Effects of acid fog on cuticular permeability and cation leaching in holly (Ilex aquifolium)

Agriculture, Ecosystems and Environment, 42 ( 1992 ) 291-306

291

Elsevier Science Publishers B.V., Amsterdam

Effects of acid fog on cuticular permeability and cation leaching in holly (Ilex aquifolium ) M.G. B a r k e r ~'b a n d

T.W. A s h e n d e n a

alnstitute of TerrestrialEcology, Bangor Research Unit, UniversityCollegeof North Wales, Deiniol Road, Bangor, GwyneddLL5 7 2 UP, UK bDivision of Biological Sciences, Institute of Environmental and Biological Sciences, University of Lancaster, Lancaster LA 1 4YQ, UK

ABSTRACT Barker, M.G. and Ashenden, T.W., 1992. Effects of acid fog on cnticular permeability and cation leaching in holly (Ilex aquifolium ). Agric. Ecosystems Environ., 42:291-306. Young holly plants were exposed for up to 9 months to simulated acid fog at a chemical composition, pH range and deposition pattern consistent with those reported for an elevated site in northwest England. Plants were exposed to acid fog at pH 5.6 (control), pH 4.5, pH 3.5 and pH 2.5. Water permeability was higher in enzyme-isolated cuticles from low-pH leaves. Foliar leaching amounts of Mg2+, Ca 2+ were higher in leaves exposed to lower pH acid fog. Similar effects for K + leaching rates were not significant. Increases in cuticular permeability and cation leaching were not accompanied by any observable changes in surface structure, as revealed by studies with wettability and extractable wax. It is suggested that any changes in cuticular functioning were the result of alterations to the intracuticular rather than epicuticular structure.

INTRODUCTION

Wet pollutant deposition may be in excess of dry deposition in some areas, including northwest England, Wales and Scotland (Fowler, 1984 ). Occult deposition consists of 10-50 #In diameter mist or fog droplets which are transferred to vegetation by turbulent transfer (Fowler, 1984). Deposition rates are difficult to estimate due to the numerous physical and biological processes involved (Jacobson, 1984; Unsworth, 1984). It is however possible that occult deposition may represent a substantial, underestimated pollutant input to vegetation (DoUard ct al., 1983). This could be particularly true for forests, which capture wind-blown droplets very efficiently. The leaf-contact time Correspondence to: M.G. Barker, School of Biological Sciences, Department of Environmental Biology, University of Manchester, Williamson Building, Oxford Road, Manchester M 13 9PL, UK.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-8809/92/$05.00

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of occult deposition can be much higher than that of acid rain. Increased duration of wetting is also likely to enhance the dissolution and dissociation of dry pollutant deposition. Fog can be much more acidic than rain, since the relative surface area of the finer droplets is large, allowing increased uptake rates of SO2 and NOx, which are oxidised to H2SO4 and HNO3 (Mengel et al., 1989). Dollard et al. (1983) report hydrogen ion contents of fog water in Cumbria, UK, which correspond with the most severe acid precipitation in Scandinavia and North America, where values in the range pH 3.0-4.5 are common (Ferenbaugh, 1976; Scherbatskoy and Klein, 1983), with peaks in the US as low as pH 2.2 (Walman et al., 1982). Leaf cuticles occur at the interface between the plant and its atmospheric environment, and they have been described as the 'principal barrier' between air pollutants and internal plant tissue (Fowler et al., 1980). Cuticles occupy all parts of the leaf surface other than stomatal openings. In astomatous regions of the leaf, for example the adaxial surfaces of most tree species, the cuticle is the only site of plant-pollutant interaction. Resultant changes in cuticular permeability can cause increased uptake of the pollutants themselves. They may also result in increased rates of water loss and leaching of nutrients from the leaf. Increases in cuticular transpiration have been reported for plants exposed to acid fog (Mengel et al., 1988, 1989), and it is suggested (Blank, 1985; Mengel et al., 1989) that combined effects of acid fog and drought may be an important stress factor in European forests. The occurrence of acid fog and that of'forest decline' in Germany often coincide (Prinz, 1987; Eiden et al., 1988). Furthermore, acid fog often occurs in high elevation sites also subjected to high 03 levels. Leaching from leaves subjected to repeated applications of simulated acid rain can occur without any visible injury (Evans et al., 1981 ), and this may be accompanied by increases in water loss per se. Acidic deposition has been shown to cause foliar injury, including lesions, at pH 3.0 and below (Wood and Bormann, 1975; Evans et al., 1981; Adams and Hutchinson, 1984). Nutrient leaching rates are higher in leaves showing injury (Tukey, 1970), though loss of nutrients may not occur in biologically significant amounts (Mitterhuber et al., 1989; Scherbatskoy and Tyree, 1990). The intention of the experiments described in this paper was to identify any functional changes in water permeability of enzyme-isolatedcuticles, and in rates of cation leaching in holly (Ilex aquifolium ) leaves subjected to prolonged acid fog and rain exposure. Wettability and amounts of extractable wax in leaves from different pH treatments were compared, to establish if there was any correlation between changes in cuticular structure and functioning.

