Tree Physiology 19, 909--916
© 1999 Heron Publishing----Victoria, Canada
Detection and quantification of changes in membrane-associated
calcium in red spruce saplings exposed to acid fog
MEI JIANG1 and RICHARD JAGELS1,2
1
Department of Forest Ecosystem Science, University of Maine, 5755 Nutting Hall, Orono, Maine 04469, USA
2
Author to whom correspondence should be addressed
Received January 14, 1999
Summary Five-year-old red spruce saplings (Picea rubens
Sarg.) were exposed to either (1) acid fog consisting of a
mixture of H2SO4 and HNO3 adjusted to pH 2.5, (2) distilledwater fog at pH 5.6, or (3) no fog (dry control) for 3.5 hours
per day, five times a week during the 1996 and 1997 growing
seasons. The effect of fog on cell membrane-associated calcium (mCa) of leaf mesophyll cells was investigated with the
fluorescence probe chlortetracycline (CTC). In both years,
mean mCa concentrations were significantly less in needles
exposed to acid fog than in needles exposed to distilled-water
fog or in untreated needles. In 1997, acid-fog treatment resulted
in 25 and 12% reductions in mCa in current-year needles, and
18 and 15% reductions in 1-year-old needles, compared with
untreated needles and needles exposed to distilled-water fog,
respectively, indicating that acid deposition induced calcium
leaching from the membranes of photosynthetic mesophyll
cells. Exposure to distilled-water fog also led to reductions in
mCa in young needles, suggesting that water films on needle
surfaces can induce losses by diffusion between the needle
interior and surface. Consistent with the chamber studies, field
data obtained from red spruce trees at two sites in Maine
showed that low mCa concentrations in needles were associated with exposure to acid fog.
Keywords: chlortetracycline, forest decline, leaching, needle
injury, Picea rubens.
Introduction
Red spruce (Picea rubens Sarg.) forests of the northeastern
United States experienced a decline in growth from the early
1960s to the 1980s (Johnson and Siccama 1983). The slowed
growth was particularly evident at high elevations where vegetation is frequently exposed to cloud water during the growing
season, and at coastal sites where trees are frequently exposed
to advective fogs (Siccama et al. 1982, Jagels et al. 1989).
Because the pH of fog or cloud water is lower, and concentra−
tions of SO 2−
4 and NO 3 are markedly higher than those in
rainwater for the same area (Weathers et al. 1988, Mohnen
1992), these observations raised concern that acidic fogs could
affect forest health.
Field and laboratory studies have shown that acidic input
causes needle injury and loss of cold hardiness in red spruce
(Jacobson et al. 1990, Sheppard 1994, Leith et al. 1995).
Hydrogen (H+) and sulfate (SO 2−
4 ) ions are believed to be
important contributors to the loss of tree health; however, the
causal mechanisms remain elusive. A prominent effect of acid
deposition on plants is enhanced foliar leaching of calcium
(Ca2+) ions (Joslin et al. 1988, Jacobson et al. 1989). It has been
proposed that H+ can replace Ca2+ through a cation exchange
process (Mecklenburg 1966, Waldman 1988, Turner and Van
Broekhuizen 1992). There are divergent views on the physiological significance of enhanced Ca leaching. Some researchers believe that the leached nutrients constitute too small
a portion of the total amount of mineral nutrients in foliage to
influence tree- or stand-level nutritional status (Pfirrmann
1990, Turner and Van Broekhuizen 1992). Other studies show
that acid deposition significantly reduces foliar Ca content
(Jacobson et al. 1989, McLaughlin et al. 1993) and leads to Ca
deficiency, which is responsible for spruce decline on base-deficient sites (Schulze 1989, McLaughlin et al. 1991). When Ca
supply is in excess, Ca is sequestered in vacuoles or pumped
out of the cell (Hanson 1984, Hepler and Wayne 1985), or
precipitated into physiologically inactive oxalate crystals in
the cuticular or epidermal or mesophyll cell walls (Fink 1991).
Losses of Ca, leached from this pool, are unlikely to be important. However, depletion under prolonged acidic input may
influence the physiologically active Ca pool. Furthermore,
when plants are growing on base-deficient sites, or have low
transpiration rates, Ca replenishment may be insufficient to
replace leached Ca.
