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Dynamics of salicylates in willows and its relationship to herbivory

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Ruuhola, Teija
Dynamics of salicylates in willows and its relation to herbivory. — University of Joensuu, 2001,
129 pp.
University of Joensuu, PhD Dissertations in Biology, n:o 6. ISSN 1457-2486.

ISBN 952-458-047-0

Keywords: Willows, Salix, salicylates, phenolic glycosides, herbivory, Phratora vitellinae, Operophtera brumata,
costs of defense, turnover, trade-off, metabolism, induction.

Secondary chemicals have ubiquitous roles in plants, and one that has been widely studied is
their role in defense against herbivores. In spite of the apparent benefits of chemical defense, the
production of defense chemicals is costly and may lower the reproductive and competitive
ability of plants. The main focus in my thesis was to study the metabolism of salicylates
(biosynthesis, degradation and metabolic turnover), and in this way to evaluate the costs of
salicylate-dependent defense in northern willows (Salix spp.). I studied also the inducibility of
salicylates in response to herbivore attack. Moreover, I studied the host use efficiency of a
generalist moth, Operophtera brumata L. (Lepidoptera: Geometridae), on three chemically
divergent willow species and the effects of induction of salicylates on the subsequent feeding of
a specialist leaf-beetle, Phratora vitellinae L. (Coleoptera: Chrysomelidae).
The study showed that salicylates were stable compounds that are not subject to marked
metabolic turnover in mature plant parts. Thus, the maintenance costs of salicylate-based defense
seems to be rather low. On the contrary, the initial construction costs appear to be high, which
was seen as a trade-off between the growth of S. pentandra plants and the synthesis of
salicylates. I propose that salicin is a precursor of higher substituted salicylates, and that the
biosynthesis of salicin in turn proceeds mainly via benzoyl-glucose and salicyloyl-glucose.
Phenylalanine (Phe) is apparently a common and limiting substrate for the synthesis of
salicylates and the growth of willows, but the majority of Phe is used for the synthesis of
phenolics.
The degradation of salicylates produced substantial amounts of catechol and salicin in the
digestive tract of O. brumata larvae. In addition, the main degradation products of salicylates
were salicin, 6-HCH (6-hydroxy-2-cyclohexenone) and catechol, both in enzymatic and in pH-
mediated decomposition. Thus, the formation of 6-HCH and catechol does not necessarily
demand enzymatic reactions. The degradation products of salicylates, saligenin and catechol,
impaired the growth of O. brumata larvae, while salicin and chlorogenic acid were inefficient in
this role. Both 6-HCH and catechol deter the feeding of generalist insect herbivores, but they
may also lower the nutritive value of leaves. Saligenin, on the other hand, is claimed to be a
toxic compound.
Salicylates of S. myrsinifolia increased as a response to attack by P. vitellinae. The induction
was systemic, appearing in young unwounded leaves, and it was apparently a consequence of an
increased rate of synthesis. In addition, high clonal variation was found both in constitutive and
in induced levels of salicylates; no trade-off was detected between constitutive and inducible
concentrations of salicylates. Induction did not influence the subsequent feeding of highly
specialized P. vitellinae, but induction may reduce the damage to foliage caused by semi-
specialist or generalist herbivores such as O. brumata whose feeding and growth were retarded
by salicylates.

Teija Ruuhola, Department of Biology, University of Joensuu. P.O.Box 111, FIN-80101

Joensuu, Finland

3
ABBREVIATIONS

AIP 2-aminoindan-2-phosphonic acid

AOPP L-α-aminooxy-β-phenyl propionic acid
BA benzoic acid
CNB Carbon/Nutrient Balance Hypothesis
DAPH 3-deoxy- D-arabinose -heptulosonate-7-phosphate
Diglucoside diglucoside of salicin
GDB Growth Differentiation Balance Hypothesis
6-HCH 6-hydroxy-2-cyclohexenone
HPLC high performance liquid chromatography
HR hypersensitive response
JA jasmonic acid
MS Murashige-Skoog medium
PAL phenylalanine ammonia-lyase
PCM Protein Competition Model
Phe phenylalanine
SA salicylic acid
SAR systemic acquired resistance
TAL tyrosine ammonia-lyase
Trp tryptophane
Tyr tyrosine
WP Woody-Plant medium

4
CONTENTS

LIST OF ORIGINAL PUBLICATIONS 6

1. INTRODUCTION 7
2. MATERIALS AND METHODS 9
2.1. Study organisms 9
2.1.1. Willows 9
2.1.2. Insects 10
2.2. Experimental procedures 11
2.2.1. Turnover and biosynthesis studies (I, II) 11
2.2.2. Induction study (III) 11
2.2.3. Operophtera brumata study (IV) 11
2.2.4. Degradation study (V) 12
2.3. Methods of analysis 12
3. RESULTS AND DISCUSSION 14
3.1. Sources of variation in willow phenolic chemistry 14
3.1.1. Chemistry of willow species 14
3.1.2. Within-plant variation 16
3.1.3. Systemic induction and clonal variation in salicylates 16
3.2. Metabolism of salicylates 18
3.2.1. Inhibition of the biosynthesis of salicylates by 2-aminoindan-
2-phosphonic acid 18
3.2.2. Biosynthesis of salicylates 19
3.2.3. Degradation of salicylates 20
3.2.4. Metabolic turnover of salicylates 22
3.2.5. Metabolic grid of salicylates 23
3.3. Trade-off between synthesis of salicylates and growth of plants 23
3.4. Dynamics of salicylates in relation to herbivory 25
4. CONCLUSIONS 28
ACKNOWLEDGEMENTS 28
REFERENCES 29

5
LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following articles, referred to in the text by their Roman numerals I-
V.

I Ruuhola, T. and Julkunen-Tiitto, R. Trade-off between the synthesis of salicylates and

the growth of micropropagated Salix pentandra plants. Manuscript.

II Ruuhola, T.M. and Julkunen-Tiitto, M-R.K. 2000. Salicylates of intact Salix myrsinifolia
plantlets do not undergo rapid metabolic turnover. Plant Physiology 122:895-905.

III Ruuhola, T.M., Sipura, M., Nousiainen, O. and Tahvanainen, J. 2001. Systemic induction
of salicylates in Salix myrsinifolia (Salisb.). Annals of Botany 88:483-497.

IV Ruuhola, T., Tikkanen, O-P. and Tahvanainen, J. 2001. Differences in host use efficiency
of larvae of a generalist moth, Operophtera brumata (Lepidoptera: Geometridae) on
three chemically divergent Salix species. Journal of Chemical Ecology 27:1595-1615.

V Ruuhola, T., Julkunen-Tiitto, R. and Vainiotalo, P. In vitro degradation of willow

salicylates. Manuscript.