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MATERIALS AND METHODS

Plant material Holly (Ilex aquifolium L. ) is a ubiquitously occurring small tree, naturally growing on both acid and alkaline well-drained soils, including upland sites. It is one of the few endemic evergreen tree species in the UK, though there have been reports (Scurfield, 1960) of holly assuming a deciduous habit in heavily polluted areas. Holly has been found to be relatively insensitive to ozone pollution (Tingey et al., 1976), though isolated holly cuticles exposed to high concentrations of 03 have been shown to increase in permeability (Kerstiens and Lendzian, 1989). Holly was used in this series of experiments because the evergreen leaves would normally be exposed to a wide range of pollutant conditions, including that of acid fog. Individual leaves are retained for up to 4 years before senescence. Holly cuticle can be removed enzymatically (Kerstiens and Lendzian, 1989), and this is not the case with most broad-leaved tree species, especially older leaves which have been subjected to air pollution (K. Lendzian, personal communication, 1989). The broad leaves of holly are also highly convenient for in vivo cuticle work, including water loss from the astomatous adaxial surface. Young, 2-year-old, 20-30 cm high holly plants, rooted in John Innes No. 2 compost in 18-cm diameter pots were used. Twelve plants were used in each treatment. The plants were placed in random positions within a group occupying part of each section of the misting tunnel. Plants from each treatment were transferred at weekly intervals, with the treatment, to diametrically opposite sections within the tunnel to avoid possible light and temperature differences brought about by the orientation of the tunnel. Stems of the plants were marked on the proximal side of emerging leaves, so that leaves which had emerged during the acid fog treatment could be identified for sampling. Plants were exposed to the acid fog treatment for up to 9 months (January-September 1990, though last samples taken June 1990).

Acidfog Plants were exposed to simulated acid fog within the misting tunnel at ITE Bangor. The tunnel was divided internally into four 1.6 m × 3.6 m treatment areas, each receiving acid fog at a different pH. The treatments were pH 5.6 (control), pH 4.5, pH 3.5 and pH 2.5. The fogs were released via atomising nozzles ('Sonicore', Lucas Dawe Ultrasonics, UK) which produced droplets in the diameter range 5-30/~m. The fog solutions were made up of equimolar concentrations of ammonium nitrate and sulphuric acid in de-ionised water.

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The S/N ratios of the solution were based on those reported for ambient fog in northwest England (Dollard et al., 1983 ). Acid fog treatments were delivered three times per week (Mondays, Wednesdays and Fridays) for periods of 4 h ( 10:00-14:00 ). The total weekly acid fog deposition per treatment was 6 mm week- i. Supplementary watering of plants in each treatment was provided manually using simulated acid 'rain' solution. The acid 'rain' consisted of sulphuric acid and nitric acid (in the ratio 7:3 v / v ) made up in distilled water to pH 4.5, and was applied at a rate of 24 m m week- i.

