Minimum cuticular conductance and cuticle features of Picea abies

Tree Physiology 22, 479–487
© 2002 Heron Publishing—Victoria, Canada
Minimum cuticular conductance and cuticle features of Picea abies and
Pinus cembra needles along an altitudinal gradient in the Dolomites
(NE Italian Alps)
T. ANFODILLO,1,2 D. PASQUA DI BISCEGLIE3 and T. URSO1
1
Dept. TeSAF, Timberline Ecology Research Unit, University of Padova, Via Romea, 16, I-35020 Legnaro (PD), Italy
2
Author to whom correspondence should be addressed ([email protected])
3
Regione del Veneto Servizio Fitosanitario Regionale, Osservatorio per le malattie delle piante, Lungadige Capuleti, 11, I-37122 Verona, Italy
Received January 16, 2001; accepted November 1, 2001; published online April 1, 2002
Summary Winter desiccation is believed to contribute to
stress in coniferous trees growing at the treeline because cuticular conductance increases with altitude. To test whether winter desiccation occurs in high-altitude conifers of the Dolomites (NE Italian Alps), we measured minimum cuticular
conductance (gmin), needle wettability (contact angle) and cuticle thickness in Picea abies (L.) Karst. and Pinus cembra L.
needles from December to August. Samples were collected
from adult trees along an altitudinal gradient from valley bottom (1050 m a.s.l.) to the treeline (2170 m a.s.l.). The treeline
site is one of the highest in the area and is characterized by a
generally low wind exposure. Altitude had no effect on gmin in
either species. In P. abies, large seasonal variations in gmin were
recorded but no changes were related to needle age class. Pinus
cembra had a low gmin and appeared to be efficient in reducing
needle water losses. There was a significant increase in gmin
with needle aging in P. cembra growing at low altitude that
could be related to a shorter needle longevity compared with
P. abies. High contact angles (> 110–120°) suggested the presence of tubular epicuticular waxes on needles of both species.
Contact angles were higher (low wettability) in high-altitude
needles than in low-altitude needles. By the end of winter, there
was no difference in contact angles between needles in the
windward and leeward positions. Wax structures transformed
toward planar shapes as demonstrated by the decrease in contact angle from winter to summer. In both species, the cuticle
was thicker in needles of high-altitude trees than in needles of
low-altitude trees and there was no correlation between gmin
and cuticle thickness. Because desiccation resistance did not
decrease with altitude in either species, we conclude that they
are not susceptible to winter desiccation at the tree line.
Keywords: epicuticular waxes, needle morphology, treeline,
winter desiccation.
Introduction
The leaf cuticle is a composite extracellular polymer membrane that interfaces with the atmosphere. Cuticular conduc-
tance is generally low—one or two orders of magnitude lower
than minimum stomatal conductance (Lendzian and Kerstiens
1991). However, it could play an important role in determining
leaf water status and in the case of severe physical or chemical
alterations of the cuticle, plant survival could be threatened,
particularly in evergreen conifers.
Elevated cuticular conductance in evergreen conifers at high
altitude can result in severe winter needle dehydration. Tranquillini (1979) proposed that winter desiccation accounted for
the altitudinal limit of the alpine treeline. Winter desiccation is
associated with two processes, often co-occurring, but independent of one another: frost-drought damage and abrasion by
strong winds and transported snow. Winter desiccation due to
frost-drought damage occurs when needles are unable to fully
develop their epidermis as a result of a lack of warmth during
the short growing season and so are inadequately protected
against cuticular transpiration during the following winter
(Michaelis 1934, Wardle 1971, Tranquillini 1979, 1982,
Sowell et al. 1982, De Lucia and Berlin 1983, Herrik and
Frieland 1991, Havranek 1993). Winter desiccation due to
strong winds and the abrasive effect of transported snow occurs because the outer layer of the needles is damaged, leading
to increased needle water losses, especially at the end of winter (Hadley and Smith 1983, 1986, 1990, Hadley et al. 1991,
Herrik and Friedland 1991).
In both cases, dehydration should be more severe in cold
winters with low snowfall because the soil and root remain
frozen longer, during which time the supply of water to the
needles is prevented. We hypothesized that, compared with
needles of lowland trees, needles of treeline trees have a thinner
cuticle layer and more severe abrasion of epicuticular waxes,
especially on the windward branches, and therefore higher cuticular transpiration.
