University of Joensuu, PhD Dissertations in Biology
No:39
The effects of drought stress
and enhanced UV-B radiation
on the growth and secondary chemistry
of boreal conifer and willow seedlings
by
Satu Turtola
Joensuu
2005
3
Turtola, Satu
The effects of drought stress and enhanced UV-B radiation on the growth and secondary
chemistry of boreal conifer and willow seedlings. –University of Joensuu, 2005, 82 pp.
University of Joensuu, PhD dissertations in Biology, No: 39. ISSN 1457-2486
ISBN 952-458-754-8
Keywords: drought stress, hybrid, phenolic, Picea abies, Pinus sylvestris, plant growth,
Salix myrsinifolia, Salix myrsinites, secondary compounds, UV-B radiation, terpene
The aim of this thesis was to study the effects of drought stress and enhanced UV-B
radiation on the growth and secondary chemistry of boreal woody species. Three studies
were carried out, two outdoor-studies and one greenhouse-study. In the former, conifer
seedlings (Pinus sylvestris and Picea abies) were exposed to drought stress and enhanced
UV-B radiation for two or three growing seasons. In the latter, willow clones (Salix
myrsinifolia and S. myrsinites × S. myrsinifolia hybrids) were subjected for four weeks to
two levels of UV-B radiation (ambient, enhanced) and two levels of watering (wellwatered, drought stressed) according to a 2 × 2 factorial design.
Drought stress reduced growth in all species, but the conifers and willows differed
with respect to the responses of secondary compounds: the concentrations of terpenes
increased in conifers, while in willows the concentrations of phenolics decreased. In
addition, the responses of willow phenolics to drought stress were clone-specific.
Enhanced UV-B radiation did not affect the growth of the conifer seedlings or their
concentrations of terpenes and phenolics, but in willows growth decreased and the
concentrations of phenolics increased. The response of willow clones to enhanced UV-B
radiation was broadly similar whether they were drought stressed or well-watered: the
only interaction effects of drought stress and enhanced UV-B were in the root/shoot ratio
and concentration of phenolic acids of hybrid willows.
The results indicate that severe drought stress may cause stronger responses than
increased UV-B radiation in boreal woody species. Boreal conifers seem to be less
susceptible to enhanced UV-B than willows, due to structural properties and differences
in their secondary compounds. The responses of secondary compounds to drought stress
and UV-B radiation also seem to depend on species, clone and the type of chemical
compound concerned. Different responses of secondary compounds, especially to
drought stress, may affect the susceptibility of different species and clones to herbivore
attack and pathogenic fungi.
Satu Turtola, Natural Product Research Laboratory, Department of Biology, University of
Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland
4
ABBREVIATIONS
CFCs
chlorofluorocarbons
CNB
carbon/nutrient balance
CO2
carbon dioxide
GC-MS
gas chromatography - mass spectrometry
GDB
growth-differentiation balance
GISS
Goddard Institute for Space Studies
HPLC
high performance liquid chromatography
PAR
photosynthetically active radiation, λ = 400-700 nm
PCM
protein competition model
UV
ultraviolet radiation (UV-A, UV-B and UV-C radiation)
UV-A
ultraviolet-A radiation, λ = 315-400 nm
UV-B
ultraviolet-B radiation, λ = 280-315 nm
UV-C
ultraviolet-C radiation, λ = 200-280 nm
5
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
6
1. INTRODUCTION
7
1.1. Climate change
7
1.2. The effects of climate change on plant growth
8
1.3. Secondary compounds
8
1.3.1. Terpenes
8
1.3.2. Phenolics
9
1.3.3. Hypotheses concerning the effects of environmental factors on secondary 10
compounds
1.4. Objectives
2. MATERIALS AND METHODS
10
11
2.1. The plant material
11
2.2. The experiments
11
2.3. Growth measurements and chemical analysis
12
3. RESULTS
3.1. The secondary compounds of the conifers and the species responses to
13
13
environmental stresses
3.2. The secondary compounds of the willows and the species responses to
15
environmental stresses
4. DISCUSSION
16
4.1. Responses to drought stress
16
4.2. Responses to enhanced UV-B radiation
18
4.3. The interaction effects of enhanced UV-B and drought stress in the willows
19
4.4. The implications for current eco-/environmental theories
19
5. CONCLUSIONS
20
ACKNOWLEDGEMENTS
21
REFERENCES
22
ORIGINAL PUBLICATIONS (I-IV)
6
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following articles. The articles are referred to in the text by their
Roman numerals I-IV.
I
Turtola S, Manninen A-M, Rikala R and Kainulainen P. 2003. Drought stress alters the
concentration of wood terpenoids in Scots pine and Norway spruce seedlings. Journal
of Chemical Ecology 29: 1981-1995.
II
Turtola S, Sallas L, Holopainen JK, Julkunen-Tiitto R and Kainulainen P. Long-term
effects of enhanced UV-B radiation on secondary compounds of outdoor-grown Scots
pine and Norway spruce seedlings. Submitted for publication.
III
Turtola S, Rousi M, Pusenius J, Yamaji K, Heiska S, Tirkkonen V, Meier B and
Julkunen-Tiitto R. 2005. Genotypic variation in drought response of willows grown
under ambient and enhanced UV-B radiation. Environmental and Experimental
Botany, in Press. Published online, doi: 10.1016/j.envexbot.2005.01.007.
IV
Turtola S, Rousi M, Pusenius J, Yamaji K, Heiska S, Tirkkonen V, Meier B and
Julkunen-Tiitto R. 2005. Clone-specific responses in leaf phenolics of willows
exposed to enhanced UV-B radiation and drought stress. Global Change Biology 11:
1655-1663.
In the studies I and II, I was responsible for extracting and analyzing secondary
compounds, processing the data and writing the articles. In the papers III and IV (study
III), I participated in planning the experiment, and was responsible for micropropagating
the plantlets, carrying out the experiment, extracting and analyzing phenolics, processing
the data and writing the articles.
Publications are reprinted with permission from the publishers. Copyrights for publication I
by Springer Science and Business Media, III by Elsevier and IV by Blackwell Publishing.
7
1. INTRODUCTION
1.1. Climate change
During the past hundred years or so
agriculture, forest destruction and the
burning of fossil fuels have led to the
accumulation of greenhouse gases such as
carbon dioxide, nitrous oxide, methane,
chlorofluorocarbons and tropospheric
ozone which permit incoming solar
radiation to reach the surface of the Earth
unhindered but restrict the outward flow
of infrared radiation (e.g. Worrest et al.
1989, Dawson 1992). Greenhouse gases
absorb and reradiate this outgoing
radiation, effectively storing some of the
heat in the atmosphere and producing a
net warming of the Earth’s surface. The
amounts of greenhouse gases are still
increasing and causing climate change,
which is predicted to produce global
warming of the Earth’s surface.
It is predicted that the warming effect
of climate change will lead to significant
changes in the frequency and nature of
precipitation (Caldwell et al. 2003). In
Finland the amount of precipitation is
expected to increase (Kuusisto et al.
1996). On the other hand, during recent
decades, both increases and decreases in
precipitation have been observed locally
in the high latitudes of the Northern
Hemisphere (Walther et al. 2002).
Moreover, the Finnish climate is
characterized by variable rainfall due to
rapid changes in the weather: Annual
rainfall may vary between lows of 200 to
300 mm and highs of 700 mm in
northern Finland, 900 to 1100 mm
elsewhere (e.g. Helminen et al. 2002,
Venäläinen et al. 2004). These annual
variations and local changes in
precipitation expose Finnish plants to
drought stress.
Climate change can also affect cloud
cover and surface albedo (e.g. McKenzie
et al. 2003), factors which, together with
aerosol gases, stratospheric ozone and the
solar zenith angle, affect the amount of
UV-B radiation at the Earth’s surface (e.g.
Németh et al. 1996, Madronich and
Flocke 1997, McKenzie et al. 2003). As
sunlight passes through the atmosphere,
all the UV-C radiation (100-280 nm) and
approximately 90 % of UV-B radiation
(280-315 nm) is absorbed, mainly by
stratospheric ozone and oxygen (Simon
1997, Madronich et al. 1998, Aucamp
2003), so that at the Earth’s surface, UVradiation consists mostly of UV-A
radiation (315-400 nm). The small UV-B
component represents less than 1 % of the
total solar energy (Webb and Weatherhead 1997), but its high energy per photon
can cause substantial damage to living
tissues (Madronich and Flocke 1997).