Permeability of enzyme-isolated cuticles Cuticles were isolated from leaves using an enzyme technique (Orgell, 1955 ) which yields structurally and functionally intact cuticles (Lendzian et al., 1986 ). Leaves were selected and removed from comparable positions on each of the plants; these leaves were of approximately the same age and had been exposed to 9 weeks of the acidic fog treatments. Smaller leaves and those showing lesions were not used. Leaf discs of 18 m m diameter were cut with a cork borer from part of the leaf, avoiding the midrib. There were three plants per treatment and up to five discs were used from each plant. Discs from different plants and treatments were placed separately in 4 ml of an enzyme isolation medium. The medium consisted of 2% cellulase and 20% pectinase (Sigma Chemicals, U K ) made up with citrate buffer, adjusted with 0.1 N HC1 to pH 3.7 (SchSnherr and Riederer, 1986; Lendzian et al., 1986). Sodium azide (1%) was added to inhibit microbial activity. The incubation was conducted at 37°C, with occasional rotary agitation, for 10-14 days. After this time, the adaxial cuticle was easily removed from the digested leaf material. The cuticles were carefully washed with distilled water and allowed to dry in Teflon vials and stored for future use. Permeability of isolated cuticles was measured by a gravimetric method, using small perspex 'transpiration chambers' (SchSnherr and Lendzian, 1981 ). The chambers incorporate a central well, and the opening is surrounded by a 15 m m diameter O-ring, which was lightly smeared with silicone grease. For each permeability experiment, 0.5 ml of distilled water was placed into the cavity of each of 10 chambers. Cuticles were rehydrated with distilled water and placed over the opening, with the physiological inner surface facing inwards. The cuticle was held in position by a lid which was screwed carefully into position, so that a good seal between the cuticle and O-ring was achieved. Cuticles mounted in the chambers were inspected using a binocular microscope; any visibly damaged cuticles were discarded. The transpiration chambers were immediately weighed (to three decimal places) on an accurate balance, then stored in an inverted position on a fine mesh support above silica gel, within a PVC air-tight box kept in an air-con-

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ditioned room at 21 ° C. The chambers were inverted so that water permeating through the cuticle was in the aqueous phase, which is the usual situation in an intact, well-hydrated leaf. The chambers were weighed at intervals for up to 4 weeks. Between five and 12 cuticles total were used from three plants per treatment. The loss in chamber mass in each case was assumed to be due to water lost by permeation; results were expressed as water lost per unit time.

Cation leaching Two leaves were selected randomly from the same relative position on each of three plants from each treatment following 13 weeks exposure to acidic fog. The cut surfaces of the petioles were lightly coated with petroleum jelly to ensure that leaching occurred through the lamina only (Brown and Roberts, 1988 ). Each leaf was immersed completely in 20 ml 0.5 m M H2804 (pH 3.0) contained in a glass vial. The containers were placed in a water bath at 25 °C and given continuous rotary agitation. The leachate was decanted from each leaf after 1.5 h and replaced with another 20 ml of 0.5 m M H/SO4. The leaching was then continued for a total of 24 h. The leaves were then removed, oven dried at 80°C for 48 h and dry weights determined. The two leachates ( 1.5 h and 24 h) from each leaf were then analysed for K + using flame photometry (EEL, Mark II, Evans ElectroSelenium, Halstead, U K ) and Ca 2+ and Mg z+ using atomic absorption spectrophotometry (SP9 Pye Unicam, Cambridge), calibrated against standard solutions. The leached cations were expressed as #g g - ~ leaf dry weight for each leaf.

Total extractable wax Total extractable wax of leaves was determined following 24 weeks exposure of plants to the acid fog treatments. Similar-sized leaves of two different age classes, 'young' and 'old' were randomly cut from equivalent positions from each of six plants in each treatment. Young leaves were those which had emerged during the acid fog treatment. Old leaves were those which were already expanded at the start of the experiment. The leaves were placed separately in accurately pre-weighed PVC vials. Five millilitres of chloroform was dispensed from a burette with a Teflon tap onto the adaxial surface of the leaf. The leaf was shaken with the chloroform for 30 s, after which the leaf was removed. Surface areas of the leaves were then determined by a Delta-T leaf area meter (Delta-T Devices, U K ) . The chloroform extract for each leaf was evaporated at 60 ° C in a water bath. A nitrogen stream was bubbled through the chloroform until a dry wax deposit was left. The vials were re-weighed. Extractable wax was calculated as mass per unit leaf area, #g cm -2 (Cape et al., 1989).