To test this hypothesis, we determined if the desiccation resistance of the two main high-altitude evergreen conifers of
the Italian Alps (Picea abies (L.) Karst., and Pinus cembra L.)
changed with altitude. To assess the relevance of the two
causes of winter desiccation, we measured the same parameters reported by Baig and Tranquillini (1976) on the same spe-
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cies. We also measured wettability of epicuticular waxes and
observed their microstructure in needles of different ages.
Materials and methods
Plant materials
Experiments were carried out at three sites in the Dolomites
(NE Italian Alps) near Cortina d’Ampezzo (46°27′ N,
12°08′ E) at different altitudes but all on a prevalently
south-facing slope. The valley-bottom site (Site 1; 1050 m
a.s.l.) is dominated by secondary successional mixed stands of
P. abies, Larix decidua L. and Pinus sylvestris L. invading the
edges of recently abandoned grassland. At the subalpine site
(Site 2; 1700 m a.s.l.), P. cembra and P. abies grow in a mixed
low-density forest. At the treeline site (Site 3; 2170 m a.s.l.),
P. cembra, L. decidua and P. abies form the typical high-altitude forests of the NE Italian Alps. The actual treeline on this
side of the Alps ranges between 2000 and 2200 m a.s.l., but it
is likely that human disturbance in previous centuries has altered the treeline is some regions. Based on the rough boulder-strewn terrain and the lack of old stumps or snags at the
treeline site, we postulate that this area escaped intense grazing and that the treeline here has been relatively stable in the
last centuries.
The species studied were P. abies at all three sites and
P. cembra at Sites 2 and 3. Samples were collected in December 1–7, 1993, February 11–17, 1994, May 5–12, 1994 and
August 1–7, 1994. Three adult trees of both species with similar features and in a windward position at Site 3 were selected.
We note that treelines in the Dolomites are seldom subjected to
strong winds. During the 1997–2000 period of continuous
measurements, maximum wind speed was about 4–5 m s –1
and peaks of 20 m s –1 were recorded during only a few days in
1999.
On each tree, four branches at 2-m height (two windward
(north-side) and two leeward (south-side)) were chosen and
marked. On each branch, two shoots were sampled, sealed in
plastic bags and taken to a laboratory at the Centre of Alpine
Environment in San Vito di Cadore (Belluno province), located near Site 1.
Shoots for needle transpiration measurements were rehydrated to full turgor at 20 °C for 12–16 h in the dark before
starting measurements. Shoots for other measurements were
sealed in bags and stored in the dark at 4 °C until used.
0.5 °C and 25 ± 3%, respectively (monitored by an MP100A
Rotronic sensor connected to a CR10 Campbell Scientific, Logan, UT). Four fans were placed in the chamber to circulate air
at 3 m s –1 over the needles to minimize boundary layer resistance (Hadley and Smith 1983).
Needles were weighed after 10 h and then at 12-h intervals.
After 70 h, needle surface area was determined by the glass
bead method of Thompson and Leyton (1971) (using beads
0.11 mm in diameter). The glass beads were then removed and
the needles dried for 24 h at 85 °C. Lengths of 150 needles in
each sample were also measured.
Cuticular conductance was calculated as needle transpiration per unit surface area (g m –2 s –1) divided by the water vapor
concentration gradient (g m –3) between needle apoplast and
experimental chamber, assuming 100% relative humidity
within the needle and similar needle and air temperatures. Cuticular conductance was then expressed in m s –1 10 –5 as recommended by Kerstiens (1996) (1 m s –1 10 –5 = 0.41 mmol m –2
s –1 at standard pressure and 25 °C).
Minimum cuticular conductance (gmin) was obtained by extrapolating the linear relationship between cuticular conductance and time after stomatal closure. A generally good direct
linear relationship was obtained (Figure 1), probably because
of the severe dehydration imposed (J.N. Cape, Institute of Terrestrial Ecology, Penicuik, Scotland, personal communication). The intercept of the relationship between cuticular
conductance and time (when t = 0) provides an unbiased estimate of gmin (van Gardingen et al. 1991), which includes cuticular conductance sensu stricto, loss of water as a result of
cuticle damage or incomplete stomatal closure. Expressing
gmin as transpiration at t = 0 facilitated comparisons between
all needle samples at the same needle water content (i.e., full
saturation), excluding errors resulting from variation in cuticular conductance with needle water content (Sowell et al.
1982).
