Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:

Tree Physiology 17, 617--626
© 1997 Heron Publishing----Victoria, Canada

Growth, leaf anatomy, and physiology ofPopulus clones in response to
solar ultraviolet-B radiation
MICHAEL A. SCHUMAKER,1 JOHN H. BASSMAN,2,4 RONALD ROBBERECHT3 and GARY
K. RADAMAKER2
1

Plant Gene Expression Center, University of California, Berkeley/U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA

2

Department of Natural Resource Sciences, Washington State University, Pullman, WA 99164-6410, USA

3

Department of Range Resources, University of Idaho, Moscow, ID 83844, USA

4

Author to whom correspondence should be addressed

Received August 9, 1995

Summary We compared the physiological and morphological responses of rooted cuttings of Populus trichocarpa Torr.
& Gray and P. trichocarpa × P. deltoides Bartr. ex Marsh.
grown in either near-ambient solar ultraviolet-B (UV-B; 280-320 nm) radiation (cellulose diacetate film) or subambient
UV-B radiation (polyester film) for one growing season. Midday biologically effective UV-B radiation was 120.6 and 1.6 mJ
m −2 s −1 under the cellulose diacetate and polyester films,
respectively. Gas exchange, leaf chlorophyll, light harvesting
efficiency of photosystem II, and foliar UV-B radiation-absorbing compounds (i.e., flavonoid derivatives) were measured in
expanding (leaf plastochron index (LPI) 5), nearly expanded
(LPI 10), and fully expanded mature (LPI 15) leaves of intact
plants of plastochron index 30 to 35. Plants were then harvested
and height, diameter, biomass allocation and leaf anatomical
attributes determined.
Net photosynthesis, transpiration, and stomatal conductance
were significantly greater in mature leaves exposed to subambient UV-B radiation than in mature leaves exposed to nearambient UV-B radiation. Concentrations of UV-B radiation-absorbing compounds (measured as absorbance of methanol-extracts at 300 nm) were significantly greater in mature
leaves exposed to near-ambient UV-B radiation than in mature
leaves exposed to subambient UV-B radiation. The UV-B radiation treatments had no effects on chlorophyll content or

intrinsic light harvesting efficiency of photosystem II.
Height, diameter, and biomass were not significantly affected by UV-B radiation regime in either clone. Leaf anatomical development was unaffected by UV-B radiation treatment
in P. trichocarpa × P. deltoides. For P. trichocarpa, leaf
anatomical development was complete by LPI 10 in the nearambient UV-B radiation treatment, but continued through to
LPI 15 in the subambient UV-B radiation treatment. Mature
leaves of P. trichocarpa were thicker in the subambient UV-B
radiation treatment than in the near-ambient UV-B radiation
treament as a result of greater development of palisade parenchyma tissue.

We conclude that exposure to near-ambient UV-B radiation
for one growing season caused shifts in carbon allocation from
leaf development to other pools, probably including but not
limited to, UV-B absorbing compounds. This reallocation curtailed leaf development and reduced photosynthetic capacity
of the plants compared with those in the subambient UV-B
radiation treatment and may affect growth over longer periods
of exposure.
Keywords: biomass accumulation, chlorophyll content, dark
respiration, deciduous trees, diameter, height, leaf anatomical
development, light harvesting, net photosynthesis, photosystem II, Populus trichocarpa, Populus trichocarpa × P. deltoides, stomatal conductance, transpiration, UV-B radiation-absorbing compounds.

Introduction
Depletion of stratospheric ozone (O3) over northern temperate
latitudes of North America and Europe is causing increased
solar ultraviolet-B (UV-B; 280--320 nm) radiation at the
ground in these regions (Madronich 1992, Kerr and McElroy
1993, Jokela et al. 1995, Zerefos et al. 1995). Because UV-B
radiation can degrade and cause conformational changes in
proteins, nucleic acids, and other macromolecules (Caldwell
1981), enhanced UV-B radiation resulting from stratospheric
O3 depletion is a potentially important factor in terrestrial and
marine ecosystems.
Crop and herbaceous species that are sensitive to solar UV-B
radiation often exhibit decreased carbon assimilation (Van
et al. 1976, Brandle et al. 1977, Sisson and Caldwell 1977,
Tevini and Iwanzik 1983, Sisson 1986, Bornman 1989), and
stomatal conductance (Bennett 1981, Negash and Björn 1986,
Tevini and Teramura 1989), accentuated mesophyll resistance
(Brandle et al. 1977, Teramura 1983) and disrupted electron
transport (Caldwell et al. 1982, Iwanzik et al. 1983, Kulandaivelu and Noorudeen 1983, Renger et al. 1989) compared to
species that are not sensitive to solar UV-B radiation. In addi-

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SCHUMAKER ET AL.