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Contact angle Leaf wettability was measured using a contact angle technique (e.g. Fogg, 1947; Hall et al., 1965; Cape, 1983; Caporn and Hutchinson, 1986). Two 'young' and 'mature' leaves were chosen (as described above) from six plants from each treatment following 20 weeks exposure to acidic fog. Part of the lamina of each leaf was dissected, and cut into three pieces approximately 4 mm × 4 mm. The midrib of the leaf was avoided, as were any lesions on the leaf. Each piece was placed, adaxial surface uppermost, onto a strip of doublesided adhesive tape, mounted on a perspex support. Care was taken to avoid contact with the adaxial surface throughout this operation. Drops of distilled water ( 1/zl) were immediately placed centrally on each piece of leaf. Groups of six leaves were then photographed. Internal contact angles of the drops were measured directly from an enlargement of the developed film. RESULTS

Permeability of enzyme-isolated cuticles The water permeability of isolated cuticles from each of the acid fog treatments is given in Fig. 1. A linear regression of these data is significant at P<0.05. 38 34 O

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Fig. 1. Mean water loss through enzyme-isolated cuticles, shown in relation to pH of acid fog treatment (n = 3 ). SE for each treatment is shown by vertical lines. Linear regression of all data is significant at P < 0.05.

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Cation leaching Cation concentrations for M g 2+ and Ca 2+ after 1.5 and 24 h leaching are shown in Fig. 2. Leaching amounts for Ca 2+ (Fig. 2a) exceeded those o f M g 1+ (Fig. 2b). Leaching amounts for K + were less than those of M g 2 + by a factor of 10, and differences between data were not significant (Fig. 2c ). Concentrations of Ca 2+ in the leachate in all pH treatments were relatively high after just 1.5 h. Leaching of Ca 2+ during 24 h occurred at a relatively slow rate. This suggests that Ca 2+ in this experiment was readily exchanged for H + from sites at or near the cuticle surface, and the rapid loss of Ca 2+ is not readily replaced during further ion exchange. The concentration of M g 2+ in the

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Fig. 2. Mean cation concentrations in leachate after 1.5 h ( • ) and 24 h (11) incubation in 0.05 mM H2SO4,shown in relation to pH of acid fog treatment. (a) calcium; (b) magnesium; (c) potassium (n = 3 ). SE for each treatment is shown by vertical lines. Significant differences (Kolomogov-Smirnov two-sample test) are shown: **P< 0.01; ***P< 0.001.

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leachate after 1.5 h was proportionally less than that after 24 h compared with Ca 2+. However, the initial leaching rates were again higher than those after 24 h. There was a non-linear relationship between pH and cation leaching. Leaching rates of Mg 2+ increased with decreasing pH in the range pH 5.6-pH 3.5. Increases were significant ( P < 0 . 0 1 ) for pH 3.5 after both 1.5 and 24 h. At pH 2.5 leaching rates decreased, although not to values at pH 4.5. A similar pattern occurred for Ca 2+ for the pH 4.5-pH 2.5 range, though leaching at pH 5.6 occurred at an amount slightly higher than that of pH 4.5. Increased Ca 2÷ leaching was significant for pH 3.5 (P<0.001) and pH 2.5 ( P < 0 . 0 1 ) after 1.5 h. The relationship between pH and leaching amounts at 24 h for K + was similar to those of Mg2+; however, differences between rates for K ÷ were not statistically significant.

Total extractable wax

Results from the extractable wax analysis for 'young' and 'old' leaves are presented in Fig. 3. There was no strong relationship between pH and the amount of extractable wax for either age class, although the mean amount of wax extracted from 'old' pH 4.5 leaves was significantly less (at P < 0.05) than that of such leaves from pH 5.6. Within each pH treatment, mean amounts of extractable wax were generally greater for the older leaves, though this was not significant except for the pH 5.6 leaves ( P < 0.01 ). vr N

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Fig. 3. Mean amounts of chloroform-extractable wax obtained from 'young' leaves (unshaded bars) and 'old' leaves (shaded bars), in relation to pH of acidic fog treatment ( n = 6 ) . SE for each treatment is shown by vertical lines. Significant differences (Kolomogov-Smimov twosample test) are shown between different acid fog treatments compared with controls of the same age class ('pollutant'), and within the same acid fog treatments for different age classes ('leaf age' ): *P< 0.05; **P< 0.01.