6
INTRODUCTION glycosides are harmless until they are
hydrolyzed by β-glucosidase activity and
toxic HCN is released (Wint, 1997).
Plants live in a world that is inhabited by
Together with oxidizing enzymes, o-
numerous enemies: plant-eating animals
substituted phenolic compounds, such as
called herbivores. In spite of the great
chlorogenic acid, produce o-quinones,
variety of herbivores, only parts of plants
which are alkylating agents binding to the
are defoliated, and the majority of plant
nucleophilic groups of amino acids and
foliage survives. This is due to the ability of
proteins (Felton et al.,1989; Constabel,
plants to tolerate herbivory by compensating
1999). This binding lowers the quality of
for resource losses (Strauss and Agrawal,
plant foliage for herbivores by inhibiting the
1999; Constabel, 1999), or to defend
assimilation of essential amino acids (Felton
themselves and thus to reduce the amount of
et al., 1989).
damage (e.g. Constabel, 1999). Plants may
Salicylates are phenolic glycosides found
use different strategies to defend themselves
in willows and poplars, members of
against herbivores. Morphological structures
Salicaceae family (e.g. Pearl, 1965; Thieme,
such as hairs, thorns and waxes may prevent
1965abc; Pearl and Darling, 1965, 1970;
the feeding of certain herbivores (Lucas et
Lindroth et al., 1987; Julkunen-Tiitto, 1985,
al., 2000). Low nutrient content of the plants
1986, 1989). These substances contain a
may retard the growth of insect herbivores
backbone structure of salicin, 2-O-β-D-
or make them unpalatable as food (Scriber
glucoside of salicyl alcohol, attached to a
and Slansky, 1981). In addition, plants
variety of substituents, such as benzyl,
contain a wide variety of secondary
acetyl and/or hydroxycyclohexenone
compounds, which may act as antifeeding
groups. Salicylates are often found in high
agents, be toxic or lower the nutritional
levels in the bark and leaves of plants, but
quality of the foliage (Scriber and Slansky,
roots do not contain salicylates (Julkunen-
1981; Constabel, 1999).
Tiitto, 1989). Salicin and salicortin are the
Secondary compounds are traditionally
most widespread salicylates in northern
divided into three groups: carbon based
willows, whereas acetysalicortin is found in
phenolics and terpenes, and nitrogen
only a few species (Julkunen-Tiitto, 1989).
containing compounds such as alkaloids
Salicin is also found in other plant families,
(Taiz and Zeiger, 1991). Simple phenolics
e.g. in Rosaceae (Vickery and Vickery,
contain an aromatic ring substituted by one
1981), but higher substituted salicylates
or more hydroxyl groups. More complicated
such as salicortin, acetylsalicortin and
phenolics contain additional functional
tremulacin, are characteristic compounds for
groups such as ester, methyl, acetyl or sugar
the Salicaceae family (Julkunen-Tiitto,
moieties. Some higher molecular weight
1989; Pierpoint, 1994).
phenolics contain several aromatic rings.
Willow bark was traditionally used in
Phenolic compounds are more or less toxic
folk medicine as cure for headache, fever
substances that often appear as glycosides,
and pain by pre-industrial cultures. Leroux
their less toxic forms, in intact plants
isolated salicin in crystalline form as far
(Hösel, 1981). These so-called phenolic
back as 1829, and thus salicin was the first
glycosides are usually stored in vacuoles as
recorded glycoside (ref. Pierpoint, 1994).
water-soluble compounds (Hösel, 1981).
The discovery of salicin eventually led to
The rupture of cells, e.g. by herbivore
the invention of its synthetic derivative,
attack, releases the glycosides from their
acetyl salicylic acid (aspirin) by Hoffman
storage site, exposing them to degrading
and Dreser, in 1897 (ref. Pierpoint, 1994).
enzymes (Hösel, 1981). The degradation of
Even nowadays, aspirin and its derivatives
defensive compounds often activates these
have maintained their status in modern
compounds by releasing toxic, deterrent or
medicine, used for their analgesic, anti-
nucleophilic, nutritive-value-lowering
inflammatory and anti-pyretic properties
moieties. For example, cyanogenic
(Pierpoint, 1994).
7
 The interaction between salicylates and
herbivores has been studied widely by many
research groups all over the world.
Salicylates are known to deter the feeding of
a great variety of generalist herbivores
including insect (Tahvanainen et al., 1985b;
Lindroth et al., 1988; Lindroth and Peterson,
1988; Lindroth and Hemming, 1990;
Clausen et al., 1989; Denno et al., 1990) and
mammal herbivores (Bryant et al., 1983,
1992; Palo, 1984; Smiley et al., 1985;
Tahvanainen et al., 1985a; Bryant, 1987;
Reichardt et al., 1990; Pass and Foley,
2000), whereas many specialist insect
herbivores are attracted by salicylates.
Salicylates may act as feeding (Matsuda and
Matsuo, 1985; Smiley et al., 1985;
Tahvanainen et al., 1985b; Rowell-Rahier
and Pasteels, 1986; Denno et al., 1990;
Rank, 1992; Kolehmainen et al., 1995) or
oviposition cues (Denno et al., 1990;
Kolehmainen et al., 1994) for such specialist
herbivores. Some of these specialists use
salicylates for their own defense against
their enemies (Pasteels et al., 1983; Smiley
et al., 1985; Rowell-Rahier and Pasteels,
1986; Denno et al., 1990; Rank 1992; Rank
et al., 1998). On the other hand, some of the
predators have evolved so that they are able
to use salicylates to find their prey (Rank
and Smiley, 1994; Rank et al., 1996).
In spite of the apparent benefits of
chemical defense for plants, the production
and maintenance of these chemicals demand
resources that are then not available for the
growth and reproduction of plants. Chemical
defense may therefore lower the competitive
ability of plants, leading to a trade-off
between growth and defense (Herms and
Mattson, 1992). In other words, the plants
have a dilemma: to grow or to defend
themselves (Herms and Mattson, 1992). The
metabolic costs are thought to provide one
explanation for the fact that all plants are not
maximally defended but there exists
considerable variation in defenses of plants
(Agrawal, 1999).
The metabolic cost of chemical defense
depends on the structure of the defensive
compound in question, the concentration of
the compound present in the plant tissues
and the stability of the compound
8
(Gershenzon, 1994). The length of the
biosynthetic pathway also influences the
cost of a compound, since the synthesis and
maintenance of the enzymes of the pathway
demand resources (Gershenzon, 1994). The
metabolic turnover of compounds may
greatly increase the costs of defense
(Fagerström, 1989; Skogsmyr and
Fagerström, 1992; Gershenzon, 1994).
Phenolic compounds were earlier
regarded as quantitative defenses that are
present at constantly high levels in plant
tissues (Feeny, 1976). More recent studies
have shown that certain phenolics may be
increased by herbivore attack or mechanical
wounding (Ke and Saltveit, 1987; Pullin,
1987; Clausen et al., 1989; Hartley and
Lawton, 1991; Brignolas et al., 1995;
Constabel, 1999). This so-called induced
defense is said to be more economical for
plants than constitutive defense, since in the
induced defense protective compounds are
synthesized as a response to herbivore attack
and the compounds need not to be
maintained at a constant, effective level as
in the constitutive defense (Herms and
Mattson, 1992; Baldwin, 1994; Gershenzon,
1994).
Generally, carbon-based phenolic
compounds are regarded as cheaper defenses
than nitrogen containing compounds, since
nitrogen is critical and often in short supply
for the growth of plants (Bryant et al.,
1983). However, the ammonium ion of Phe,
a precursor of a variety of carbon-based
compounds, is recycled (Van Heerden et al.,
1996; Razal et al., 1996), and thus the
synthesis of carbon-based compounds, such
as pheylpropanoids and salicylates, does not
need nitrogen directly. On the other hand,
salicylates, being fairly low molecular
weight phenolic glycosides, are assumed to
be mobile defenses that are subject to
metabolic turnover (Reichardt et al., 1991).
Furthermore, the concentration of salicylates
can be very high, and thus the synthesis and
maintenance costs of a salicylate-based
defense may appear to be substantial.
The main focus of my study was to
investigate the metabolism of salicylates:
the biosynthesis, degradation, metabolic
turnover and induction of salicylates, and
thus to evaluate the potential costs of a
salicylate-dependent defense. Another focus
of the investigation was the trade-off
between the synthesis of defenses and
growth of plants, a consequence of
metabolic costs of defense. Further objects
of the study were the effects of salicylates
on the feeding and growth of larvae of a
generalist moth and on the feeding choice of
adults of a specialist leaf-beetle. The
specific aims of the study were:
3. To study a potential trade-off
between the synthesis of salicylates
and the growth of plants (I).
4. To study the effects of salicylates and
their degradation products on the
feeding of a generalist herbivore (IV).
5. To study the effects of induction of
salicylates on the subsequent feeding
of a specialist leaf-beetle, Phratora
vitellinae L. (Coleoptera:
Chrysomelidae) (III).

1. To investigate biosynthesis of salicin 2. MATERIALS AND METHODS

and higher substituted salicylates (I)
and the inducibility of salicylates by 2.1. Study organisms
herbivore attack (III). 2.1.1. Willows
2. To study the metabolic turnover of
salicylates in intact plants (I, II) and I used four willow species, Salix
the degradation of salicylates in vivo myrsinifolia, Salix pentandra, Salix
and in vitro (IV,V). phylicifolia and Salix repens in my study
(Table 1).

Table 1. Willow and herbivore species used in different studies.

STUDIES
SPECIES
I II III IV V
Salix Leaves collected
phylicifolia in nature, 11
individuals
Salix In vitro –plants, Shoot sprouts Leaves collected Greenhouse, 1
myrsinifolia clone V8 grown in in nature, 10 clone, leaves; in
-leaves, stems greenhouse, 6 individuals vitro –plants,
and shoot-tips clones clone V6, leaves
-leaves and buds
Salix pentandra In vitro –plants, Leaves collected Greenhouse,
clone RM1 in nature, 10 clone RM7,
-leaves, stems, individuals leaves; in vitro –
shoot-tips and plants, clone
roots RM1, leaves
Salix repens Leaves collected
in nature, 1
individual
Operophtera Laboratory
brumata stock of larvae,
(Lepidoptera: 4th instars
Geometridae)
-generalist
Phratora Adult beetles
vitellinae collected in
(Coleoptera: nature
Chrysomelidae)
-specialist