tion, UV-B radiation induces production of UV-B-absorbing
compounds (Biggs et al. 1981, Caldwell et al. 1983b, Warner
and Caldwell 1983, Flint et al. 1985, Schnabl et al. 1986), and
damage to photosynthetic pigments (Vu et al. 1982a, Teramura
1983, Day and Vogelmann 1995). The main site for damage
appears to be photosystem II (PS II) (Melis et al. 1992, van
Leewen and van Gorkom 1992). Photosynthetic effects vary
with leaf ontogeny (Teramura and Caldwell 1981); younger
leaves or those of maximum photosynthetic activity are more
sensitive to UV-B radiation than older leaves. Solar UV-B
radiation can also increase dark respiration (Sisson and
Caldwell 1976), reduce transpiration (Tevini and Teramura
1989) and induce the closure of stomata (Negash and Björn
1986). Ultraviolet-B radiation reduces plant height and
biomass (Tevini and Teramura 1989, Teramura and Sullivan

1994), leaf area, and can retard leaf maturation in many sensitive species (Tevini and Teramura 1989). High UV-B irradiance increases leaf weight:leaf area ratios (Teramura 1983)
reflecting increased carbon allocation to leaves. Root:shoot
ratios can also fluctuate with UV-B dose, although to a smaller
degree than leaf weight:leaf area ratios (Teramura 1980a,
Biggs et al. 1981).
Although trees, unlike most crop and herbaceous plants, are
subject to long-term, accumulated effects of environmental
stress, the responses of trees and other perennial plants to
UV-B radiation are not well understood. Substantial interspecific growth responses to enhanced UV radiation have been
demonstrated in some conifers (Cline and Salisbury 1966,
Kaufmann 1978, Kossuth and Biggs 1981, Sullivan and Teramura 1988, Stewart and Hodinott 1993, Sullivan and Teramura
1994). In loblolly pine (Pinus taeda L.), enhanced UV-B radiation had minimal effects on photosynthesis, dark respiration,
stomatal conductance, chlorophyll concentrations and apparent quantum efficiency (PS II) (Sullivan and Teramura 1989,
1992, Naidu et al. 1993); however it caused reductions in
needle elongation, total leaf area and biomass over a 3-year
period suggesting cumulative effects with additive growth
reductions.
Recently, several studies have focused on the effects of
UV-B radiation on angiosperm tree species (Lovelock et al.
1992, Sullivan et al. 1992, DeLucia et al. 1994, Searles et al.

1995). Angiosperm trees may be more sensitive to UV-B
radiation than conifers because they may have a less effective
UV-B screen (Day et al. 1992, 1993). In angiosperms, UV-Babsorbing compounds occur primarily in vacuoles of epidermal cells (Weissenböck et al. 1984, Schnabl et al. 1986,
Weissenböck et al. 1986, Schmelzer et al. 1988), whereas in
conifers these compounds occur both in vacuoles and are
conjugated in cell walls (Strack et al. 1988, 1989). Angiosperms allow considerable penetration of UV-B through anticlinal epidermal cell walls to sensitive mesophyll tissue below,
whereas in conifers the epidermis attenuates a large proportion
of incoming UV-B radiation (Day et al. 1993).
Solar UV-B radiation may affect anatomical development in
leaves (Caldwell 1981, Barnes et al. 1990). It may also result
in protein degradation (Jordan et al. 1992) and reductions in
ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco)

and PEP carboxylase activities (Vu et al. 1982a, 1982b, 1984).
Thus, exposure to UV-B radiation may inhibit development of
photosynthetic capacity in expanding leaves and increase the
rate of senescence in mature leaves. As a result, the contributions of both young and old leaves to the carbon balance of
trees may be severely reduced.
We have evaluated the effects of solar UV-B radiation on a
fast-growing angiosperm tree species (Populus) by comparing

responses under near-ambient UV-B radiation to those where
UV-B was excluded from natural sunlight. Specific objectives
were to quantify effects of UV-B radiation regime on: (1)
growth and carbon allocation; (2) gas exchange and internal
leaf CO2 concentrations; (3) leaf chlorophyll contents and light
harvesting efficiency of photosystem II; (4) accumulation of
UV-B radiation-absorbing compounds; and (5) leaf anatomy.
We also determined how these responses varied with leaf
ontogeny.
Materials and methods
Plant material
Ramets from trees of Populus trichocarpa Torr. & Gray (Clone
1A) and Populus trichocarpa × P. deltoides Bartr. ex Marsh.
(Hybrid 5 [11-5]; hereafter Clone HY5) were studied. Clone
1A is native black cottonwood originating in the Palouse River
drainage of eastern Washington (45°50′ N, 117°05′ W). Clone
HY5 is a cross between western Washington state P. trichocarpa and a variety of eastern cottonwood (P. deltoides) developed at Stoneville, MS (Stettler 1968). This clone
demonstrates considerable heterosis, having much larger
leaves, greater growth and biomass accumulation than either
parent.

