University of Joensuu, PhD Dissertations in Biology
No:11
Impact of elevated ultraviolet-B radiation
on three northern deciduous woody plants
by
Riitta Tegelberg
Joensuu
2002
Tegelberg, Riitta
Impact of elevated ultraviolet-B radiation on three northern deciduous woody plants.
– University of Joensuu, 2002, 86 pp.
University of Joensuu, PhD Dissertations in Biology, n:o 11. ISSN 1457-2486
ISBN 952-458-131-0
Keywords: Betula pendula, biomass, condensed tannins, diameter growth, flavonoids, height
growth, phenolic acids, salicylates, Salix, silver birch, soluble sugars, terpenoids, UV-B
radiation, willows.
The aim of this thesis was to assess the impact of elevated ultraviolet-B radiation (UV-B, 280320 nm) on growth and phytochemicals of three northern deciduous woody species. Therefore,
silver birch (Betula pendula Roth) seedlings were exposed for three growing seasons and
clonal shoots of dark-leaved willow (Salix myrsinifolia Salisb.) and tea-leaved willow (Salix
phylicifolia L.) for one growing season to elevated UV-B radiation in a modulated irradiation
system outdoors. In addition, clonal plantlets of dark-leaved willow were exposed to short-term
elevated UV-B radiation in a growth chamber.
Elevated UV-B radiation significantly increased the concentrations of UV-B-absorbing
flavonoids, such as quercetin-3-arabinoside, quercetin-3-glucose+glucuronide and kaempferol3-rhamnoside, and a few phenolic acids in silver birch leaves during the first and second
growing seasons of the field study. During the third growing season, the contents of phenolics
in leaves were not affected by the treatments; but compared with the controls, the stem
diameter growth of the saplings treated with elevated UV-B radiation was significantly
reduced. It was also found that with long-term elevated UV-B treatment the concentrations of a
phenolic acid, 3,4’-dihydroxypropiophenone-3-glucopyranoside, and two soluble sugars,
sucrose and glucose, increased in the bark of silver birch saplings. These results indicate that if
exposure is long-term, the growth of field-grown silver birch saplings is affected by elevated
UV-B radiation. The symptoms of UV-B stress also included changes in the metabolism of
carbohydrates and phenolic compounds.
Indoors, with elevated UV-B treatment the leaves of dark-leaved willow clones
accumulated UV-B-screening luteolin glycosides, myricetin glycoside and a hydroxycinnamic
acid derivative, while the low-UV-B-absorbing salicylates, salicin and saligenin, decreased in
concentration. Similarly, in the field, with elevated UV-B radiation certain flavonoids and
phenolic acids accumulated in the leaves of dark-leaved willow clones and tea-leaved willow
clones, while the low-UV-B-absorbing phenolics, i.e. condensed tannins, gallic acid derivatives
and salicylates, either decreased or remained unaffected. These results show that under higher
UV-B exposure, willow leaves accumulated only those phenolics that screen UV-B radiation
efficiently. The results also indicate that the chemical responses in willows were more clonespecific than species-specific.
Despite high constitutive concentrations of UV-B-protective flavonoids in the leaves, both
growth and biomass of one field-grown tea-leaved willow clone were sensitive to elevated
levels of UV-B radiation. In contrast, the growth of dark-leaved willows grown outdoors or
indoors was not affected by elevated UV-B radiation, even though the concentrations of UV-Bscreening flavonoids in the leaves were low. Consequently, the secondary chemical
background of a native willow species or clone does not necessarily predict its sensitivity to
elevated UV-B radiation.
Riitta Tegelberg, Natural Product Research Laboratories, Department of Biology, University of
Joensuu, P.O.Box 111, 80101 Joensuu, Finland
4
ABBREVIATIONS
DHPPG
3,4’-dihydroxypropiophenone-3-glucoside
GC
gas chromatography
HPLC
high performance liquid chromatography
PAR
photosynthetically active radiation
UV-A
ultraviolet-A radiation, λ = 320-400 nm
UV-B
ultraviolet-B radiation, λ = 280-320 nm
UV-BBE
biologically effective UV-B radiation
UV-BCIE
erythemally effective UV-B radiation
5
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
6
1. INTRODUCTION
7
2. MATERIALS AND METHODS
9
2.1. Plant material and experimental conditions
2.2. Growth measurements and chemical analyses
3. RESULTS AND DISCUSSION
3.1. Effects of elevated UV-B radiation on growth
3.1.1. Diameter and height growth
11
12
12
12
3.1.1.1. Relationship between growth and sugars
12
3.1.1.2. Relationship between growth and carbon allocation
13
3.1.1.3. Ecological implications
13
3.1.2. Biomass
3.2. Effects of elevated UV-B radiation on secondary metabolism
3.1.2. Phenolics in leaves
14
16
16
3.1.2.1. Flavonoids
16
3.1.2.2. Phenolic acids
17
3.1.2.3. Low-UV-B-absorbing phenolics
19
3.1.2.4. Variation in phenolic-related strategies
19
3.2.2. Phenolics in the bark of silver birch saplings
21
3.2.3. Terpenoids in the bark of silver birch saplings
21
3.3. Effects of elevated UV-B radiation on silver birch leaf litter
22
3.3.1. Loss of mass and litter quality
4.
9
22
CONCLUSIONS
23
ACKNOWLEDGEMENTS
25
REFERENCES
26
ORIGINAL PUBLICATIONS (I-IV)
6
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following articles and previously unpublished results. The articles
are referred to in the text by their Roman numerals I-IV.
I
Tegelberg, R., Julkunen-Tiitto, R. and Aphalo, P.J. 2001. The effects of long-term
elevated UV-B on the growth and phenolics of field-grown silver birch (Betula
pendula). Global Change Biology 7: 839-848.
II
Tegelberg, R., Julkunen-Tiitto, R. and Aphalo, P.J. The effects of long-term elevated
ultraviolet-B radiation on phytochemicals in the bark of silver birch (Betula pendula).
Accepted for publication in Tree Physiology.
III
Tegelberg, R. and Julkunen-Tiitto, R. 2001. Quantitative changes in secondary
metabolites of dark-leaved willow (Salix myrsinifolia) exposed to enhanced
ultraviolet-B radiation. Physiologia Plantarum 113: 541-547.
IV
Tegelberg, R., Veteli, T., Aphalo P.J. and Julkunen-Tiitto, R. Clonal differences in
growth and phenolics of willows exposed to elevated ultraviolet-B radiation.
Submitted for publication.
Publications are reprinted with permission from the publishers. Copyrights for publication I by
Blackwell Science and III by Blackwell Munksgaard.
7
1. INTRODUCTION
Our nearest star, the sun, emits short
wavelength radiation that is incident on the
earth’s atmosphere. Most of the radiation in
the atmosphere is infrared radiation (λ =
700-3000 nm, 67 % of the photons) and
visible light (400-700 nm, 28 %; Nobel,
1983). Ultraviolet radiation (UV, 200-400
nm), on the other hand, reaches the
atmosphere in smaller amounts (5 % of the
photons). The biologically most hazardous
part of UV radiation, i.e. UV-C (200-280
nm) and UV-B (280-320 nm) below 290
nm, are completely absorbed by the
stratospheric ozone (O3) layer and by other
oxygen molecules in the atmosphere (see
review by Frederick, 1993). In addition, the
ozone layer absorbs some longer-wave UVB and UV-A radiation (320-400 nm).
Consequently, of the photons at the earth’s
surface, only about 2 % are in the
ultraviolet range (Nobel, 1983). However,
of the total solar energy reaching the earth’s
surface, UV-B radiation comprises about
1.5 % and UV-A radiation about 6.4 %
(Frederick et al., 1989).