9
 S. myrsinifolia, S. pentandra and S.
phylicifolia are common willows throughout
Scandinavia (Hämet-Ahti et al., 1998;
Skrotsov, 1999). These willows grow in
moist and nutrient rich habitats such as old
fields, banks of ditches, rivers and lakes. S.
repens is morphologically and ecologically
very distinct from the three other species. S.
repens is a low, creeping shrub whereas the
three other species are usually tall shrubs or
small trees (Hämet-Ahti et al., 1998;
Skrotsov, 1999). Moreover, S. repens grows
in nutrient poor environments such as sandy
and peaty damp meadows, sand dunes,
marshes and the edges of wetlands (Hämet-
Ahti et al., 1998; Skrotsov, 1999). S.
myrsinifolia, S. phylicifolia and S. repens
belong to the subgenus Vetrix of Salix
(Skrotsov, 1999).
S. phylicifolia and S. myrsinifolia often
grow in mixed stands, but S. myrsinifolia is
normally the less abundant species (Sipura,
2000). The appearance of these two willows
is rather similar, but the chemical
composition of the leaves and consequently
the composition of herbivore fauna is very
distinct.
The very high level of non-acyled
salicylates characterizes the chemistry of the
leaves of S. myrsinifolia (Julkunen-Tiitto,
1989; Rank et al., 1998). The majority of
generalist herbivores normally rejects these
bitter-tasting leaves (Sipura 1999, 2000).
The leaves of S. phylicifolia, on the other
hand, contain a very low level or are totally
lacking of salicylates but are rich in
flavonoids (Rank, 1998; Sipura et al., 2001).
The leaves of this mild-tasting willow are
palatable food for a variety of generalist
herbivores, and in some places S.
phylicifolia populations suffer from heavy
defoliation (Sipura, 1999, 2000).
Salix pentandra belongs to the subgenus
Salix (Skrotsov, 1999), and the chemistry of
the leaves of this “old” species are
characterized by a considerable level of
acetylated salicylates, (Julkunen-Tiitto,
1989; Julkunen-Tiitto et al., 1995; Rank et
al., 1998). Both S. myrsinifolia and S.
pentandra are favored hosts of specialized
insect herbivores such as the leaf beetle
Phratora vitellinae (Kolehmainen et al.,
10
1995; Rank et al., 1998) and the shoot-
galling sawfly Euura amerinae
(Kolehmainen et al., 1994).
2.1.2. Insects
I used two herbivore species in my study, a
generalist moth Operophtera brumata L.
(Lepidoptera: Geometridae) and a specialist
leaf-beetle Phratora vitellinae L.
(Coleoptera: Chrysomelidae) (Table 1). P.
vitellinae is a highly specialized leaf-beetle
whose larvae use host-plant derived
salicylates for their own defense against
predators (Pasteels et al., 1983). The larvae
convert salicylates into a volatile, strong
smelling salicylaldehyde, which is repellent
to many of the natural enemies of P.
vitellinae larvae (Pasteels et al., 1983;
Denno et al., 1990; Rank et al., 1998)
apparently providing an enemy-free space
for these larvae (Denno et al., 1990).
On the other hand, the higher survival
rate of larvae on salicylate-rich willows
might rather be a consequence of the higher
growth rate of larvae, as larvae are exposed
for a shorter time to predation, than a
consequence of the effective repellence of
natural enemies (Rank et al., 1989). Larvae
of P. vitellinae are able to use a glucose
moiety of salicylates, cleavaged by β-
glucosidase activity of the exertile glands,
for their growth (Pasteels et al., 1983;
Rowell-Rahier and Pasteels, 1986), and thus
a salicylate-containing diet may increase the
growth rate of the larvae (Rowell-Rahier
and Pasteels, 1986; Rank et al., 1998).
In addition, the adult beetles prefer to
feed on salicylate rich species (Rowell-
Rahier, 1984ab; Tahvanainen et al., 1985b;
Rank et al., 1989), and the oviposition of
females is also stimulated by salicylates
(Denno et al., 1990).
Operophtera brumata is a polyphagous,
spring-feeding defoliator of deciduous
shrubs and trees that can use a great variety
of plant species as hosts, including willows
and poplars. However, Querqus robur L.
and Prunus padus L. are its main hosts in
Southern Finland (Tikkanen, 2000). The
larvae of O. brumata hatch early in the
spring at the time of leaf-flush of the host
plants. The timing of hatching is very
critical for these larvae since, if the hatching
occurs too early, there is no food for the
larvae, and if they hatch too late, the quality
of the leaves is inferior (Tikkanen and
Lyytikäinen-Saarenmaa, 2001).
Females do not actively select the species
where they lay their eggs (Tikkanen, 2000),
but neonate larvae are able to disperse by
“ballooning” with a silk thread on air
currents (Holliday, 1977). O. brumata has to
acquire all the resources for reproduction
during the larval stage. The pupal weight
correlates directly with the fecundity of
females (Holliday, 1977; Wint, 1983). The
winter moth, on the other hand, can use
numerous plant species as hosts, and there is
great variability in larval growth between
host species (Kirsten and Topp, 1991;
Tikkanen et al., 1999). Thus, the quality of
the host plants on which the larvae settle
influences the subsequent fecundity of
females.
2.2. Experimental procedures
2.2.1. Turnover and biosynthesis studies (I,
II)
In the turnover study (II), S. myrsinifolia
(clone V8) plantlets were micropropagated
by using axillary shoot multiplication
(Julkunen-Tiitto, 1996). The plantlets were
cultivated on a solidified Murashige-Skoog
(MS) medium (Murashige and Skoog, 1962)
with 3% sucrose content, and no hormones
were added. For the biosynthesis study (I),
S. pentandra was chosen, since it contained
both acyled and non-acyled salicylates.
Seeds were collected from the adult S.
pentandra trees growing in the vicinity of
Joensuu, Eastern Finland. The seeds were
sterilized and germinated in sterile
conditions. Small seedlings were
micropropagated by using axillary shoot
multiplication, one seedling corresponding
to one clone. Plantlets were cultivated on a
solidified Woody-Plant (WP) medium
(Lloyd and McCown, 1980) with 3%
sucrose content and without hormones.
A powerful and specific inhibitor of
phenylalanine ammonia lyase (PAL; EC
11
4.3.1.5.), 2-aminoindan-2-phosphonic acid
(AIP) (Zón and Amrhein, 1992) was used
for inhibiting the biosynthesis of salicylates
(I, II). In the turnover study (II), the six-
week old plantlets were transferred into
culture vessels on cotton pads moistened
with liquid MS-medium. To the half of the
vessels, 30 µM AIP was added. The
concentration of salicylates and aromatic
amino acids was monitored during the 10-
day experiment.
In the biosynthesis study (I), three-
weeks-old S. pentandra plantlets were
transferred into a liquid WP-medium
containing 30 µM AIP. Presumable
precursors of salicylates, benzoic acid (BA),
salicylic acid (SA), helicin and salicin, were
applied to the vessels at the final
concentration of 2.5 mM, and the plantlets
were cultivated for three weeks in the
presence of these precursors. Two kinds of
controls were used in this study: +AIP and –
AIP control plants.
2.2.2. Induction study (III)
In the induction study (III), shoot-sprouts of
six clones of S. myrsinifolia were used.
Leafless shoot-cuttings were potted in pre-
fertilized peat and the shoot-sprouts were
over-wintered in the field. Next spring,
before leaf-flush, the plants were taken into
a growth chamber, and the surface layer of
peat was replaced with new peat fertilized
with chicken manure.
Twenty six-week-old individuals of each
clone were used in the experiment. The
mature leaves of the main shoots were
enclosed in a gauze-bag (Figure 1). Then, 9–
16 adult leaf-beetles (Phratora vitellinae)
per bag were enclosed in half of the bags,
and the beetles were allowed to eat for three
days. The sampling was conducted in blocks
through three consecutive days, one week
after the injury (Figure 1).
2.2.3. Operophtera brumata study (IV)
The leaves of two-year-old S. myrsinifolia,
S. pentandra and S. phylicifolia plants were
collected from Siikalahti, in Parikkala,
eastern Finland (IV). A leaf was split in two

1 reared on the basic diet for 12 hours, after
2 which they were weighed and transferred for
3
4 a
5 b
B.
Figure 1. Experimental procedure and sampling of
the induction study. Woolen yarn (a) was tied
carefully under the present shoot tip (1). Mature
leaves of the main shoot were enclosed in a gauze
bag (b). 9-16 adults of P. vitellinae were added to the
half of the bags. After herbivore treatment, the plants
were allowed to grow for one week. Five separate
samples were taken: A new shoot-tip with two to
three upper leaves (1), a young immature leaf just
above of the woolen yarn (2), a young leaf just under
the woolen yarn (3), a second, young mature leaf
under the woolen yarn (4) and a collected sample of
older mature leaves from the main shoot (5).
halves and one half was offered to larvae of
O. brumata while the other was preserved
for analysis. The larvae of laboratory stock
were raised on the leaves of Prunus padus
until they reached the 4th instar. The larvae
were reared on the leaf-halves of
experimental plants for 36 hours at 16°C
with an18:6 h light:dark cycle.
In an artificial diet experiment, the 4th
instars of O. brumata were reared on a diet
containing four different concentrations of
chlorogenic acid and the degradation
products of salicylates, catechol, saligenin
and salicin, in equimolar ratios with the
concentrations found in the “real leaf”
experiment. The diet was based on the
recipe of Goujet and Guilbort (Singh, 1985),
12
with some modifications. The larvae were
different treatments. The larvae were reared
for 24 hours at 16°C and were weighed
again.
2.2.4. Degradation study (V)
Dry leaves of S. myrsinifolia (clone V8), S.
pentandra (clone RM7) and S. repens
(collected in nature from one individual)
were extracted in 100% methanol. The
samples were dissolved in 0.1 M potassium
buffer (Sambrook et al., 1989) at pH 5.5, pH
7.0, pH 9.0 or 50% MeOH. The samples
were allowed to stabilize for a few hours at
+4°C prior to HPLC analysis. Authentic
samples of salicortin and 6-HCH (Julkunen-
Tiitto and Meier, 1992b) were treated and
analyzed similarly.
For enzymatic degradation, fresh leaves
of in vitro cultured willows, S. myrsinifolia
(clone V6), S. pentandra (clone RM 1) and
S. repens (clone H251) were cut and
weighed. The leaves were homogenized in
0.1 M potassium buffer. The supernatants
were incubated for 15 min at +37°C, after
which the samples were centrifuged. Half of
the supernatant was dissolved to 100%
MeOH and analyzed by HPLC. The
salicylate content of intact plants was
established by small volume extraction
followed by HPLC analysis.
2.3. Methods of analysis
All plant material and also larvae frass were
extracted in 100% methanol by small (III,
V) or large volume extraction (I-V). Fresh
plant material was placed in methanol
immediately after the preparation of the
plants. Both dried and fresh samples were
incubated for 15 min in methanol in an ice
bath prior to clipping with a homogenizer
(Janke and Kunkel, IKA Labortechnik). The
extractions were repeated three times, after
which the samples were dried in a vacuum
evaporator and/or with gaseous nitrogen.
The dried samples were stored at -20°C
before analysis.
 The samples were analyzed by high
performance liquid chromatography, HPLC
(Hewlett-Packard, Palo Alto). HPLC device
consisted of quaternary solvent delivery and
an autosampler system. A HP Hypersil ODS
II column was used for separating the
compounds. A photodiode array detector
coupled with a data system/personal
computer recorded the UV-Vis spectra of
compounds.
Compounds were identified by
comparing their retention times and spectra
with those of reference compounds. The
response factor of salicin was used for
quantification of diglucoside of salicin and
acetylsalicin, the response factor salicortin
for the salicortin derivatives, the response
factor of 2’-O-acetylsalicortin for
acetylsalicortin derivatives and the response
factor of tremulacin for acetyltremulacin.
The unknown salicylates were tentatively
identified by HPLC/API-ES mass
spectrophotometry (HP 1100 Series
LC/MSD, Hewlett-Packard, Palo Alto) (I,
III, IV, V). HP Hypersil ODS columns were
used for separating the substances. The
fragmentation voltages used were 60-200 V,
depending on the substance. An authentic 6-
HCH sample was checked by mass selective
gas chromatography (GC/MS; JEOL D300)
(V).
To confirm the identification of benzoyl-
and SA-glucoside, the root samples of BA
and SA treated plants were hydrolyzed with
almond β-glucosidase (I). In the reaction
mixture (0.1 M KH2 PO4), 5 U of enzyme (7
U/mg protein, Sigma) per 0.1 µmol
glucoside was used. The incubation time
was 15 min at +37°C, and the products of
hydrolysis were analyzed by HPLC.