Contact angle measurements and micromorphology of
epicuticular waxes
Contact angle (CA) is defined as the angle between the surface
of the leaf and the tangent plane of a water droplet at the point
Determination of minimum cuticular conductance
Transpiration rates were determined gravimetrically on fully
saturated excised needles. Samples of current-year (5–
13 months old), 2- (17–25 months old) and 3-year-old (29–
37 months old) needles were carefully removed with tweezers
from each selected branch at the midpoint of the shoot length,
giving 90 subsamples for each measurement period. After
sealing each excision with silicon grease, eight needles of
P. abies and two needle fascicles of P. cembra were placed on
the fine-net lids of pre-weighed plastic boxes. They were
weighed and then placed in a darkened air-conditioned chamber with temperature and relative humidity maintained at 28 ±
Figure 1. Variation in cuticular conductance during the desiccation
period (one sample shown) and regression lines used to estimate gmin.
TREE PHYSIOLOGY VOLUME 22, 2002
CUTICULAR CONDUCTANCE IN CONIFERS AT DIFFERENT ALTITUDES
of contact between air, liquid and leaf (Martin and Juniper
1970). In theory, CA on a surface can vary from 0° (completely flat) to 180°. The CA of a water droplet on the needle
surface was determined as indicated by Cape (1993). One µl of
distilled water was placed at the center of the needle surface
and CA measured with the aid of a binocular microscope
equipped with a goniometer (40×). Measurements were made
at room temperature (20 °C) within 36 h of collection on 15
P. abies needles per subsample. Previous studies demonstrated
no significant differences among CA on different needle surfaces (Anfodillo et al. 1994). For P. cembra needles, 15 CAs
on the abaxial surface (where stomata are located) and 15 CAs
on the adaxial surface were measured per subsample.
Observations of the micromorphology of epicuticular
waxes were made on the same samples as those used for anatomical measurements. Samples were kept at 4 °C until prepared for scanning electron microscopy (SEM). Fresh needles
were metallized with gold (Anfodillo et al. 1994) and then examined with a Cambridge Stereoscan II SEM (Bio-Rad
Digilab Division, Cambridge, MA) at 20 kV.
Anatomical measurements
Anatomical measurements were made on current-year and
2-year-old needles collected in December 1993 and August
1994, from windward branches of both P. abies and P. cembra.
For each sample, 10 needles were chosen from the middle part
of the shoot. Midpoint needle cross sections (thickness about
10 µm) were obtained with a freezing microtome. The technique to highlight cuticle and cutinized cell wall layer was derived from Baig and Tranquillini (1976). Sections were placed
in 10% chromic acid for 10 min, then washed in water and
placed in Sudan III (saturate solution in ethanol 70%) for 1 h.
After washing in alcohol they were mounted in glycerine and
analyzed immediately. Images, which were recorded with a
video camera connected to a microscope (1000×), were digitized. Software specifically designed for anatomical measurements (NIH-Image, National Institutes of Health, Bethesda,
MA) was used, allowing 0.18 µm resolution.
In both species, cuticular membrane (cutin layer and epicuticular waxes) and cutinized cell wall thickness were measured as described by Baig and Tranquillini (1976) and
Vanhinsberg and Colombo (1990).
Multiple comparisons were computed based on one-way
analysis of variance (Scheffé test) where data were normally
distributed and variances among groups were homogeneous,
otherwise the differences were tested based on a non-parametric multiple comparison (Zar 1999).
Results
Minimum cuticular conductance
Timing of shoot extension and needle development shifted
from April–May at the lowland site to June–July at the treeline
site. Air temperature significantly affected needle length (Table 1). Picea abies needles produced in relatively cold years
(e.g., 1991) were shorter than needles produced in warmer
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years (e.g., 1992). Interannual variations in air temperature
had no significant effect on needle length at low altitude
(Site 1). Differences in needle length of P. abies did not account for variations in gmin at any of the sites (Figure 2). Variations in gmin were coupled to air temperature only in P. abies
trees at the treeline, but gmin of the three needle age classes was
not significantly different (P = 0.29, Kruskal-Wallis test).