Plant culture
Twenty-four dormant hardwood cuttings of each clone were
removed from cold storage, soaked in water for 72 h at room
temperature, and then planted in 11-liter pots containing standard nursery potting medium. After 24 h in a greenhouse, pots
containing planted cuttings were placed in a second pot to
prevent root-binding with substrate outside the pots. The entire
unit was set in the ground (to maintain root temperatures near
normal) inside a 3.0 × 1.2 × 2.0-m gable-end plastic-covered
house oriented with the long axis east and west at the E. H.
Steffen Center in Pullman, WA (46°44′ N, 117°10′ W). Each
house contained two trees of each clone that were randomly
arranged in a single row to minimize shading. Plants were
grown from late June until mid-August 1994. The ground area
within the houses was covered with tan bark to minimize soil
temperature variations.
The 12 houses were constructed from 19-mm 40-PVC pipe
and covered with transparent plastic film. Six houses were
covered with 0.13 mm cellulose diacetate (near-ambient UV-B
radiation; transmittance down to 300 nm), and six were covered with 0.13 mm polyester film (subambient UV-B radiation;
transmittance down to 315 nm). Because of weathering of the

materials, the cellulose diacetate film was changed every 14-28 days and the polyester film was changed every 28 days. No

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RESPONSES OF POPULUS TO UV-B RADIATION

619

appreciable solarization of the cellulose diacetate film was
detected after 28 days of use in the field.

ment. Variable to maximal fluorescence ratios (Fv /Fm ) were
calculated with variable fluorescence (Fv) equal to Fm − Fo.

Ultraviolet-B radiation treatment

Gas exchange

Midday spectral irradiance under cloudless conditions was
measured in mid-August under the cellulose diacetate and

polyester films with a spectroradiometer (Optronic Laboratories 754, Optronic Laboratories, Orlando, FL). Unweighted
UV-B irradiance was 2.33 W m −2 nm −1 under the cellulose
diacetate and 0.14 W m −2 nm −1 under the polyester film. The
absolute spectral irradiance, weighted using the generalized
plant response function (Caldwell 1971) normalized to
300 nm, was equivalent to 200.1 and 0.08 mW m −2 biologically effective UV-B radiation (UV-BBE) under the cellulose
diacetate and polyester films, respectively. Midday solar spectral irradiance under cloudless conditions was calculated according to Björn and Murphy (1985).
Environmental conditions were similar in the cellulose
diacetate and polyester houses. Photosynthetic photon flux
density (PPFD; 400--700 nm) measured around solar noon at
the top of the plant canopies ranged from 150 µmol m −2 s −1 on
cloudy days to 2050 µmol m −2 s −1 on sunny days. The PPFD
under the plastic films ranged between 90 and 95% of ambient.
Noon temperatures at 1.0 m above the ground within the
houses ranged from 29 to 40 °C and were typically 1 to 3 °C
higher than temperatures recorded outside the houses. Houses
were vented by leaving the lower 0.3 m uncovered at the base
and 48 cm2 uncovered at the peak of the gable ends.
Plants were watered to maintain soil water near field capacity and fertilized at regular intervals with N,P,K (20,20,20)
commercial liquid fertilizer containing micronutrients. To

minimize herbivory by insects, plants were periodically
sprayed with Xclude PT 1600A (Whitmire Research Laboratories, Inc., St. Louis, MO).

Immediately following chlorophyll fluorescence measurements, gas exchange was measured on the same leaves with an
open, compensating gas exchange system (Bingham Interspace BI-7, Bingham Interspace Co., Logan, UT) connected to
an infrared gas analyzer (225 MK, Analytical Development
Company, Ltd., Hoddesdon, U.K.). Portions of intact leaves
were enclosed in a 300 cm3 stainless steel and glass cuvette.
Because of absorption of UV-B radiation by the cuvette glass,
gas exchange measurements represent cumulative rather than
instantaneous effects of UV-B radiation. Temperature within
the cuvette was controlled thermoelectrically, and air was
circulated by three fans. Fluctuations in water vapor concentration within the cuvette were monitored with a dewpoint
sensor (General Eastern Instruments Corp., Watertown, MA),
and leaf temperature was measured by a small copper-constantan thermocouple appressed to the abaxial leaf surface. Irradiance was supplied by a metal halide lamp and photosynthetically active radiation (PAR) was measured at the leaf
surface with a Li-Cor 190-SB quantum sensor (Li-Cor, Inc.,
Lincoln, NE). During measurements, radiation was filtered
through a water bath to reduce infrared heating within the
cuvette. Foliage outside the cuvette was covered with cheesecloth to prevent heat damage from the lamps. All measurements were made at saturating PAR (≥ 1100 µmol m −2 s −1),
optimal leaf temperature (25.0 °C), and minimal leaf-to-air
vapor density difference (VDD ≤ 6.0 g cm −3). Net photosynthesis, dark respiration, transpiration, water-use efficiency, stomatal conductance, and leaf internal CO2 concentrations were
calculated according to Ball (1987). Leaf area enclosed in the
cuvette was measured with a leaf area meter (Delta-T Devices,
Cambridge, U.K.).

Height and diameter
Chlorophyll analysis

Height and diameter (10 mm above the intersection with the
cutting) were measured weekly during the growing period. The
plastochron index (PI) and leaf plastochron index (LPI) (Larson and Isebrands 1971) were used as the morphological time
scales. A reference length of 40 mm for the lamina of the index
leaf was used. Thus at PI 30, the index leaf (LPI 0), the 30th
leaf from the base, would be exactly 40 mm long. When plants
reached a plastochron index (PI) of 30 to 35 (7 to 9 weeks for
Clone 1A; 9 to 10 weeks for Clone HY5), one plant of each
clone was randomly selected from each house for final growth
measurements.