Table 2. Salicylates found in four willow species in five different studies. These results consist of the data from in
vitro (I, II, V) and in vivo (III, IV, V) plants. S. phylicifolia: in vitro n=0, in vivo n=12; S. myrsinifolia; in vitro
n=2, in vivo n=16, S. pentandra; in vitro n=2, in vivo n=12, S. repens; in vitro n=1, in vivo n=1. N refers to number
of clones (laboratory/greenhouse studies) or individuals (field studies).
WILLOW SPECIES

SALICYLATES S. phylicifolia S. myrsinifolia S. repens S. pentandra

Type of Compound In vitro In vivo In vitro In vivo In vitro In vivo In vitro In vivo
compound
Salicin — 0 X X X X X X
Non- Diglucoside — 0 X X 0 X X X
acyled of salicin
glucose Salicortin — 0 X X X X X X
moiety Disalicortin — 0 X X 0 0 0 0
Salicortin-2 — 0 0 X X X X X
Salicortin-3 — 0 0 X 0 X 0 0
2’-O-acetyl- — 0 0 0 0 0 X X
Acetylated salicin
glucose 2’-O-acetyl- — 0 0 0 0 0 X X
moiety salicortin
Diacetyl- — 0 0 0 0 0 X X
salicortin
Acetylcyclo- — 0 0 0 0 0 X 0
hexenone
salicortin
Benzoy- Tremuloidin — 0 0 0 X X X X*
lated Tremulacin — 0 0 0 X X X X*
glucose
moiety
Acetylated Acetyl- — 0 0 0 0 0 0 X*
+ benzoy- tremulacin
lated
glucose
moiety
— = not studied, 0 = not detected, X = detected, * = detected in one genotype.

