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LEAF CUTICLES AS POTENTIAL MARKERS OF AIR POLLUTANT EXPOSURE IN TREES Virginia Seymour Berg Biology Department University of Northern Iowa Cedar Falls, IA 50614 ABSTRACT Because leaf cuticle covers a large area and is continually exposed to the environment, it might serve as a sensitive and early indicator of exposure to air pollutants. Some investigations have shown alterations of the ultrastructure of surface waxes with field or laboratory exposure to air pollutants, while others have not detected significant differences. It is possible that the changes in the surface structure are largely a product of pollutant-induced stress, rather than a direct consequence of exposure of the cuticle itself. The healthiest trees in polluted zones may show less alteration of cuticle than the least healthy trees of less polluted zones. Laboratory studies have demonstrated that prolonged exposure of isolated cuticle to acid can increase studies have the changes of IS air air the permeability of the cuticle to the acid, but such not yet been carried out on intact leaves. Because seen for cuticles are nonspecific, resembling an acceleration natural aging processes or exposure to other stresses, it currently difficult to use them as specific markers of pollution exposure, especially if the object is to distinguish pollution from other sources of stress. The cuticle as a barrier Plant cuticles present the main barrier between the interior of the leaf and the external environment. They are in constant contact with air pollutants, including gaseous pollutants, particulates and acid precipitation. If the cuticle is altered in a specific and predictable manner by exposure to air pollutants, it could serve as a diagnostic marker of air pollution exposure. Here we will look first at the structure of the cuticle, then at changes in the outer surface associated with air pollution exposure, and finally at some information on the movement of acid through cuticles and changes in permeability of isolated cuticles caused by acid exposure. Cuticle structure The innermost part of the cuticle is characterized by interspersed pectin, cellulose and lipids (Martin and Juniper 1970~. The portion of the cuticle exterior to this layer can typically be enzymatically separated from the outer walls of the epidermal cells using a pectinase/cellulase solution (Schoenherr 1976~. The next layer (moving toward the outside) consists primarily of cutin and waxes. The cutin, which provides much of the 333

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334 mechanical integrity of the cuticle, consists of esterified fatty acids which may be further crosslinked by ether bridges (Kolattukudy 1980~. There are some free acidic (dissociable) groups on the cutin polyester matrix, and these may be important in transport of substances through the cuticle. The term "waxes" is used loosely here, indicating members of a large collection of saturated straight-chain hydrocarbons and their derivatives, chiefly primary and secondary alcohols, ketones and aldehydes, fatty acids and wax esters (Kolattukudy 1976~. The waxes are embedded in the cutin matrix and form a continuous layer on top of it. It is this final, uninterrupted, wax layer that forms the primary barrier between the internal and external environments of the leaf (Schoenherr 1976~. On top of this wax layer may be crystalline deposits of epicuticular wax; these play an important role in interactions between acid precipitation and leaf surfaces, and may serve to collect or retain pollutants deposited in dry form, as they can substantially increase the area of the wax surface. Epicuticular wax: an indicator of pollutant exposure? The structure of epicuticular waxes changes with age (Reicosky and Hannover 1976, Franich et al. 1977, Crossley and Fowler 1986, Grill et al. 1987~. Although they are normally crystalline, the material is rather soft and can be altered or removed by the impact of rain or other precipitation (Baker and Hunt 1986, Mayeux and Jordan 1987), by abrasion from wind-blown particulates (Rotem 1965), or by contact with other leaves or adjacent surfaces (Wilson 1984~. In addition, the crystals appear to degrade slowly over time, fusing into amorphous masses and eventually into a largely continuous layer (Reicosky and Hannover 1976, Crossley and Fowler 1986, Grill et al. 1987~. These changes are accompanied by a modification of the appearance of the leaf surface (blue spruce, for instance, turns green; Reicosky and Hannover 1978) and by alteration of the wettability of the surface. The wettability determines how readily a drop of liquid is retained by the leaf, and how well it contacts the surface, and, therefore, it indicates the potential degree of interaction between precipitation (including acid precipitation) and the leaf. This contact may be important in the process of