Three leaf discs (1.0 cm2) were cut from each leaf immediately
after gas exchange measurements and placed in a light-proof
vial containing 5.0 ml N,N-dimethylformamide (DMF). The
vials were stored in the dark for 24 h at 4 °C. Absorbance of
the chlorophyll extract was determined at 647 and 664.5 nm
(Bausch and Lomb Spectronic 21, Bausch and Lomb, Woburn,
MA). Total chlorophyll (total Chl), chlorophyll a (Chl a),
chlorophyll b (Chl b), and the ratio of chlorophyll a to b (Chl
a/b) were calculated as described by Inskeep and Bloom
(1985).

Chlorophyll fluorescence

Ultraviolet-B radiation-absorbing compounds

Chlorophyll fluorescence measurements were made following
illumination of each plant with a sodium halide lamp. Minimal
fluorescence (Fo) and maximum fluorescence (Fm) were determined for a vertical series of leaves consisting of LPI 5 (developing leaf), LPI 10 (nearly expanded leaf), and LPI 15 (fully
expanded leaf) for each plant with a modulated fluorometer
(Opti-Sciences OS-500, Opti-Sciences, Inc., Tewksbury, MA).
Leaves were placed in the dark for 10 min before measure-

Three leaf discs (1.0 cm2) were excised from each leaf used in
the gas exchange measurements, dried at 95 °C for at least
24 h, and then ground in a mortar with a pestle in 10 ml of
methanol/H2O/HCl solution (70/29/1 v/v). The extract was
centrifuged at 600 g for 10 min. The absorbance of the supernatant was measured at 5-nm increments between 260 and
310 nm with a Perkin-Elmer Lambda 1 UV/Visible spectrophotometer (Perkin-Elmer Corp., Wilton, CT). Absorbance at

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SCHUMAKER ET AL.

300 nm was used as an index of the relative content of UV-B
radiation-absorbing pigments. Values were calculated on a leaf
area basis (Robberecht and Caldwell 1986).
Leaf anatomy
Transverse sections of fresh leaf tissue, taken at a point approximately one-third of the lamina length from the tip of
leaves used for the physiological measurements, were fixed in
2.5% (v/v) glutaraldehyde, and stored at 4 °C. Fixed specimens
were washed three times with a 0.5 M phosphate buffer
(pH 7.2) and dehydrated in a graded ethanol series. Samples
were then embedded in LR White resin (1/3, 1/2, 1/2, 2/1, resin
to ethanol, pure resin three times, 18 to 24 h each). The resin
was polymerized at 60 °C for 20 to 24 h within gelatin capsules. Sections 1.0 µm thick were cut from each sample with a
glass knife (Sorvall MT2-B ultra-microtome, DuPont Co.,
Newtown, CT), mounted on gelatin-coated slides and stained
with either 0.5% aqueous Stevenel’s blue or 1% aqueous
Safranin O. Total leaf thickness and thickness of the upper and
lower epidermal, palisade parenchyma, and spongy mesophyll
layers of each section were quantified by means of an image
analysis program (NIH Image 1.56, National Institute of
Health, Washington, DC), high-resolution video camera (Pulnix America TM-7CN, Pulnix America, Inc., Sunnyvale, CA)
and light microscope (Olympus BH-2, Olympus Corp., Lake
Success, NY).

rates, which were linear and added approximately 4--5 plastochrons weekly. At harvest, plants were between 0.85 to 1.07 m
tall with diameters of 11 to 15 mm. Total dry weight and
shoot:root ratio of plants of Clone 1A were about 50 g and
2.74, respectively. Total dry weight of plants of Clone HY5
was 160 to 180 g and shoot:root ratios were 1.59 in the
near-ambient UV-B radiation treatment and 1.39 in the subambient UV-B radiation treatment. There were no statistically
significant differences between UV-B radiation treatments for
either clone in any of these variables.
Gas exchange, chlorophyll fluorescence, and chlorophyll
pigments
Net photosynthesis ranged from about 10 µmol CO2 m −2 s −1
for immature leaves (LPI 5) to > 22 µmol CO2 m −2 s −1 for fully
expanded mature leaves (LPI 15). The UV-B radiation treatments had no statistically significant effect on gas exchange in
expanding leaves (LPI 5 and 10) of either clone (Figure 1).
Although the near-ambient UV-B radiation treatment decreased net photosynthesis, stomatal conductance and transpi-