13
 The carbon and nitrogen contents of leaf
and frass samples of the O. brumata study
(IV) were analyzed by a CHN-analyzer
(Elemental Microanalysis – Mod. 1106,
Carbo Erba Stumentazione). Acetanilide
OAS (Elemental Microanalysis Limited)
was used as a standard (C= 71.09%;
H=6.71%; O=11.84%). The levels of
condensed tannins were determined from
methanol extracts by means of the butanol-
HCl test (Hagerman, 1995).
3. RESULTS AND DISCUSSION
3.1. Sources of variation in willow
phenolic chemistry
3.1.1. Chemistry of willow species
S. myrsinifolia, S. pentandra and S. repens
all contained three common salicylates;
salicin, salicortin and diglucoside of salicin
(I-V; Table 2). S. phylicifolia was totally
devoid of salicylates (Table 2), but its leaves
were very rich in ampelopsin and its
derivatives (IV). The salicylate pool of S.
myrsinifolia (II-V) and S. repens (V) were
quite similar (Table 2). Salicortin was the
main salicylate in both species and novel
salicortin derivatives, salicortin-2 and –3
were found in the leaves of both species.
Salicortin derivative-2 had a molecular ion
peak at m/z 871 [M+Na]+, and it has been
suggested that it consists of two salicortin
moieties (Julkunen-Tiitto and Sorsa, 2001).
Salicortin-3 had the same molecular weight
than salicortin-2, and it was considered to be
an isomer of salicortin-2 (III).
Tremulacin and tremuloidin were found
in the leaves of S. repens (V), but not in
those of S. myrsinifolia (II-V). S.
myrsinifolia leaves contained another novel
salicortin derivative: disalicortin (III, IV).
The mass spectra of the compound has a
putative molecular ion peak at m/z 585
[M+Na]+, and it has been suggested that it
consists of two 1-hydroxy-6-oxo-2-
cyclohexen-1-carbonyl moieties attached to
a backbone of salicin (Tegelberg and
Julkunen-Tiitto, 2001). The compound that
was identified as 2’-O-acetylsalicortin in the
turnover study (II) was re-identified by MS-
14
HPLC as a disalicortin not as an
acetylsalicortin (III, IV,V).
The diversity of salicylates was greatest
in the leaves of “old willow”, S. pentandra
(I, IV,V; Table 2). The main salicylate of S.
pentandra was 2’-O-acetylsalicortin, and 2’-
O-acetylsalicin was also detected. The S.
pentandra clones (RM1 and RM7) used in
the biosynthesis and degradation studies
were unique, containing tremulacin (I, V),
and a novel tremulacin derivative,
acetyltremulacin was found in the clone
RM7 (V; Figure 2). Neither of these
compounds have been detected in S.
pentandra in earlier studies (Julkunen-
Tiitto, 1989; Julkunen-Tiitto et al., 1995;
Rank et al., 1998).
Acetyltremulacin was identified
according to the molecular ion peak (at m/z
593 [M+Na]+) and the fragmentation pattern
of the compound (V). In addition, two novel
derivatives of acetylsalicortin were found.
These compounds had putative molecular
ion peaks at m/z 531 [M+Na]+ and m/z 627
[M+Na]+. The first compound was identified
by its molecular ion and fragmentation
pattern as diacetylsalicortin containing two
acetyl groups attached to the glucose moiety
(I). The acetyl groups were assumed to be in
positions 2’ and 6’, so the compound would
then be 2’,6’-O-diacetylsalicortin (I, Figure
2).
The second compound was identified by
its molecular ion and fragmentation pattern
as acetylhydroxycyclohexenone salicortin
having both an acetyl group and
a 1-hydroxy-6-oxo-2-cyclohexen-1-carbonyl
group attached to the glucose moiety (I,
Figure 2). In the degradation study, two
isomers of 2’-O-acetylsalicin, 2’-O-
acetylsalicortin and diacetylsalicortin were
detected (V). The degradation products of
diacetylsalicortins, diacetylsalicins, were
also found (V). Diacetylsalicins have a
putative molecular ion peak at m/z 393
[M+Na]+.
The main phenolic compounds of
micropropagated S. pentandra (I, V), S.
myrsinifolia (II, V) and S. repens (V) were
salicylates. These in vitro -plantlets
contained only minor amounts of other
Figure 2. The putative structures of new compounds, 2’,6’-O-diacetylsalicortin, acetylhydroxycyclohexenone
salicortin and acetyltremulacin, found in Salix pentandra. The compounds were identified by MS-HPLC according
to molecular ion peaks and fragmentation patterns.
compounds, especially flavonoids (data not
shown). This is obviously due to the
artificial light, which is devoid of UV-
radiation. UV-radiation is known to promote
the synthesis of flavonoids in many plants,
also in S. myrsinifolia (Tegelberg and
Julkunen-Tiitto, 2001). Surprisingly, the
leaves of S. myrsinifolia collected in nature
contained low levels of flavonoids;
quercetin (about 4 mg/g DW) and luteolin
(about 2 mg/g DW) derivatives (IV). The
leaves contained also cinnamic acid
derivatives and moderate levels of
chlorogenic acids (about 50 mg/g DW), but
only small amounts of condensed tannins
(under 1 mg/g DW).
S. phylicifolia leaves collected in nature
contained minor levels of other flavonoids
beside ampelopsin; myricetin, luteolin and
apigenin derivatives and small amounts of
(+)-catechin and chlorogenic acids (IV). In
addition, the level of condensed tannins was
low in these young leaves of S. phylicifolia.
The leaves of S. pentandra collected in
nature contained moderate levels of
15
chlorogenic acids and flavonoids, mainly
quercetin and myricetin derivatives, as well
as some cinnamic acid derivatives (IV).
Micropropagated S. pentandra plantlets
contained negligible amounts of condensed
tannins (I), whereas the leaves collected in
nature contained more considerable levels of
tannins (IV).
Acetylated and benzoylated salicylates
are not generally found in the same willow
species (see e.g. Julkunen-Tiitto, 1989),
suggesting that these two pathways compete
for a common precursor, or that the willow
species have evolved along separate
phylogenetic lines: willows contain an
enzyme that either acetylates or benzoylates
the glucose moiety. However, S. pentandra,
which is one of the oldest willow species
(Skrotsov, 1999), contained both acetylated
and benzoylated salicylates, which proves
that metabolic pathways leading to non-
acyled, acetylated or benzoylated salicylates
can be found in the same species. The
pathways leading to certain types of
salicylates might be inactivated during the
evolution of younger willow species.
3.1.2. Within-plant variation
Salicylate concentration has been shown to
vary among individual plants (e.g. Julkunen-
Tiitto, 1989; Julkunen-Tiitto and Meier,
1992a; Hakulinen et al., 1995, 1999; III) and
within plants, depending on the plant part
(Julkunen-Tiitto, 1989; Julkunen-Tiitto and
Meier, 1992a; I-III) and the age of the plant
part (Julkunen-Tiitto, 1989; III). In addition,
some seasonal variation also occurs in the
salicylate contents (Thieme, 1965b;
Julkunen-Tiitto, unpublished results).
However, salicylates are not withdrawn
from the abscising leaves in autumn time
(Julkunen-Tiitto, 1989). The condensed
tannins of S. pentandra and S. myrsinifolia
increase during the ontogeny of the plants,
while simultaneously the level of salicylates
decreases (Julkunen-Tiitto, unpublished
results).
In my studies, the highest concentration
of salicylates was found in the shoot-tips
and young leaves of S. myrsinifolia and S.
pentandra and the lowest in the mature
stems (I, II). Also in an earlier study, similar
results were obtained with these species
(Julkunen-Tiitto, 1989). In Populus
deltoides (Salicaceae), the total level of
phenolics was three times higher in the
youngest leaf than in the oldest leaf, and
subsequently the growth of generalist gypsy
moth larvae was 85% lower on the youngest
leaf than on the oldest one (Meyer and
Montgomery, 1987). However, contrary
results are also reported. For example,
Denno et al. (1990) showed that mature
leaves of S. dasyclados and S. fragilis
contain higher levels of salicylates than
young ones.
The synthesis of secondary compounds is
restricted to a definite developmental stage
of a plant or plant organ (Wierman, 1981;
Matsuki, 1996). According to Matsuki
(1996), the synthesis of phenolics occurs
after the exponential growth phase, at the
stationary phase of leaf development, before
leaf maturation, whereas the protein-
competing model (PCM) predicts that
16
phenolics are synthesized at the bud and
unfurling stages of leaf development and
when the leaf is hardening off, but not
during the expansion phase of a leaf (Jones
and Hartely, 1999). The growth-
differentiation-balance hypothesis (GDB)
predicts that the synthesis of phenolics
occurs mainly at the differentiation phase of
cells, after the cells have developed
vacuoles. However, synthesis may also
occur during the growth phase of cells,
when secondary compounds are stored in
small vesicles that later fuse to developed
vacuoles (Herms and Mattson, 1992). In S.
myrsinifolia and also in S. pentandra, the
synthesis of salicylates is apparently
restricted to the bud and unfurling stages of
leaf development (I-III), but salicylates are
also synthesized in the expansion phase in
response to herbivory exposure (III).
3.1.3. Systemic induction and clonal
variation in salicylates
Induced resistance refers to changes in plant
quality that reduce herbivory on plant
foliage. Induced resistance may be due to an
increased concentration of defensive
phytochemicals or proteinous defenses such
as proteinase inhibitors, peroxidases and
polyphenol oxidases (Constabel, 1999).
Induction may be systemic, appearing in the
unwounded tissues as distinct from the
wounded site, or induction might occur
locally, in the vicinity of the injury
(Constabel, 1999).
Long-term induction leads to consistent
changes in plant quality and extends one or
more years after severe defoliation (Hartley
and Lawton, 1991). In short-term induction,
the changes in plant quality occur
immediately or several days after the
herbivore injury (Clausen et al., 1991).
Chewing of leaves by insect herbivores
usually causes a stronger response in plants
than mechanical wounding (Baldwin, 1990;
Korth and Dixon, 1997). Insect regurgitants
apparently contain factors that mediate the
induced response in plants (Korth and
Dixon, 1997; Paré and Tumlinson, 1997).
Systemic response is more costly than a
localized one, but may benefit plants more
than the localized response (Zangerl, 1999).
The wounding of older mature leaves of S.
myrsinifolia plants by a specialist leaf
beetle, Phratora vitellinae, led to a systemic
induction of salicylates in young,
unwounded leaves (III). No local response
was detected in older wounded leaves.
The wounding also caused an
accumulation of aromatic amino acids, Phe,
Tyr and Trp, in the young unwounded
leaves, but led to their decline in the
wounded leaves, suggesting that enzymes of
the shikimate pathway were activated in
young leaves but deactivated in older
wounded leaves. The first enzyme of the
shikimate pathway, 3-deoxy-D-arabinose-
heptulosonate-7-phosphate synthase
(DAPH-synthase; EC 4.1.2.15), as well as
PAL are induced by wounding (Ke and
Saltveit, 1987; Dyer et al., 1989; Hartley
and Lawton, 1991). The induction of these
enzymes is tightly co-regulated (Dyer et al.,
1989).
The increased levels of aromatic amino
acids were obviously a consequence of the
activation of DAPH-synthase and the
increased level of salicylates due to
subsequent induction of PAL. Jasmonic acid
(JA) and its derivatives are signal molecules
that participate in wound responses in plants
(Bennet and Wallsgrove, 1994). JA induces
many defensive pathways and enzymes,
including PAL (Gundlach et al., 1992) and
oxidative enzymes such as PPO (Constabel
et al., 1995). It is expected that induced
defense, also in S. myrsinifolia, was JA-
mediated.
Induction was dependent on the type of
compound: the levels of the most abundant
