et al., 1982). In fact, maintenance respiration is frequently correlated negatively with production (Amthor, 1989). The northern population’s greater investment in maintenance could offset its greater investment in aerial biomass, resulting in the lower growth rate found in this population. One of the most noteworthy effects of the SO treatment 2 was its almost total incapacity to alter the individual’s architecture, suggesting that the arboreal nature of the species studied makes it less flexible than that of the herbaceous species studied by other authors (Taylor, 1989). It should be noted that, in the northern population, the fumigated individuals keep the same leaf area as that of the control seedlings in spite of the significant decrease in leaf weight. This lack of response of LA was the direct consequence of a significant decrease in SLW in response to SO . In the 2 southern population, SLW did not change at all; so the decrease in leaf weight in response to the SO treatment 2 resulted in a strong and almost significant (P"0.10) decrease in the leaf area of the individuals. In contrast, the treatment induced a significant decrease in the values of all the physiological parameters studied—in particular, that of the leaf photosynthetic rate, which decreased by some 60% in the southern population (Table 3). This considerable decrease in photosynthetic rate explains the marked decreases in weight of each of the organs considered, which were, in general, about 30% and resulted in an equivalent decrease in growth rate (Table 1). In the northern population, the effect on the photosynthetic rate was lower (a decrease of 30%), resulting in a smaller decrease in the weight of the organs (about 8%), and, consequently, in a smaller decrease in growth rate (7%). The difference between the strong treatment-induced decrease in the mean photosynthetic rate in each population and the small decrease observed in relative growth rate could be explained, in part, by the decrease in maintenance respiration, which fell by some 23—25% in the two populations as a result of the treatment. Additionally, in the northern population, the observed lessening of the treatment’s effect on growth rate appears to be the result of the operation of compensatory processes involving LA, LWR, and SLW interactions (Table 2), similar to those described in other SO fumigation studies (Mooney et al., 1988). 2 Finally, despite the low value reached by the photosynthetic rate as a result of the treatment in the southern population compared with that in the northern one (3.9 lmol CO /m2/s and 7.6 lmol CO /m2/s, respectively), 2 2 the lower contribution of the aerial parts to the total weight of the fumigated plants in the former population (S/R values of 2.4 and 2.7 for the southern and northern populations, respectively), and the compensatory mechanisms described earlier, the mean relative growth rate in the southern population was virtually identical with that in the northern one (18.5 and 16.0 mg/g/day, respectively).

As heretofore discussed for the nonfumigated plants, this apparent paradox can be explained by the difference in the maintenance cost between the two populations. This difference is sustained under fumigation conditions, maintenance respiration being some 28% higher in the northern population (Table 3), which possibly offsets the relatively high photosynthetic rate and the higher S/R ratio of the fumigated individuals in this population. Physiological Variables The results discussed so far underline the importance of the values of the physiological parameters (photosynthesis, respiration) and that of their response to SO treatment as 2 main determinants of the differences detected in the overall response (growth rate) of the two populations studied. The mean decrease in the photosynthetic rate induced by the treatment in the two populations was 46% with respect to the control. When the relatively high level of fumigation (0.23 ppm), the length of the experiment (130 days), and the number of hours of fumigation per day (14 h) are taken into account, Q. rotundifolia can be classed as a very resistant species, above all in comparison with the results published for other species in which the decrease in photosynthetic rate under similar conditions reached 84% of its initial value (Barton et al., 1980). The resistance was even greater in the northern population, because the decrease in this case was only 30%. The results of the present study do not allow evaluation of the degree to which the decrease observed in the photosynthetic rate is a result of the direct effect of SO on the 2 stomatal apparatus and degree to which it is a consequence of damage to the mesophyll. The non-significant alteration of the slope of the stomatal conductance—photosynthesis curves by the effect of the treatment (Table 3) and the maintenance of the initial values of the internal concentration of CO in the fumigated plants of the two populations 2 studied (Table 3) suggest that the decrease observed in the photosynthetic rate is basically a consequence of damage to the mesophyll rather than a result of stomatal closure as a direct effect of the treatment. The low stomatal conductances observed (Table 3) could explain, at least partly, the high resistance to fumigation observed in this species. Stomatal conductance is one of the characteristics determining a species’ resistance to pollution from SO (Reich and Amudson, 1985), because the real dose 2 (i.e., the amount of pollutant gas reaching the leaf mesophyll) is a function of the degree of stomatal opening. In general terms, the Mediterranean woody species, in their actual locations, present low conductances compared with species of temperate climate regions where the problems related to the use of water resources are usually fewer (Larcher, 1980). It must also be emphasized that most of the available data on the effect of SO treatment refer to 2