The intensity of UV-B radiation, in
particular, is affected by the thickness of
the ozone layer, which in turn varies
periodically as a consequence of natural
processes such as seasons, winds and solar
cycles. In addition, latitude, time of year
and time of day determine the length of the
path of a UV-B photon through the
absorptive ozone layer (Caldwell et al.,
1980). UV-B irradiance also varies due to
clouds, air moisture, aerosols and
tropospheric ozone molecules (Johanson,
1997 and references therein). Furthermore,
the gases and particles in the atmosphere
and the surface albedo (e.g. snow) scatter
almost half of the UV radiation; therefore at
ground level only half of the UV-B is direct
radiation.
On average, the ozone concentration in
the stratosphere is low, i.e. about ten ozone
molecules per million molecules of air
(Graedel and Crutzen, 1993); and it is
highly dynamic because the ozone
molecules are created and destroyed
continuously. However, since the 1970’s,
human activities have disrupted the natural
balance between synthesis and breakdown
of ozone. Depletion of the ozone layer has
repeatedly been reported to occur over
Antarctica (e.g. Farman et al., 1985), but in
the 1990’s there were also frequent
occurrences of major spring-time ozone
depletion over the Arctic (von der Gathen et
al., 1995; Taalas et al., 1996). It has been
found that the main man-made compounds
responsible for enhancing ozone breakdown
are the chlorofluorocarbons (CFC) and
nitrogen oxides (Crutzen, 1972; Molina and
Rowland, 1974). Recently, it was also
found that the increasing concentrations of
greenhouse gases result in stratospheric
cooling, thus creating suitable conditions
for breakdown of ozone molecules
(Shindell et al., 1998). Therefore, the most
recent predictions based on stratospheric
chemistry and climate-change models
estimate that in the northern areas (60-90°
N), compared with the long-term means,
the maximum springtime UV-B radiation
will increase up to 50-60 % in 2010-2020
(Shindell et al., 1998; Taalas et al., 2000).
Even present-day levels of UV-B
radiation affect the growth and
development of plants (Ballaré et al., 1996;
Day et al., 1999; Ruhland and Day, 2000).
In addition, hundreds of studies undertaken
in the past two decades indicate that if the
dose of UV-B radiation is elevated, this will
have many direct and indirect effects on
plants (e.g. review by Caldwell et al.,
1998). The direct effects of UV-B radiation
on plant cells are mostly damaging, because
UV-B photons have enough energy to
create lesions in important UV-B-absorbing
biomolecules such as nucleic acids and
proteins (e.g. Greenberg et al., 1989;
Caldwell, 1993; Davies, 1995; Taylor et al.,
1997). It is known that the photoproducts of
DNA formed by UV-B radiation,
cyclobutane pyrimidine dimers and
pyrimidine (6-4) pyrimidone and (6-4)
photoproducts, are all toxic and mutagenic
(Taylor et al., 1997). In addition, the altered
DNA and RNA structures may interfere
with transcription and replication; and
therefore protein synthesis may be slowed
down during UV-B stress (Jordan et al.,
8
1994; Taylor et al., 1997). In order to avoid
the effects of DNA damage, plants have
efficient systems for DNA repair, including
photoreactivation and excision repair,
which are involved in restoring the
structure of genetic material during
exposure to UV-B radiation (Sancar and
Sancar, 1988). However, the indirect effects
of UV-B on plant cells can also be
damaging: UV-B radiation may cause
oxidative damage in chlorophylls and
polyunsaturated lipids by increasing the
formation of free radicals and peroxides
(Brandle et al., 1977; Bornman et al., 1983;
Kramer et al., 1991; Jordan, 1996). To
prevent oxidative damage, cells contain
antioxidants, e.g. phenolic compounds, that
scavenge the free radicals (Takahama,
1988; Takahama and Oniki, 1997;
Yamasaki et al., 1997). Phenolic
compounds have variable antioxidant
properties (e.g. review by Rice-Evans et al.,
1997); and several studies have shown that
during UV-B exposure, the production of
compounds with efficient antioxidant
structures, such as additional hydroxyl
groups on ring B of the flavonoid skeleton,
is favoured (e.g. Liu et al., 1995; Reuber et
al., 1996; Markham et al., 1998; Ryan et al.,
1998). Plant cells also contain enzymes,
e.g. superoxide dismutase (SOD) and
catalase, which scavenge superoxide
radicals and protect the cells against H2O2,
respectively (e.g. Takeuchi et al., 1996).
In addition to damaging plant cell
components, UV-B radiation often exerts
its effects through altered patterns of gene
activity (review by Caldwell et al., 1998);
e.g. the effects of UV-B radiation on
photosynthesis, UV-B-screening phenolics,
growth, reproductive processes, plant form
and timing of life phases, are all caused by
altered gene action. The mechanisms by
which plants perceive UV-B radiation are
not fully understood, but it has been
suggested that direct absorption of UV-B
by DNA could result in the formation of a
“signal” that regulates the transcription of
genes (Jenkins et al., 1997). It is also
possible that reactive oxygen species (ROS)
or nitric oxides that form during exposure
to UV-B radiation, could be triggers for
altered gene action (Green and Fluhr, 1995;
A.-H.-Mackerness et al., 1998; A.-H.Mackerness et al., 2001). In addition, it has
been hypothesized that in plant cells,
specific UV-B photoreceptor-mediated
signalling processes regulate gene
expression (review by Jenkins et al., 2001).
However, the characteristics of a UV-B
photoreceptor and how the signals are
transduced after UV-B perception, are not
yet known. There is some evidence that
certain UV-B signalling pathways that
induce gene expression overlap with
wound-response and pathogen-defence
pathways. For example, distinct pathways
involving salicylic acid, jasmonic acid and
ethylene, all of which function as woundand/or defence-signalling molecules,
mediate the UV-B-caused induction of
several genes in plants (Surplus et al., 1998;
A.-H.-Mackerness et al., 1999).
In order to avoid UV-B radiation, plants
have developed several mechanisms for
UV-B exclusion. Thicker leaves may
decrease the internal fluence of UV-B
radiation (Johanson et al., 1995; Newsham
et al., 1996). This additional leaf thickness
in field-grown silver birch has been
associated with a slight increase in the
thickness of the upper epidermis, spongy
parenchyma and spongy intercellular space
(Kostina et al., 2001). In addition, optical
structures in the leaf, such as epidermal
wax and leaf hairs, scatter and reflect UV-B
radiation (Karabourniotis et al., 1993;
Vogelmann, 1993; Karabourniotis et al.,
1999; Kinnunen et al., 2000), but in
general, the reflectance of the UV-B
radiation reaching leaf surface is only about
10 % (Robberecht et al., 1980; Grant,
1997). Apparently, the most efficient
mechanism of exclusion is the
accumulation of UV-B-screening phenolics
in the epidermal cells of leaves (e.g. Viestra
et al., 1982; Cen and Bornman, 1993; Day,
1993; Day et al., 1994; Ålenius et al., 1995;
Burchard et al., 2000). Consequently, the
penetration of UV-B radiation through the
epidermis has been shown to be nearly zero
in conifer needles, 3-12 % in the leaves of
deciduous trees and grasses, and 18-41 % in
9
the leaves of herbaceous plants (Day et al.,
1992).
One of the most common responses of
field-grown plants to elevated UV-B
radiation is an increase in UV-B-absorbing
phenolics in the leaves (Searles et al.,
2001). In fact, accumulation of certain
phenolic filters with UV-B levels above the
ambient level has been found to be a
continuation of the response within the
ambient range (de la Rosa et al., 2001).
UV-B radiation stimulates the expression of
genes that encode phenylalanine
ammonialyase (PAL) and chalcone
synthase (CHS), which are the key
regulatory enzymes in the phenylpropanoid
and flavonoid pathways (e.g. Hahlbrock
and Griesebach, 1979; Chapell and
Hahlbrock, 1984). Recently, it was also
found that UV light selectively induces
several primary metabolic activities that are
directly or indirectly required for flavonoid
formation (Logemann et al., 2000). This
implies complex regulation in the different
branches of the phenylpropanoid
biosynthesis pathway during UV-B stress.