Membrane-associated calcium (mCa) is an important type
of physiologically active Ca. It binds to anionic sites of membrane phospholipids and proteins to form bridges between
lipid molecules and between lipid and protein molecules. This
binding helps to tighten the plasma membrane and reduce
membrane permeability (Hanson 1984, Hepler and Wayne
1985). Acid deposition has also been shown to reduce photosynthesis, increase dark respiration, alter carbon allocation and
mineral nutrition, and reduce cold hardiness in red spruce
(DeHayes 1992, McLaughlin et al. 1991,1993). For many of
these deleterious effects to occur, the components in acid
deposition must modify the physiology of the plasma mem-
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JIANG AND JAGELS
brane in order to enter the cytosol. Because mCa is essential
for maintaining the integrity of the plasma membrane, a disturbance of mCa may precede the loss of control over membrane
permeability. It has been shown that a loss of mCa can cause
increased membrane leakage, leading to initiation and progression of freezing injury in corn (Zocchi and Hanson 1982),
tomato (Woods et al. 1984), and onion (Arora and Palta 1988)
cells. Furthermore, Ca binding to the membrane is not favored
at low pH (Hanson 1984). Hydrogen ion is able to displace
Ca2+ by protonating the head groups (pK values 3.0--4.0) of
membrane phospholipids, thereby reducing the negative
charges to which Ca2+ binds (Van Dijck et al. 1978, Watts et al.
1978).
The focus of the present study was to ascertain whether mCa
in mesophyll cells of red spruce could be disturbed by longterm exposure of leaf surfaces to acidic fog deposition. To
separate the effects of leaf wetting and acidity, a subset of trees
was exposed to distilled-water fog. We used chlortetracycline
(CTC), a fluorescent chelate dye with specific affinity for Ca
associated with biological membranes (Caswell and Hutchison
1971). This dye has been used previously in both animal and
plant systems to localize and quantify the distribution and
alteration of mCa in response to environmental and developmental changes (e.g., Saunders and Hepler 1981, Woods et al.
1984, Timmers et al. 1996, Borer et al. 1997, DeHayes et al.
1997).
Materials and methods
Plant materials and treatments
In 1992, red spruce seedlings from a single seed source were
obtained from the Tree Breeding Center, Department of Natural Resources, Debert, Nova Scotia, and replanted in 12-liter
pots filled with a 2:1:1 (v/v) mixture of peat, vermiculite and
sand. The seedlings were kept well-watered and fertilized
annually with controlled-release fertilizer (Osmocote:
18:6:12, N,P,K). Seedlings were kept outdoors at the University of Maine, Orono campus until subjected to the experimental treatments in their fourth and fifth years (1996--1997). Plant
height was 1.4--1.6 m at the beginning of the 1996 growing
season.
The first study was carried out in three outdoor chambers,
arranged in a North--South axis, during the 1996 growing
season. Eighteen trees were randomly assigned to the chambers (six per chamber). Three treatments: (1) untreated ‘‘dry’’
control (DC); (2) distilled-water fog at pH 5.6 (DWF); and
(3) acid fog at pH 2.5 (AF) were randomly assigned to the three
chambers. We faced a dilemma in dealing with the chamber
effects. Moving trees from one chamber to another during the
experiment would have disturbed salt deposits and possibly
water films and cuticular waxes on needle surfaces. We compensated for this design impediment by (1) placing the trees on
a turntable base that was gently rotated one-sixth revolution
each day, and (2) repeating the experiment a second year
(1997) with the treatments repositioned in the chambers,
which themselves were reassembled differently.
Fog treatments
The chambers were open-top cylinders, with a diameter of
1.2 m and a height of 1.8 m, made of wooden frames and
transparent polyethylene sheets. Each chamber had one door
and two windows, which were closed only during fogging.
Watertight plastic tarps were used during fogging to cover the
tops of the chambers. Fogging was carried out at night to avoid
chamber overheating and rapid evaporation of fog droplets
before deposition on needles. We placed plastic shields around
the tree bases to exclude fog from the soil. Trees were rotated
on the turntable base each day to equalize fog deposition over
the season. A layer of transparent fiberglass was installed, two
feet above the chambers, to exclude rainfall. Shade cloth was
used to provide uniform light among chambers. Temperature
and solar irradiances were monitored and recorded by a data
logger (21X Micrologger, Campbell Scientific, Inc., Logan,
UT).