The stratospheric ozone layer has been
partly depleted by the catalytic effect of
man-made halogenated chemicals, and
this has led to a rise in UV-B radiation
(Madronich et al. 1998). Although the
production and consumption of ozonedepleting chemicals (e.g. CFCs, halons)
has fallen, thanks to the Montreal
Protocol, ozone recovery may be delayed
by the increasing concentrations of
greenhouse gases (Shindell et al. 1998,
McKenzie et al. 2003), which cool the
stratosphere, causing enhanced chemical
depletion and reduced transport of ozone
into high latitudes (Shindell et al. 1998,
Taalas et al. 2000, McKenzie et al. 2003).
The worst ozone depletion at northern
high latitudes is expected to occur during
2010-2020 (Shindell et al. 1998). GISS
model results indicate that springtime
erythemal UV-B doses will increase by up
to 90% in the Northern hemispheric (6090°N), corresponding to a 14 % increase
8
in the annual UV-B dose (Shindell et al.
1998, Taalas et al. 2000).
1.2. The effects of climate change on
plant growth
changes
in
species
abundance,
composition and interactions with
herbivores and pathogens, thus affecting
the biodiversity of forests.
1.3. Secondary compounds
The levels of precipitation and UV-B
radiation are known to affect plant
growth. Drought stress causes stomata
closure and reduced CO2 diffusion into
leaves, limiting photosynthesis (Hsiao
1973), and it reduces cell division,
enlargement and differentiation (Begg
1980), resulting in a reduction of the leaf
area and biomass of the plant (e.g.
Osório et al. 1998, Rhodenbaugh and
Pallardy 1993, Guarnaschelli et al.
2003).
UV-B radiation can reduce a plant’s
photosynthesis,
leaf
area,
shoot
elongation and biomass production (e.g.
Caldwell et al. 2003, Hofmann et al.
2003). It has also been shown to increase
epidermal thickness (e.g. Laakso et al.
2000, Kostina et al. 2001) and promote
wax synthesis (Björn et al. 1997, Manetas
et al. 1997), changes that may help the
plant to maintain critical leaf water
content and protect it from drought stress
(e.g. Petropoulou et al. 1995, Manetas et
al. 1997). A combination of stresses can
also have the opposite effects: i.e.
drought stress may cause biochemical
and/or anatomical changes, which
reduce the damaging effect of enhanced
UV-B radiation (Tosserams et al. 2001).
Previous studies have shown that plants
exposed to drought stress can be less
sensitive to UV-B radiation than wellwatered ones (e.g. Balakumar et al.
1993, Hofmann et al. 2003). The effects
of one stress factor can, therefore, be
modified by other environmental factors.
Besides, the responses of different plant
species to environmental stresses are
highly variable, and could result in
Plant metabolites can be divided into
two major groups: primary compounds
and secondary compounds. The former
are those produced by and involved in
primary metabolic processes such as
growth, respiration and photosynthesis
(e.g. Seigler 1998). The role of
secondary compounds has been less
clear. Many of them used to be regarded
as waste products, since they did not
seem to have any clear function in the
organism that produced them (see e.g.
Seigler 1998), but it is now known that
they are needed in plant defense against
herbivores and pathogens (e.g. Bennett
and Wallsgrove 1994, de Groot and
Turgeon 1998, Seigler 1998) and that
they make a major contribution to the
specific odours, tastes and colours of
plants (e.g. Bennett and Wallsgrove
1994). They may also be involved in
storage
and
protection
against
environmental stresses (e.g. Bennett and
Wallsgrove 1994). Two carbon-based
secondary compound groups are widely
distributed in higher plants: terpenes and
phenolics.
1.3.1. Terpenes
Terpenes are the largest and most
diverse group of plant secondary
compounds, occurring in almost all
plants (e.g. Obst 1998). They are
composed of units of five carbons
(isoprenoids) and are classified as hemi(C5), mono- (C10), sesqui- (C15), di(C20), tri- (C30), tetra- (C40) and
polyterpenes (e.g. Lichtenthaler 1997).
9
They are produced via two separate and
biochemically different isopentenyl
diphosphate biosynthesis pathways: 1)
the acetate-mevalonate pathway in the
cytosol-endoplasmic reticulum and 2)
the
pyruvateglyceraldehyde-3phosphate pathway, which occurs in
plastids (e.g. Lichtenthaler et al. 1997,
Phillips and Croteau 1999). Mono-, diand tetraterpenes are synthesized via this
second pathway and sesqui- and
triterpenes via the first pathway (e.g.
Lichtenthaler et al. 1997, Phillips and
Croteau 1999).
Conifers produce resin, which is a
complex mixture of mono-, sesqui- and
diterpenes (Croteau and Johnson 1985,
Phillips and Croteau 1999). The bestdeveloped resin canal systems in all
conifer genera are found in the
gymnosperm genera Pinus and Picea
(Price et al. 1998, Phillips and Croteau
1999). Species of these genera such as
Scots pine and Norway spruce
constitutively produce and store large
amounts of primary (constitutive) resin
as a result of normal physiological
processes, while secondary (induced)
resin forms as a response to the
wounding of the trees (Croteau and
Johnson 1985). Resin is an important
component in the defense response of
conifers to herbivore and pathogen
attack (e.g. de Groot and Turgeon 1998,
Phillips and Croteau 1999) and both
constitutive and inducible resins are
involved (Lombardero et al. 2000): If the
constitutive resin system fails, conifers
can respond with an induced defense
reaction (de Groot and Turgeon 1998).
However, these defenses can be affected
by environmental factors such as
drought (e.g. Kainulainen et al. 1992,
Croisé and Lieutier 1993) and
fertilization (e.g. Anttonen et al. 2002,
Turtola et al. 2002), which can alter the
levels of constitutive and inducible
terpenes.
1.3.2. Phenolics
Phenolic compounds are characterized
by an aromatic ring (C6) bearing one or
more hydroxyl substituents, and these
compounds can be found in all plant
tissues (e.g. Harborne 1980, Strack
1997). The majority of plant phenolics
originate from shikimate via the
shikimate
and
phenylpropanoid
pathways (e.g. Harborne 1980, Strack
1997). Most phenolics occur in
conjugated forms such as water-soluble
glycosides, because free forms are
potentially toxic to plant tissues (e.g.
Harborne 1980).
It has been suggested that the role of
many plant phenolics is to defend the
plant against herbivores (e.g. Fraenkel
1959, Tahvanainen et al. 1985) or to
protect it from ultraviolet radiation (e.g.
Li et al. 1993, Close and McArthur
2002). Phenolic glycosides (salicylates)
have been shown to play a significant
role in the herbivore resistance of
willows (e.g. Tahvanainen et al. 1985,
Ruuhola et al. 2001), while other
phenolics such as flavonoids and
phenolic acids are well known for their
UV-B-absorbing (e.g. Lavola et al. 1997,
Turunen et al. 1999) and antioxidant
properties (e.g. Larson 1988, Grace et al.
1998). The protective function of
flavonoids is demonstrated in flavonoiddeficient Arabidopsis thaliana mutants
(e.g. Li et al. 1993), which show greater
UV-B sensitivity and oxidative damage
than wild-type plants (Landry et al.
1995).
Environmental stresses such as
drought and UV-B increase the
production of active oxygen species with
the result that antioxidative defense
10
systems can become overwhelmed (e.g.
Foyer et al. 1994). Plant resistance to
various stresses is associated with
antioxidant capacity and increased levels
of antioxidants may prevent stress
damage (e.g. Monk et al. 1989).
1.3.3. Hypotheses concerning the
effects of environmental factors
on secondary compounds
Primary compounds and secondary
compounds share common precursors
and intermediates (e.g. Berenbaum 1995,
Haukioja et al. 1998). The former
require high levels of limited plant
resources and during intense growth the
synthesis of secondary compounds may
be substrate- and/or energy-limited (e.g.
Coley et al. 1985, Herms and Mattson
1992, Jones and Hartley 1998). Several
hypotheses have been put forward to
explain the effects of environmental
factors on the defensive chemistry of
plants, including the carbon/nutrient
balance (CNB) hypothesis (Bryant et al.
1983), growth-differentiation balance
(GDB) hypothesis (Herms and Mattson
1992), the protein competition model
(PCM) (Jones and Hartley 1998, 1999)
and the photoinhibition hypothesis
(Close and McArthur 2002).