The most important factor determining variations in gmin of
P. abies needles was season (Figure 3). Highest gmin values (up
to 4.5 m s –1 10 –5) were measured in needles collected in August at Site 2, and the lowest values were found in needles collected in February at Site 2 (< 0.5 m s –1 10 –5). At Site 1, gmin
increased more than fourfold from December (5-month-old
needles) to August (13-month-old needles). Similarly, trees at
Site 2 had a maximum gmin at the end of summer and a minimum in February. Trees at the treeline (Site 3) had a maximum
gmin in February and a minimum in December. Seasonal differences in gmin of P. abies needles were smaller at Site 3 than at
the other sites but a significant increase in mean gmin occurred
at the end of winter (February). The effect of altitude on winter
gmin appeared to differ between pre- and post-winter conditions: in December gmin was highest in trees at Site 2, but in
February gmin increased with altitude (Figure 3). There were no
significant differences in gmin between windward and leeward
P. abies branches, except for current-year needles at Site 1 in
August, when leeward needles had higher gmin than windward
needles.
The pattern of gmin variation in P. cembra differed from that
in P. abies. At both sites, gmin increased significantly with
needle age (Figure 4a), but changes in gmin were not coupled to
variations in air temperature during needle extension growth.
Seasonal variations in gmin were small (Figure 4b) within a
needle age class and only in 1-year-old needles at Site 2 and
2-year-old needles at Site 3 were the differences significant.
Mean gmin did not differ significantly between treeline (Site 3)
and subalpine stands (Site 2), except in 2-year-old needles. In
general, P. cembra had a lower gmin than P. abies (gmin of
P. cembra was not measured in December 1994).
Needle wettability, epicuticular wax structure and cuticle
thickness
Values of CA varied widely among sites and seasons. Picea
abies showed highest CA values in the youngest needles of
trees growing at the treeline in February (Figure 5). Needles of
lowland trees (Site 1) had a relatively low CA (high wettability) throughout the year, with a significant increase in May and
August. Needle age had no significant effect on CA. Picea
abies trees at Site 2 had generally less wettable needles than
trees at the other sites, and needle aging led to a significant decrease in CA. At the treeline (Site 3), a significant increase in
CA was observed during the winter months, followed by a
marked decrease. As at Site 2, there was a progressive decrease in CA from current-year to 2-year-old needles of
P. abies trees at Site 3, but this trend was not correlated to environmental conditions or to needle length at either Site 2 or 3
(see Table 1). In August, CA was higher in windward needles
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ANFODILLO, PASQUA DI BISCEGLIE AND URSO
Table 1. Mean air temperature at Site 1 during periods of needle development at all three sites (Site 1: April–May; Site 2: May–June; Site 3:
June–July), estimated mean air temperature at Site 3 during the period of needle development, and mean length of P. abies needles. Within a site,
values of needle length followed by different letters are significantly different (P < 0.05; n = 150).
Year/period
Mean air temperature (°C)
At Site 1
Mean 1966–2000
1991 (2 year)
1992 (1 year)
1993 (current year)
1
Needle length (mm)
At Site 3
April–May May–June
June–July
June–July1
8.5
5.2
9.5
9.6
15.2
14.3
15.9
14.6
8.9
10.6
9.3
12.3
9.5
13.5
13.2
Site 1
Site 2
Site 3
17.9 ± 2.0a
17.8 ± 1.9a
17.5 ± 1.6a
16.0 ± 1.7a
16.9 ± 1.5b
16.0 ± 1.9a
13.8 ± 1.7a
14.9 ± 1.8b
14.3 ± 1.3c
Temperatures at Site 3 estimated by linear regression of data measured at Site 1 and close to Site 3 (r 2 = 0.9) during 1997–2000.
(north facing) than in leeward needles (south facing) (P <
0.001) at all three sites. In May, windward needles had a lower
CA than leeward needles only at Site 3, but during the rest of
the year no significant differences were recorded at any site.
In P. cembra, the adaxial needle surface had a significantly
lower CA (75°) than the abaxial needle surface (96°) irrespective of site and needle age class. Within a site, annual trends
and variations were similar to those measured in P. abies and
there was a significant decrease in CA with needle age (Figure 6), especially on the abaxial needle surface. Highest variations in CA during the year were recorded on the abaxial
surface of needles in P. cembra trees at Site 3 (more than 35°).
Changes in CA were less on the adaxial surface than on the
abaxial surface and the difference between maximum and
minimum mean values was always less than 20°. Differences
between windward and leeward needles of P. cembra were
similar to those in P. abies, except that there was no significant
difference at Site 3 in August.
In both species, epicuticular waxes appeared as fine tubes
on both interstomatal and intrastomatal zones (Figure 7a).