Acid fog (AF) was made up of H2SO4, HNO3 and Na2SO4 in
deionized water, with final pH of 2.5. The final concentrations
−
of H+, SO 2−
4 and NO 3 were 3.16, 1.20 and 1.04 mM, respectively, which closely approximated the more acidic fogs measured along the coast of Maine (Kimball et al. 1988).
Distilled-water fog (DWF) was a simulation of an idealized,
low ionic strength fog or dew. Fog was generated by ultrasonic
fog generators (Bionaire 201 Ultrasonic Humidifiers; Biotech
Electronics, Ltd. Montréal, Québec, Canada), with fog droplet
size ranging from 0.5 to 31 µm (Jagels 1991). Saplings were
exposed to fog for 3.5 hours, providing 1 mm precipitation of
water in the chambers, on five consecutive days per week from
May 26 to September 27 in 1996 and 1997. Control trees
received no fog and are designated dry controls.
Fluorescence microscopy
Membrane-associated Ca was visualized and quantified with
chlortetracycline (CTC, Sigma/Aldrich, St. Louis, MO) by the
method of Reiss and Herth (1978). Needles from six trees per
chamber were sampled and tested every two weeks. In 1996,
only current-year needles were sampled. In 1997, both currentyear and 1-year-old needles were sampled. Fresh needles were
detached and cut into 120 µm-thick cross sections with a
sliding microtome (Model 860, American Optical Company,
Buffalo, NY). Some sections were transferred immediately to
100 µM CTC (pH 7.5) and incubated for 30 min. Other sections were incubated in distilled water to determine background fluorescence. After staining, cross sections were
mounted on slides and observed with the aid of a Zeiss Axioskop microscope equipped with epifluorescence objectives
(Carl Zeiss, Jena, Germany). Interference filters (Chroma
Technology Corp., Brattleboro, VT) were used to isolate the
420 nm peak of the high-pressure mercury lamp output for
excitation and 530 nm peak of emission specific for the Ca2+CTC complex (Woods et al. 1984). Fluorescent images were
recorded with a video camera (Cohu 4910, High-Performance,
Monochrome CCD Camera, Cohu Inc., San Diego, CA)
mounted on the microscope tube. The video contrast and gain
control were held constant throughout the experiments. The
video signals were acquired, digitized and stored as computer
TREE PHYSIOLOGY VOLUME 19, 1999
EFFECT OF ACID FOG ON MEMBRANE-ASSOCIATED CALCIUM
images by a frame-grabber (DT 3152, Data Translation,
Marlboro, MA). Image processing and analyses were performed with the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on
the Internet at http://rsb.info.nih.gov/nih-image/). The Ca2+CTC fluorescence intensities were represented by a numerical
continuum of brightness from 0 (black) to 255 (white). The
relative amounts of mCa in mesophyll cells were determined
by measuring mean pixel brightness values of the plasma
membrane region, with higher numbers reflecting larger quantities of mCa. For each tree, four needle cross sections were
examined and recorded. To minimize the auto-fluorescence
interference from the cuticle and the vascular tissue, a fixedsized frame that included only mesophyll cells was selected in
each cross section. The location of the frame was standardized
for the same relative area of each cross section to minimize
variability.
Fluorescence microscopy reference standards were used to
test the accuracy and reproducibility of the imaging system
(InSpeckTM Microscope Image Intensity Calibration Kits,
I-7219, Molecular Probes, Eugene, OR). In quantitative fluorescence microscopy, the intensity of fluorescence should be
proportional to the amount of fluorescence dye (Rost 1991).
Based on a fluorescence standard with known concentrations,
we obtained a linear relationship between fluorescence intensity and fluorescence dye concentration (Figure 1), indicating
that our system met this requirement. The linear regressions
from three replicated tests made on separate dates were highly
consistent (Figure 1).
A specific Ca chelator, ethylene glycol-bis (β-aminoethyl
ether) N,N,N′,N′-tetraacetic acid (EGTA), was used to confirm
that the fluorescence was from Ca2+, not from other divalent
ions. Cross sections were incubated in 1 mM EGTA for 30 min
before CTC staining. The marked reduction in fluorescence
intensity (Figure 2) when Ca was chelated by EGTA indicates
that Ca2+ was the primary source of the fluorescence.