leaching of substances from leaves, and is necessary for the movement of acid into leaves through the cuticle. There are many reports of differences in the structure and wettabillity of epicuticular wax between polluted and unpolluted sites (Percy and Riding 1 97S, Huttunen and Laine 1983, Crossley and Fowler 1986, Grill et al. 1987~. Typically the symptoms are similar to those seen with increased leaf age: fusion of wax crystals eventually to form continuous layers of wax on top of the normal uninterrupted wax layer. This wax may completely cover stomates, or may largely occlude antestomatal chambers. Other differences in the waxes over stomates have been observed (Trimble et al. 1982~. There is considerable uncertainty as to the mechanism by which the normal aging process takes place, and more still as to how pollutants might accelerate it. It is possible that there is some direct chemical effect of pollutants on the waxes themselves, or that there is a mechanical effect associated with wetting and drying of surface. The wetting and drying of the surface could be altered by the presence of particulate matter on the cuticle, a feature of many field sites with high pollution levels. There are two reports which further complicate this issue. Jean Fincher of the Boyce Thompson Institute (pers. comm.) has examined cuticles of outdoor-grown red spruce saplings which have repeatedly received experimental treatments of acid rains of nitrate or sulfate, along with cuticles of ozone-treated saplings. She has seen no evidence of direct effects of these treatments on the cuticle, and no clear indication of accelerated aging. (Ozone-treated plants did sometimes show increased insect damage to the cuticle, however.) Grill and colleagues (1987) examined the cuticles of relatively vigorous trees and declining trees from polluted and relatively clean sites in Germany. The epicuticular wax on vigoro,us

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335 trees (selected visually, based on quantity and quality of foliage) appeared to be in good condition, while the trees showing symptoms of decline had needle surfaces which appeared very much older than their chronological age. The authors suggest that these results show that the condition of the cuticle is an indicator of the general vigor of the tree, rather than specifically of exposure of the outside surface of the leaf to pollutants. This leads to the question of whether changes in the cuticle are an indication of direct effects of air pollutants on the cuticle waxes, or an indication of changes in the metabolism of the leaf, eventually leading to alteration of the surface. A change in the rate or duration of wax production could have such an effect, as could changes in the chemical composition of the waxes. There is some evidence that deficiencies of mineral nutrients also lead to changes in the cuticle and its Nettability (Seymour 1976~. Preliminary studies of acid exposure of waxes isolated from cuticles do not show obvious changes in the compound classes (Berg, unpublished). Nonetheless, some untested effects, such as that of S02, which, being quite soluble in the wax, might disrupt its crystal structure, could provide a mechanism for accelerated aging. The compounds that make up the epicuticular waxes are chemically stable, but the crystal structures they form are not. With the current state of knowledge, it is impossible to use the condition of epicuticular waxes as an accurate predictor of tree decline due to pollution exposure, although the two are clearly correlated at many field sites. The state of the epicuticular waxes is important because these crystals act to reduce contact between precipitation and the leaf surface. An increase in Nettability has been observed for leaves of plants artificially exposed to acid precipitation (Percy and Baker 1988), and for needles from plants in polluted sites (Cape 1983, McIlveen, Ontario Department of the Environment, pers. comm.), but the increased Nettability of needles in the more polluted sites is of the same general magnitude as that observed for increased needle age. An additional difficulty in using the condition of epicuticular wax as an indication of pollution exposure is the variation seen from plant to plant due to environmental factors such as light level (Baker 1974), temperature (Armstrong and Whitecross 1976, Haas 1977) and humidity. This may mean that the cuticle can serve as a general marker of plant stress, but not a specific one. Movement through cuticles While the epicuticular waxes are important, the final barrier to non-stomata! entry of air pollutants is the continuous wax layer upon which the epicuticular wax lies. When the wax components are removed from isolated cuticles, the cutin matrix that supports and anchors the wax has a greatly increased permeability to water, indicating that at least for water, the wax layer is