Among sites, density of interstomatal epicuticular waxes was
Figure 2. Variation in gmin in current-year, 1- and 2-year-old needles
of P. abies at Sites 1, 2 and 3. Vertical bars are +1 SE (n = 24). Differences between needle age classes within a site were not significantly
different (Kruskal-Wallis test).
Figure 3. Variation in gmin in current-year needles of P. abies at Sites
1, 2 and 3 throughout the season (December to August). Vertical bars
are +1 SE. Within a site, different letters indicate significant differences (P < 0.05) between sampling periods.
Figure 4. (a) Variation in gmin in current-year, 1- and 2-year-old needles of P. cembra at Sites 2 and 3. Vertical bars are +1 SE. Within a site,
different letters indicate significant differences (P < 0.05) between
needle age classes. (b) Seasonal variations in gmin in current-year
needles of P. cembra needles at Sites 2 and 3. Vertical bars are +1 SE.
Within a site, differences were not significant (Kruskal-Wallis test).
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CUTICULAR CONDUCTANCE IN CONIFERS AT DIFFERENT ALTITUDES
Figure 5. Variation in contact angle in current-year (N), 1- (N–1) and
2- year-old (N–2) needles of P. abies at Sites 1, 2 and 3 throughout the
season (February to August). Vertical bars are +1 SE. Within a needle
age class, different letters indicate significant differences (P < 0.05).
much higher in needles from Site 3. Needles from Site 1 had a
very sparse wax architecture. Marked degradation of the original wax structure was evident in needles of different age
classes at Sites 2 and 3, but particularly at Site 3, leading to a
smooth wax structure in 2-year-old needles (Figure 7b).
Once needle elongation had finished, seasonal morphological changes occurred. In P. abies (Figure 8), changes in cuticle
thickness were site-related. At low altitude (Site 1), winter
needles had a significantly thicker cuticle than summer needles in both age classes measured. Seasonal variations in cuticle
thickness were small and not significant at Sites 2 and 3. Cuticle thickness did not differ significantly between Site 2 and
Site 3 needles but it was thicker than at Site 1. Thickness of the
cutinized cell wall showed similar variations to cuticle thickness. Compared with P. abies, P. cembra had a more differentiated epicuticular wax layer reaching a thickness of 3–4 µm.
Differences between adaxial and abaxial surfaces were not
significant. A large increase in quantity of wax was observed
in both current-year and 2-year-old needles of P. cembra trees
at Site 3 during summer (Figure 9), leading to maximum cuticle thickness in August. No significant variations in cuticle
483
Figure 6. Variation in contact angle of the adaxial needle surface and
the abaxial needle surface of current-year (N), 1- (N–1) and 2-yearold (N–2) needles of P. cembra at Sites 2 and 3 throughout the season
(February to August). Vertical bars are +1 SE. Within a needle age
class, different letters indicate significant differences (P < 0.05).
thickness were related to needle age class, but cuticle thickness increased significantly with altitude in both species.
Discussion
Minimum cuticular conductance
Mean gmin (between 0.5 and 5.6) was of the same order of magnitude as reported for other conifers including P. abies (see
Sowell et al. 1982, Kerstiens 1996, Heinsoo and Koppel
1999). Minimum cuticular conductance is an overall measure
of needle water loss; i.e., gmin measures the efficiency with
which a leaf avoids water loss independently of the path of water movement. From an ecological point of view, a species that
restricts water loss has to exert strong control on both cuticular
permeability and stomata closure when water deficits occur.
Large intra-annual variations in gmin were recorded on needles from P. abies trees at Sites 1 and 2. The large increase of
gmin during the growing season (more than four times) points
to defective stomatal closure rather than to macroscopic
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Figure 8. Variation in cuticle thickness in current-year (N) and
2-year-old (N–2) needles of P. abies at Sites 1, 2 and 3 in December
(open bars) and in August (solid bars). Vertical bars are +1 SE.