The CNB (e.g. Bryant et al. 1983)
and GDB (Herms and Mattson 1992)
hypotheses say that there is a trade-off
between growth and differentiation (the
production of carbon-based secondary
compounds). In conditions of high
resource
availability,
growth
is
dominant, but when shading and nutrient
deficiency (CNB) or any resource
limitations (GDB) restrict growth more
than photosynthesis, differentiation is
dominant.
The protein competition model
(PCM) (Jones and Hartley 1998, 1999)
suggests that protein synthesis and
phenolic synthesis compete for the use
of the precursor phenylalanine (Jones
and Hartley 1998, 1999), while the
biosynthesis of terpenoids appears to
proceed without direct competition with
protein synthesis (Haukioja et al. 1998).
Consequently, any environmental factor
that affects plant growth and protein
synthesis also affects the availability of
phenylalanine for phenolic synthesis.
The
photoinhibition
hypothesis
(Close and McArthur 2002) says that
any factor that increases oxidative
pressure causes an increase in phenolic
levels. It also says that the primary role
of many plant phenolics is to protect
leaves from photodamage, although they
can also protect them from herbivores.
1.4. Objectives
There have been many studies of the
impact of UV-B on plant growth and
secondary chemistry, but less work has
been done on the responses of secondary
compounds to drought stress and, in
particular, on the interactive effects of
drought stress and UV-B. The main aim
of the present studies was to investigate
the effects of drought stress and
enhanced UV-B radiation on the growth
and secondary compounds of slowgrowing conifer seedlings and fastgrowing willow clones in order to see
how these common boreal species
respond to these climate change stresses.
The studies were designed in such a way
that the effects of each individual stress
and of their interaction could be
investigated. More specifically, the
following questions were raised: whether
drought stress affects the growth and
terpenes of conifers (Article I); whether
enhanced UV-B radiation affects the
growth and secondary compounds
(terpenes and phenolics) of conifers
11
(Article II); and what the effects of
drought stress, enhanced UV-B and the
combination of these stresses are on the
growth (Article III) and phenolics
(Article IV) of clones of pure willow
species
(S.
myrsinifolia
×
S.
myrsinifolia) and hybrids (S. myrsinites
× S. myrsinifolia).
2. MATERIALS AND METHODS
2.1. The plant material
The conifers used in the studies
consisted of 4-year-old Scots pine (Pinus
sylvestris L.) and 3-year-old Norway
spruce seedlings (Picea abies (L.)
Karst.) (I, II). Both species have a wide,
continuous distribution in Europe and
Asia (e.g. Boratyński 1991, Grossnickle
2000). Both are wind-pollinated,
predominantly outcrossing species. Scots
pine is a shade-intolerant pioneer
species, while Norway spruce is a fairly
shade-tolerant secondary colonizer (e.g.
Szaniawski and Wierzbicki 1978,
Hämet-Ahti et al. 1992). Pines typically
grow on dry sites that are subject to
drought stress (Boratyński 1991, de
Groot and Turgeon 1998), while Norway
spruce grows on wet, eutrophic sites
(e.g. Hämet-Ahti et al. 1992).
The deciduous species consisted of
eight-week-old, micropropagated willow
clones (III, IV). Eleven of them were
hybrids of whortle-leaved and darkleaved willows (Salix myrsinites L. ×
Salix myrsinifolia Salisb.) and four of
them were pure dark-leaved willows (S.
myrsinifolia × S. myrsinifolia) (II, III). S.
myrsinifolia is one of the most
widespread Salix species in Finland and
S. myrsinites is common in northern
Finland (Hämet-Ahti et al. 1992).
Hybridization between these species is
common in nature (Newsholme 1992).
Most Salix species thrive in moist
habitats, but they are also pioneer
species and they are frequently found in
the most unfavorable places, since they
are able to tolerate drought stress.
2.2. The experiments
The effects of drought stress and
enhanced UV-B radiation were studied
in three different experiments (Table 1).
Two of the experiments were carried out
outdoors (I, II) and one indoors (III, IV).
Table 1. A summary of the plant material and a description of the three experiments (IIV). (o = outdoor, gh = greenhouse, gs = growing seasons, wk = weeks)
article
Plant material
I
Scots pine
Norway spruce
Scots pine
Norway spruce
II
III and
IV
S. myrsinifolia and
hybrid (S.myrsinites ×
S. myrsinifolia) clones
site Treatments
(levels)
o Drought (3)
o
UV-B (2)
gh
Drought (2)
UV-B (2)
Duration
2 gs
2 gs
2 gs
3 gs
4 wk
4 wk
Analysed
chemistry
wood terpenes
wood terpenes
needle terpenes
and phenolics
leaf phenolics
12
The two outdoor experiments, which
were carried out at the experimental field
of the University of Kuopio (62°13´ N,
27°35´E), used pot-grown seedlings of
Scots pine and Norway spruce (I, II).
The drought stress experiment (I) lasted
for two growing seasons and involved
three treatments: a control group of
plants was well-watered, (60% of the
amount of water in the pore space), a
second group was subjected to medium
drought (33 % less water than the
controls) and a third group was subjected
to severe drought (66 % less water than
the controls). The watering was based on
pilot experiments and measurements
made with a ThetaProbe (Delta-T
Devices, Cambridge, UK). The seedlings
were watered 3-4 times a week
according to need. In the UV-B
experiment (II), Scots pine seedlings
were exposed for two growing seasons
and Norway spruce seedlings for three
growing seasons to supplemental UV-B
radiation, corresponding to a 30%
increase in the ambient UV-B radiation
(weighted according to the erythemal
action spectrum). The experiment also
included appropriate controls for UV-A
and ambient radiation. The lamp output
was controlled electronically, varying
the enhanced UV-B treatment according
to the ambient UV-B level.
The indoor experiment, which was
carried out in the greenhouse of the
Forest Research Institute of Finland at
Punkaharju (61°41´ N, 29°20´ E), used
pot-grown willow clones (III, IV). It
included both drought and UV-B
treatments and lasted for four weeks.
The treatments were: 1) ambient UV-B
+ well watered (control), 2) enhanced
UV-B + well-watered, 3) ambient UV-B
+ drought stressed and 4) enhanced UVB + drought stressed. Drought stressed
willows received 50 % less water than
well-watered ones. The watering was
based on pilot experiments and
measurements made with a ThetaProbe.
The level of UV-B radiation given in the
ambient UV-B treatment corresponded
to that in Joensuu on 15 June (= 3.6 kJ
m-2 day-1), and the level of enhanced
UV-B radiation (twice that of ambient
UV-B) resembled the maximum values
of springtime UV-B radiation.
2.3. Growth measurements
chemical analysis
and
The growth of the conifer seedlings (I,
II) was followed by measuring the height
and stem-base diameter of the seedlings
and the current year shoots at the end of
each growing season. The height growth
of the main stems of the willow clones
(III) was measured every week, while
the biomass of their leaves, stems and
roots was determined at the end of the
experiment.
Samples were taken from the second
or third annual growth of the main stems
of the conifers and the current year
needles in order to determine the
monoterpene and resin acid (diterpene)
concentrations. Monoterpenes were
extracted from fresh samples with nhexane and resin acids from freezedried, ground samples with petroleum
ether- diethyl ether (I, II). The extracts
were analyzed by gas chromatography –
mass spectrometry (GC-MS) (I, II).
Nutrients (I) and phenolics (II) were
analyzed from current year needles. The
nutrients were analyzed from oven-dried
needles, using atomic absorption
spectrometry (K, Ca, Mg) and a
spectrophotometer (P) (I). The phenolics
were extracted from freeze-dried, ground
needles with methanol and analyzed by
high performance liquid chromatography
13
with diode array detection (HPLC/DAD)
(II).
The youngest fully expanded willow
leaves were collected and air-dried at
room temperature for phenolic analyses.
They were extracted with methanol and
analyzed by HPLC (IV). Individual
phenolic compounds were identified by
their retention times, UV-vis spectra and
mass spectrometry (HPLC-MS) (IV).
3. RESULTS
3.1. The secondary compounds of the
conifers and the species responses to
environmental stresses
The needles and wood of Scots pine and
Norway spruce seedlings contained
Tricyclene
α-pinene
Camph en e
several different mono- and diterpenes
(I, II) (Fig. 1a,b). The terpenes in the
wood and needles of Scots pine were the
same as those in Norway spruce, except
that the needles of the latter contained
some
monoterpenes
(eucalyptol,
linalool, camphor and borneol) (Fig.1a)
that were not found in those of the
former or in the woody tissues of either
species (II). In both species the
concentration of diterpenes in the woody
tissue was higher than that in the
needles, while the needles contained
more monoterpenes than the wood (II).