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Fig. 4. Mean contact angle values for adaxial leaf surfaces of 'young' leaves (unshaded bars) and 'old' leaves (shaded bars), in relation to pH of acidic fog treatment (n = 6). SE for each treatment is shown by vertical lines. Significant differences (Kolomogov-Smirnov two-sample test) are shown between different acid fog treatments compared with controls of the same age class ('pollutant'), and within the same acid fog treatments for different age classes ('leafage'): *P< 0.05; **P< 0.01; ***P< 0.001.

Contact angle Mean contact angle values for 'young' and 'old' leaves from each pH treatment are presented in Fig. 4. Low contact angles indicate high wettability. The generally low values (below 70 ° ) obtained here indicate that holly leaves do not have a distinct layer of crystalline epicuticular wax, and this was confirmed by SEM studies (results not included here ). There was no clear treatment pH × wettability correlation; there is some indication of increasing contact angle (i.e. decreasing wettability) with decreasing pH, but this was only significant for pH 3.5 (P< 0.05 ). There was a strong, consistent trend of lower contact angles (equalling higher wettability ) in 'old' leaves. This was significant at P < 0.001 for leaves from pH 5.6 and pH 4.5, and P<0.01 for pH 2.5 leaves. DISCUSSION

Permeability of enzyme-isolated cuticles Variation within each treatment was quite large, and this has been observed by others in work with isolated cuticles (Haas and SchiSnherr, 1979; Sch6nherr and Schmidt, 1979). This may have been due to the heterogeneity of cuticles, and to difficulties in working with the relatively low rates of water loss from a small area ( 1.77 cm 2) of cuticle. However, evidence from isolated cuticles provides tentative support for increased cuticular permeability. Moreover, since cuticles with visible damage (mostly those from pH 2.5) were discarded, the results for isolated cuticle permeability may represent an underestimation. Sch/Snherr and Schmidt ( 1979 ) have pointed out that the high variation even between isolated cuti-

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cles taken from the same leaf effectively precludes studies, for instance, ofpH effects on permeability. However, the intention in this experiment was to examine the influence of long-term exposures on in vivo cuticles, including any developmental changes of leaves which expanded during the acid fog exposure. The effect of acidic fog on cuticular permeability has not been widely studied; interactions with gaseous pollutants are much better understood (Lendzian and Kerstiens, 1988). It is difficult to argue a case for a direct effect of fog acidity on raising cuticular transpiration rates. Work with isolated cuticles (SchSnherr, 1976b) has shown that increases in cuticular permeability occur with increasing pH of the diffusing.solution, owing to pH-dependent increases in the number of polar pores per unit cuticle area. Using intact leaves, H~irtel ( 1951 ) showed that maximum cuticular transpiration rates occur at a critical pH, around pH 7. However, these transpiration rates also depend on ionic concentrations, although not those of Ca 2÷ (H~irtel, 1951 ). It may be that pH-related changes in cuticular permeability to water operate by an alternative, indirect process. Such a mechanism might include changes in the biosynthesis of different types and/or amounts of waxes. Water permeability is completely determined by the composition and distribution of waxes (Grncarevic and Radler, 1967; SchSnherr, 1976a). Changes to epicuticular waxes may correspond with pH (Percy and Baker, 1987), and this may in turn affect transpiration rates (Cape and Fowler, 1981 ). However, no such changes were observed in this study (Figs. 3 and 4). In any case, the relationship between wax degradation and cuticular resistance is not simple (Barnes et al., 1988).

Cation leaching There was a general increase in leaching rates of Ca 2+, Mg 2+ and K + with decreasing pH in the range pH 5.6-pH 3.5. Foliar losses of these cations occurred at a lower rate at pH 2.5 compared with pH 3.5, but higher than pH 4.5 and pH 5.6. Ions are readily lost from all wetted leaves, and Ca 2+, Mg 2+ and. K + are lost more readily than most ions (Kramer, 1957). These cations are leached by water at the rate of 1-10% of leaf content per 24 h (Tukey, 1970). The term 'leaching' is often used to include cation loss which takes place as a neutralisation process, when leaves are subjected to low pH treatments. In practice, it is difficult to separate the two processes of cation exchange and leaching (Cape, 1985). The neutralisation process can be regarded as an appropriate physiological response to acidification at the leaf surface. Leaching may not confer any particular advantage and is not likely to be physiologically significant unless it results in short- or long-term nutrient deficits. The leaves in this experiment were subjected to the same leaching conditions (i.e. 0.05 mM H2SO4), though leaching rates tended to be higher in those leaves