Figure 7. (a) An SEM image of epicuticular waxes of 2-year-old needles of P. abies collected in August at Site 2. (b) An SEM image of
epicuticular waxes of current-year needles of P. abies collected in August at Site 2. Note the marked degradation of epicuticular wax microstructure of 2-year-old needles in Panel a.
changes in cuticle permeability (Heinsoo and Koppel 1999),
although Riolo (1999) reported a two- to threefold increase in
wax in winter compared with summer in needles at our study
sites. These findings indicate that, in the southern alpine environment, good control of water loss during summer is not an
essential requirement for survival of P. abies trees. We note
that summer droughts seldom occur in this region because
summer is the wettest season with mean precipitation of
400–500 mm and the soil has a relatively high water-holding
capacity. However, trees have to reduce gmin during winter,
when low soil and air temperatures associated with relatively
high vapor pressure deficits limit water uptake and storage
(Larcher 1985). The intraannual pattern of gmin shown by trees
at Site 3 is consistent with an enhanced water storage capacity
of trees at the treeline compared with lowland ecotypes
(Barton and Teeri 1993), which might have evolved as a result
of the low water-holding capacity of alpine soils inducing potential drought conditions (Anfodillo et al. 1998).
Interannual variations in gmin in P. abies showed that needles
did not undergo degradation of cuticular properties with needle age, at least until the third year. This finding contrasts with
that reported by Schreiber (1994). We conclude that, in
P. abies, changes in the content or constituents of cuticular
waxes (Prügel et al. 1994) and physical degradation (van
Gardingen at al. 1991) are unimportant or are counterbalanced
by new synthesis of some cuticular constituents that maintain
the permeability unchanged. A significant increase in gmin was
detected in 3-year-old needles of P. abies (Heinsoo and Koppel
1999).
Mean values of gmin in P. cembra (Figure 3) confirmed its
high water storage capacity (Tranquillini 1979), especially in
trees on Site 3. The small intraannual variations in gmin indicate
that P. cembra is well adapted to environmental changes. If an
Figure 9. Variation in cuticle thickness in current-year (N) and
2-year-old (N–2) needles of P. cembra at Sites 2 and 3 in December
(open bars) and in August (solid bars). Vertical bars are +1 SE.
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increase in gmin with needle age can be interpreted as a negative
factor for plant water status, we conclude that trees on Site 3
are better adapted to the environment than trees on Site 2. The
enhanced senescence of trees at Site 2 is consistent with previous observations (Nebel and Matile 1992).
The frequently observed trend of an increase in gmin with increasing altitude (Baig and Tranquillini 1976, Sowell et al.
1982, Herrick and Friedland 1991, W. Platter cited in
Havranek 1993) was not wholly confirmed. The relatively
high gmin of P. abies needles in February is inconsistent with a
lack of maturing tissue, because if this is the case, gmin should
also be high in December. Cuticle damage caused by some environmental factor (e.g., wind, snow) may account for the high
gmin in February. Presumably needle damage was effectively
repaired, because gmin returned to pre-winter values in May.
Needle wettability and cuticle thickness
The width of the CA depends primarily on the nature and arrangement of exposed surface atoms, but it is also strongly affected by surface roughness. Contact angles wider than 110°
indicate that the surface is covered entirely by an epicuticular
wax ultrastructure. The widest CAs are found when epicuticular waxes are tube-shaped (Martin and Juniper 1970) as is the
case in the species examined (Günthardt 1985). Alterations in
the structure of epicuticular waxes, which can be assessed by
measuring CA, can be induced by pollutants or physical damage (e.g., wind). These changes in CA have been correlated to
an increase in gmin (MacKerron 1976, Turunen and Huttunen
1990, van Gardingen et al. 1991), suggesting that changes in
gmin can be predicted from CA, which is easier to measure.
The density and amount of epicuticular waxes change with
environmental factors (Cape and Percy 1993). For example,
low temperatures and a decrease in radiant energy rate increase wax deposits in Brassica oleracea L. (Baker 1974).
Temperature also determines wax shape. Rods and tubes are
formed at low temperatures, whereas large plates are more
common at high temperatures (Baker 1974). Picea abies trees
growing in a greenhouse (higher temperature) had more wax
compared with outdoor trees (lower temperature); however,
shaded plants had significantly more wax than unshaded
plants (Cape and Percy 1993).