Biomass
Following physiological determinations and collection of anatomical samples, plants were harvested and partitioned into
leaf (main stem leaves, lateral branch leaves and petioles),
stem (main stem and lateral branches), and root components.
All biomass components were dried separately at 70 °C to
constant mass, and weighed. Shoot (sum of stem and leaf dry
weights) to root ratio was calculated for each plant. For each
leaf used in the physiological and anatomical studies, leaf area
was measured with a leaf area meter (Delta-T Devices), leaf
length was measured to the nearest mm, and leaf dry weight
per unit of leaf area (g m −2) was calculated.
Experimental design
The experiment was a split-plot design with UV-B radiation
treatments as a whole-plot factor and Populus clones as a
sub-plot factor. Data were subjected to analysis of variance
(ANOVA) using the SYSTAT statistical package (SYSTAT,
Inc., Evanston, IL). Whole-plot factor was analyzed as a completely randomized design. Sub-plot means were pooled and
compared by least significant difference (LSD) tests when no
interaction was detected. Differences were considered significant at P ≤ 0.05 or 0.10.
Results
The UV-B radiation treatments had no effect on the physical
appearance of the Populus trichocarpa and P. trichocarpa ×
P. deltoides plants during the growing period. There were no
clonal or UV-B radiation treatments effects on plant growth

Figure 1. Relationships between leaf age and net photosynthesis (A),
stomatal conductance (B), and transpiration (C) in Populus grown in
near-ambient UV-B radiation or subambient UV-B radiation. Vertical
lines represent ± SE of the mean. The data represent the mean of n = 6
for LPI 5 and 15; n = 5 for LPI 10.

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RESPONSES OF POPULUS TO UV-B RADIATION

ration more in mature leaves (LPI 15) of plants of Clone 1A
than in mature leaves of plants of Clone HY, neither the clonal
nor treatment effects were statistically significant. However,
when data for both clones were pooled, net photosynthesis,
stomatal conductance, and transpiration were decreased by
24% (P ≤ 0.05), 20% (P ≤ 0.10) and 28% (P ≤ 0.05), respectively, in plants in the near-ambient UV-B radiation treatment
compared with plants in the subambient UV-B radiation treatment. Rates of dark respiration decreased with leaf age in both
clones from about 3 µmol CO2 m −2 s −1 for immature leaves
(LPI 5) to < 2 µmol CO2 m −2 s −1 for fully expanded mature
leaves (LPI 15) with no effects by UV-B radiation regime (data
not shown). Leaf internal CO2 concentrations were consistently between 275 and 283 µl l −1 irrespective of leaf age, clone
and UV-B treatment. In both clones and UV-B radiation regimes, water-use efficiency (WUE) was about 4.2 in immature
leaves and 5.5 in mature leaves (data not shown). The UV-B
radiation regime had no effect on the maximum intrinsic light
harvesting efficiency of photosystem II. Chlorophyll fluorescence (Fv /Fm ) was about 0.75 in both clones and for both UV-B
treatments. Chlorophyll a content ranged from about 6 mg l −1
cm −2 in immature leaves to 10.6 mg l −1 cm −2 in mature leaves,
and the corresponding values for chlorophyll b were 1.75 and
3.7 mg l −1 cm −2, respectively. There were no effects of UV-B
radiation regime on chlorophyll a and b or total chlorophyll
content, or on chlorophyll a/b ratios (data not shown).
Ultraviolet-B radiation-absorbing compounds
Leaf extracts from plants in both treatments showed a peak of
absorbance at 270 nm (Figure 2). At all LPIs, absorbances of
leaf extracts from plants in the near-ambient UV-B treatment
were somewhat higher than those of leaf extracts from plants
in the subambient treatment over the range 260--290 nm. At
wavelengths greater than 290 nm, absorbances of extracts from
LPI 15 leaves were significantly greater (P ≤ 0.10) for plants
grown in near-ambient UV-B radiation than for plants grown
in subambient UV-B radiation (Figure 2). For LPI 15 leaves,
absorbances at 300 and 310 nm were slightly greater in
Clone 1A than in Clone HY5, and for both clones they were
higher in the near-ambient UV-B radiation treatment than in
the subambient UV-B radiation treatment (Figure 3).

621

Leaf morphology and anatomy
Clones 1A and HY5 differed in rates of leaf expansion, leaf
lengths, leaf weight:leaf area ratios, and leaf area; however,
there were no statistically significant treatment differences at
any leaf developmental stage (Table 1). Both clones exhibited
similar leaf anatomical organization.
Exposure to UV-B radiation did not affect leaf anatomical
development in Clone HY5 (Tables 1 and 2). In Clone 1A, the
near-ambient UV-B radiation treatment resulted in leaves that
were 11 and 10% (P ≤ 0.10) thicker at LPIs 10 and 15,
respectively, than equivalent leaves in the subambient UV-B
radiation treatment (Table 1). Leaves attained maximum thickness at LPI 10 in the near-ambient UV-B regime, whereas leaf
thickness continued to increase through to LPI 15 in the
subambient UV-B radiation regime (Table 1), resulting in
LPI 15 leaves that were significantly thicker in the subambient
UV-B radiation treatment than in the near-ambient UV-B radiation treatment (Table 1).
Differences in palisade parenchyma layer thickness accounted for most of the treatment differences in overall leaf
thickness for Clone 1A (Table 2). Palisade parenchyma thickness was 17% greater (P ≤ 0.05) at LPI 5 and 10% greater
(P ≤ 0.10) at LPI 10 for plants grown in near-ambient UV-B
radiation compared with plants grown in subambient UV-B
radiation. At LPI 15, palisade parenchyma thickness was 15%
greater (P ≤ 0.05) for plants grown in subambient UV-B radiation than for plants grown in near-ambient UV-B radiation
(Table 2). Generally, there were no differences in other leaf
tissues between the UV-B treatment regimes with the exception of lower epidermal thickness in LPI 5 leaves (Table 2).