The needles and wood of Scots pine
seedlings
contained
higher
concentrations of terpenes than those of
Norway spruce (I, II).
β-pinene
Sabinen e
Myrcene
O
3-caren e
Limo nene
β-phelland rene
α-terpin olene
O
Bo rn yl acetate
OH
OH
O
Camph or*
O
Linalool*
Eucalyptol*
Borneol*
Figure 1a. The structures of monoterpenes detected in Scots pine and Norway spruce
wood and needles, (* found only in spruce needles).
14
CO 2 H
CO 2 H
CO 2 H
CO 2 H
Pimaric acid
Sand araco pim aric acid
Isopimaric acid
Levop im aric acid
CO 2 H
CO 2 H
CO 2 H
Palustric acid
Dehydroabietic acid
CO 2 H
Abietic acid
Neoabietic acid
Figure 1b. The structures of diterpenes detected in Scots pine and Norway spruce wood
and needles.
Phenolics, including different flavonoid
compounds, were found in the needles of
both species (II) (Fig. 2). The needles of
Norway spruce also contained high
amounts of acetophenones (picein) and
stilbenes (piceatannol), which were not
found in those of Scots pine (II). The
concentrations of phenolics were higher
in the needles of Norway spruce than in
those of Scots pine (II).
R
OH
OH
O
HO
O
OH
H3C C
O glucose
O
HO
Picein*
OH
OH
O
Flavonols:
Kaempferol
Quercetin
Isorhamnetin
OH
OH
OH
HO
OH
(+)-Catechin
R=H
R = OH
R = OCH3
OH
Piceatannol*
Figure 2. Basic structures of typical phenolics detected in the needles of Scots pine and
Norway spruce (* found only in spruce needles).
15
The effect of drought stress was different
from that of enhanced UV-B radiation (I,
II). Drought stress reduced growth in
both species (I), while enhanced UV-B
radiation had no significant effect on
growth (II) (Table 2). In addition,
drought
stress
increased
the
concentrations of terpenes (I), while
enhanced UV-B radiation had no effect
on terpenes and phenolics (II) (Table 2).
Table 2. The effect of drought stress, enhanced UV-B radiation and their combination on
growth and secondary compounds of different woody species.
Symbols: terp = terpenes, phen = phenolics, + = increased, − = decreased, 0 = unaffected,
nm = not measured
Species
Scots pine
Norway spruce
Hybrid willows
S. myrsinifolia
Treatments
Drought
Enhanced UV-B
growth terp phen growth
terp
phen
+
nm
0
0
0
−
+
nm
0
0
0
−
nm
0
nm
+
−
−
nm
nm
+
−
−
−
3.2. The secondary compounds of the
willows and the species responses to
environmental stresses
The main phenolic group in the willow
leaves consisted of salicylates (IV). The
concentrations of phenolic acids were
higher in the leaves of S. myrsinifolia
clones than in those of hybrid clones,
while the concentrations of flavonoids
were
lower
(IV).
Quantitative
differences were found also between
families and between clones within each
group of plantlets (i.e. the S. myrsinifolia
and the hybrids) with respect to the
concentrations of individual and total
phenolic groups (IV).
There were qualitative differences
between the hybrids and the pure species
with respect to salicylates and
flavonoids, while phenolic acids were
qualitatively similar (IV). Hybrids
UV-B x D
growth phen
nm
nm
nm
nm
+
−
0
0
contained luteolins and apigenins, while
the flavonoids in the leaves of S.
myrsinifolia
clones
consisted
of
quercetins (IV). Typical phenolics of
willows are shown in Fig. 3.
Both drought stress and enhanced
UV-B radiation reduced the growth of
the willows (III, Table 2), but the effects
on phenolics were different: drought
stress reduced the concentrations of
phenolics, and especially phenolic acids,
while enhanced
UV-B
radiation
increased their concentrations (IV, Table
2). Interaction effects of enhanced UV-B
and drought stress were found only in
the concentrations of phenolic acids and
the root/shoot ratio of hybrid willows
(III, IV, Table 2). Otherwise, the
response of willow clones to enhanced
UV-B radiation was similar in both wellwatered and drought-stressed conditions
(III, IV).
16
O
CH2OH
H2 C O glucose
HO
O C
O glucose
O glucose
O
Diglucoside of salicyl alcohol
CH2
Salicin
O glucose
O
CH 2OH
CH2OH
O
O
CH2
OH
O
Salicortin
CH 2 OH
O
C
O
O
OH
HO
O
HO
O
HO
O C
OH
O
Tremuloidin
O
R
Cinnamic acid
C
OH
O
HO
HO
Tremulacin
COOH
O
HO
OH
O
Flavones:
Luteolin
Apigenin
O
OH
Chlorogenic acid
OH
OH
R = OH
R=H
Figure 3. The basic structures of phenolics detected in the willow leaves.
4. DISCUSSION
4.1. Responses to drought stress
Drought stress reduced the growth of the
conifers and willows (I, III), and had a
greater impact on all the species studied
than enhanced UV-B radiation. It had a
greater effect on the growth of Scots
pine than on that of Norway spruce (I),
although Scots pine might have been
expected to be more tolerant of drought,
since it typically grows on dry sandy
soils (Boratyński 1991, de Groot and
Turgeon, 1998), while Norway spruce
grows on wet eutrophic soils (e.g.
Hämet-Ahti et al. 1992). The reason for
this unexpected result might be that
during the first summer it was more
difficult to subject the spruce seedlings
to severe drought than pine seedlings
because different growing media were
used in order to optimize moisture
conditions in the control treatments for
both species (I). However, the present
data shows that severe drought stress did
17
not significantly affect the growth of
either species during the first summer
(I).
Reduced growth may cause the
accumulation of secondary compounds,
because more carbon becomes available
for their synthesis, since photosynthesis
is less affected, and/or there is less
biomass to dilute these compounds (e.g.
Mattson and Haack 1987). Reduced
growth also lowers the potential of
seedlings to compensate for damage by
herbivores and consequently the
amounts of defensive compounds are
increased (e.g. Coley et al. 1985). In the
present study drought stress caused the
accumulation of constitutive terpenes in
the wood of both conifer species (I).
Drought stress has been shown to
increase the occurrence of resin pockets
in Norway spruce (Temnerud 1999) and
the production of axial resin canals in
the wood of Norway spruce (Wimmer
and Grabner 1997) and Scots pine
(Rigling et al. 2003). Drought stress
seems, therefore, to increase the number
of
specialized
terpene
secretory
structures, which are thought to be more
important than carbon availability in
limiting the production of terpenes (e.g.
Gerschenzon 1994). Drought stress has
also been shown to increase the flow of
constitutive resin (e.g. Lombardero et al.
2000), whereas the concentration and
flow of wound-induced resin is reduced
(e.g. Croisé and Lieutier 1993, Cobb et
al. 1997, Lombardero et al. 2000). It
seems that under drought conditions
conifer species with high constitutive
resin
levels
and
well-developed
secretory systems, such as Scots pine
and Norway spruce, invest more in
constitutive defense than in woundinduced defense, because constitutive,
preformed resin is the primary defense
against pests (Lewinsohn et al. 1991).
Wound-induced resin is needed when
the constitutive resin system fails to
defend the plant against initial pest
attack (de Groot and Turgeon 1998,
Rosner and Hannrup 2004), but it may
be less effective in drought stressed trees
because the pests may have become
acclimatized to high concentrations of
constitutive resin (Mattson et al. 1988).
Drought stress clearly decreased the
growth of the willows (III). The fastgrowing S. myrsinifolia clones were
more susceptible to drought stress than
the slower-growing S. myrsinites × S.
myrsinifolia hybrid clones (III). The
difference in susceptibility is probably
because the leaves of the hybrid willows
were smaller, thicker and shinier than
those of S. myrsinifolia (personal
observation), possibly making them
better able to acclimatize to drought
stress.
Drought causes oxidative stress and
an increase in the amounts of flavonoids
and phenolic acids in willow leaves was
expected, because they are the active
antioxidants in quenching reactive
oxygen radicals (e.g. Larson 1988, Foyer
et al. 1994). The effect of drought stress
on flavonoids and phenolic acids was,
however, variable: the concentrations of
total phenolic acids decreased in all
willow clones, but the concentration of a
single individual flavonoid increased in
hybrid clones (IV). The effect of drought
seems to be highly dependent on the
particular species, because it has been
shown to have no effect on the
concentrations of flavonoids or phenolic
acids in the leaves of fast growing
willow cultivars (S. viminalis (L.) × S.
dasyclados (L.)) (Glynn et al. 2004). The
concentrations of phenolics in European
privet (Ligustrum vulgare L.) have been
shown to decrease (e.g. Tattini et al.