Although previous studies have shown that epicuticular
waxes increase in P. abies needles with increasing altitude
(Günthardt 1984, Riolo 1999), we were unable to infer anything about epicuticular wax quantity from our study. However, the high CA values in needles of high-altitude trees of
both species suggest that these needles have a dense structure
of rod-shaped wax crystals and this was confirmed by SEM
analysis (Figure 7a). The main component of epicuticular
waxes in P. abies (also abundant in P. cembra) is a secondary
alcohol ((S)-nonacosan-10-ol) (Prügel et al. 1994). A particular feature of this wax compound is that it crystallizes in the
form of a tubule. A tubular crystal shape has a large surface
area to volume ratio and is therefore thermodynamically unstable (Jetter and Riederer 1994). The kinetic and metastable
tubular form transforms spontaneously into the thermodynamically more favorable compact planar form of the alcohol,
485
which probably accounts for the large decrease in CA from
winter to summer months, especially on needles with more articulated tubules (Figures 5–7). Needles with sparse epicuticular waxes (Site 1) underwent small changes between seasons and years. Needles on south-facing branches had a higher
degradation of epicuticular waxes during May and August
(significantly lower CA) than north-facing branches, presumably because rearrangement to planar crystals was accelerated
on sun-exposed needles (Kim 1985).
An alternative explanation for the decrease in CA from winter to summer may be associated with age-related changes in
needles. In general, a decrease in (S)-nonacosan-10-ol occurs
in older needles (Prügel et al. 1994). The occurrence of recrystallized wax tubes (shown by the increase in CA in 1- and
2-year-old needles on trees at Sites 2 and 3 in winter) is enhanced in needles undergoing strong transition to planar forms
(Site 3), or more evident physical damage (BermadingerStabentheiner 1995). However, redevelopment of tubes never
replaced the quantity and structure of the original epicuticular
wax, leading to decreased CA. This suggests that re-crystallization and changes in epicuticular wax structure can also occur when the needle is fully expanded some years after its
formation.
In P. abies needles from Site 3, the highest values of gmin
(Figure 3) corresponded to the highest CA, especially in current-year needles, and a decrease in CA throughout the season
paralleled the decrease in gmin. However, relationships between CA and gmin seemed to be uncoupled in needles from
Site 2. In P. cembra, a significant decrease in CA in current-year needles from Site 3 (both sides) throughout the season (Figure 6) did not correspond to changes in gmin (Figure
4b). Thus, overall, variations in CA were not correlated to gmin
in either species, indicating that variations in CA cannot be
used to infer changes in gmin and that changes in outer surface
properties cannot always be correlated to changes in water
conservation capacity of the needles.
Measured values of cuticle thickness were similar to those
reported by Baig and Tranquillini (1976), with cuticular membrane thickness ranging from 2.1 to 9.1 µm in P. abies and
from 2.0 to 10.0 µm in P. cembra. A decrease in cuticle thickness with increasing altitude has been reported for Abies
balsamea (L.) Mill. (DeLucia and Berlyn 1983) and P. abies
and P. cembra (Baig and Tranquillini, 1976), which is in
agreement with the frost-drought theory. Our data suggest an
opposite trend. Günthardt and Wanner (1982) found an increase in total amount of waxes in high-elevation P. cembra
and P. abies trees and Sase et al. (1998) showed a similar trend
in Cryptomeria japonica (L.f.) D. Don. An increased amount
of wax in needles of high-altitude trees may minimize the effect of UV radiation because the wax layer has a peak absorption at 320 nm (Sase et al. 1998).
The effect of a large amount of waxes on water storage is
more controversial. Cape and Percy (1996) demonstrated that
water loss rate is independent of the amount of surface wax in
spruce needles. Becker et al. (1986) and Schreiber and Riederer (1996) showed that plant species with thin cuticles have
lower cuticular conductances than species with thick cuticles,
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
486
ANFODILLO, PASQUA DI BISCEGLIE AND URSO
Figure 10. Relationship between gmin and cuticular layer thickness in
P. abies (䊊) and P. cembra (䊉). Horizontal bars are ±1 SE.
refuting the premise that a thick cuticle is more efficient in reducing water loss. Our data confirm the lack of a significant relationship between gmin and cuticle thickness (Figure 10).
Overall, given the position of the treeline in the Dolomites,
our data indicate that trees at the treeline are not susceptible to
winter desiccation. This finding supports the conclusion of
Körner (1998) that winter desiccation may be a problem for
young trees in some parts of the temperate zone but cannot explain the world-wide alpine treeline.
Acknowledgments
The research was funded by CNR 94.00966.CT06 and by the Italian
Ministry of University and Scientific Research (MURST ex 40%). We
are grateful to Fausto Fontanella, Roberto Menardi and Giuseppe Sala
of the Ecology Laboratory in San Vito di Cadore for technical help
and to Giovanna Capizzi and Guido Masarotto for the helpful discussion on data analysis. We thank Satu Huttunen and Manfred Havranek
for helpful suggestions.
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