Discussion
The UV-B radiation treatments had no significant effects on
height, diameter, total biomass and biomass allocation of
plants of either clone. In contrast, Bogenrieder and Klein
(1982) found that biomass was significantly greater for Fraxinus excelsior L., Carpinus betulus L., Fagus sylvatica L., Acer
platanoides L., and Acer pseudoplatanus L. trees grown with
exclusion of ambient solar UV-B radiation than with ambient
UV-B radiation. However, in the study of Bogenrieder and

Figure 2. Absorbance between 260
and 310 nm of leaf extracts of Populus grown in near-ambient UV-B radiation or subambient UV-B
radiation. The data represent the
mean of n = 12 for LPI 5 and 15;
n = 10 for LPI 10.

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SCHUMAKER ET AL.

Figure 3. Relationship between absorbance at 300 and 310 nm of leaf
extracts in Populus grown in nearambient UV-B radiation or subambient UV-B radiation. Vertical lines
represent ± SE of the mean. The
data represent the mean of n = 6 for
LPI 15.

Table 1. Relationships between leaf development and total leaf thickness and leaf age (LPI) in Populus trichocarpa (1A) and P. trichocarpa ×
P. deltoides (HY5) plants grown in near-ambient UV-B radiation or subambient UV-B radiation. Values are mean ± 1 SE of n = 6 for LPI 5 and
15; n = 5 for LPI 10. Asterisks indicate significant differences at P ≤ 0.10 LSD (*).
LPI
5

Clone

UV-B Treatment

Leaf length
(cm)

1A

Near-ambient
Subambient
Near-ambient
Subambient

11.3 ± 0.6
11.4 ± 0.5
12.4 ± 0.9
12.1 ± 1.1

35.3 ± 3.9
34.8 ± 3.5
71.6 ± 13.6
67.2 ± 11.7

78.0 ± 5.4
78.6 ± 1.9
92.8 ± 4.1
83.8 ± 4.2

257.7 ± 11.4
227.0 ± 8.8
205.7 ± 16.7
208.7 ± 9.8

Near-ambient
Subambient
Near-ambient
Subambient

13.2 ± 0.4
14.3 ± 0.7
13.5 ± 0.5
15.4 ± 1.2

54.7 ± 2.2
61.5 ± 4.8
93.4 ± 4.7
122.3 ± 20.4

93.2 ± 7.8
90.2 ± 2.6
102.5 ± 6.5
94.2 ± 3.7

305.3 ± 7.4
272.6 ± 19.2*
247.5 ± 7.4
250.2 ± 3.6

Near-ambient
Subambient
Near-ambient
Subambient

13.9 ± 0.4
14.9 ± 0.5
18.0 ± 0.6
18.6 ± 0.6

56.8 ± 3.9
66.1 ± 3.2
150.9 ± 9.8
157.1 ± 6.8

93.9 ± 7.2
100.9 ± 2.0
106.0 ± 3.3
106.8 ± 1.8

305.3 ± 9.0
330.9 ± 6.8*
278.0 ± 10.3
289.4 ± 5.9

HY5
10

1A
HY5

15

1A
HY5

Leaf area
(cm2)

Leaf weight:
leaf area (g m −2)

Leaf thickness
(µm)

Table 2. Relationships between leaf anatomical development and leaf age (LPI) in Populus trichocarpa (1A) and P. trichocarpa × P. deltoides
(HY5) plants grown in near-ambient UV-B radiation or subambient UV-B radiation. Values represent mean ± 1 SE of n = 6 for LPI 5 and 15; n = 5
for LPI 10. Asterisks indicate significant differences at P ≤ 0.05 LSD (**) or at P ≤ 0.10 LSD (*).
LPI