2004) while in hawthorn species
18
(Crataegus laevigata (Poir.) and C.
monogyna Jacq.) some phenolics
increased
and
others
decreased
(Kirakosyan et al. 2004).
Drought stress can also affect plantinsect relationships (e.g. Mattson and
Haack 1987, Sipura et al. 2002).
Drought-stressed leaves are warmer
(Begg 1980) and contain more nitrogen
than the leaves of well-watered plants
(e.g. Glynn et al. 2004), and this may
stimulate insect feeding and growth
(Mattson and Haack 1987). On the other
hand low leaf water content and high
concentrations of secondary compounds
can cause reductions in insect abundance
(e.g. Ikonen et al. 2002, Sipura et al.
2002), because the composition and
concentration of salicylates affect the
food selection of insect herbivores (e.g.
Tahvanainen et al. 1985, Ruuhola et al.
2001, Ikonen et al. 2002, Glynn et al.
2004). In the present study there was
high clone-specific variation in the
concentrations of salicylates and in the
response of these compounds to drought
stress (IV), and this might cause the
willow clones to vary in their resistance
to insect herbivores.
4.2. Responses to enhanced UV-B
radiation
Enhanced levels of UV-B can inhibit
plant
growth,
development
and
physiological processes. Most conifer
species are relatively tolerant of UV-B
(e.g. Petropoulou et al.1995, Manetas et
al. 1997, Laakso and Huttunen 1998),
while some conifer species show an
increase in growth in response to UV-B
radiation and others a decrease (e.g.
Sullivan and Teramura 1988, 1992). In
the present study, it had no effect on the
growth of Scots pine and Norway spruce
(II). This might be because both species
can acclimatize to direct sunlight,
although they frequently grow in
different light conditions: Scots pine
needs direct sunlight, while Norway
spruce survives in fairly shady
conditions. The needles of Norway
spruce had higher amounts of UV-B
absorbing phenolics than those of Scots
pine (II), suggesting that the former
species has better screening against UVB radiation.
While the growth of conifer species
was unaffected by enhanced UV-B
treatment (II), that of willows was
reduced (III). Conifer needles are known
to be more tolerant of UV-B, because
they have a thicker waxy cuticle, a
smaller surface area and higher amounts
of UV-B absorbing compounds per
surface area than deciduous (willow)
leaves (Sullivan and Teramura 1988,
Day 1993). Moreover, the UV-Babsorbing compounds found in conifer
needles are more effective in screening
UV-B radiation than those in deciduous
trees (Day 1993). In the present studies,
the needles of Norway spruce and Scots
pine contained acylated flavonol
glucosides (II), while the flavonoids in
the leaves of willows were non-acylated
(IV). Acylation enhances the UV-Babsorbing capacity of flavonoids (e.g.
Fischbach et al. 1999, Turunen et al.
1999). The willow leaves also contained
high amounts of salicylates, which are
not very effective in absorbing the UV-B
radiation (IV). Since different phenolic
compounds vary substantially in their
UV-absorbance spectra, there are
relative differences in screening
effectiveness between species. In the
needles of evergreen conifers nearly all
of the UV-B is absorbed by the
epidermis:
the
mean
epidermal
transmittance of the radiation is only 1
%, as compared to about 21 % in the
19
leaves of deciduous trees (Day 1993,
Day et al. 1994).
The conditions and durations of the
UV-B experiments with the conifers
differed from those with the willows.
The increased UV-B radiation in the
study with conifers was 30 % higher
than the ambient value (II), while for the
willows the increase was 100 % (IV) and
this is probably partly responsible for the
differences in their responses in growth
and phenolics. The phenolics in conifer
needles were unaffected by enhanced
UV-B radiation (II), while flavonoids
and phenolic acids in willow leaves
increased (IV). Moreover, the willows
were located in a greenhouse (IV), in
which the amount of PAR was much
lower than that in the outdoor-study of
conifers (II). Lower irradiances of PAR
have been shown to increase the
sensitivity of plants to UV-B radiation
(e.g. Cen and Bornman 1990, Caldwell
et al. 1998) and several studies have
shown that UV-B-absorbing compounds
are higher in outdoor-grown plants than
in indoor-grown ones (e.g. Kuokkanen et
al. 2001, Warren et al. 2003), thus
suggesting that the former are better
protected against increased UV-B
radiation than the latter.
Previous studies have shown that UVB promotes terpene production in
aromatic plants (e.g. Karousou et al.
1998, Johnson et al. 1999), suggesting
that monoterpenes may have a role in
UV-B-protection. However, enhanced
UV-B radiation did not affect the
concentrations of mono- and diterpenes
in the wood and needles of Scots pine
and Norway spruce (II). The only
significant effect was caused by the UVA control treatment, which reduced the
concentrations of some individual
diterpenes in Scots pine wood (II).
4.3. The interaction effects of
enhanced UV-B and drought stress in
the willows
Enhanced UV-B can cause changes in
growth and biomass allocation, such as
increases in epidermal thickness and
decreases in leaf area, that may reduce
water loss (transpiration rate) and thus
improve drought resistance (e.g.
Petropoulou et al. 1995, Manetas et al.
1997, Nogués et al. 1998, Bassman et al.
2001). However, in the present study,
although enhanced UV-B radiation
reduced the growth of willows, the effect
of drought stress was similar in both
ambient and enhanced UV-B conditions
(III). The only interaction effects of
enhanced UV-B and drought stress were
found in the root/shoot ratio of hybrid
willows. In well-watered conditions the
root/shoot ratio was unaffected by
enhanced UV-B, while in drought
stressed conditions it was significantly
increased. The only effect of enhanced
UV-B and drought stress interactions on
phenolics was on the total concentrations
of phenolic acids in hybrid clones and no
interaction effects were observed in pure
S. myrsinifolia clones (IV). Thus it
seems that short-term drought stress had
only a very small effect on the response
of these willow clones to enhanced UVB radiation. However, use of
transformed data in the statistical
analysis of the willow study (III, IV)
may have affected the significance of the
UV-B × Drought interactions.
4.4. The implications for current eco/environmental theories
According to the CNB and GDB
hypotheses, when growth decreases
more than photosynthesis, more carbon
is allocated to the production of
20
secondary compounds (Herms and
Mattson 1992). In the present studies,
drought stress decreased the growth of
conifers and willows (I, III), and
increased the concentrations of terpenes
in conifers (I). However, in willow
clones the concentrations of total
phenolics were reduced or remained
unaffected (IV). The results of enhanced
UV-B radiation treatments were also
variable: in conifers neither growth nor
the
concentrations
of
secondary
compounds were affected (II, Table 2),
while in willows growth decreased and
the concentrations of phenolics increased
(III, IV). Thus some results seem to
support the hypotheses, while others do
not.
The
photoinhibition
hypothesis
(Close and McArthur 2002) suggests
that both UV-B and drought stress
increase the amounts of phenolics, but in
the present study flavonoids and
phenolic acids increased only in
response to UV-B and thus the
hypothesis was not supported (IV).
The PCM hypothesis (Jones and
Hartley 1998, 1999) claims that severe
drought stress and UV-B radiation have
variable effects across species, causing
increases, reductions and no changes in
phenolic concentrations. In the present
studies the responses of willow and
conifer phenolics to these stresses
varied: drought stress did not affect the
total phenolics of hybrid willows, while
in
S.
myrsinifolia
clones
the
concentrations of total phenolics
decreased (IV). Conifer phenolics were
not affected by enhanced UV-B
radiation (II), whereas the amounts of
willow phenolics increased (IV). It may
be concluded that PCM is not very
useful in predicting the effects of
drought stress and enhanced UV-B on
the secondary chemistry of these boreal
conifer and willow species.
Another limitation of the CNB, GDB
and PCM hypotheses is that they do not
make specific predictions regarding
carbon allocation at the level of the
individual compounds (e.g. Koricheva et
al. 1998). As is known, the carbon
allocated to secondary compounds is
split into several alternative pathways
and the distribution of carbon among
these pathways and among branches of
the same pathway is often unequal.