The direction and severity of changes in
plant processes during elevated UV-B
radiation have been found to vary
considerably among species, varieties and
clones, but also among plant parts and
developmental stages (e.g. Day, 1993;
Jordan, 1996; Lavola, 1998). Long-lived,
slow-growing species such as trees may
also show cumulative effects of higher
doses of UV-B radiation (Sullivan and
Teramura, 1992). However, up to now,
there have been only a limited number of
studies in which northern deciduous woody
plants, in particular, and their populations
and clones have been exposed to elevated
UV-B radiation. Thus, the objective of the
present study was to determine the effects
of elevated UV-B radiation on three
northern deciduous woody species: silver
birch, dark-leaved willow and tea-leaved
willow. Attention was paid to (1) the
sensitivity of growth and biomass
production to long-term (I) and short-term
(III, IV) elevated UV-B radiation, (2) the
concentrations of phenolic filters in leaves
(I, III, IV), (3) the interspecific (IV) and
intraspecific (III, IV) variation in response
to elevated UV-B radiation, (4) the effects
of long-term elevated UV-B radiation on
the concentrations of phytochemicals in the
bark of silver birch saplings (II) and (5) the
effects of elevated UV-B radiation on the
decomposition of silver birch leaf litter.
2. MATERIALS AND METHODS
2.1. Plant material and experimental
conditions
The plant material used was seed-originated
silver birch (Betula pendula Roth) seedlings
and saplings (I, II, litter study),
micropropagated clonal plantlets of darkleaved willow (Salix myrsinifolia Salisb.)
(III) and clonal shoots of dark-leaved
willow and tea-leaved willow (S a l i x
phylicifolia L.) originating from cultivated
stands (Julkunen-Tiitto and Meier, 1992)
and randomly chosen trees from natural
environments (IV).
The silver birch seedlings and willow
shoots were subjected to elevated UV-B
radiation in a UV-B irradiation field at the
Botanical Gardens of the University of
Joensuu (I, II, IV). The UV-B exposure
lasted either for three summers (I, II) or one
summer (IV). The outdoor system of UV-B
irradiation (Aphalo et al., 1999) consisted
of 18 (I), 21 (I, II) or 24 (IV) lamp frames,
which were arranged in a randomised block
design with UV-A + UV-B radiation
treatment, UV-A radiation control and an
ambient control within each of the six (I),
seven (I, II) or eight (IV) blocks (Fig. 1).
The UV-A + UV-B radiation treatment was
obtained by covering the UV-B lamps with
cellulose diacetate filters, which transmitted
UV-B and UV-A radiation but absorbed
radiation below 290 nm. To maintain a
constant 50 % increase in UV-BCIE (UVB CIE based on the erythemal action
spectrum (McKinlay and Diffey, 1987)) in
comparison to that of sunlight, the lamps
were adjusted once a minute. The increase
in UV-B radiation corresponded to a 20-25
% reduction in the ozone column above
central Finland (Björn, 1990). Compared to
sunlight, the increase in UV-A radiation
10
Figure 1. Plan of the UV-B irradiation field at the Botanical gardens of
the University of Joensuu in 1997-1999, showing the location of lamp
frames, data logger and fence. Treatments: 0, ambient radiation; UVA, UV-A radiation control; UV-B, elevated UV-A and UV-B radiation.
was about 0.75 – 1.54 %. The control for
this UV-A emitted by the lamps was
obtained by covering the UV-B lamps with
polyester filters, which absorbed the UV-B
radiation below 313 nm but transmitted the
UV-A radiation; the increase in UV-A
radiation was about 0.56 – 1.12 %
compared to sunlight. The ambient control
was provided by the same set-up with
unenergized lamps. During the first
summer, the silver birch seedlings were
grown in pots filled with soil and prefertilized peat (1:1 v/v) (see cover page);
they were then planted in the soil of the
experimental field (I, II). In the onesummer experiment (IV), the cuttings of
willow shoots were planted in pots filled
with pre-fertilized peat and sand (4:1 v/v);
commercial fertilizer was added to the pots
five times. On each cutting, one bud was
allowed to grow into a shoot. In both
studies the plants were placed under lamp
frames in an area within which the
variation in UV-B irradiance was less than
10%.
For the decomposition study silver
birch leaves were collected from 17months-old seedlings that were growing in
the irradiation area at the end of October
1998, when most of the leaves had fallen.
The leaf samples (about 20 g fresh weight)
were stored at –18º C in plastic bags so
that one bag contained all the leaves from
11
under one frame. The next spring the
leaves from each bag separately were
divided, weighed (fresh weight) and placed
in 12 polyester fabric bags (12 x 10 cm)
with a mesh size of 1.3 mm2 . The fabric
bags were then taken to the irradiation area
and distributed under the lamp frames
(four bags per frame) of the block where
the leaves originated and attached to the
soil surface with stainless steel wires. The
fresh weight of the leaf litter in each bag
was determined after the 111-day
irradiation treatment. For analysis of dry
weight, 3-4 leaves were taken before and
after the litter-bag experiment.
Plantlets of dark-leaved willow were
exposed to enhanced UV-B radiation in a
growth chamber at the Department of
Biology of the University of Joensuu for 10
days (III). The plantlets were exposed
either to ambient time-integrated irradiance
of UV-BBE (3.6 kJ m-2 day-1) or to twice the
ambient time-integrated irradiance of UVBBE (7.18 kJ m-2 day -1 ). To remove the
radiation below 290 nm, the UV-B lamps
were covered with cellulose diacetate filters
during the experiment. The UV-B
irradiation was centered around noon; all
the lamps were on for one hour, but after 30
min, those that provided lower UV-B
irradiance were covered with polyester
film. All the plantlets received very similar,
low
UV-A
irradiance.
The
photosynthetically active radiation (PAR) at
plant level was about 25% of the solar PAR
at noon in early summer in Joensuu. During
the experiment, the plantlets were grown in
pots in a mixture of unfertilized peat and
perlite (9:1).
2.2. Growth measurements and chemical
analyses
The height growth and diameter growth of
the stems were measured (I, IV). In
addition, the air-dried biomass of the
leaves, stems and roots (III) or the biomass
of the air-dried leaves and stems (I, IV) was
determined. The formation of wintering
buds, bud growth and bud dry weights, leaf
area and the rust-frequency index in silver
birch seedlings were also determined (I).
Chemical analyses were made from
fresh leaves (III), air-dried leaves (I, IV,
litter study) or from fresh frozen bark (II).
From the leaves, leaf disks (I, III, IV, litter
study) or one-half of a leaf blade (I) were
excised and homogenized in methanol with
a glass rod (III) or with a clipping
homogenizer (I, IV, litter study). From the
stems of silver birch saplings, a 10 cm
section was cut 40 cm from the shoot tip,
and the outer bark and phloem were
separated from the xylem with a sharp
knife, cut into small pieces and
homogenized in methanol and ethanol with
a homogenizer (II). The solvents of all
samples were evaporated in a rotavapor.
The phenolics in leaves, bark and litter
were analysed by high performance liquid
chromatography (HPLC) according to the
methods of Julkunen-Tiitto (1996) and
Julkunen-Tiitto et al. (1996), and the
terpenoids and soluble sugars (sucrose,
glucose, raffinose) in bark were determined
by capillary gas chromatography (GC). For
HPLC-analyses, the samples were
dissolved in water:methanol (1:1). The
identification of phenolics was based on (1)
retention time, (2) UV-spectra monitored at
220, 270, 280, 320 and 360 nm and (3)
mass spectrum (III). For GC-analyses (II),
the dried samples were dissolved in
dimethylformamide and then derivatized
with trimethylsilylimidazole in pyridine
(7:3). Identification of the trimethylsilyl
derivatives of terpenoids and soluble sugars
as based on (1) retention time and (2) mass
spectra (m/z) using selected ion monitoring
(SIM). The composition of the individual
flavonoids, phenolic acids, terpenoids and
soluble sugars in leaves and stems was
consistent with earlier studies (e.g.