911
Figure 2. Digitally acquired images showing the effect of EGTA on
Ca2+-CTC fluorescence intensity. (A) Cross section stained with CTC
× 130; and (B) cross section treated with EGTA before staining with
CTC × 130.
lyzed for mineral nutrients. Elemental analysis was performed
by the Analytical Laboratory at the Agricultural and Forest
Experiment Station, University of Maine. Calcium was measured by ashing dried and ground needles and dissolving the ash
in dilute HCl. Solution analysis was carried out by plasma
emission method (Model 975 Plasma Atomcomp, Jarrell-Ash
Company, Franklin, MA).
Field sampling
Foliar nutrient analysis
At the end of the 1997 experiment, current-year and 1-year-old
needles from six trees per chamber were harvested and ana-
Figure 1. Relationship between fluorescence intensity and concentrations of fluorescence dye standard. Lines are linear regressions
(P < 0.0001, r 2 = correlation coefficient) for three replicated experiments at three dates.
To compare the results obtained from the chamber studies with
field data, we analyzed mCa in mesophyll cells of needles of
red spruce trees at two field sites in Maine. Previous soil
analyses had revealed comparable soils at these sites (Jagels
et al. 1989). A red spruce stand at a coastal site (Isle Au Haut
Island) is exposed regularly to advectively generated acid fog,
and fogs with pHs as low as 2.3 have been recorded (Kimball
et al. 1988, Jagels et al. 1989). A site in Old Town, Maine
contains red spruce trees that are not exposed to acid fog and
are only rarely exposed to radiational fogs (Jagels et al. 1989).
At each site, four trees, 15--20 years old, were selected and
sampled in mid-August, 1997. Needles were collected from
the south quadrant of the upper crown (Meyer et al. 1994).
Current-year and 1-year-old needles were tested by the CTC
staining and fluorescence microscopy procedures.
Statistical analyses
Data on mCa and foliar nutrients were subjected to analysis of
variance (ANOVA) by the general linear model procedures
(SAS 1985). The ANOVA assumptions were tested by means
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912
JIANG AND JAGELS
of Levene’s test for homogeneity of variances and the χ2 test
of normality, respectively. Duncan’s multiple range test was
used to assess the significance of differences among treatment
means. In the chamber studies, mean mCa was the mean of 24
measurements (four needle cross sections × six trees). In the
field study, mean mCa was calculated from 16 measurements
(four needle cross sections × four trees).
Results
In unstained cross sections of red spruce needles, only autofluorescence from cutinized and lignified tissues such as the
cuticle, hypodermal and epidermal cell walls, guard cells and
vascular bundles was visible (Figure 3A). Figure 3B shows
Ca2+-CTC fluorescence from mesophyll and epidermal cells of
cross sections of needles stained with CTC. Evidence that the
Ca2+-CTC fluorescence was from membrane-associated Ca
(mCa), not from Ca that was associated with other cell components, is presented in Figure 3C. In plant cells, about 67% of
total calcium is in an associated form, whereas the other 33%
is in the soluble fraction. Among the associated forms of Ca,
about 90% is in the cell wall, mostly deposited in the middle
lamella as Ca pectate, whereas only about 10% is associated
with the plasma membrane (Demarty 1984, Hanson 1984).
The diagram in Figure 3C shows that fluorescence was highest
on the plasma membranes of neighboring mesophyll cells and
was minimal at the middle lamella, indicating that the large
amount of cell-wall Ca did not contribute to the observed
Ca2+-CTC fluorescence.
There were highly significant (P < 0.0001) treatment effects
on the amount of mCa in mesophyll cell membranes (Table 1).
In both years, exposure to fog decreased the amount of mCa in
needles. In 1996, there was a 27% decrease in mean mCa in
AF-treated current-year needles compared with DC needles,
and a 19% decrease compared with DWF-treated needles. The
DWF treatment resulted in a 10% decrease in mean mCa
compared with the DC treatment. In 1997, we sampled both
current-year and 1-year-old needles and found similar treatment effects to those observed in 1996. The AF-treated current-year needles showed 25 and 12% reductions in mCa
compared with DC and DWF-treated needles, respectively,
and mean mCa in DWF needles was 14% lower than in DC
needles (Figure 4). In 1-year-old needles, mean mCa in AFtreated needles was 18 and 15% lower than in DC and DWFtreated needles, respectively, but the difference between DC
and DWF-treated needles was not significant.