salicylates, salicortin and disalicortin,
increased, whereas other salicylates did not
respond to the injury (III). There was also
great variability in responses between
individual clones; the clones having the
highest level of salicylates were also the
most capable of increasing this level. In
other words, no trade-off between constant
and induced levels was found. Similar
results were obtained by Havill and Raffa
(1999) who reported marked variation in
both constitutive and inducible resistance in
Populus clones. They did not find a trade-
17
off between constitutive and inducible
resistance either. These results are
inconsistent with the hypothesis that plants
with high levels of resistance show low
levels of inducible resistance (Herms and
Mattson, 1992).
In earlier studies, slight wounding caused
a short-term local induction of salicylates in
S. myrsinifolia (Julkunen-Tiitto et al., 1995)
and in Populus tremuloides (Clausen et al.,
1989). On the other hand, heavy defoliation
of quaking aspen caused long-term
induction in the salicylate levels of new
flush foliage (Clausen et al., 1991). There
was also great variability in the responses
among Populus clones (Clausen et al.,
1989).
Many internal and external factors may
influence the induced responses of plants.
First, the developmental stage of a plant or
plant part may affect the response (Coleman
and Jones, 1991; Baldwin, 1994; Constabel,
1999). The youngest leaves are very strong
sinks, and they should be more rapidly
induced by the systemically mobile wound
signal than older leaves (Constabel, 2000).
Secondly, the severity of injury affects the
response, and there may be genotypic
variation between individuals at threshold
levels of damage before the plants exhibit an
induced response (Coleman and Jones,
1991). Finally, the internal and external
resources of plants may affect the response
(Coleman and Jones, 1991; Baldwin, 1994;
Honkanen, 1995).
Environmental factors are shown to
strongly affect the levels of many secondary
compounds and also the levels of salicylates
(e.g. Larsson et al., 1986; Julkunen-Tiitto
and Meier, 1992a; Lindroth et al., 1993;
Hakulinen et al., 1995; Hakulinen, 1998;
McDonald et al., 1999). In earlier studies,
nitrogen fertilization decreased the salicylate
concentration of S. myrsinifolia (Hakulinen
et al., 1995), although high clonal variation
was detected both in growth and in
salicylate concentrations (Hakulinen et al.,
1995). In the induction study (III), the
constitutive levels of salicylates of S.
myrsinifolia were very high (as much as
over 10% of DW in some clones),
apparently as a result of excess light supply
and slight fertilization following the
predictions of the carbon/nutrient balance
(CNB) hypothesis (Bryant et al., 1983). This
hypothesis predicts that in nutrient limited
conditions growth is more restricted than
photosynthesis, and overflow carbon is used
for the production of carbon-based
compounds. Nitrogen fertilization increases
the growth of plants, and concomitantly the
concentrations of carbon based secondary
compounds are assumed to decrease, since
carbon is thought to be used preferentially
for growth (Bryant et al., 1983).
In spite of similar light conditions and
fertilization, there was high clonal variation
in the constitutive and induced levels of
salicylates in the leaves of S. myrsinifolia
and the effect of the clone on the salicylate
levels was stronger than the effect of
induction (III). Also, the salicylate
concentration of Salix sericea (Nichols-
Orians et al., 1993) and Populus tremuloides
(Hwang and Lindroth, 1997) varied
extensively between clones. These results
show that environmental factors strongly
affect levels of salicylates, while the
genotype of the plant dictates the limits
within which the variation occurs and how
the plant responds to external stimuli.
3.2. Metabolism of salicylates
3.2.1. Inhibition of the biosynthesis of
salicylates by 2-aminoindan-2-phosphonic
acid
Aromatic amino acids of plants, bacteria and
fungi are synthesized via the shikimate
pathway. For animals, Phe and Trp are
essential amino acids that have to be
acquired from food, while Tyr is synthesized
directly from Phe (Herrmann, 1995).
DAPH-synthase is the first enzyme of the
shikimate pathway, controlling the carbon
flow into the shikimate pathway (Herrman,
1995).
PAL catalyzes the first reaction in plant
phenylpropanoid metabolism, an elimination
of ammonia from Phe forming t-cinnamic
acid (Jones, 1984; Strack, 1997). The PAL
of many monocotyledons and fungi also
shows tyrosine ammonia-lyase (TAL)
18
activity (Jones, 1984). TAL catalyzes the
deamination of Tyr producing p-coumaric
acid, a precursor of phenylpropanoids
(Strack, 1997).
Salicylates are shown to be derived from
t-cinnamic acid via the shikimate pathway
(Zenk, 1967). 2-Aminoindan-2-phosphonic
acid (AIP) is said to be a specific and
competitive PAL inhibitor (Zón and
Amrhein, 1992) that effectively inhibits the
biosynthesis of Phe-derived phenolics (e.g.
Schmutz et al., 1992; Mösli Waldhauser and
Bauman, 1996; I, II). The new shoot-tips of
S. myrsinifolia and S. pentandra developed
after the transfer of plants to the AIP
medium contained a low level of salicylates
(I, II), indicating efficient inhibition of the
biosynthesis of salicylates by AIP. In S.
myrsinifolia, the inhibition of major
salicylates was 70-80%, when the salicylate
content of shoot-tips of AIP plants was
compared to that of control plants. In S.
pentandra, the respective values were 50-
90% depending on the individual
compound.
A high accumulation of Phe both in S.
myrsinifolia and in S. pentandra confirmed
that PAL was efficiently inhibited by AIP.
In S. myrsinifolia a slight but marked
accumulation of Tyr was detected,
indicating that AIP also inhibited TAL
activity. In earlier studies, another PAL
inhibitor, L-α-aminooxy-β-phenyl propionic
acid (AOPP), caused a marked accumulation
of Phe (Amrhein and Holländer, 1979;
Holländer et al., 1979; Nóe et al., 1980;
Havir, 1981; Amrhein et al., 1983;
Holländer-Czytko and Amrhein, 1983) and
also inhibited the functioning of TAL
(Holländer et al., 1979). The most
unexpected result of my study was the
accumulation of Trp in response to AIP
treatment (II), which has not been reported
in earlier inhibitor studies. These results
suggest that AIP is not a specific inhibitor of
PAL, but that it may also inhibit other
enzymes of the shikimate pathway, such as
TAL.
Accumulating intermediates or end
products may inhibit their own synthesis by
what is called feedback inhibition (Lewin,
1995). For example, t-cinnamic acid is
known to inhibit PAL activity (Jones, 1984).
The down-regulation of PAL by t-cinnamic
acid has been shown to occur via a decrease
in the transcription activity of PAL-
encoding genes (Mavandad et al., 1990).
Marked accumulation of Phe in so many
studies suggests that there is no feedback
control of the synthesis of Phe in plants. In
addition, the accumulation of Tyr and Trp
(II) shows that their synthesis is not
controlled by feedback inhibition either.
DAPH-synthase of Escherichia coli is
regulated by feedback inhibition and by
repression of transcription (Herrmann,
1995). No feedback inhibition of DAPH-
synthase by aromatic amino acids has been
detected in plants (Herrman, 1995). On the
other hand, in Spinacia oleracea, Phe and
Tyr controlled their own synthesis by
feedback inhibition and Trp controlled the
synthesis of all three aromatic amino acids
(Bickel and Schultz, 1978). It was suggested
that the control point is between shikimate
and chorismate; the synthesis was not
controlled via down-regulation of DAPH-
synthase. These deviant results suggest that
feedback inhibition depends on the plant
species in question, but it is obviously not
found in willows.
3.2.2. Biosynthesis of salicylates
The biosynthesis of salicin has been studied
by a few researchers (e.g. Ibrahim and
Towers, 1959; Pridham and Saltmarsh,
1962; Zenk, 1967), and different pathways
and intermediates have been proposed. Zenk
(1967) proposed that the synthesis of salicin
might occur via two pathways in S.
purpurea: 1) t-cinnamic acid – o-coumaric
acid – o-coumaryl-CoA – salicyloyl-CoA –
salicylaldehyde – helicin – salicin. 2) BA –
benzyl alcohol – benzaldehyde –
salicylaldehyde – helicin – salicin.
Surprisingly, the biosynthesis of more
substituted salicylates have remained
unresolved, and no studies have been
published to date. I studied the biosynthesis
of salicin and higher substituted salicylates
by applying putative precursors to plants
cultured in the presence of a PAL inhibitor.
In my preliminary experiments, BA was a
19
more efficient precursor of salicylates than
t-cinnamic acid or o-coumaric acid. The
biosynthesis of the closely related SA, an
important mediator of defense responses of
plants in pathogen infections, has been
shown to proceed via either o-coumaric acid
or BA (El-Basyouni et al., 1964; Ellis and
Amrhein, 1971; Chadha and Brown, 1974),
although the BA-route seems to be the more
important one (Klämbt, 1962; Yalpani et al.,
1993; Léon et al., 1993, 1995; Coquoz et al.,
1998).
BA is hydroxylated to SA by benzoic
acid 2-hydroxylase or BA2H (Léon et al.,
1993, 1995; Yalpani et al., 1993). SA is
often metabolized further e.g. to the
glucoside of SA by SA-UDP-glucosyl
transferase (Yalpani et al., 1990; Eneydi and
Raskin, 1993). On the other hand, Chong et
al. (2001) suggested that BA is not the major
intermediate in the synthesis of SA but that
biosynthesis proceeds via benzoyl-glucose
synthesized either from cinnamoyl-CoA or
from cinnamoyl-glucose.
In my study, BA was glucosylated to
benzoyl-glucose in the roots; no free BA
was detected in either roots or shoots (I).
Benzoyl-glucose was rapidly further
metabolized, since in the shoots only a trace
amount of the compound was detected. At
the same time, the levels of salicylates
increased, suggesting that benzoyl-glucose
was converted to salicylates. In addition, the
roots, which are normally devoid of
salicylates, contained small but detectable
amounts of salicylates deriving from BA
treatment, confirming that benzoyl-glucose
was indeed a precursor of salicylates in S.
pentandra. Furthermore, in my preliminary
experiment, the labeled BA [14C] was
incorporated in salicylates (data not shown).
A compound whose spectrum was close
to that of SA-glucoside was detected in the
BA-fed roots, and it was suggested that it
might be salicyloyl-glucose ester, the
precursor of SA and SA-glucoside (Chong
et al., 2001). SA was converted to SA-
glucoside in the roots, but the further
metabolism of SA-glucoside was slower
than that of benzoyl-glucose, and
concomitantly the increase in the
concentration of salicylates was smaller (I).
 I suggest that the synthesis of salicin
proceeds mainly via benzoyl-glucose and
salicyloyl-glucose, which is possibly further
glucosylated to salicyloyl-diglucose ester
(see Pierpoint, 1994). Salicyloyl-diglucoside
has three putative fates: it might be
converted to SA-glucoside, hydrolysed to
helicin or reduced directly to diglucoside of
salicin, which would be the most intimate
precursor of salicin (Figure 9 in I). SA-
glucoside may act as a precursor of salicin
(see Ibrahim and Towers, 1959; Pridham
and Saltmarsh, 1962), but free SA is not
very likely to be a precursor of salicin in the
endogenous synthesis of salicylates. In
addition, I suggest that neither
salicylaldehyde nor benzaldehyde are
precursors in the endogenous pathway of
salicylates due to their highly unstable and
toxic nature.
Several mechanisms are proposed for the
side-chain shortening of t-cinnamic acid,
which produces BAs. For example, an
oxidative, CoA-dependent pathway that
proceeds via 3-hydroxy-3-phenylpropanoic