the principal limiting structure (Schoenherr 1976~. The wax layer appears to be less important for the movement of ions (Schoenherr 1976~. There are two alternative views of the movement of polar or dissociated compounds through the cuticle, one involving relatively polar pores and the other involving relatively polar areas throughout the wax. There is support for both of these models, in different species (Schoenherr 1976, Seymour 1980~. The polar pores or areas are thought to be aggregations (continuous for polar pores and discontinuous for polar areas) of the more polar parts of the molecules that form the wax, possibly including acids and alcohols. This model does not envision pores as "holes" to be filled, but rather an area of the cuticle into which water molecules may pass, forming a channel. In either case, a material moving through the cuticle might pass either through the hydrophobic, nonpolar portion of the wax, as would be the case for SO2, which has a substantial solubility in

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336 wax (Len~zian 1984), or through the pores or other polar sites, as would be the case for ions, such as sulfate, nitrate or cations. Permeability of cuticles Pollutants in acid precipitation can pass through intact cuticles. Damage to the tissue is first apparent in the epidermal cells, which can collapse, along with mesophyll cells, in the absence of any obvious damage to the cuticle (Smith and Davis 1 97S, Paparozzi and Tukey 1983, Adams et al. 1984~. This has been observed frequently in experimental systems (Evans et al. 1 977a,b, Paparozzi and Tukey 1983, Adams et al. 1984, Musselman 1988), but seldom in the field. In part the difference may be due to the frequent use of greenhouse-grown plants in experimental systems. The wax of these plants typically is different in quantity (and possible in quality) from that of outdoor- grown plants. Damage of leaf tissue indicates the failure of the cuticle as a barrier, not necessarily damage to the cuticle. The importance of the quantity of wax can be seen from the sensitivity of cabbage cotyledons to acidity, contrasted with the resistance of cabbage true leaves to acid-induced damage (Caporn and Hutchinson 1986~. The quantity of wax on the cotyledons is orders of magnitude less than that on the true leaves (Overholtzer and Berg 1987~. The permeability of isolated cuticles of Berberis aquifolium to a variety of acids has been measured (Dreyer et al. 1981~. Acids do pass through cuticles, but many hours are required for substantial changes in the pH on the "inside" of the cuticle. Acids that were less completely dissociated passed through the cuticle more readily, indicating that they were probably moving through the wax itself, rather than through pores in the wax. Recent experiments involving HC1 with grapefruit leaf cuticles (Berg and Overholtzer 1987), and H2SO4 with pear and lemon leaf cuticles (Berg and Overholtzer 1987), showed a more complicated pattern of permeability (Heuser and Berg 1988~. After an initial very low permeability lasting from several hours to a day or more, the permeability increased substantially. The original permeability could not be restored by removing the acid, soaking the cuticle in water overnight, or soaking it in base (KOH) overnight. The presence of Ca2+ at physiological levels ( 1 to 10 mM) on the "inside" of the cuticle makes it highly impermeable to acid. This may account for the lack of visible acid precipitation damage in the field, despite rain and fog at pH 3 or below. The Ca2+ effect described here may be due to the ion bridging between negatively charged sites in the cutin, perhaps creating a bottleneck where the wax meets the cutin. Because it is impossible to isolate cuticles and measure permeability for most plants, it is not possible to measure these properties for many of the plants involved in forest decline. At this time we cannot make general statements about changes in the properties of isolated, intact cuticles due to the action of air pollutants. Studies of neutralization of acid droplets have shown that much, but not all, of the neutralization of acids is due to particulates on the leaf surfaces (Adams and Hutchinson 1984~. Calcium nutrition has not been shown to be important in determining acid drop neutralization. The current state of knowledge concerning the interactions between plant cuticles and air pollutants does not suggest the use of cuticles as a sensitive marker of air pollutant exposure. At present, the cuticle best serves as an indicator of, rather than a predictor of, forest decline. In the future, however, it may be possible to use subtle changes in cuticle properties, including wettability, to indicate exposure to certain pollutants. If we wish to use and understand such techniques, we must have a better understanding of the natural changes that occur in all cuticles in the field.