Thus, it may be that although the total
amount of secondary compounds is not
affected, there are significant effects on
individual compounds, as were found in
the present studies (I, IV), and changes
in these individual compounds may be as
important as or even more important
than changes in the total concentration of
secondary compounds.
5. CONCLUSIONS
Some caution is needed in drawing
general conclusions because the
treatments applied to conifers and
willows were not identical. Nevertheless,
it is evident that
1. drought stress causes stronger
responses in boreal conifers and
willows than enhanced UV-B
radiation;
2. in
conifers,
terpene
concentrations increase after
drought treatment, while in
willows drought induces variable
changes in the individual
phenolics;
3. Scots pine and Norway spruce
are so well-protected against UVB radiation, that exposure to
higher doses of UV-B radiation
does not significantly affect their
21
growth or their levels of
phenolics and terpenes;
4. willows are more susceptible to
enhanced UV-B radiation than
conifers and their growth and
secondary chemistry may be
affected even by short term UVB radiation;
5. there is considerable genetic
variation in the response of
plants to environmental stresses.
ACKNOWLEDGEMENTS
I want to thank everyone, who has
helped me with this thesis. I am grateful
to my supervisors Docent Matti Rousi,
Prof. Riitta Julkunen-Tiitto, and Docent
Pirjo Kainulainen for their advice,
support and
encouragement
and
especially for their different views on the
studies, which gave me new ideas, when
I did not know how to continue working
with the articles. Thanks also to Pirjo for
allowing me to use the two studies done
at Kuopio University in my thesis, which
gave me a good starting-point to this
work. I also want to thank Kenneth
Meaney and Joann von Weissenberg for
revising the English of my manuscripts.
Prof. Takayoshi Koike and Docent Pedro
Aphalo kindly pre-examined this thesis.
I also want to express my thanks to
my colleagues Sarita, Susanne, Riitta T.,
Riitta K., Marja-Leena, Jaana, Ria, Outi,
Sinikka, Maija, Keiko and all the others
I worked with. Thanks to Sarita for her
comments and advice and for the long,
cheerful lunchtimes. I want to thank
Riitta T. for her good advice and help,
especially
when
referees
were
particularly critical. It was fun to work
with Susanne, especially during the first
two summers in the big, hot willow
field. Thanks also to the others in “The
Science Community”, and especially
Ursula for stimulating conversations. My
thanks also go to the staff of the Finnish
Forest Institute at Punkaharju for all
their help during my greenhouseexperiment, and especially Hanni. I also
thank all the co-authors, especially those
from the Kuopio University, who did a
lot of work before I joined the studies. I
thank the Academy of Finland (projects
No: 43159, 52784 and 64308), the
University of Joensuu (Faculty of
Science) and the Finnish Cultural
Foundation for supporting this study
financially.
Special thanks go to my parents,
sisters and brother for their love. Really
warm thanks to my goddaughters and
their families, and all my friends, who
gave me other things to think about,
enabling me to forget about this thesis
during evenings and weekends. I want to
thank my dearest friends from Joensuu –
Minna, Elina, Mimmi, Heli and all the
others – for discussions and sharing their
freetime with me. Minna and Mimmi
gave me a lot of support when my life
seemed to be a disaster and I want to
thank them especially for their prayers,
which gave me strength to go on. Really
warm thanks also go to my dear friends
from my days in Kuopio – Mari Ke.,
Milla, Mari Ko., Anna-Mari, Virve and
Anu – who I often called up and visited
while working on my thesis. Thanks for
listening and supporting me. I also want
to thank Heli A. for our long friendship.
In fact, I want to thank all my friends for
being there to talk to when I want to
share the joys and sorrows of my life. I
am also grateful for Joensuu OPKO for
the Student Evenings and to the invited
speakers for their good teaching about
the gospel. Thanks to all my brothers
and sisters in Jesus Christ for the
wonderful times at OPKO evenings,
which gave me renewed strength to
22
continue this work. Thanks also to
members of the gospel-chorus Arepa for
our rehearsals and performances; it has
been always a great relaxation and joy to
sing with you. Finally, and most of all, I
want to express my loving thanks to our
Heavenly Father for His endless love
and help during the struggles and joys of
my life. Doing this thesis was mentally
very demanding, and without the faith
and all the wonderful things in my life,
which God has given to me; I would
never have had the strength to finish this
thesis. With God’s guidance and care it
is good to continue living this life and I
trust my life into His hands.
REFERENCES
Anttonen S, Manninen A-M, Saranpää P,
Kainulainen P, Linder S and Vapaavuori E.
2002. Effects of long-term nutrient
optimisation on stem wood chemistry in
Picea abies. Trees 16: 386-394.
Aucamp PJ. 2003. Eighteen questions and
answers about the effects of the depletion of
the ozone layer on humans and the
environment.
Photochemical
&
Photobiological Sciences 2: ix-xxiv.
Balakumar T, Vincent VHB, Paliwal K. 1993.
On the interaction of UV-B radiation (280315 nm) with water stress in crop plants.
Physiologia Plantarum 87: 217-222.
Bassman JH, Robberecht R and Edwards GE.
2001. Effects of enhanced UV-B radiation on
growth and gas exchange in Populus
deltoides Bartr. Ex Marsh. International
Journal of Plant Sciences 162: 103-110.
Begg JE. 1980. Morphological adaptations of
leaves to water stress. In Turner NC and
Kramer PJ (eds). Adaptation of plants to
water and high temperature stress. John
Wiley & Sons, Inc. USA, pp. 31-42.
Bennett RN and Wallsgrove RM. 1994.
Secondary metabolites in plant defence
mechanisms. New Phytologist 127: 617-633.
Berenbaum MR. 1995. The chemistry of
defence: Theory and practice. Proceedings of
the National Academy of Sciences USA 92:
2-8.
Björn LO, Callaghan TV, Johnsen I, Lee JA,
Manetas Y, Paul ND, Sonesson M, Wellburn
AR, Coop D, Heide-Jørgensen HS, Gehrke
C, Gwynn-Jones D, Johanson U, Kyparissis
A, Levizou E, Nikolopoulos D, Petropoulou
Y and Stephanou M. 1997. The effects of
UV-B radiation on European heathland
species. Plant Ecology 128: 253-264.
Boratyński A. 1991. Range of natural
distribution. In Giertych M, Mátyás C (eds),
Genetics of Scots pine, Académiai Kiadó,
Hungary, pp. 19-30.
Bryant JP, Chapin FS and Klein DR. 1983.
Carbon/nutrient balance of boreal plants in
relation to vertebrate herbivory. Oikos 40:
357-368.
Caldwell MM, Björn LO, Bornman JF, Flint SD,
Kulandaivelu G, Teramura AH and Tevini
M. 1998. Effects of increased solar
ultraviolet radiation on terrestrial ecosystems.
Journal of Photochemistry and Photobiology
B: Biology 46: 40-52.
Caldwell MM, Ballaré CL, Bornman JF, Flint
SD, Björn LO, Teramura AH, Kulandaivelu
G and Tevini M. 2003. Terrestrial
ecosystems, increased solar ultraviolet
radiation and interactions with other climatic
change
factors.
Photochemical
&
Photobiological Sciences 2: 29-38.
Cen Y-P and Bornman JF. 1990. The response of
bean plants to UV-B radiation under different
irradiances of background visible light.
Journal of Experimental Botany 232: 14891495.
Close DC and McArthur C. 2002. Rethinking the
role of many plant phenolics – protection
from photodamage not herbivores? OIKOS
99: 166-172.
Cobb NS, Mopper S, Gehring CA, Caouette M,
Christensen KM and Whitham TG. 1997.
Increased moth herbivory associated with
environmental stress of pinyon pine at local
and regional levels. Oecologia 109: 389-397.
Coley PD, Bryant JP, Chapin FS III. 1985.
Resource availability and plant antiherbivore
defence. Science 230: 895-899.
Croisé L and Lieutier F. 1993. Effects of drought
on the induced defence reaction of Scots pine
to bark beetle-associated fungi. Annales des
sciences forestières. 50: 91-97.
Croteau R and Johnson MA. 1985. Biosynthesis
of terpenoid wood extractives. In: Higuchi T
(ed) Biosynthesis and Biodegradation of
Wood Components. Academic Press,
Orlando, pp 379-439.
Dawson A. 1992. Global Climate Change.
Oxford University Press, UK. 48 p.