acid (Jarvis et al., 2000), a non-oxidative
pathway that proceeds via benzaldehyde
(Schnitzler et al., 1992), or phenylpropanoid
glucose ester serving as activated
intermediates in side-chain shortening (Funk
and Brodelius, 1990). Since most phenolics
appear as their glycosides in plant cells due
the toxicity of aglycones, it is possible that
intermediates of salicylates are also present
as their glycosides.
Salicin and helicin were both partly
converted to isosalicin, a non-natural isomer
of salicin, in the roots. In the shoot tissue,
this isomer was metabolized, probably to
salicin. Exogenously applied salicin failed to
increase the concentrations of salicortin,
acetylsalicortins and tremulacin, although
salicin is chemically the only rational
structure where the esterification of
hydroxycyclohexenone carboxylic acid
might occur (Professor Vainiotalo, personal
communication). However, the precursors of
salicin, helicin, BA and also SA increased
the levels of these more substituted
salicylates. The experiments with cell
suspension cultures failed to produce higher
substituted salicylates by precursor
20
treatments (Dombrowski, 1993; Julkunen-
Tiitto, unpublished results). It seems that the
esterification step needs some activator that
is not present in the non-green tissue such as
cell suspensions or roots, and furthermore,
AIP inhibits this step. However, small
molecular weight phenolics such as BA, SA
and helicin activate this step, or they may
even act as precursors of
hydroxycyclohexenone carboxylic acid.
I suggest that the biosynthesis of more
substituted salicylates proceeds via salicin
esterified to salicortin (Figure 10 in I).
Salicortin for its parts is the most intimate
precursor of tremulacin and
acetylsalicortins. Generally, the synthesis of
a diversity of higher substituted salicylates
needs three basic reactions: esterification of
hydroxycyclohexenone carboxylic acid,
acetylation and/or benzoylation of glucose
moiety.
To conclude, the biosynthesis of
salicylates seems to be complex, and parallel
pathways may exist. In addition,
exogenously supplied precursors are not
necessary intermediates in endogenous
pathways, even if they are metabolized in
plant cells. These reactions can be simple
detoxifying reactions that do not need
specific enzyme activity. For example, cell
suspension cultures glycosylate a variety of
phenolics to non-naturally occurring
glycosides such as isosalicin (Tabata et al.,
1976, 1988; Mitzukami et al., 1983).
2.2.3. Degradation of salicylates
Salicortin and other salicylates that contain a
labile 1-hydroxy-6-oxo-2-cyclohexen-1-
carbonyl moiety are easily degraded to
salicin during the preservation of samples or
extraction procedures (Pearl and Darling,
1970, 1971; Lindroth and Pajutee, 1987;
Julkunen-Tiitto and Tahvanainen, 1989;
Julkunen-Tiitto and Gebhardt, 1992; Orians,
1995; Lindroth and Koss, 1996; Julkunen-
Tiitto and Sorsa, 2001). In addition, the
rupture of leaf compartmentalization, e.g. by
herbivores, leads to a degradation of these
labile salicylates producing 6-hydroxy-2-
cyclohexenone (6-HCH) and salicin
(Clausen et al., 1989, 1991). The released 6-
HCH acts as a feeding deterrent to generalist
herbivores (Clausen et al., 1989, 1991;
Reichardt et al., 1990) and thus may lower
the herbivore damage to foliage.
Salicylates of S. myrsinifolia and S.
pentandra were degraded to salicin and
catechol in the digestive tract of the
generalist larvae, O. brumata, whereas
degradation of salicin further to salicyl
alcohol (saligenin) was rather slow,
indicating low β-glucosidase activity in the
midgut of larvae (IV). In addition, in vitro
degradation of salicylates of S. myrsinifolia,
S. pentandra and S. repens produced salicin,
6-HCH and catechol (V).
The high level of catechol found in the
larvae frass (IV) and also in pH mediated
degradation (V) was apparently produced by
the oxidation of 6-HCH in alkaline pH (see
Clausen et al., 1989, 1991). Catechol is a
substrate of PPO (Catechol oxidase: EC
1.10.3.2) and produced o-quinone (Mayer,
1987; Constabel, 1999), as well as 6-HCH
itself, is an electrophile (Clausen et al.,
1989) that is capable of binding nucleophilic
groups of amino acids and proteins
(Constabel, 1999).
The 1-hydroxy-6-oxo-2-cyclohexen-1-
carbonyl (hydroxycyclohexenone) moiety of
salicortin, acetylsalicortin and tremulacin,
which is released by esterases is thought to
be converted first to respective carboxylic
acid, which is further decarboxylated to
enol, which is in turn isomerized to 6-HCH.
These reactions are claimed to be enzymatic
(Mattes et al., 1987). However, 6-HCH is
also found as a result of degradation of
salicylates in in vitro-conditions (Julkunen-
Tiitto and Meier, 1992b; authors personal
observation), suggesting that this step is not
necessarily enzymatic.
In my study, authentic salicortin was
rather stable in MeOH and in acidic (pH
5.5) and neutral (pH 7.0) conditions, but was
totally degraded to salicin in alkaline
conditions (pH 9.0). Degradation also
produced 6-HCH and catechol, confirming
that production of 6-HCH can also occur
non-enzymatically. In addition, salicortin,
tremulacin and 2’-O-acetylsalicortin of leaf
extracts were degraded in alkaline
conditions to salicin, 6-HCH and catechol.
21
However, the amount of catechol was higher
than that of 6-HCH, thus according with
earlier observations that 6-HCH is converted
to catechol by alkaline pH (Clausen et al.,
1989, 1991; IV).
Salicortin derivatives, disalicortin,
salicortin-2 and –3 of S. myrsinifolia were
more labile than salicortin, already
disappearing at neutral pH (V). 2’-O-
Acetylsalicortin, 2’-O-acetylsalicin and
diacetylsalicortin of S. pentandra were
isomerized at neutral and alkaline pH,
producing two isomers. This was apparently
due to migration of the acetyl group. Also
tremulacin of S. repens leaf extract
isomerized at alkaline pH (V). Pearl and
Darling (1963) observed similar migration
of the benzoyl group of tremuloidin from
position 2’ to position 6’ as a result of mild
alkali or elevated temperature.
Degradation of tremulacin was not
followed by an increase in the level of
tremuloidin, but salicortin was detected at
alkaline pH, apparently as a result of the
degradation of tremulacin.
Degradation of salicylates by foliar
enzymes produced salicin, 6-HCH and
catechol in all willow species (V). The
proportion of 6-HCH was higher than in
non-enzymatic degradation. In both S.
myrsinifolia and S. pentandra, the lowest
level of salicin was detected at pH 5.5, and
diglucoside of salicin was absent in S.
pentandra leaves at this pH. Saligenin was
found only at this pH indicating that the
optimum pH of β-glucosidase was near pH
5.5.
In S. pentandra, marked amount of 2’-O-
acetylsalicin and unknown salicylate were
also found at pH 5.5; at pH 9.0 both
compounds disappeared probably due to
esterase activity (see Julkunen-Tiitto and
Meier, 1992b). 2’-O-Acetylsalicortin and
diacetylsalicortin of S. pentandra degraded
totally at all pH regimes, but tremulacin and
tremuloidin were found at pH 5.5 and 7.0. In
alkaline conditions, both compounds
disappeared, and salicortin appeared,
supporting the idea that salicortin is a
degradation product of tremulacin.
However, tremulacin was degraded to
tremuloidin not to salicortin in earlier
studies (Clausen et al., 1989; Julkunen-
Tiitto and Meier, 1992b; Julkunen-Tiitto and
Sorsa, 2001). These deviant results could be
due to the different experimental procedures
and conditions.
In S. repens, tremulacin and tremuloidin
were found at all three pH regimes,
suggesting that enzymes of S. repens leaves
are not capable of degrading these
compounds efficiently. Julkunen-Tiitto and
Meier (1992b) showed that almond β-
glucosidase does not degrade tremulacin or
tremuloidin, while the compounds were
decomposed slowly by porcine liver
esterase. Salicortin and salicortin-3 were
decomposed totally at all pH regimes (V).
On the contrary, the amount of salicin was
identical at all pH regiemes and no saligenin
was detected suggesting that β-glucosidase
activity in S. repens leaves was low or that
the conditions were not suitable for
supporting enzyme activity.
To summarize, I suggest that the
degradation of acetylsalicortins and
salicortins begins with a cleavage
of the 1-hydroxy-6-oxo-2-cyclohexen-1-
carbonyl moiety that produces acetylsalicin
and salicin, respectively. The acetylsalicins
are degraded further to salicin in reactions
that are catalyzed by esterases or caused by
an alkaline pH. Salicin in turn is degraded to
saligenin by β-glucosidase activity.
Tremulacin is degraded via salicortin to
salicin by alkaline pH or in reaction
catalyzed by esterases, or via tremuloidin to
salicin. This model accords in broad outline
with the models presented by Clausen et al.
(1989, 1991), Mattes et al., (1987),
Julkunen-Tiitto and Meier (1992b) and
Julkunen-Tiitto and Sorsa (2001). These
models disagree with the model presented
by Lindroth (1989), who proposed that the
decomposition of salicortin and tremulacin
begins with the hydrolysis of the sugar
moiety by β-glucosidase, producing
cyclohexenone saligenin ester, which is the
effective component of salicylates in
defense against herbivores. Cyclohexenone
saligenin ester is detoxified by
carboxyesterases (Lindroth, 1989).
The more detailed degradation pattern of
salicortin suggests that the degradation
22
releases salicin and a 1-hydroxy-6-oxo-2-
cyclohexene carboxylic acid anion (Figure 8
in V). Due to rearrangement of electrons,
CO2 is released and a negative ion of 2-
hydroxy-3-cyclohexenone is produced. This
is reduced to enol, which in turn can be
converted either to 6-HCH, 2-hydroxy-3-
cyclohexenone or to catechol (Figure 8 in
V). Quite a similar model was proposed by
Pearl and Darling (1971) who suggested that
degradation salicortin produces 1-hydroxy-
6-oxo-2-cyclohexene carboxylic acid, which
eliminates CO2 and dehydrogenates in the
presence of air to catechol.
The enzyme activities of the larval
digestive tract and the pH of the midgut
could have a considerable impact on the
degradation of secondary compounds (see
Lindroth, 1989). Furthermore, the properties
of foliar enzymes (pH-optimum, specificity)
probably also have a marked influence on
the degradation and thus defensive
properties of compounds.
3.2.4. Metabolic turnover of salicylates
Many secondary compounds have been
shown to undergo metabolic turnover
(Gershenzon, 1994, and references therein).
However, in many turnover studies, a
detached plant or plant parts have been used,
which may produce unnaturally high
turnover rates (Gershenzon, 1994; Mihaliak
et al., 1991). Salicylates, which are very
labile compounds in vitro (Pearl and
Darling, 1971; Lindroth and Pajutee, 1987;
Meier, 1988; Julkunen-Tiitto and
Tahvanainen 1989; Julkunen-Tiitto and
Gebhardt, 1992), were surprisingly stable in
intact willow plants (I, II). In S.
myrsinifolia, the salicylate pools of mature
leaves were not subject to turnover at all,
and the salicylate pools of stems turned over
slowly, with half-lives of 11 to 25 days (II).
In addition, concentrations of salicylates
were low in the stems, indicating the
insignificant role of turnover at level of the
whole plant: only 0.6% of total salicylates of
mature shoots turned over per day.
The salicylate concentrations of the
mature leaves and stems of S. pentandra did
not decrease during the three week
cultivation of plants in the presence of AIP,
indicating that the salicylate pools did not
turnover to any marked degree (I). As water-
soluble glycosides, salicylates are obviously
stored in vacuoles, and thus they are
protected from degradative enzymes such as
esterases and β-glucosidases in intact plant