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previously subjected to lower pH conditions. This suggests that the low-pH acid fog treatment may have predisposed leaves to a more 'leaky' condition. This could have important implications in terms of both nutrient loss and increased water permeability. Results for Mg 2÷ and Ca 2÷ indicate generally higher leaching rates at lower acid fog pH, within the range pH 5.6-pH 3.5. A similar trend exists for K ÷, though this was not significant. The apparent decrease in cation leaching from pH 2.5 leaves might be explained by reduced cation content in these leaves, due to much higher previous leaching activity, though the mineral content of the leaves in this experiment was not determined. Increased cation leaching would be expected from pH 2.5 leaves as (a) cuticular transpiration and permeability is higher in these leaves (trans-cuticular migration of cations occurs through aqueous pathways), and (b), pH 2.5 leaves showed a high level of visible injury including lesions, which would provide additional routes for cation loss. Relative leaching rates after 1.5 and 24 h provide some indication of the source of cations lost into the leachate. A greater proportion of those cations lost after 1.5 h would be expected to have come from the cuticle surface or within the cuticle, from exchange sites, compared with those lost over the full 24 h. Surface and near-surface ions are thought to be particularly important in the neutralisation process (Hutchinson and Adams, 1987 ). Several workers have reported increases in foliar leaching with decreasing pH (e.g. Fairfax and Lepp, 1975; Wood and Bormann, 1975; Adams and Hutchinson, 1984; Leonardi and Fliickiger, 1989; Mitterhuber et al., 1989; Takemoto et al., 1989). However, Scherbatskoy and Tyree (1990) argue that a simple mechanism for this does not exist. Reported leaching rates of Ca 2÷ always exceed those of Mg 2+ and K ÷ with acid treatment. The relative leaching rates of the three cations analysed in this experiment are Ca2+> Mg2+> K ÷. This sequence has been found by other workers (Adams and Hutchinson, 1984; Takemoto et al., 1989). The actual differential of the leaching rates depends on the pH, other treatment conditions (Tukey, 1970; Scherbatskoy and Klein, 1983; Adams and Hutchinson, 1984 ), and also the species used (Wood and Bormann, 1977; Richardson and Dowding, 1988). The leaching behaviour of K ÷ is much less consistent than that of Mg 2+ and Ca 2÷. Mitterhuber et al. ( 1989 ) observed K ÷ leaching rates higher than those for Mg 2÷, whilst Scherbatskoy and Klein ( 1983 ) noted no pH effect on K ÷ loss, and Leonardi and FliJckiger ( 1989 ) found a decrease in K ÷ leaching at lower pH. One possible explanation for the range of leaching response observed for K ÷ is that it is mostly accumulated in living tissue. This is not the case for Ca 2÷ and Mg 2÷, which are concentrated mainly in the apoplast, from which they are more readily leached (Mecklenberg et al., 1966; Miller, 1984 ). The predominantly intracellular location of K ÷ may be a critical factor, as higher K ÷ losses might indicate a pH-induced disruption to cell

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membranes (Wood and Bormann, 1975). However, there was no evidence for this in the results presented here. Nutrient deficiency, particularly of Ca: + and Mg2+, has been implicated in forest decline (Prinz, 1987). A proportion (perhaps 10%) of certain leached ions (including Mg2+ and Ca 2÷ ) may be reabsorbed by roots (Tukey and Mecklenberg, 1964). The problem may be exacerbated by insufficient uptake by poorly developed root systems (e.g. by A13+ toxicity) of plants growing in acidified soil (Prinz, 1987; Richardson and Dowding, 1988 ). There has been much recent debate concerning the possible effects of foliar leaching on nutrient deficiency. Someworkers have argued that losses can be potentially harmful (Wood and Bormann, 1977; Miller, 1984). However, it has been argued that foliar leaching does not occur at biologically significant rates (Richter et al., 1983; Mengel et al., 1987; Mitterhuber et al., 1989; Scherbatskoy and Tyree, 1990). However, 'suboptimal' foliar nutrient levels may develop 'over years', particularly if the cuticle is damaged. Deficiencies of Mg2+ will result in chlorosis. Insufficient Ca 2÷ (and possibly also Mg2÷ ) could interfere with stomatal functioning (Miller, 1984; Leonardi and Fliickiger, 1989). Total extractable wax