23
Day TA. 1993. Relating UV-B radiation
screening effectiveness of foliage to
absorbing compound concentration and
anatomical characteristics in a diverse group
of plants. Oecologia 95: 542-550.
Day TA, Howells BW and Rice WJ. 1994.
Ultraviolet absorption and epidermal
transmittance spectra in foliage. Physiologia
plantarum 92: 207-218.
de Groot P and Turgeon JJ. 1998. Insect –pine
interactions. In Richardson DM (ed.) Ecology
and Biogeography of Pinus. Cambridge
University Press, UK, pp. 354-380.
Fischbach RJ, Kossmann B, Panten H,
Steinbrecher R, Heller W, Seidlitz HK,
Sandermann H, Hertkorn N and Schnitzler JP. 1999. Seasonal accumulation of
ultraviolet-B screening pigments in needles
of Norway spruce (Picea abies (L.) Karst).
Plant, Cell and Environment 22:27-37
Foyer CH, Lelandais M, Kunert KJ. 1994.
Photooxidative stress in plants. Physiologia
Plantarum 92: 696-717.
Fraenkel GS. 1959. The raison d´ệtre of
secondary plant substances. Science 129:
1466-1470.
Gerschenzon J. 1994. The cost of plant chemical
defense against herbivory: a biochemical
perspective. In: Insect-Plant Interactions, Vol
V (ed. Bernays EA), pp. 108-116. CRC
Press, Boca Raton.
Glynn
C,
Rönnberg-Wästljung
A-C,
Julkunen.Tiitto R, Weih M. 2004. Willow
genotype, but not drought treatment, affects
foliar phenolic concentrations and leaf beetle
resistance. Entomolologia Experimentalis et
Applicata 113: 1-14.
Grace SC, Logan BA and Adams III WW. 1998.
Seasonal differences in foliar content of
chlorogenic
acid,
a
phenylpropanoid
antioxidant, in Mahonia repens. Plant, Cell
and Environment 21: 513-521.
Grossnickle SC.2000. Ecophysiology of northern
spruce species: The performance of planted
seedlings. NRC. Research Press, Ottawa,
Ontario, Canada, 409 pp.
Guarnaschelli AB, Lemcoff JH, Prystupa P and
Basci SO. 2003. Responses to drought
preconditioning in Eucalyptus globulus
Labill. provenances. Trees 17: 501-509.
Harborne JB. 1980. Plant phenolics. In: Bell EA
and Charlwood BV (eds.) Secondary Plant
Products, Springer-Verlag, Germany, pp.
329-402.
Haukioja E, Ossipov V, Koricheva J, Honkanen
T, Larsson S and Lempa K. 1998.
Biosynthetic
origin
of
carbon-based
secondary compounds. Cause of variable
responses of woody plants to fertilization?
Chemoecology 8: 133-139.
Helminen J, Nordlund A, Karlsson P and
Kersalo J. 2002. Ilmastokatsaus 12/2002.
Finnish Meteorological Institute, Helsinki.
Herms DA and Mattson WJ. 1992. The dilemma
of plants: to grow or defend. The Quarterly
Review of Biology 67: 283-335.
Hsiao TC. 1973. Plant responses to water stress.
Annual Review of Plant Physiology 24: 519570.
Hofmann RW, Campbell BD and Fountain DF.
2003. Sensitivity of white clover to UV-B
radiation depends on water availability, plant
productivity and duration of stress. Global
Change Biology 9: 473-477.
Hämet-Ahti L, Palmer A, Alanko P and
Tigerstedt PMA 1992. Woody flora of
Finland. Publications of the Finnish
Dendrological Society 6:1-373.
Ikonen A, Tahvanainen J and Roininen H. 2002.
Phenolic
secondary
compounds
as
determinants of host plant preferences of the
leaf beetle, Agelastica alni. Chemoecology
12: 125-131.
Johnson CB, Kirby J, Naxakis G and Pearson S.
1999. Substantial UV-B-mediated induction
of essential oils in sweet basil (Ocimum
basilicum L.). Phytochemistry 51: 507-510.
Jones CG and Hartley SE. 1998. Global change
and plant phenolic concentrations: species
level predictions using the protein
competition model. In: De Kok LJ and Stulen
I (eds) Responses of plant metabolism to air
pollution and global change. Backhuys
publishers, Leiden, pp. 23-50.
Jones CG and Hartley SE. 1999. A protein
competition model of phenolic allocation.
OIKOS 86: 27-44.
Kainulainen P, Oksanen J, Palomäki V,
Holopainen JK and Holopainen T. 1992.
Effect of drought and waterlogging stress on
needle monoterpenes of Picea abies.
Canadian Journal of Botany 70:1613-1616.
Karousou R, Grammatikopoulos G, Lanaras T,
Manetas Y and Kokkini S. 1998. Effects of
enhanced UV-B radiation on Mentha spicata
essential oil. Phytochemistry 49: 2273-2277.
Kirakosyan A, Kaufman P, Warber S, Zick S,
Aaronson K, Bolling S and Chang SC. 2004.
Applied environmental stresses to enhance
the levels of polyphenolics in leaves of
hawthorn plants. Physiologia Plantarum 121:
182-186.
24
Koricheva, J, Larsson S, Haukioja E and
Keinänen M. 1998. Regulation of woody
plant secondary metabolism by resource
availability: hypothesis testing by means of
meta-analysis. OIKOS 83: 212-226.
Kostina E, Wulff A and Julkunen-Tiitto R. 2001.
Growth, structure, stomatal responses and
secondary metabolites of birch seedlings
(Betula pendula) under elevated UV-B
radiation in the field. Trees 15: 483-491.
Kuokkanen K, Julkunen-Tiitto R, Keinänen M,
Niemelä P and Tahvanainen J. 2001. The
effect of elevated CO2 and temperature on the
secondary chemistry of Betula pendula
seedlings. Trees 15: 378-384.
Kuusisto E, Kauppi L and Heikinheimo P. (eds.).
1996. Climate change and Finland. Helsinki
University Press, Helsinki.
Laakso K and Huttunen S. 1998. Effects of the
ultraviolet-B radiation on conifers: a review.
Environmental Pollution 99:319-328
Laakso K, Sullivan JH and Huttunen S. 2000.
The effects of UV-B radiation on epidermal
anatomy in loblolly pine (Pinus taeda L.) and
Scots pine (Pinus sylvestris L.). Plant, Cell
and Environment 23:461-472
Landry LG, Chapple CC, Last RL. 1995.
Arabidopsis mutants lacking phenolic
sunscreens exhibit enhanced ultraviolet-B
injury and oxidative damage. Plant
Physiology 109: 1159-1166.
Larson RA. 1988. The antioxidants of higher
plants. Phytochemistry 27: 969-978.
Lavola A, Julkunen-Tiitto R, Aphalo P, de la
Rosa T and Lehto T. 1997. The effect of u.v.B radiation on u.v.-absorbing secondary
metabolites in birch seedlings grown under
stimulated forest soil conditions. New
Phytologist 137: 617-621.
Lewinsohn E, Gijzen M and Croteau R. 1991.
Defence mechanisms of conifers. Differences
in
constitutive
and
wound-induced
monoterpene biosynthesis among species.
Plant Physiology 96: 44-49.
Li J, Ou-Lee T-M, Raba R, Amundson RG, Last
RL. 1993. Arabidopsis flavonoid mutants are
hypersensitive to UV-B irradiation. The Plant
Cell 5: 171-179.
Lichtenthaler HK, Rohmer M and Schwender J.
1997.
Two
independent
biochemical
pathways for isopentenyl diphosphate and
isoprenoid biosynthesis in higher plants.
Physiologia plantarum 101: 643-652.
Lombardero MJ, Ayres MP, Lorio PL Jr and
Ruel JJ. 2000. Environmental effects on
constitutive and inducible resin defences of
Pinus taeda. Ecology letters 3: 329-339.
McKenzie RL, Björn LO, Bais A and Ilyasd M.
2003. Changes in biologically active
ultraviolet radiation reaching the Earths´s
surface. Photochemical & Photobiological
Sciences 2: 5-15.
Madronich S and Flocke S. 1997. Theoretical
estimation of biologically effective UV
radiation at the Earth’s surface. In Zerefos
CS and Bais AF (eds). Solar ultraviolet
radiation: modelling, measurements and
effects. NATO ASI series: Ser. I, Global
environmental change; Vol. 52, pp. 23-48.