Vainiotalo et al., 1991; Julkunen-Tiitto and
Meier, 1992; Julkunen-Tiitto et al., 1996;
Ossipov et al., 1996; Hakulinen, 1998;
Keinänen and Julkunen-Tiitto, 1998).
Condensed tannins (proanthocyanidins)
were determined from the extract (I, II, III,
IV) and from the residue (IV, litter study)
by means of a butanol-HCl test (Hagerman,
1995), which was standardized with
purified tannins from leaves of Betula nana
L. (I, II, litter study) or from leaves of Salix
12
purpurea L. (III, IV). The concentrations of
chlorophylls (chlorophyll a, chlorophyll b
and total chlorophyll) were determined
from leaf samples of silver birch (I)
according to the method of Inskeep and
Bloom (1985).
Table 1. Plant material, duration and location of the UV-B experiments and analysed metabolites in each study.
Study
Species
Duration of Location
exposure
Plant
part
Analysis
Phenolics
Terpenoids Sugars
Chlorophylls
I
Betula
pendula
Long-term
Outdoors
Leaves
+
—
—
+
II
Betula
pendula
Long-term
Outdoors
Bark
+
+
+
—
III
Salix
Short-term
myrsinifolia
Growth
chamber
Leaves
+
—
—
—
IV
Salix
Short-term
myrsinifolia,
Salix
phylicifolia
Outdoors
Leaves
+
—
—
—
Betula
pendula
Outdoors
Leaf
litter
+
—
—
—
Litter
study
Long-term
3. RESULTS AND DISCUSSION
3.1. Effects of elevated UV-B radiation
on growth
3.1.1. Diameter and height growth
Earlier outdoor studies have shown that, in
general, tree growth is insensitive to
elevated UV-B radiation (Petropoulou et
al., 1995; Newsham et al., 1996; Weih et
al., 1998; Liakoura et al., 1999; Newsham
et
al.,
1999b).
Because
the
photoreactivated repair of DNA damage
uses solar UV-A and blue light, natural
irradiation conditions have strong
ameliorating effects on plant responses to
UV-B radiation (e.g. Stapleton, 1992).
Previous measurements in most field
studies have also shown that
photosynthesis is not significantly affected
by elevated UV-B radiation (review by
Allen et al., 1998; meta-analysis by Searles
et al., 2001). Similarly, during the field
study (I) the concentrations of chlorophylls
in silver birch leaves were not affected by
elevated UV-B radiation in any of the
growing seasons. However, certain studies
of long duration, i.e. lasting for several
growing seasons, have indicated that
growth, biomass allocation and carbon
fixation of field-grown tree seedlings may
be susceptible to elevated UV-B radiation
(Sullivan and Teramura, 1992; Sullivan et
al., 1994; Newsham et al., 1999b; Keiller
and Holmes, 2001). A reduction in growth
was also found in the long-term study with
silver birch saplings: during the third
growing season, the stems of silver birch
saplings treated with elevated UV-B
radiaton were significantly thinner than the
stems of all other saplings grown in the
experimental area (I). In addition, the
height of the saplings tended to be reduced
after long-term exposure to UV-B. These
delayed responses may indicate that the
effects of elevated UV-B radiation
accumulated gradually.
3.1.1.1. Relationship between growth and
sugars
Over the long-term, a shortage of sugars,
which are used in respiration and for
synthesis of other biomolecules, may
reduce the growth of plants. It has been
shown that with elevated UV-B radiation
the ratio of storage starch to chloroplast
13
area in field-grown silver birch leaves
decreases (Kostina et al., 2001). In
addition, it was recently found that after
five years of exposure to elevated UV-B
radiation, the carboxylation efficiency in
leaves of certain broad-leaved trees had
decreased significantly (Keiller and
Holmes, 2001), which might be due to the
UV-B-induced loss of soluble Calvin cycle
enzymes (Allen et al., 1997). This kind of
response may lead to the production of
lower concentrations of sugars. In the
long-term study with silver birch, the
diameter growth of saplings treated with
UV-B radiation slowed down, especially
during the mid- and late summer of the
third growing season (I), which implies
that a shortage of sugars may have
developed gradually. However, the results
also show that growth early in the season,
which is determined mainly by the
previous year’s carbon production and
nutrient storage, was not significantly
affected by exposure to UV-B radiation.
Apparently, the UV-B-induced reduction
in leaf-to-shoot weight ratio from the
second growing season did not affect the
carbon reserves for the third growing
season (I).
3.1.1.2. Relationship between growth and
carbon allocation
In addition to possible changes in the
carbohydrate metabolism of the source, the
reduction in diameter growth under
elevated UV-B radiation may result from
disturbances in the carbon allocation to
sinks. Most of the diameter growth in
woody plants is comprised of the
production of secondary xylem. Therefore,
the reduced diameter in the third growing
season implies that wood formation in
silver birch saplings was sensitive to longterm elevated UV-B radiation. It is known
that the formation of latewood can be
stimulated by auxin-transport inhibitors
(Lauchaud, 1989). However, auxin is an
UV-B-absorbing compound, and
supplemental doses of UV-B radiation
may decrease its concentration (Huang et
al., 1997), which could thus enhance the
formation of latewood. Therefore, it is
possible that the reduction in diameter
growth was related to the UV-B-induced
accumulation of growth precursors,
sucrose and glucose, which were left
unused in silver birch stems (II). The
concentrations of soluble sugars in leaves
have been reported to increase (Newsham
et al., 2001b), decrease (Yue et al., 1998)
or remain unaffected by elevated UV-B
radiation (Gehrke et al., 1995; Rozema et
al., 1997a). However, the concentrations of
soluble sugars in stems not only reflect the
production of sugars but also the rate of
transport and allocation of carbon to sinks.
The formation of thick cell walls of latesummer wood cells, in particular, uses
large amounts of transportable sugars as
components of cellulose and other cell
wall materials. Apparently, the metabolic
processes that use the assimilates in the
formation of cell walls might have been
gradually slowed down by elevated UV-B
radiation, leading to the observed
accumulation of soluble sugars (II). Earlier
it was found that the content of α-cellulose
in leaves was lower under enhanced UV-B
radiation than under control irradiation
(Gehrke et al., 1995), but it has also been
reported to increase following exposure to
elevated UV-B radiation (Rozema et al.,
1997a). Thus, it is also possible that the
diameter growth in silver birch was
affected by some other UV-B-sensitive
mechanisms, and that lower sink demand
(reduced growth rate) led to the
accumulation of soluble sugars in the
stems.
3.1.1.3. Ecological implications
The reduction in wood production may
have marked ecological and economic
consequences, but eventually it may also
affect the tree’s height growth because, in
general, sturdier stems enable the tree to
grow taller. Exposure to elevated UV-B
radiation in the field was found to lead into
significantly decreased height growth of a
clone of tea-leaved willow after one
growing season (IV). According to the
action spectrum for inhibition of shoot
14
elongation, this inhibition is more effective
at shorter wavelengths in the UV-range
(Steinmetz and Wellmann, 1986).
Therefore, the effect of UV-B radiation on
height growth could be caused by damage
to DNA and proteins or by oxidative stress
(Ballaré et al., 1996; Mazza et al., 1999b).
In addition, changes in the concentrations
and distribution of flavonoids by elevated
UV light may lead to changes in growth
(both diameter and height) and
morphology, because the transport of the
plant hormone auxin is negatively
regulated by flavonoids, such as quercetin
and kaempferol, in areas of organ
transition and maturation (Murphy et al.,
2000; Brown et al., 2001). However, it was
found that at least in the leaves of tealeaved willows, elevated UV-B radiation
had only minor effects on quercetins (IV).
According to Barnes et al. (1996), the
reduction in height under higher doses of
UV-B radiation can influence the ability of
plants to compete for light in dense and
mixed vegetation. Thus, the reduced
interception of photosynthetic active
radiation (PAR) by shorter willow shoots
may amplify the effects on shoot growth.