Seasonal changes in mCa in red spruce needles exposed to
fogs in 1997 are plotted in Figure 5. Current-year needles were
sampled first on June 30, 1997, about a week after buds
opened. In untreated (DC) needles, mCa increased during
needle elongation and stabilized when needle elongation was
completed in August (Figure 5A). In September, amounts of
mCa were twice those in June. In AF-treated needles, mCa
increased during needle development from June to July; however, it reached a plateau in July after 5 weeks of fogging,
whereas in DWF-treated needles, mCa did not begin to plateau
Table 1. Mesophyll cell mCa in red spruce needles following different
exposures. Relative mCa levels are expressed in Ca2+-CTC fluorescence brightness values. Data represent means (SE in parenthesis)
over the whole growing season. Within rows, treatment means followed by different letters are significantly different (P < 0.05), according to the Duncan’s multiple range test, following significant ANOVA
results. DC = dry control; DWF = distilled water fog; and AF = acid
fog.
Figure 3. Digitally acquired fluorescent images of cross sections of red
spruce needles. (A) Unstained control × 65; (B) stained with CTC ×
65; and (C) mesophyll cell portion of cross section stained with CTC
× 130. The diagram in (C) shows fluorescence intensity along the short
line that crosses two neighboring mesophyll cells. Abbreviations:
RC = resin canal; E = epidermal cell; H = hypodermal cell; C = cuticle;
GC = guard cell; M = mesophyll cell; S = stoma; and V = vascular
bundle.
Treatment
1996 experiment
Current-year
1997 experiment
Current-year
1-year-old
TREE PHYSIOLOGY VOLUME 19, 1999
DC
DWF
AF
20.9(4.44) a
18.7(3.09) b
15.1(2.77) c
26.8(6.51) a
26.6(2.79) a
22.8(3.88) b
25.6(2.78) a
19.9(3.84) c
21.8(3.81) b
EFFECT OF ACID FOG ON MEMBRANE-ASSOCIATED CALCIUM
913
Figure 5. Seasonal changes (1997) in mCa in current-year (A) and
1-year-old (B) red spruce needles. Data represent means of 24 measurements (four cross sections × six trees). Letters (a--c) indicate
significant differences (P < 0.05) between treatments, according to
Duncan’s Multiple Range test, following a significant ANOVA result.
Figure 4. Digitally acquired images showing treatment effects on mCa
(Ca2+-CTC fluorescence intensity) in current-year needles sampled on
September 27, 1997. Cross sections were stained in CTC for 30 min.
(A) DC × 130; (B) DWF × 130; and (C) AF × 130.
until after 7 weeks of fogging. The increase in mCa in AF- and
DWF-treated needles was less than in DC needles. In September, amounts of mCa in DWF- and AF-treated needles were
1.5- and 1.1-fold higher, respectively, than the amounts in
June.
In all treatments, there was less seasonal variation in mCa in
1-year-old needles than in current-year needles (Figure 5B).
There was a slight increase in mCa in 1-year-old DC needles
beginning around mid-August. By September, mCa of 1-yearold DC needles was 1.2-fold higher than in June. In contrast,
mCa in 1-year-old needles in the DWF treatment remained
stable throughout the season. In 1-year-old needles exposed to
AF, mCa remained stable from June to July; however, it decreased after 7 weeks of fogging, and by September, it was
only 0.8-fold of the amount in June.
There were significant treatment differences in foliar Ca
concentration in current-year needles (P = 0.039). Calcium
concentration was highest in DC needles (2088 mg kgdw−1),
being 1.8- and 1.3-fold higher than the concentrations in DWF(1171 mg kgdw−1) and AF-treated (1558 mg kgdw−1) needles,
respectively (Figure 6). In 1-year-old needles, Ca concentration was highest in DC needles and lowest in AF-treated
Figure 6. Calcium concentrations of current-year and 1-year-old red
spruce needles harvested at the end of the 1997 experiment. Means and
standard errors (error bars) from six trees are shown.
needles, but the differences were not statistically significant
(P > 0.05). The Ca concentrations of 1-year-old needles were
2870, 2416, and 2170 mg kgdw−1 in the DC, DWF, and AF
treatments, respectively.