Madronich S, Mckenzie RL, Björn LO and
Caldwell MM. 1998. Changes in biologically
active ultraviolet radiation reaching the
Earth´s surface. Journal of Photochemistry
and Photobiology B: Biology 46: 5-19.
Manetas Y, Petropoulou Y, Stamatakis K,
Nikolopoulos D, Levizou E, Psaras G and
Karabourniotis G. 1997. Beneficial effects of
enhanced UV-B radiation under field
conditions: improvement of needle water
relations and survival capacity of Pinus pinea
L. seedlings during the dry Mediterranean
summer. Plant Ecology 128: 101-108.
Mattson WJ and Haack RA. 1987. Role of
drought stress in provoking outbreaks of
Phytophagous insects. In Barbosa P, Schultz
JC (eds.) Insect outbreaks. Academic Press,
Inc., New York, pp. 365-407.
Mattson WJ, Lawrence RK, Haack RA, Herms
DA and Charles P-J. 1988. Defensive
strategies of woody plants against different
insect-feeding guilds in relation to plant
ecological strategies and intimacy of
association with insects. In Mattson WJ,
Levieux J and Bernard-Dagan C (eds.)
Mechanisms of woody plant defences against
insects, pp. 3-38. Springer-Verlag, New
York.
Monk LS, Fagerstedt KV and Crawford RMM.
1989. Oxygen toxicity and superoxide
dismutase as an antioxidant in physiological
stress. Physiologia Plantarum 76: 456-459.
Németh P, Tóth Z and Nagy Z. 1996. Effect of
weather conditions on UV-B radiation
reaching the earth´s surface. Journal of
Photochemistry and Photobiology B: Biology
32: 177-181.
Newsholme C. 1992. Willows: the genus Salix.
BT Batsford Ltd, London, 224 p.
Nogués S, Allen DJ, Morison JIL, Baker NR.
1998. Ultraviolet-B radiation effects on water
relations,
leaf
development
and
25
photosynthesis in droughted pea plants. Plant
Physiology 117: 173-181.
Obst JR. 1998. Special (secondary) metabolites
from wood. In Bruce A and Palfreyman JW
(eds) Forest products Biotechnology. Taylor
& Francis, London, pp. 151-165.
Osório J, Osório ML, Chaves MM and Pereira
JS. 1998.Water deficits are more important in
delaying growth than in changing patterns of
carbon allocation in Eucalyptus globulus.
Tree Physiology 18: 363-373.
Petropoulou Y, Kyparissis A, Nikolopoulos D
and Manetas Y. 1995. Enhanced UV-B
radiation alleviates the adverse effects of
summer drought in two Mediterranean pines
under field conditions. Physiologia Plantarum
94: 37-44.
Phillips MA and Croteau RB. 1999. Resin-based
defences in conifers. Trends Plant Sci. 4/5:
184-190.
Price RA, Liston A and Strauss SH. 1998.
Phylogeny and systematics of Pinus. In
Richardson DM (ed.) Ecology and
Biogeography
of
Pinus.
Cambridge
University Press, UK, pp. 49-68.
Rigling A, Brühlhart H, Bräker OU, Forster T,
Schweingruber FH. 2003. Effects of
irrigation on diameter growth and vertical
resin duct production in Pinus sylvestris L.
on dry sites in the central Alps, Switzerland.
Forest Ecology and Management 175: 285296.
Rhodenbaugh EJ and Pallardy SG. 1993. Water
stress, photosynthesis and early growth
patterns of cuttings of three Populus clones.
Tree Physiology 13: 213-226.
Rosner S and Hannrup B. 2004. Resin canal
traits relevant for constitutive resistance of
Norway spruce against bark beetles:
environmental and genetic variability. Forest
Ecology and Management 200, 77-87.
Ruuhola T, Tikkanen O-P, Tahvanainen J. 2001.
Differences in host use efficiency of larvae of
a generalist moth, Operophtera brumata on
three chemically divergent Salix species.
Journal of Chemical Ecology 27: 1595-1615.
Seigler DS. 1998. Plant secondary metabolism.
Kluwer Academic Publishers, Boston.
Shindell DT, Rind D, Lonergan P. 1998.
Increased polar stratospheric ozone losses
and delayed eventual recovery owing to
greenhouse-gas concentrations. Nature 392:
589-592.
Simon PC. 1997. Extraterrestrial solar irradiance
in the near and medium UV ranges. In
Zerefos CS and Bais AF (eds). Solar
ultraviolet
radiation:
modelling,
measurements and effects. NATO ASI series:
Ser. I, Global environmental change; Vol. 52,
pp.1-12.
Sipura M, Ikonen A, Tahvanainen J and
Roininen H. 2002. Why does the leaf beetle
Galerucella lineola F. attack wetland
willows? Ecology 83: 3393-3407.
Strack D. 1997. Phenolic metabolism. In Dey
PM and Harborne JB (eds.) Plant
Biochemistry. Academic press, San Diego,
USA. Pp. 387-416.
Sullivan JH and Teramura AH. 1988. Effects of
ultraviolet-B irradiation on seedling growth
in the Pinaceae. American Journal of Botany
75:225-230
Sullivan JH and Teramura AH. 1992. The effects
of UV-B radiation on loblolly pine. 2.
Growth of field-grown seedlings. Trees
6:115-120.
Szaniawski RK and Wierzbicki B. 1978. Net
photosynthetic rate of some coniferous
species at diffuse high irradiance.
Photosynthetica 12, 412-417.
Taalas P, Kaurola J, Kylling A, Shindell D,
Sausen R, Dameris M, Grewe V, Herman J,
Damski J, Steil B. 2000. The impact of
greenhouse gases and halogenated species on
future solar UV-radiation doses. Geophysical
Research Letters 27: 1127-1130.
Tahvanainen J, Julkunen-Tiitto R, Kettunen J.
1985. Phenolic glycosides govern the food
selection pattern of willow feeding leaf
beetles. Oecologia 67: 52-56.
Tattini M, Galardi C, Pinelli P, Massai R,
Remorini D and Agati G. 2004. Differential
accumulation
of
flavonoids
and
hydroxycinnamates in leaves of Ligustrum
vulgare under excess light and drought stress.
New Phytologist 163: 547-561.
Temnerud E. 1999. The occurrence of resin
pockets in sawlog populations of Picea abies
(L.) Karst. From five geographic regions in
Sweden. Scandinavian Journal of Forest
Research 14: 143-155.
Tosserams M, Smet J, Magendans E and
Rozema J. 2001. Nutrient availability
influences UV-B sensitivity of Plantago
lanceolata. Plant Ecology 154: 159-168.
Turtola S, Manninen A-M, Holopainen JK,
Levula T, Raitio H and Kainulainen P. 2002.
Secondary metabolite concentrations and
terpene emissions of Scots pine xylem after
long-term forest fertilization. Journal of
Environmental Quality 31: 1694-1701.
26
Turunen M, Heller W, Stich S, Sandermann H,
Sutinen M-L and Norokorpi Y. 1999. The
effects of UV exclusion on the soluble
phenolics of young Scots pine seedlings in
the subarctic. Environmental Pollution 106:
219-228.
Venäläinen A, Nordlund A, Karlsson P and
Kersalo J. 2004. Ilmastokatsaus 12/2004.
Finnish Meteorological Institute, Helsinki.
Walther G-R, Post E, Convey P, Menzel A,
Parmesaon C, Beebee TJC, Fromentin J-M,
Hoegh-Guldberg O and Bairlein F. 2002
Ecological responses to recent climate
change. Nature 416: 389-395.
Warren JM, Bassman JH, Fellman JK, Mattinson
DS, Eigenbrode S. 2003. Ultraviolet-B
radiation alters phenolic salicylate and
flavonoid
composition
of
Populus
trichocarpa leaves. Tree Physiology 23: 527535.
Webb AR and Weatherhead EC. 1997. Current
status of UV measurements. In Zerefos CS
and Bais AF (eds). Solar ultraviolet radiation:
modelling, measurements and effects. NATO
ASI series: Ser. I, Global environmental
change; Vol. 52, pp. 267-278.
Wimmer R and Grabner M. 1997. Effects of
climate on vertical resin duct density and
radial growth of Norway spruce (Picea abies
(L.) Karst). Trees 11: 271-276.
Worrest RC, Smythe KD and Tait AM. 1989.
Linkages between climate change and
stratospheric ozone depletion. In: White JC
(Ed.) Global climate change linkages, acid
rain, air quality and stratospheric ozone.
Elsevier Science Publishing Company, Inc.,
USA, pp. 67-78.