On the other hand, the dose of UV
radiation received by the shorter seedlings
may be decreased by the taller, shading
plants (Grant, 1997). However, when the
total irradiance decreases, the UV-B/PAR
ratio often increases - even though the
dose of UV-B radiation is low - which can
be damaging to shaded plants (Deckmyn et
al., 1994).
In conclusion, the growth of northern
deciduous woody plants can be reduced by
elevated UV-B radiation. However,
depending on the species, clone and
individual, the degree of sensitivity to
radiation varies, which may affect the
competition balance in forests and bushy
habitats. In the future, the growth
responses of woody plants may also be
modified by the indirect effects of UV-B;
for example, elevated UV-B radiation may
affect herbivory (e.g. Mazza et al., 1999a),
decomposition of litter by microorganisms (e.g. Gehrke et al., 1995) and
abundance of plant pathogens (e.g. Levall
and Bornman, 2000). In natural habitats,
the growth of plants will also be
influenced by the interactions of higher
doses of UV-B radiation with other abiotic
stress factors such as temperature (e.g.
Mark and Tevini, 1997), water stress (e.g.
Nogués and Baker, 2000), nitrogen
deficiency (e.g. Deckmyn and Impens,
1997; Pinto et al., 1999), increased ozone
level (e.g. Zeuthen et al., 1997) and
increased atmospheric CO2 (e.g. Visser et
al., 1997; Lavola et al., 2000).
3.1.2. Biomass
Biomass accumulation is considered to be
a reliable indicator of the sensitivity of a
plant to UV-B radiation (Smith et al.,
2000). However, a reduction in shoot
biomass has often been found to occur
only when the levels of simulated ozone
depletion are greater than 20 % (Searles et
al., 2001). The biomass of the silver birch
saplings and most of the clonal willow
shoots exposed to doses of UV-B
radiation, simulating 20-25 % reduction in
the ozone column, showed no sensitivity to
UV-B radiation (I, IV). In addition,
indoors, the biomass of the dark-leaved
willow plantlets was not affected by a
short-term exposure to a doubled amount
of UV-B radiation (III). These results
imply that the woody species studied here
were relatively tolerant to UV-B radiation.
Alternatively, the periods of UV-B
exposure were probably not long enough
to reveal all the responses of these longlived species. Nonetheless, in one fieldgrown tea-leaved willow clone the
biomass was significantly reduced by
elevated UV-B radiation (IV). Recently, it
was found that higher accumulation of
quercetins, a group of UV-B-absorbing
flavonoids, may correlate with the
tolerance of plants to UV-B-induced
growth reduction (Hofmann et al., 2000).
The leaves of the tea-leaved willow clone
contained constitutively moderate
concentrations of quercetins; but when
growth was reduced by elevated UVradiation, none of the quercetins or other
UV-B-absorbing phenolics increased (IV).
15
This may indicate the lack of a trade-off
between biomass and chemical protection
in willows during UV-B stress. Generally,
a UV-B-induced reduction in biomass
production in field conditions is associated
with a reduced ability to intercept light due
to the smaller leaf area (see review by
Allen et al., 1998). Therefore, in sensitive
woody species or clones, the effects of
UV-B radiation on leaf morphology may
depress the accumulation of biomass. In
addition, the extra input in repair during
UV-B stress may reduce the resources
available for biomass accumulation.
In general, plants that display a high
relative growth rate under optimum growth
conditions are the ones damaged most
when environmental conditions become
suboptimal (Grime, 1977). Accordingly,
plants that accumulate more biomass are
more likely to show UV-B sensitivity, i.e.
be more susceptible to UV-B damage
(Barnes et al., 1993; Smith et al., 2000;
Hofmann et al., 2001). This was also
exemplified in the field study (IV), where
the biomass was reduced by elevated UVradiation in one of the most productive
clones of tea-leaved willow. However, the
results also show that although native
willows are considered to be rapidlygrowing plants, they do not necessarily
show great sensitivity to UV light. This
could be expected because in nature
willows are well-adapted to open habitats
with high ambient levels of irradiance,
including UV radiation.
Reduction in the productivity of
sensitive plants in plant communities has
been suggested to lead to increased
production of more tolerant plants, which
have access to more resources (e.g. light,
moisture and nutrients; Caldwell et al.,
1998). This may cause changes in the
clonal and species composition of the plant
communities and affect other trophic
levels as well. However, UV-B radiation
may also change the allocation of plant
biomass, which leads to changes in plant
form but not necessarily in total biomass
(e.g. Deckmyn and Impens, 1999). Even
subtle changes in plant form have been
found to be sufficient to change the
balance of two species in competition for
sunlight (Barnes et al., 1995). In the longterm study (I), silver birch saplings
showed no clear effects of UV-B radiation
on leaf number, leaf area or branching; but
in the second growing season the leaf-toshoot ratio was significantly reduced under
elevated UV-B radiation. Changes in
biomass allocation were also observed in
the indoor study with dark-leaved willows:
in the plantlets under elevated UV-B
radiation the root-to-shoot ratio decreased
(III). The willow plantlets may have
produced thicker leaves in response to
elevated UV-B radiation, which may partly
explain the change in biomass allocation.
Previously, the shoot growth of silver
birch seedlings was found to be stimulated
indoors by enhanced UV-B radiation,
which likewise led to a decreased root-toshoot ratio (Lavola et al., 2000). On the
whole, UV-B-induced changes in aerial
and below-ground biomass of field-grown
tree seedlings have been found to vary
among species and growing seasons
(Sullivan and Teramura, 1992; Sullivan et
al., 1994; Newsham et al., 1999b).
During plant development the UVA/blue light photoreceptors, the
cryptochromes, have been found to
mediate a wide range of responses such as
extension of the hypocotyl, stem, leaf
petioles and the leaf lamina (review by
Jenkins et al., 2001). In study IV it was
found that the leaf-to-shoot ratio in the
shoots of one dark-leaved willow clone
and one tea-leaved willow clone
significantly increased under the UV-A
control treatment compared with the
control treatment. When both UV-B and
UV-A radiation were elevated, plants
showed no changes in morphology (IV).
However, it was also found that the
biomass and height growth of one tealeaved willow clone was reduced both by
the UV-A control treatment and by
elevated UV-B and UV-A radiation (IV).
Because in both treatments the UV-A
radiation increase in terms of percentage
was small, it may be that in addition to the
possible photomorphogenic effects of UVA radiation, the high number of statistical
16
tests performed increased the possibility to
obtain significant results. It is also possible
that, as suggested by Newsham et al.
(1996) and Newsham et al. (1999b), some
unidentified factor associated with the
polyester-filtered UV-lamps modulated the
plant responses. Furthermore, it has been
found that ageing of both cellulose
diacetate and polyester filters affects the
transmission and spectral distribution
(Adamse and Britz, 1992), which may
cause some potential errors in results.
In summary, the northern deciduous
woody species studied here were relatively
tolerant to elevated UV-B radiation in
terms of changes in biomass. However, the
observed effects of UV-exposure on
biomass allocation could affect their
competition abilities in forest ecosystems.
3.2. Effects of elevated UV-B radiation
on secondary metabolism
3.2.1. Phenolics in leaves
Earlier studies have shown that phenolic
compounds, especially flavonoids, located
in the epidermis of leaves are important in
protecting terrestrial plants against UV-B
radiation (see review by Rozema et al.,
1997b). Solar UV-B radiation can cause
damage to DNA, but e.g. the number of
pyrimidine dimerphotoproducts is clearly
lower when the production of UVabsorbing phenolics is induced in leaves
(Mazza et al., 1999b; Mazza et al., 2000).
Without the epidermal flavonoids, plants
are very sensitive to natural sunlight (Li et
al., 1993; Reuber et al., 1996). In addition
to the flavonoids, phenolic acids may be
involved in the attenuation of UV-B
radiation in leaves (Landry et al., 1995;
Sheahan, 1996; Lavola et al., 1997; BooijJames et al., 2000; Burchard et al., 2000).