The results from the field samples are presented in Figure 7.
For both needle-age classes, amounts of mCa of red spruce
from the coastal Isle Au Haut (IAH) site were significantly
lower than those of red spruce from the Old Town (OT) site
(P = 0.0001). Amounts of mCa in current-year and 1-year-old
needles of OT red spruce were 1.3- and 1.5-fold higher, respectively, than those of IAH red spruce.
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914
JIANG AND JAGELS
Figure 7. Comparison of mean mCa in current-year and 1-year-old
needles of red spruce trees from two field sites: IAH = Isle Au Haut,
Maine, and OT = Old Town, Maine. Data represent means and standard errors (error bars) of 16 measurements (four cross sections × four
trees).
Discussion
Acid precipitation enhances Ca leaching from forest canopies
(Cronan 1984, Joslin et al. 1988). Leaching increases with
decreasing pH and increasing exposure time in many species,
indicating a role for H+ exchange in the removal of Ca2+ from
the forest canopy (Mecklenburg 1966, Puckett 1990, Turner
and Van Broekhuizen 1992). Although the source of the removed Ca2+ is not known, it is generally believed that H+
exchanges for Ca2+ from the apoplast (Mitterhuber et al. 1989,
Puckett 1990, Turner and Van Broekhuizen 1992, Turunen et
al. 1995, Wulff et al. 1996). We found that physiologically
active Ca associated with the plasma membrane of photosynthetic mesophyll cells was displaced as a consequence of
acidic deposition.
Plant cuticles are relatively permeable to acids, and permeability increases significantly after prolonged immersion
(Hauser et al. 1993). The H+ ion is mobile within cuticles, with
permeability greater than all other ions (Tyree et al. 1991).
Cuticles account for most of the resistance of the leaf surface
to exchange between the leaf interior and exterior (Lendzian
and Kerstiens 1991). Once H+ ions pass through the cuticle,
they can easily migrate by diffusion through the apoplast and
access the plasma membrane. There, they can protonate the
head groups (pK values 3.0--4.0) of membrane phospholipids
and, thus, reduce the negative charges to which Ca2+ binds
(Hanson 1984, Gennis 1989). The pH of the bathing solutions
of synthesized or biological membranes greatly modifies
membrane surface charges and the degree of Ca2+ binding (Van
Dijck et al. 1978, Watts et al. 1978, Landau and Leshem 1988).
Reduction of negative charges in the membrane as a result of
protonation also reduces electrostatic repulsion of the membrane surface against anions (Gennis 1989). Enhancement of
SO 2−
4 uptake at low pH might explain why high acidity is
necessary for SO 2−
4 to produce needle injury and a loss of cold
hardiness in red spruce exposed to acid mist (Sheppard 1994,
Leith et al. 1995).
It has been suggested that acid-induced Ca leaching is insignificant in relation to the total apoplast cation exchange capacity (Mitterhuber et al. 1989, Puckett 1990, Turner and Van
Broekhuizen 1992). However, ion exchange between H+ and
membrane-associated Ca2+ may be more important than the
effect of acidity on total foliar Ca content. If displaced Ca2+
ions are physiologically active and perform critical functions
in plants, then their loss may pose significant consequences
before a reduction in total foliar Ca content can be detected. In
Norway spruce (Picea abies (L.) Karst.) and Sitka spruce
(Picea sitchensis (Bong.) Carrière), acid mist caused reductions in apoplast Ca, but had no significant effects on total
foliar Ca (Turunen et al. 1995, Wulff et al. 1996). DeHayes
et al. (1997) observed more dynamic seasonal variations in
mCa than in total foliar Ca in current-year needles of red
spruce. These authors found no correlation between mCa and
total foliar Ca and concluded that total Ca was a physiologically less meaningful indicator than mCa (DeHayes et al.
1997). In our study, mCa was significantly lower in AF-treated
current-year needles than in DC and DWF-treated needles,
whereas the differences in total Ca between DC and AF-treated
needles and between DWF- and AF-treated needles were not
statistically significant. In 1-year-old needles, a significant
reduction in mCa occurred in response to AF treatment,
whereas AF-induced differences in total Ca were not statistically significant.