5

Clone

1A
HY5

10

1A
HY5

15

1A
HY5

UV-B Treatment

Leaf tissue thickness (µm)
Palisade
parenchyma

Spongy
mesophyll

Upper
epidermis

Lower
epidermis

Near-ambient
Subambient
Near-ambient
Subambient

105.3 ± 5.0
87.0 ± 2.8**
72.9 ± 5.6
73.4 ± 3.5

126.3 ± 5.7
118.0 ± 6.6
107.7 ± 9.3
112.9 ± 6.3

14.2 ± 1.0
13.2 ± 0.7
13.2 ± 0.9
12.3 ± 0.3

14.0 ± 0.6
11.8 ± 0.6*
11.6 ± 0.9
10.3 ± 0.2

Near-ambient
Subambient
Near-ambient
Subambient

121.9 ± 1.5
109.8 ± 7.8*
95.5 ± 4.1
95.5 ± 2.9

154.3 ± 6.2
140.5 ± 11.7
130.3 ± 5.8
130.8 ± 1.0

16.6 ± 1.1
14.0 ± 0.8
11.0 ± 2.5
12.9 ± 0.5

13.9 ± 0.5
12.2 ± 1.1
10.7 ± 0.4
12.2 ± 0.7

Near-ambient
Subambient
Near-ambient
Subambient

127.3 ± 5.7
149.8 ± 2.2**
120.8 ± 5.7
119.0 ± 3.0

147.0 ± 6.7
153.7 ± 5.3
131.7 ± 5.1
145.6 ± 7.8

16.3 ± 1.5
14.7 ± 0.6
14.7 ± 1.2
13.6 ± 0.9

14.7 ± 1.1
13.3 ± 0.5
11.7 ± 0.4
11.7 ± 0.4

TREE PHYSIOLOGY VOLUME 17, 1997

RESPONSES OF POPULUS TO UV-B RADIATION

Klein (1982), the Plexiglas (transmittance down to 380 nm)
used to exclude UV-B also absorbed a large percentage of
ambient UV-A radiation, which is known to moderate the
effects of UV-B radiation (Middleton and Teramura 1993). In
the temperate angiosperm Liquidambar styraciflua L. (sweetgum), Sullivan et al. (1994) found that supplemental UV-B
applied for two years to seedlings grown in the field had no
effect on total plant biomass; however, small changes were
observed in leaf physiology, carbon allocation and growth.
DeLucia et al. (1994) reported that supplemental UV-B radiation had no effect on growth of Amelanchier arborea
(Michx. f.), Betula papyrifera Marsh., Nyssa sylvatica Marsh.,
and Robinia pseudoacacia L., whereas total biomass, plant
height and leaf area were significantly reduced in Cercis canadensis L. and Morus alba L. Searles et al. (1995) reported
that exclusion of UV-B radiation improved height growth of
the tropical trees Tetragastris panamensis Kuntze and Calophyllum longifolium Willd., but had no effect on height of
Cecropia obtusifolia Bertol., and Swietenia macrophylla King.
Total plant biomass was reduced only in Cecropia.
We found that the shoot:root ratio was unaffected by UV-B
radiation regime. Similar findings have been reported for other
angiosperm trees (DeLucia et al. 1994, Searles et al. 1995) and
for some crop plants (Teramura 1980a, Biggs et al. 1981,
Teramura et al. 1991). It is possible that effects of near-ambient
UV-B radiation on height and biomass of Populus were too
small to detect over the 7 to 10-week growing period. Sullivan
and Teramura (1992) found that sensitivity of loblolly pine
(Pinus taeda) seedlings from several seed sources to enhanced
UV-B radiation varied considerably after one field season but
growth reductions of 12--20% were observed for all seed
sources after three field seasons. They concluded that assessment of UV-B radiation damage over one growing season was
not adequate to determine tolerance to UV-B radiation.
Leaf growth and anatomical development appeared to vary
between treatments in Clone 1A but not in Clone HY5. In
Clone 1A, although palisade parenchyma tissue was slower to
develop in the subambient UV-B radiation treatment than in
the near-ambient UV-B radiation treatment, the additional time
over which development proceeded resulted in significantly
more palisade parenchyma tissue (Table 2), which in turn
contributed to greater overall leaf thickness of plants in the
subambient UV-B radiation treatment compared with plants in
the near-ambient UV-B radiation teatment (Table 1). Because
palisade parenchyma cells are responsible for a major fraction
of carbon fixation within the leaf, the increased rates of photosynthesis observed in the subambient UV-B radiation treatment (Figure 1A) may be the result of increased palisade
parenchyma tissue.
Duration of leaf expansion and palisade tissue development
in the near-ambient UV-B radiation treatment could have been
curtailed because of demands from competing sinks for carbon, in particular, enhanced production of UV-B-absorbing
compounds (Figure 3). Alternatively, UV-B radiation could
cause greater turnover of proteins involved in cell wall expansion thereby resulting in earlier termination of palisade parenchyma cell elongation. Damage to proteins by UV-B radiation