In contrast, the role of other phenolic
compounds such as lignin, salicylates,
condensed tannins and hydrolysable
tannins in UV-B-protection is less clear.
For example, the efficiency of salicylates
and condensed tannins in absorbing UV-B
radiation is low compared with the UV-B-
absorbing efficiency of certain flavonoids
(I, III).
3.2.1.1. Flavonoids
In this study, the leaves of silver birch
seedlings and saplings contained high
constitutive concentrations of UV-Babsorbing flavonoids. Most of the
flavonoids were identified as flavonol
glycosides (Fig. 2 and 3; I), which are
water-soluble compounds and are often
stored in the vacuoles of plant cells
(Strack, 1997). In previous indoor studies
the UV-B-induced increase in these
vacuolar flavonols has been demonstrated
in silver birch leaves (e.g. Lavola et al.,
1997; Lavola, 1998; Lavola et al., 2000; de
la Rosa et al., 2001). Similarly, in the first
summer of the field study, with elevated
UV-B radiation the concentrations of
quercetin glycosides and kaempferol-3rhamnoside in silver birch leaves were
found to increase (I). In other plant
species, quercetins and kaempferols also
have been found to accumulate selectively
in response to UV-B radiation (e.g. Möhle
et al., 1985; Olsson et al., 1998; Ryan et
al., 1998; Wilson et al., 1998; Hofmann et
al., 2000). Differences between the
screening capacities of quercetin-3galactoside and myricetin-3-rhamnoside
were found to be slight (I), and therefore it
is unlikely that the quercetin and
kaempferol derivatives provided more
efficient UV-B-absorption than the
myricetins did. However, the quercetins
and kaempferols may be better able to
dissipate the energy of UV-B radiation,
rendering it harmless (Smith and
Markham, 1998). In addition, differences
in the potential of flavonols to act as
radical-scavenging antioxidants may lead
to specific accumulation during UV-B
stress (Cooper-Driver and Bhattacharya,
1998). Quercetins with an orthodihydroxyl group in ring B of the
flavonoid skeleton have been shown to
have increased antioxidant activity
compared with compounds that do not
contain the ortho-dihydroxyl group
(reviews by Larson, 1988 and Rice-Evans
17
et al., 1997). For example, quercetin-3galactoside was induced by elevated UV-B
radiation in leaves of the three woody
species studied (I, IV), which suggests that
certain advantageous phenolics may be
“universally” induced by UV-B. Because
UV-B radiation has been found to generate
free radicals in plant cells (Hideg and
Vass, 1996), an increase in the
concentrations of efficient flavonoid
antioxidants would be beneficial. Recently,
a strong negative correlation has been
found between a quercetin glycoside and
lipid peroxidation levels in silver birch
leaves exposed to enhanced UV-B
radiation (Kostina et al., 2001). However,
whether the flavonoids function as
antioxidants in plant cells depends on
several factors. For example, the main
location of flavonol glycosides in the
vacuoles does not favour their
participation in the prevention of oxidative
processes in the chloroplasts. In general,
the antioxidant activity is lowered by
glycosylation of the flavonoid (Rice-Evans
et al., 1997); and different sugars may
specifically modify the activity (Wang et
al., 1997). This may have consequences in
UV-protection in birch leaves, because the
concentrations of individual flavonols
showed clear changes during seedling
ontogeny: e.g. the constitutive quantity of
quercetin-3-galactoside in leaves increased
with the age of the seedling and sapling
(I).
The efficiency of flavonoids in UVscreening may also be affected by their
location in the leaves. Burchard et al.
(2000) suggested that if flavonoids are
located in the mesophyll, they do not
contribute to UV-filtering. In contrast, the
UV-B-absorbing compounds in the leaf
cuticle may be increased by enhanced UVB radiation, which suggests that they are
actively involved in UV-B screening
(Stephanou and Manetas, 1997). The
actual location of flavonol glycosides and
phenolic acids in silver birch leaves is not
known, and thus no conclusions can be
drawn about their importance in UVscreening. However, the concentrations of
flavone aglycones that are located on the
surface of silver birch leaves (Keinänen
and Julkunen-Tiitto, 1998) did not increase
during UV-B exposure (I).
Preferential accumulation of B-ringdihydroxylated flavonoids, quercetins and
luteolins, was displayed in willow leaves
treated with UV-B radiation (III, IV). In
contrast, the concentrations of apigenins,
which are B-ring-monohydroxylated
flavonoids and thus less ideal structures for
radical-scavenging (Rice-Evans et al.,
1997), did not show any UV-B-induced
increase (III, IV). This is in accordance
with earlier studies, which have reported
an increase in the ratio of luteolin:apigenin
in plants grown with enhanced UV-B
radiation (Liu et al., 1995; Reuber et al.,
1996; Markham et al., 1998). The
secondary chemistry, especially in tealeaved willow leaves, also allowed
comparisons between the effects of UV-B
radiation on different flavonoid groups
(flavonols, flavones, dihydroxyflavonols
and flavanones). These groups differ not
only in their chemical structures but also in
their position in the flavonoid biosynthetic
pathway (Strack, 1997). For example, in
tea-leaved willow leaves, eriodictyol
glycoside (a flavanone) was not induced
by UV-B radiation (IV), possibly due to its
role as an intermediate in the biosynthesis
of other flavonoids, condensed tannins and
anthocyanidins. In addition, the absorption
maxima of eriodictyol glycoside is shifted
towards the UV-C region of the spectrum
(pers.obs.). Nor were the high
concentrations of dihydromyricetins
affected by elevated UV-B radiation,
which may be partly explained by their
lack of structural arrangements, such as
ortho-dihydroxyl moiety in the B ring and
a 2,3-double bond in the A ring of the
flavonoid skeleton, that impair antioxidant
activity (Rice-Evans et al., 1997).
3.2.1.2. Phenolic acids
According to Rozema et al. (1997b), the
main UV-B filters in terrestrial nonvascular plants are phenolic acids. In
addition, in the epidermis of certain higher
plants the most important UV-screeners
18
19
may be hydroxycinnamic acid derivatives
(Landry et al., 1995; Sheahan, 1996).
Although phenolic acids may serve as
intermediates in the biosynthesis of other
phenolics, in many plant species they are
thought to provide at least constitutive
filtering (e.g. Burchard et al., 2000), due to
their absorption maximas in the UV-B
region of the spectrum (Lavola et al.,
1997). In leaves of silver birch and darkleaved willow, the constitutive
concentrations of phenolic acids were
relatively high and were specifically
induced by UV-B radiation (I, III, IV).
This indicates that phenolic acids in
deciduous woody plants can also be
actively involved in UV-B-screening.
Furthermore, phenolic acids such as
chlorogenic acid have been repeatedly
implicated as active antioxidants (review
by Larson, 1988), providing additional
protection from UV-B radiation.
Previously, the pigment accumulation in
leaves has been shown to be dependent on
leaf age (Day et al., 1996; Burchard et al.,
2000). The long-term study showed that in
silver birch, the induction and contribution
of different UV-B-absorbing phenolics
may be dependent also on seedling age,
developmental stage or duration of
exposure. In the first growing season,
elevated UV-B radiation induced the
production of flavonols, while in the
second growing season it induced
production of phenolic acids. Apparently,
the different phenolics in silver birch
leaves may have been integrated
temporally to provide multiple
mechanisms for protecting the plant as
efficiently as possible from UV-B stress.
which may be the reason why the tannin
content was not sensitive to UV-B
radiation. Results from other field studies
have also shown that in leaves, condensed
tannins are not affected by changes in UVB radiation (Gehrke et al., 1995; Rozema
et al., 1997a). Therefore, tannins may only
be part of the constitutive UV-protection.