Results from this experiment did not show any obvious pH-related effect on amounts of extractable wax. There was a slight, non-significant trend of increasing amounts of wax in 'young' leaves in the treatment range pH 5.6pH 3.5, but a reduction (again, not significant) occurred at pH 2.5. All treatments showed an increase (though significant only with pH 5.6 leaves, at P< 0.05 ) in extractable wax in older leaves, suggesting that biosynthesis of total waxes was not affected by the acidic fog treatment. The amount of wax extracted from 'old' leaves at pH 4.5 was significantly lower than those at pH 5.6. No explanation is offered for this phenomenon. The technique used in this experiment does not provide any information on the relative amounts of epicuticular and intracuticular wax present. These components are difficult to distinguish by any convenient extraction method (J.N. Cape, personal communication, 1990). However, the short (30 s) extraction time would be expected to mostly remove surface waxes. Cape et al. (1989) have argued that a 10 s extraction would be expected to remove surface waxes but not a significant proportion of internal waxes. Cuticular resistance to permeability is determined by several different components. The relative contribution of each one to the cuticular pathway is difficult to quantify (Martin and Juniper, 1970; Sch/Snherr and Schmidt, 1979 ). Epicuticular ('superficial') wax may b e more important in determining leaf wettability, whilst intracuticular ('cuticular') wax influences permeability, especially to water.

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Although it is widely agreed that acid deposition can alter cuticular characteristics, it is not clear whether this is by reduced wax production or increased weathering (McLaughlin, 1985 ), or indeed by other mechanisms. The relationship between surface wax degradation and cuticular resistance is not simple (Barnes et al., 1988). Other workers have found no correlation between extractable wax content and permeability (Norris, 1974), or between amounts of epicuticular wax and contact angle (Cape, 1983 ). An inverse correlation of wax production and foliar injury found by some workers (Sweicki et al., 1982 ) has not been observed in this experiment.

Contact angle There were no apparent effects of acid fog on wettability in this study, although comparable investigations with acid rain and acid fog have shown reductions in contact angle (increases in wettability) (Cape and Fowler, 198 l; Riding and Percy, 1985; Haines et al., 1985; Baker and Hunt, 1986; Percy and Baker, 1987) and erosion of epicuticular wax (e.g. Huttunen and Laine, 1983 ). Any change in wettability of poUuted leaves is likely to be due to alterations of the surface wax rather than the cuticle itself (Cape, 1983). Furthermore, surface wax may also be important in water conservation (Kolattukudy, 1968; Mengel et al., 1989). However, in most angiosperm leaves there is little or no surface wax (Schieferstein and Loomis, 1959). Evidence from this study suggests that crystalline epicuticular wax may not be a functionally significant component of holly leaf cuticle. SEM and wettability observations did not provide evidence of a highly hydrophobic crystalline layer. Contact angles in this experiment were all less than 70 °; angles less than about 110 ° are thought to occur on leaves with little or no epicuticular wax, i.e. with hydrophilic surfaces exposed (Hall et al., 1965). ACKNOWLEDGEMENTS

We are grateful for the valuable help provided by Leena Rapponen (AAS analysis), Phil Smith (transpiration chamber construction), Ray Rafarel and Sam Bell (acid fog facilities ). Acknowledgement is made of the valuable discussions with Professor T.A. Mansfield, Dr. K. Lendzian, Dr. G. Kerstiens and Dr. J. Wolfenden. Financial support for one author (MB) was provided by a NERC CASE award. REFERENCES Adams, C.M. and Hutchinson, T.C., 1984. A comparison of the ability of leaf surfaces of three species to neutralize acidic rain. New. Phytol., 97: 463-478.

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