623

has been well documented (Caldwell 1971), including the
protein xyloglucan endotransglycosylase (XET), which plays
a major role in cell expansion (McCann and Roberts 1994,
Tomos and Pritchard 1994). Another possible explanation is
that auxin mediated the inhibition of palisade parenchyma cell
elongation, because auxin concentrations in plant tissue can be
reduced by UV-B radiation (Witztum et al. 1978).
Leaf expansion and anatomical development are altered by
UV-B in other agronomic and tree species. For example,
Staxen and Bornman (1994) reported that Petunia × hybrida
plants grown without UV-B irradiance had thicker leaves than
UV-B-irradiated plants, whereas enhanced UV-B radiation
caused increased leaf thickness in Brassica napus L. (Cen and
Bornman 1993). Supplemental UV-B applied to sweetgum
(Liquidambar styraciflua) seedlings had no effect on final leaf
size but rates of leaf elongation and accumulation of leaf area
was slower in leaves exposed to daily UV-B supplementation
rates of 3 kJ UV-B (Dillenburg et al. 1995). In field studies of
loblolly pine (Pinus taeda), enhanced UV-B radiation caused
reductions in needle lengths ranging from 4 to 25% (Naidu
et al. 1993).
Ontogenetic patterns of gas exchange paralleled those of
leaf development in Clone 1A (Figure 1A). In response to
near-ambient UV-B radiation, net photosynthesis reached
maximum rates at LPI 10 and then remained constant with
further leaf development, whereas photosynthetic rates continued to increase through LPI 15 in the subambient UV-B regime. Because the UV-B radiation treatment had no significant
effects on either chlorophyll content or photosystem II (as
determined by chlorophyll fluorescence), we postulate that the
increase in net photosynthesis in response to the subambient
UV-B radiation treatment was partly a consequence of increased photosynthetic capacity resulting from increased
amounts of palisade parenchyma tissue.
Increased net photosynthetic rates in response to the subambient UV-B radiation treatment may also be partly the result of
increased flux of CO2 to the leaf mesophyll (Figure 1B),
because stomatal conductance was greater for plants in the
subambient UV-B regime than for plants in the near ambient
UV-B regime (Figure 1). Both stomatal conductance and
mesophyll conductance can be reduced by UV-B radiation
(Brandle et al. 1977, Bennett 1981, Teramura 1983, Negash
and Björn 1986) with concomitant reductions in net photosynthesis (Middleton and Teramura 1993, Musil and Wand 1993).
The absence of effects of near-ambient UV-B radiation on
chlorophyll content and chlorophyll fluorescence is consistent
with the findings of other field studies involving exclusion
(Tevini et al. 1988, Searles et al. 1995) or supplementation of
UV-B radiation (Ziska et al. 1992, Naidu et al. 1993, Caldwell
et al. 1994). These findings contrast with those of several
greenhouse and growth chamber studies where plants were
grown in enhanced UV-B radiation (Brandle et al. 1977, Vu
et al. 1982a, Iwanzik et al. 1983, Teramura 1983); however, the
UV-B/PAR ratios used in the greenhouse and chamber studies
were artificially high and probably served to amplify the deleterious effects of UV-B radiation (Teramura 1980b, Warner and
Caldwell 1983).

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624

SCHUMAKER ET AL.

The near-ambient UV-B radiation either stimulated the production of, or retarded the degradation of, UV-B radiation-absorbing compounds, typically flavonoids or their derivatives
(Figures 2 and 3), but in both treatments absorbance decreased
with leaf age (Figure 2). Leaves exposed to enhanced UV-B
radiation frequently show increased production of flavonoids
and related compounds that reduce UV-B radiation penetration
to sensitive mesophyll tissues thereby mitigating the damaging
effects of UV-B radiation (Sisson 1981, Robberecht and
Caldwell 1983, Flint et al. 1985, Lois and Buchanan 1994).
Despite the significant increase in UV-B radiation-absorbing
compounds, net photosynthesis was significantly reduced in
leaves at LPI 15 grown in near-ambient UV-B radiation compared to leaves grown in subambient UV-B radiation (cf. Figures 1A, 2 and 3).
The two clones differed in their response to the exclusion of
UV-B radiation, implying some degree of genetic adaptation
to ambient UV-B radiation. The environment where Clone 1A
originated (eastern Washington state) is characterized by comparatively clear skies during the growing season with concomitantly high solar radiation. In contrast, Clone HY5 derives
from a cross between western Washington Populus trichocarpa and a Mississippi source of Populus deltoides. Native
climates for both parents have considerably more cloudy periods during the growing season. Moreover, clear days are frequently accompanied by considerable water vapor and other
particulates in the air that may attenuate or scatter incoming
solar UV-B radiation. Thus, Clone 1A is from a location with
comparatively higher ambient UV-B radiation compared with
Clone HY5. Therefore, the exclusion of UV-B radiation might
be expected to have a larger effect on Clone 1A than on Clone
HY5. Such an effect was evident for leaf development and gas
exchange, although not for many of the other parameters
evaluated. Clone 1A tended to have slightly higher concentrations of UV-B-absorbing compounds than Clone HY5, which
is consistent for a genotype evolving under conditions of high
UV-B radiation (Robberecht et al. 1980).
The Populus clones responded to near-ambient solar UV-B
radiation primarily through small alterations in leaf anatomy
and morphology rather than large reductions in growth or
biomass accumulation (cf. Barnes et al. 1988, Caldwell and
Flint 1994, Sullivan et al. 1994, Searles et al. 1995). The
ontogenetic effects support our hypothesis that UV-B radiation
inhibits development of photosynthetic capacity in expanding
leaves.

Acknowledgments
The authors thank Dr. R.A. Black and Dr. G.E. Edwards for early,
critical review of the manuscript. This material is based on work
supported by the Cooperative State Research Service, U.S. Department of Agriculture, under agreement 94-37100-0312. Research was
also supported by the Agriculture Research Center, College of Agriculture and Home Economics, Washington State University under
Project 0113 and by the Northwest Scientific Association. Any opinions, findings, conclusions, or recommendations expressed in this
publication are those of the authors and do not necessarily reflect the
view of the U.S. Department of Agriculture.

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