Salicylates, whose efficiency to absorb
UV-B radiation is also low, may play a
similar minor role in UV-protection
despite their high concentrations in darkleaved willow leaves. In the indoor study
the concentrations of salicin and saligenin
in the leaves of dark-leaved willows
decreased under enhanced UV-B radiation,
while the flavonoids and phenolic acids
accumulated (III). In the field study the
same trend was observed; in willow leaves
the low-UV-B-absorbing phenolics
(salicylates, tannins and gallic acid
derivatives) were either decreased or were
unaffected by elevated UV-B radiation
(IV). These results indicate that during
elevated UV-B radiation, the leaves of
deciduous woody-plant seedlings
accumulate those phenolics that screen
UV-B radiation efficiently. These changes
in the concentrations among different
phenolic groups require specific regulation
in the branching routes of the
phenylpropanoid pathway, and it has been
suggested that the transcription rates of the
regulatory enzymes of different branches
change when the plant experiences stress
(Dixon and Paiva, 1995). Redirection of
the flow of phenolic precursors during
UV-B exposure may lead to changes in the
composition of phenolic compounds
(Booij-James et al., 2000).
3.2.1.3. Low-UV-B-absorbing phenolics
3.2.1.4. Variation in phenolic-related
strategies
In field conditions the content of
condensed tannins was high in all species
(I, IV). Because only the methanolextractable tannins were measured, the
total concentration, including the
compounds bound in the cell wall, was
probably even higher. However, the
efficiency of birch tannins in absorbing
UV-B radiation was found to be low (I),
None of the UV-B-absorbing phenolics in
silver birch leaves did increase during
elevated UV-B radiation in the third
growing season, which may have caused
damage in leaf cells (I). Kinnunen et al.
(2001) suggested that accumulated effects
of UV-B radiation inhibit the synthesis of
phenolics. If so, flavonoid production
20
could have been affected already in the
second growing season, when only
phenolic acids were induced by UV-B
radiation (I). In fact, it was calculated that
even during the first summer, the clear
induction of UV-B-absorbing phenolics
was not enough to maintain UV-B
radiation in the mesophyll of UV-B-treated
silver birch leaves at the same level as in
ambient leaves (I). If the UV-B filters were
not sufficient to compensate for the higher
intensity in UV-B radiation, it may have
eventually led to damage and reduction in
growth (I). However, the ontogenic
changes in phenolic metabolism (I) and
possible changes in cell-wall-bound
phenolics (e.g. Hutzler et al., 1998; Laakso
et al., 2000), in the density of phenoliccontaining glandular trichomes at the
surface of silver birch leaves (Fig. 3), and
in leaf anatomy (Kostina et al., 2001)
complicate interpretation of the results on
the efficiency of UV-filtration.
Figure 3. Scanning electron micrograph of leaf from a wintering bud
of silver birch, showing glandular trichomes and leaf hairs, x 20.
The phenolics in leaves of the species
studied here varied in quantity and quality
(I, III, IV), which implies that their
chemical defence strategies against UV-B
radiation differed. In addition, the
individual clones of both willow species
varied greatly in terms of their constitutive
and UV-B-induced phenolic chemistry
(III, IV). It might thus be expected that this
chemical variation affects the efficiency of
deciduous trees in filtering UV-B radiation
and also their adaptation to changing
irradiation environments. However, neither
the content nor the quality of phenolics in
willow leaves predicted the sensitivity to
elevated UV-B radiation (IV). The changes
in flavonoids and phenolic acids in willow
leaves with elevated UV-B radiation were
small, which may indicate that other UV-B
protection mechanisms contributed to the
overall protection of these clones.
However, as in the study by Hofmann et
al. (2000), also the willow clones that
accumulated certain flavonoids and
phenolic acids already contained these
compounds constitutively in higher
concentrations than the other clones did
(IV). Therefore, these clones may have
been better adapted to high levels of
sunlight irradiation.
In conclusion, the leaves of fieldgrown silver birch, tea-leaved willow and
dark-leaved willow contained high
constitutive concentrations of UV-B-
21
absorbing phenolics. In addition, they were
able to accumulate specifically those
phenolics that are efficient in screening
UV-B radiation during exposure.
However, the UV-B-induced accumulation
of phenolics was dependent on the species,
clone, individual, age and developmental
stage as well as the duration of exposure,
which might have caused variation in the
degree of UV-B damage.
3.2.2. Phenolics in the bark of silver birch
saplings
The bark of silver birch seedlings and
saplings has been found to contain smallmolecular-weight phenolic glycosides,
flavonoids and condensed tannins
(Tahvanainen et al., 1991; Vainiotalo et
al., 1991; Julkunen-Tiitto et al., 1996). It
has been suggested that the function of
these phenolics is to hinder the utilization
of birch stems as food by herbivorous
mammals (Sunnerheim et al., 1988;
Tahvanainen et al., 1991 and references
therein). However, whether the phenolics
in the bark are also UV-inducible was not
known. When the soluble phenolics in the
bark of the 28-month-old silver birch
saplings were measured, it was found that
most phenolics were not induced by
elevated UV-B radiation (II). Apparently,
the main function of these phenolic
compounds is not in UV-B avoidance
mechanisms. Most of the living, possibly
photosynthetic, cells are located in the
inner cell layers and are probably thus
protected from UV-radiation by the outer
cork layers and other anatomical
structures.
However,
the
higher
concentration
of
3,4’-dihydroxypropiophenone-3-glucoside
(DHPPG) in bark treated with UV-B
radiation suggests that it may have been
actively involved in screening elevated
UV-B radiation. This accumulation may be
due to the ability of DHPPG to screen UVB radiation efficiently at around 300 nm
(Lavola et al., 1997) and its possible
location in the upper layers of the bark.
The slight increase in UV-A radiation
led into significant increase in the main
flavonoids, (+)-catechin and (-)epicatechin, in silver birch bark (II). The
contents of certain phenolic compounds in
leaves have also been found to change by a
small increase in UV-A radiation (I; IV;
Kostina et al., 2001; Newsham et al.,
2001b).
The
UV-A/blue
light
photoreceptor, cryptochrome, has been
found to mediate the induction of several
genes involved in flavonoid biosynthesis
(see review by Jenkins et al., 2001).
However, the flavonoids have also been
shown to decrease in a dose-dependent
manner in response to UV-A radiation
(Wilson et al., 2001). Nonetheless, it is
known that between 300-368 nm the
absorbance of e.g. (+)-catechin is
relatively low (Hoque and Romus, 1999),
which suggests that also in protection
against UV-A radiation, the flavonoids
may play roles other than absorption of
excess radiation. Thus, although the levels
of UV-A radiation are not expected to
increase significantly in the future, the
UV-A mediated effects on phenolics in the
bark of silver birch may lead to complex
changes in their concentrations. For
example, expression of the chalcone
synthase gene is synergistically enhanced
by UV-B and UV-A radiation (Fuglevand
et al., 1996). In contrast, the possible
elevation in UV-B radiation alone is not
likely to affect the phenolics in silver birch
bark significantly.
3.2.3. Terpenoids in the bark of silver
birch saplings
In the stems and twigs of silver birch
seedlings and saplings, terpenoid
compounds, which are secreted as resin
droplets onto the surface of the bark, help
defend the plant against mammalian
browsing (Rousi et al., 1991; Tahvanainen
et al., 1991). Although the resin droplets
may cause some reflection and scattering
(Vogelmann, 1993), terpenoids do not
participate in the screening of UV-B
radiation. The spatial and temporal
restrictions may also decrease the
importance of terpenoids in UVprotection; terpenoids are synthesized and
22
secreted by active resin glands only during
the period when the apical growth takes
place in that segment of the shoot (Taipale
et al., 1993). In addition, in older parts of
the shoots, the droplets gradually wear off
mechanically. In fact, terpenoids are costly
to produce (Gershenzon, 1994), and it can
be hypothesized that the induced
production of chemofilters and other UVprotection mechanisms during UV-stress
may limit the allocation of resources to
terpenoid production. However, no tradeoff was found between the production of
terpenoids and phenolic filters in study II.
The terpenoids appeared to be controlled
more by the genetic background of an
individual seedling or by other
environmental variables than by elevated
UV-B radiation. In short, a higher dose of
UV-B radiation is not a strong modulator
of the terpenoid contents in silver birch
bark.
