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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
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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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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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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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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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[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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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.
Representative terms from entire chapter:
epicuticular waxes