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337 REFERENCES Adams, C.M., N.G. Dengler and T.C. Hutchinson. 1984. Acid rain effects on foliar histology of Artemesia tilesii. Canadian Journal of Botany 62, 463-474. Adams, C.M. and T.C. Hutchinson. 1984. A comparison of the ability of leaf surfaces to neutralize acid rain drops. New Phytologist 97, 463-478. Armstrong, D.J. and M.I. Whitecross. 1976. Temperature effects on formation and fine structure of Brassica napus leaf waxes. Australian Journal of Botany 224, 309-318. Baker, E.A. 1974. The influence of environment on leaf wax developemot in Brassica oleracea var. Gemmifera. New Phytologist 73, 955-966. Baker, E.A. and G.M. Hunt. 1986. Erosion of waxes from leaf surfaces by simulated acid rain. New Phytologist 102, 161 - 173. Berg, V. and K. Overholtzer. 1987. Movement of acidity through grapefruit leaf cuticles. Plant Physiology, 83 (suppl.), 85. Cape, J.N. 1983. Contact angles of water droplets on needles of Scots pine (Pinus sylvestris) growing in polluted atmospheres. New Phytologist, 93, 293-299. Caporn, S.J.M. and T.C. Hutchinson. 1986. The contrasting response to simulated acid rain of leaves and cotyledons of cabbage (Brassica oleracea L.) New Phytologist 103, 311-324. Crossley, A. and D. Fowler. 1986. The weathering of Scots pine epicuticular wax in polluted and clean air. New Phytologist 103, 207-218. Dreyer, S.A., V. Berg Seymour and R.E. Cleland. 1981. Low proton conductance of plant cuticles and its relevance to the acid-growth theory. Plant Physiology 6S, 664- 667. Evans, L.S., N.F. Gmur, F. Da Costa. 1977a. Leaf surface and histological perturbations of leaves of Phaseolus vulgaris and Helianthus annuus after exposure to simulated acid rain. American lournal of Botany 64, 903-913. Evans, L.S., N.F. Gmur and J.J. Kelsch. 1977b. Perturbations of upper leaf surface structures by simulated acid rain. Environmental and Experimental Botany 17, 145- 149. Franich, R.A., L.G. Wells and J.R. Barnett. 1977. Variation with tree age of needle cuticle topography and stomata! structure in Pinus radiata needles. Phytochemistry 17, 1617-1623. Grill, D., H. Pfeifhofer, G. Halbwachs and H. Waltinger. 1987. Investigations on epicuticular waxes of differently damaged spruce needles. European Journal of Forest Pathology 17, 246-255.

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338 [Iaas, K. 1977. Einfluss von Temperatur und Blattaelter auf das Cuticularwachs van Hedera helix. Biochemie und Physiologie der Pflanzen 171, 26-31. Hauser, H.D. and V.S. Berg. 1988. Effect of repeated acid exposure on acid permeability of pear leaf cuticle. Plant Physiology, 86 (suppl.), 59. Huttunen, S. and K. Laine. 1983. Effects of air-borne pollutants on the surface wax structure of Pinus sylvestris needles. Annales Botanici Fennici 20, 79-86. Kolattukudy, P.E. 1976. Introduction to natural waxes. Pp. 1-15 in Kolattukudy, P.E. (ed.), Chemistry and biochemistry of natural waxes. Elsevier Sci. Pub. Co., Amsterdam and New York. Kolattukudy, P.E. 1980. Biopolyester membranes of plants: cutin and suberin. Science 208, 990-1000. LendzIan, K.J. 1984. Permeability of plant cuticles to gaseous air pollutants. In Kohoil, M.J. & F.R. Whatley, (eds.) Gaseous air pollutants and plant metabolism. Butterworths, London. Martin, J.T. and B.E. Juniper. 1970. The cuticle of plants. St. Martin's Press, New York. Mayeux, Jr., H. and W. Jordan. 1987. Rainfall removes epicuticular waxes from Isocoma leaves. Botanical Gazette 14S, 420-425. Musselman, R.C. 1988. Acid neutralizing capacity of leaves exposed to acid fog. Environmental and Experimental Botany 2S, 27-32. Overholtzer, K.D. and V.S. Berg. 1987. Surface waxes and sensitivity to acid precipitation: cabbage cotyledons and leaves. Proceedings of the Iowa Academy of Science 94, abstract 62. Paparozzi, E.T. and H.B. Tukey, Ir. 1983. Developmental and anatomical changes in leaves of yellow birch and red kidney bean exposed to simulated acid precipitation. lournal of the American Society of Horticultural Science 10S, 890-898. Percy, K.E. and R.T. Riding. 1978. The epicuticular waxes of Pinus strobus subjected to air pollutants. Canadian Journal of Forest Resources 8, 474-477. Percy, K.E. and E.A. Baker. 1988. Effects of simulatec! acid rain on leaf wettability, rain retention and uptake of some inorganic ions. New Phytologist 10S, 75-82. Reicosky, D.A. and J.W. Hannover. 1976. Seasonal changes in leaf surface waxes of Picea pungens. American Journal of Botany 63, 449-456. Reicosky, D.A. and J.W. Hannover. 1978. Physiological effects of surface waxes. I. Light reflectance of glaucous and non-glaucous Picea pungens. Plant Physiology 62, 101- 104. Rotem, I. 1965. Sand and dust storms as factors leading to Alternaria blighta epidemics on potatoes and tomatoes. Agricultural Meterology 2, 281-288.