3.3. Effects of elevated UV-B radiation
on silver birch leaf litter
3.3.1. Loss of mass and litter quality
The phytomass production of plants is
often limited by the rate of mineralization
of nutrients from decomposing litter.
Therefore, any change in the decomposing
processes could affect plant growth. It has
been found that UV-B radiation is able to
influence the decomposition of leaf litter
through direct effects on organic
compounds and microbes (review by Zepp
et al., 1998). However, most of the UV-B
radiation in deciduous forests is absorbed
or reflected by the foliage (Brown et al.,
1994; Grant, 1997); thus the
decomposition of e.g. silver birch leaves is
more likely affected indirectly by UV-B
radiation. For example, the UV-B-induced
Table 2. Effects of elevated UV-B radiation on dry weight and concentrations of flavone aglycones in silver birch leaf
litter.
Irradiation trt
Irradiation trt
during growth
during
Dry weight loss
Apigenin dimethylether
Luteolin der
Apigenin der
(%)
(mg/g dw)
(mg/g dw)
(mg/g dw)
decomposition
Ambient
Ambient
24.4 ± 3.2
0.44 ± 0.12
0.51 ± 0.14
1.10 ± 0.28
Ambient
UV-A co
28.9 ± 3.9
0.40 ± 0.09
0.48 ± 0.09
1.08 ± 0.27
Ambient
UV-B+
23.4 ± 3.4
0.28 ± 0.03
0.43 ± 0.05
0.76 ± 0.09
UV-A co
Ambient
19.9 ± 3.5
0.36 ± 0.08
0.45 ± 0.09
0.80 ± 0.19
UV-A co
UV-A co
26.4 ± 4.0
0.43 ± 0.06
0.48 ± 0.06
0.93 ± 0.10
UV-A co
UV-B+
23.8 ± 3.1
0.41 ± 0.09
0.49 ± 0.10
0.86 ± 0.12
UV-B+
Ambient
22.3 ± 3.2
0.41 ± 0.12
0.52 ± 0.08
0.79 ± 0.21
UV-B+
UV-A co
24.9 ± 3.6
0.34 ± 0.08
0.47 ± 0.08
0.70 ± 0.11
UV-B+
UV-B+
17.1 ± 3.4
0.25 ± 0.04
0.36 ± 0.04
0.47 ± 0.08
changes in leaf quality may modify the
rate of decomposition (review by
Hättenschwiler and Vitousek, 2000).
Earlier experiments have shown that the
rate of loss of leaf-litter dry mass can be
either accelerated (Newsham et al., 1999a;
Newsham et al., 2001a) or reduced
(Rozema et al., 1997a) by enhanced UV-B
radiation. Our measurements showed that
the dry weight loss of silver-birch leaf
litter was smallest in samples that had been
exposed to elevated UV-B during the
growth and litter experiment (Table 2).
Apparently, the rate of decomposition was
23
reduced by both indirect and direct effects
of elevated UV-B. However, the litter dry
weights in our experiments did not differ
statistically, which may be partly due to
the relatively short UV-B exposure during
decomposition. Recently, it was found that
the effects of UV-B on decomposition of
leaf litter may persist for more than four
years (Newsham et al., 2001a). Phenolic
compounds are released from leaf litter
into the soil, where they have been shown
to inhibit nutrient cycling processes by
affecting the activity of fungal and
bacterial communities and the availability
of nitrogen. To determine whether soluble
flavonoids, phenolic acids and condensed
tannins in silver birch leaves are
decomposed at different rates under
elevated
UV-B
radiation,
the
concentrations of phenolics were
determined before and after the 111-day
litter-bag experiment. It was found that
most of the phenolic compounds had been
leached from the decomposing silver-birch
leaf litter and only the flavone aglycones,
which are located in the epidermal cuticle,
and some cell-wall-bound tannins were
present in the litter samples (Fig. 4). The
analysis showed that the concentrations of
flavones in the litter did not change
significantly by the irradiation treatments,
but in all cases they were lowest in leaves
grown and decomposed under elevated
UV-B radiation (Table 2). Most of the
earlier data show that elevated UV
radiation has no significant effect on
polyphenolics in leaf litter during
decomposition (Gehrke et al., 1995;
Newsham et al., 1997; Rozema et al.,
1997a; Newsham et al., 1999a). Therefore,
the decomposition of silver birch leaves
will probably not be greatly affected by the
effects of elevated UV-B radiation on
flavonoids, phenolic acids and tannins.
4. CONCLUSIONS
The following conclusions can be drawn:
1 . Exposure to higher doses of UV-B
radiation can reduce the growth of
young silver birches and tea-leaved
2 .
3.
4.
5.
6.
willows. In the long run, diameter
growth in particular, i.e. wood
formation, in silver birch saplings is
sensitive to elevated UV-B radiation.
Some phenolic compounds are
specifically induced in the leaves of
silver birch and willows and in the
bark of silver birch by elevated UV-B
radiation. Most of the compounds
accumulated are directly involved in
UV-B protection: they are either
efficient in filtering excess radiation
or in scavenging radicals.
Both the constitutive and the UV-Binduced concentrations of UV-Babsorbing phenolic compounds in
leaves vary greatly among the
deciduous woody species and clones
studied.
However,
these
concentrations do not necessarily
correlate with the level of UV-B
tolerance.
Species and clones of willows differ
in their degree of UV-B susceptibility.
Long-term elevated UV-B radiation
may induce changes in the primary
metabolism of young woody plants. In
UV-B-treated stems of silver birch,
soluble carbohydrates accumulated,
which implies disturbances in the
ability to store and/or metabolize
sugars during UV-B stress.
Decomposition of silver birch leaves
is not significantly affected by
exposure to elevated UV-B radiation.
Figure 4. An HPLC-chromatogram of silver birch (Betula pendula) leaf extract (A) and leaf litter extract (B) at 320 nm. Peaks: DHPPG = 3,4’dihydroxypropiophenone-3-glucopyranoside, 5-CQA = 5-coumarylquinic acid, 3-CQA = 3-coumarylquinic acid, myr = myricetin, que =
quercetin, kae = kaempferol, gal = galactoside, glu = glucoside, glucu = glucuronide, arap = arabinopyranoside, araf = arabinofuranoside, rha
= rhamnoside, der = derivative.
24
25
ACKNOWLEDGEMENTS
I would like to thank all the people who have contributed to this thesis. I am especially grateful
to my supervisors Prof. Riitta Julkunen-Tiitto and Ph.D. Pedro J. Aphalo for their support and
expertised guidance during my studies in Joensuu. I also want to express my gratitude to Prof.
Jorma Tahvanainen and other fellow collegues in the “Plant-Herbivore Research Group” for
their help and co-operation. I am also grateful to Dr. Tarja Lehto, who helped me in the litter
decomposition study.
The staff in the Natural Product Research Laboratories, especially Ms. Outi Nousiainen,
has been a great help to me both in the laboratory and in the UV-B irradiation field, which I
gratefully acknowledge. Mr. Matti Savinainen and the personnel at the Botanical Gardens and
in the University workshop are thanked for their valuable help during the construction of the
UV-B experimental area. I am also very grateful to Mr. Kenneth Meaney and Mrs. Joann von
Weissenberg, who worked hard with the English language in my manuscripts. Doc. Elina
Oksanen and Doc. Kurt Fagerstedt kindly pre-examined the thesis. My roommates in 173b and
the other doctoral students in the Department of Biology are thanked for all the non-scientific
and cheerful discussions.
This thesis was financed by the Academy of Finland; project numbers 38059 and 45591,
and Finnish Centre of Excellence Program, project number 64308. The working facilities were
provided by the Department of Biology and the Plant-Herbivore Research Project.
I dedicate this thesis to my parents, who endlessly have supported me and believed in me,
to my sister Jaana and her family, who took care of me during my student years in Helsinki,
and finally to Petteri, who has stood by me for all these years.
26
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