47

RESPONSE OF Quercus rotundifolia TO SO 2

species of agricultural or forestry interest, often characterized by high stomatal conductances, which are essential to sustain a high production. The relatively low stomatal conductances observed in this study results in lower real doses of pollutants, and, therefore, in a greater resistance to air pollution, which helps to explain the lower sensitivity observed in the present study in comparison with the results obtained by other authors. At the same time, because of the occurrence of frequent stomatal closure associated with water shortage, Mediterranean species appear to evolve defense mechanisms addressed to minimizing the effects of molecular oxidative species generated under the (very common) situations of high light intensity and low substrate (CO ) availability 2 deriving from stomatal closure (Faria et al., 1996). Besides, it should be noted that free radicals are also generated as a result of the primary reactions to SO inside the leaf cells 2 (Pell and Dan, 1991). So, if resistance to diverse stresses involves common underlying biochemical mechanisms (Hochachka and Somero, 1984), such mechanisms could minimize the effect of SO on the leaf mesophyll. In fact, 2 some studies in crop plants have demonstrated that resistance to SO appears to be strongly related to free radical 2 scavenging enzymes and endogenous antioxidant compounds (Lee et al., 1992).

The notion that there are strong relationships between the biogeographic location of the populations, their resistance to stress, and the use that individuals make of energy, is becoming more and more accepted (Parsons, 1990). Studies carried out in animal species found that, owing to the higher overall levels of stress in populations originating from the boundaries of their area of distribution, evolution seems to have favored more abundant defensive biochemical mechanisms in these populations, with the result of individuals inherently more resistant. The synthesis and operation of greater (and almost certainly more complex) defensive endowments mean a higher maintenance cost and thus a higher maintenance respiration rate (Parsons, 1993). The higher maintenance cost results in a lower availability of energy for growth processes and thus in lower growth rates in the individuals originating from populations at the area boundaries. The syndrome described above matched very well the results obtained in the present study (low sensitivity of the photosynthetic process, higher maintenance cost, and lower growth rates in the northern populations), all of which underlines the need to consider the biogeographic origin of the populations in studies of resistance to air pollutants.

CONCLUSION

The authors thank R. Villar, J. L. Garcı´ a, and J. Escudero, for their help during the experiment, and ‘‘Junta de Andalucı´ a-Monte de Piedad,’’ for a fellowship to D. Garcı´ a. This project was financed by grants from the DGICYT (AMB 95-0443) and MOPTMA.

Thus Q. rotundifolia and possibly many other Mediterranean woody evergreen species could be considered species preadapted to SO pollution, given both their low mean 2 stomatal conductances and their low mesophyll sensitivity because of the presence of biochemical detoxification mechanisms. Besides, northern population individuals are more resistant (have a less-sensitive mesophyll) than the southern ones. Also, they appear to be more plastic because they are able to change their SLWs and thus keep their leaf areas under SO fumigation—all resulting in a comparatively 2 smaller decrease in RGR in response to the treatment. It is difficult to explain the different sensitivities of the two populations to the treatment with SO . In principle, given 2 that the slopes of the photosynthesis-to-conductance curves and the internal concentrations of CO have almost the 2 same values in the two populations, it could be concluded that the differences observed in the photosynthetic rates of the fumigated individuals (7.6 lmol CO /m2/s in 2 the northern population and 3.9 lmol CO /m2/s in the 2 southern one) are probably not due to the different sensitivities of their respective stomatal apparatus, but possibly to the different sensitivities of the mesophyll in the two populations. The basic determinants of this difference in sensitivity might be related to the biogeographic origin of the populations.

ACKNOWLEDGMENTS

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