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339 Schoenherr, J. 1976. Water permeability of isolated cuticular membranes: the effect of cuticular waxes on diffusion of water. Planta 131, 159-164. Seymour, V. Berg. 1976. Leaf wet/ability--a mechanism for the increased foliar leaching of nutritionally stressed plants. M.S. Thesis, University of Washington, Seattle, WA. Seymour, V. Berg. 1980. A study of water movement through plant cuticles. Ph.D. Dissertation, University of Washington, Seattle, WA. Smith, H.J. and D.D. Davis. 1978. Histological changes induced in Scotch pine needles by sulfur dioxide. Phytopathology 68, 171 1-1716. Trimble, J.L., J.M. Skelly, S.A. Tolin and D.M. Orcutt. 1982. Chemical and structural characterization of the needle epicuticular wax of two clones of Pinus strobus differing in sensitivity to ozone. Phytopathology 72, 652-656. Wilson, J. 1984. Microscopic features of wind damage to leaves of Acer pseudoplatanus L. Annals of Botany, 53, 73-82.

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338 lIaas, K. 1977. Einfluss von Temperatur und Blattaelter auf das Cuticularwachs von Hedera helix. Biochemie und Physiologie der Pflanzen 171, 26-31. Hauser, H.D. and V.S. Berg. 1988. Effect of repeated acid exposure on acid permeability of pear leaf cuticle. Plant Physiology, 86 (suppl.), 59. Huttunen, S. and K. Laine. 1983. Effects of air-borne pollutants on the surface wax structure of Pinus sylvestris needles. Annales Botanici Fennici 20, 79-86. Kolattukudy, P.E. 1976. Introduction to natural waxes. Pp. 1-15 in Kolattukudy, P.E. (ed.), Chemistry and biochemistry of natural waxes. Elsevier Sci. Pub. Co., Amsterdam and New York. Kolattukudy, P.E. 1980. Biopolyester membranes of plants: cutin and suberin. Science 208, 990-1000. LendzIan, K.J. 1984. Permeability of plant cuticles to gaseous air pollutants. In Kohoil, M.J. & F.R. Whatley, (eds.) Gaseous air pollutants and plant metabolism. Butterworths, London. Martin, J.T. and B.E. Juniper. 1970. The cuticle of plants. St. Martin's Press, New York. Mayeux, Jr., H. and W. Jordan. 1987. Rainfall removes epicuticular waxes from Isocoma leaves. Botanical Gazette 14S, 420-425. Musselman, R.C. 1988. Acid neutralizing capacity of leaves exposed to acid fog. Environmental and Experimental Botany 2S, 27-32. Overholtzer, K.D. and V.S. Berg. 1987. Surface waxes and sensitivity to acid precipitation: cabbage cotyledons and leaves. Proceedings of the Iowa Academy of Science 94, abstract 62. Paparozzi, E.T. and H.B. Tukey, Ir. 1983. Developmental and anatomical changes in leaves of yellow birch and red kidney bean exposed to simulated acid precipitation. lournal of the American Society of Horticultural Science 10S, 890-898. Percy, K.E. and R.T. Riding. 1978. The epicuticular waxes of Pinus strobus subjected to air pollutants. Canadian Journal of Forest Resources 8, 474-477. Percy, K.E. and E.A. Baker. 1988. Effects of simulatec! acid rain on leaf wettability, rain retention and uptake of some inorganic ions. New Phytologist 10S, 75-82. Reicosky, D.A. and J.W. Hannover. 1976. Seasonal changes in leaf surface waxes of Picea pungens. American Journal of Botany 63, 449-456. Reicosky, D.A. and J.W. Hannover. 1978. Physiological effects of surface waxes. I. Light reflectance of glaucous and non-glaucous Picea pungens. Plant Physiology 62, 101- 104. Rotem, I. 1965. Sand and dust storms as factors leading to Alternaria blighta epidemics on potatoes and tomatoes. Agricultural Meterology 2, 281-288.