A reduction in mCa may affect the ability of red spruce to
withstand winter injury. DeHayes et al. (1997) found that mCa
in mesophyll cells of current-year needles of red spruce increased during late September--early October, coinciding with
the cold acclimation process. We observed similar increases in
mCa in DC needles in late August--September, but not in AFor DWF-treated needles.
Although red spruce needles in the field are never continuously dry for the duration of the growing season, our dry
‘‘control’’ provided a useful reference for comparing the effects
of fogs at different pHs. By exposing trees to DWF and AF, we
created surrogate water film conditions on leaf surfaces that
simulate occult, ‘‘unpolluted’’ (including dew) deposition versus polluted fog deposition in the field. Simple wetting of
needle surfaces can facilitate exchange, by diffusion, of substances between the water film and the interior of plant leaves
along ion concentration gradients (Burkhardt and Eiden 1994);
and Ca is particularly susceptible to such non-pH driven leaching (Cronan, 1984). Thus, in current-year DWF-treated needles, the reduction in mCa may be a consequence of simple
diffusion of ions from the plant interior to a low ionic strength
surface water film, whereas the acidic fog treatment resulted in
the development of ion exchange along an H+ gradient. An
interesting follow-up experiment would be to maintain fogs of
increasing ionic strength at a pH of 5.6, to determine if increasing ionic strength differentially affects mCa.
Some leaching of salts from leaves, especially leaves retained for several years, as is the case for red spruce needles,
has adaptive value, because transpiration leads to salt accumulation in leaves. Some of these salts are metabolically important, but others can accumulate to toxic concentrations
(e.g., NaCl, aluminum salts). Osmotically-driven leaching to
surface water films probably occurs in most plants, and is
sufficient, except for extreme environments where halophytes
have adapted more specific mechanisms for shedding, secret-
TREE PHYSIOLOGY VOLUME 19, 1999
EFFECT OF ACID FOG ON MEMBRANE-ASSOCIATED CALCIUM
ing or excluding high salt concentrations (Waisel 1972, Jagels
and Barnabas 1989).
Both the field and chamber studies indicated that low
amounts of mCa were associated with acid fog. For both
needle age-classes, mean amounts of mCa in needles of red
spruce from the coastal site, where acid fog frequently occurs
(Kimball et al. 1988), were significantly lower than in needles
from the site where trees are not exposed to acid fog. However,
we were unable to establish a clear cause--effect relationship
between acid fog and amounts of mCa in field-grown red
spruce, because of the possible confounding effects of genetics, rain-induced leaching, tree phenology, soil nutrition, or
wind and sea salt effects at the two sites.
Compared with the field sites, the chamber experiments
probably provided for longer residence time of water films on
needle surfaces. Furthermore, the chamber trees were not subjected to soil water deficits, which often occur when red spruce
trees are growing in thin organic soils over hardpan or bedrock
(Jagels et al. 1989)----conditions that favor water uptake
through leaf surfaces (Boucher et al. 1995, Yates and Hutley
1995). Because of these differences, the potential for leaching
by diffusion may be less in the field than in the chamber;
however, the potential for acidic solution uptake (mass flow)
could be greater in the field than in the chamber.
In summary, exposure to distilled-water fog led to reduced
amounts of mCa in young needles. This finding implies that in
vegetation that is frequently immersed in fogs or cloud water,
or subject to heavy and prolonged dew deposition, even if
unpolluted, some loss of Ca by diffusion will probably occur.
The results of the field and chamber studies were consistent
with reduced amounts of mCa occurring in needles exposed to
acid-fog deposited water films, indicating disturbance to membrane function under conditions of acid deposition.
Acknowledgments
We thank Dr. Christopher Cronan, Dr. Michael Greenwood, Dr. William Livingston, and Dr. John Tjepkema for their critical reading of
this manuscript. Thanks are also extended to Jobie Carlisle for his
technical support in chamber design and construction, Dr. William
Halteman for his statistical advice, and Dr. Laurence Mott and Dr.
Steve Shaler for permitting us to use their video equipment during an
early stage of this study. This research is supported by McIntire-Stennis funds, University of Maine. Experimental Station report No. 2320.
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