cells.
Secondary compounds are divided into
mobile and immobile defenses (Coley et al.,
1985; Basey and Jenkins, 1993). High
molecular weight phenolics such as tannins
are considered to be immobile defenses,
representing high construction costs but low
turnover rates and maintenance costs, and
the compounds remain in senescent leaves
(Coley et al., 1985).
Phenolic glycosides such as salicylates
are considered to be mobile defenses, which
have low construction costs but rapid
turnover rates and high maintenance costs.
In addition, mobile defenses are typically
withdrawn during leaf senescence (Coley et
al., 1985).
The results with northern willows suggest
that salicylates should rather be considered
as immobile defenses than as mobile
defenses. The synthesis of salicylates is
costly (I) and salicylates are stable
compounds that are not subject to marked
metabolic turnover (I, II), and salicylates are
not withdrawn from the abscising leaves in
fall (Julkunen-Tiitto, 1989).
Immobile defenses are said to be found in
inherently slow-growing plants inhabiting
nutrient-poor environments (Coley et al.,
1985). However, willows, such as S.
myrsinifolia and S. pentandra, are
inherently fast-growing plants that usually
inhabit resource-rich environments and thus
do not support this generalization.
3.2.5. Metabolic grid of salicylates
Salicin is a degradation product of higher
substituted salicylates such as salicortin,
acetylsalicortin and tremulacin (IV), but it is
obviously chemically the only rational
structure, in which the esterification of
23
hydroxycyclohexenone carboxylic acid
might occur (I). This suggests that there
exists a metabolic grid of salicylates in
willows (Figure 3), in which salicylates
might be converted to each other while their
total concentration remains unchanged.
Salicin would be the key intermediate in this
metabolic grid of salicylates (Figure 3). This
metabolic grid could be one reason why the
salicylate pools of mature plant parts are not
subject to turnover.
3.3. Trade-off between synthesis of
salicylates and growth of plants
Plants have limited resources, which are
competed by the growth and differentiation
processes, and a physiological trade-off is
thus to be expected especially in high-
resource environments (Herms and Mattson,
1992). Thus, it has been suggested that
inherently fast-growing plants inhabiting
resource-rich environments produce lower
amounts of defensive compounds than
inherently slow-growing ones inhabiting
resource-poor environments (Coley et al.,
1985).
Willows are fast-growing species, often
inhabiting resource-rich environments such
as old fields and banks of ditches,
suggesting a potential trade-off between
growth and defense. Investments in defense
may lead to a decrease in the growth and
reproduction of plants, lowering their
competitive ability (Coley et al., 1985;
Herms and Mattson, 1992). However, under
herbivore pressure the investments in
defense would be paid back by increased
plant fitness compared with more weakly
defended individuals (sensu Coley et al.,
1985). Natural selection should favor
synthesis of defenses if the costs of
production are less than the benefits of
protection against herbivores (Coley et al.,
1985).
Salicylates were the major secondary
compounds of micropropagated willows,
indicating that the enhancement of growth
Figure 3. Metabolic grid of salicylates. Salicin is the key intermediate in the metabolic grid of salicylate, acting as a
degradation product and a precursor of higher substituted salicylates.
was due to inhibition of salicylate synthesis.
In addition, the precursor treatments
increased the levels of salicylates and
substantially decreased the growth of plants.
Moreover, the biomass of the whole shoots
and roots were increased by AIP treatment.
Growth and biomass correlated markedly
but negatively with the concentration of
salicylates, suggesting a trade-off between
growth and synthesis of salicylates (I). In an
earlier experiment with S. myrsinifolia, a
significant negative correlation was found
between leaf phytomass and the amount of
total phenolics, indicating a similar trade-off
between growth and secondary metabolism
(Hakulinen et al., 1995). In addition, a
significant genetic trade-off was found
between the salicylate concentration and the
24
growth of P. tremuloides (Hwang and
Lindroth, 1997) and S. sericea (Nichols-
Orians et al., 1993): faster-growing clones
produced a lower concentration of
salicylates.
Notwithstanding the many correlative
results obtained by many researchers
(Herms and Mattson, 1992 references
therein), there is still little direct evidence
for trade-off. Such evidence was presented
by Baldwin and Hamilton (2000) who
showed that induced synthesis of nicotine
reduced the growth and competitive ability
of Nicotiana sylvestris in the absence of
herbivore pressure. However, induced
defense benefited plants under herbivore
pressure (Baldwin, 1988). In addition, JA-
induced defense imposed significant costs
on tomato plants, reducing the reproductive
ability of the plants (Redman et al., 2001).
Haukioja et al. (1988) suggested that
trade-off is to be expected when growth and
defense compete for common precursors. In
my study, a high accumulation of Phe
caused by inhibitor treatment could explain
the enhanced growth of plants in the
presence of AIP (I). Phe is said to be a
common and limiting resource of protein
and phenolic synthesis, which are inversely
correlated (Herms and Mattson, 1992;
Haukioja et al., 1998; Jones and Harley,
1999). Meta-analysis of 35 woody plant
species showed that fertilization decreased
the concentration of phenylpropanoids,
which was assumed to be due to competition
between the growth and synthesis of
phenylpropanoids for a common precursor,
Phe (Haukioja et al., 1998).
Precursor treatments that retarded the
growth of plants also decreased the
accumulation of Phe, further supporting the
growth-enhancing role of Phe (I). An
extremely high accumulation of Phe in the
presence of a PAL-inhibitor in my study
indicates that a very marked amount of Phe
is used for the synthesis of salicylates and
other phenolics in S. pentandra (I) and also
in S. myrsinifolia (II).
It has been proposed that proteins are
minor sinks for Phe, since the majority of
labeled Phe was found in phenolics (Hall
and Yeoman, 1991). Furthermore, the high
accumulation of Phe in AIP-treated plants (I,
II) indicates that Phe is not the only growth-
limiting factor in willows; growth was only
slightly enhanced compared to the enormous
level of available Phe. In addition, the
accumulation of Phe under herbivore
exposure (III) challenges the role of Phe as
the only limiting factor in the biosynthesis
of phenolics (Margna, 1977; Da Cunha,
1987; Jones and Harley, 1999).
To summarize, Phe may limit the growth
of plants and the synthesis of phenolics, but
when the Phe demand is satisfied, other
factors become the limiting factors.
In indeterminately growing plants such as
willows, it would be most profitable to
defend young leaves, since the value of
young leaves is higher than that of older
25
ones (Herms and Mattson, 1992). Since
resources are needed for both defense and
leaf production, this leads to a trade-off
between growth and defense (Herms and
Mattson, 1992).
Salicylate-rich willows are able to grow
fast and at the same time protect newly
produced shoot parts by producing a high
level of salicylates. In addition, the synthesis
of salicylates was induced by herbivory
exposure in the young expanding leaves of
S. myrsinifolia (III). In other words, these
species seem to maintain a high level of
production of salicylates and proteins
simultaneously, also at exponential growth
phase, in spite of an apparent trade-off.
CNB (Bryant et al., 1983), PCM (Jones
and Harley, 1999) and GDB (Herms and
Mattson, 1992) hypothesis predict that
growth takes priority over the synthesis of
phenolics in resource allocation. However,
phenolics play a significant role in the
growth of plants, providing mechanical
support such as lignin for developing
organs, and thus the synthesis of phenolics
can be considered an important part of
growth.
I suggest, therefore, that there is a
delicate balance in the allocation of Phe to
the simultaneous synthesis of phenolics and
proteins in willows, and the majority of the
Phe is used for the synthesis of phenolics
(see also Schmid and Amrhein, 1995). This
allocation could be altered by extrinsic
factors such as fertilization, light or
wounding, towards either phenolic or
protein synthesis. In other words, the
priority in Phe allocation is largely
determined both by the genotype of the plant
in question and by the environment where
the plant lives (see also Coley et al., 1985).
3.4. Dynamics of salicylates in relation to
herbivory
The 4th instars of a generalist Operophtera
brumata used the salicylate-free leaves of S.
phylicifolia efficiently, but the growth of
larvae was reduced on the leaves of S.
pentandra containing a moderate level of
salicylates and chlorogenic acids (IV).
Larvae fed and grew very poorly on the
leaves of salicylate- and chlorogenic acid-
rich S. myrsinifolia. Differences in the
nutritive status of leaves or assimilation
efficiencies cannot explain the differences in
the growth rates of larvae on these willow
species. However, the feeding of larvae
correlated strongly but negatively with the
growth of larvae, indicating that the
decrease in the growth was a consequence of
feeding deterrence. Both salicylate and
chlorogenic acid concentrations correlated
negatively with the feeding and growth of
larvae.
Earlier experiments have shown that both
salicylates (e.g. Tahvanainen et al., 1985b;
Lindroth et al., 1988; Lindroth and Peterson,
1988; Lindroth and Hemming, 1990; Denno
et al., 1990) and chlorogenic acid (Isman
and Duffey, 1982; Felton et al., 1989; Felton
and Duffey, 1991) decrease the growth of
insect herbivores. However, the artificial
diet experiment showed that the degradation
products of salicylates, saligenin and
catechol, were responsible for the decrease
of growth in O. brumata larvae, but neither
salicin nor chlorogenic acid had any
negative impact on larval growth (IV).
Similar results have been obtained with
other Lepidopteran species. Clausen et al.
(1989) showed that 6-HCH and catechol
both significantly reduced the pupal weights
of Choristroneura conflictana (Lep.) reared
on an artificial diet. Salicortin and
tremulacin reduced the growth of
Spodoptera eiridania (Lep.) by decreasing
consumption; neither salicin nor chlorogenic
acid had deleterious effects (Lindroth and
Petersson, 1998). The growth rates of
Papilio glaucus glaucus were decreased by
salicortin and tremulacin due to feeding
inhibition; salicin had no negative effects
(Lindroth et al., 1988). On the other hand,
tremulacin reduced the growth of Lymantria
dispar (Lep.) larvae through the reduction of
approximate digestibility and food
conversion efficiency, but not via feeding
deterrence (Lindroth and Hemming, 1990).
In the artificial diet experiment,
saligenin decreased the growth of O.
brumata larvae efficiently, but no feeding
deterrence as was found in the case of
catechol, was detected (IV). Saligenin could
26
be a toxic agent that may cause gut lesions
such as those observed by Lindroth and
Peterson (1988).
The absence of foliar PPO and
peroxidases in the artificial diet experiment
may explain the inefficiency of chlorogenic
acid in affecting O. brumata larval
performance. Felton and Duffey (1991)
demonstrated that plant peroxidases together
with H2O2 enhanced the growth-retarding
effects of chlorogenic acid.
The age of larvae may also affect their
sensitivity to chlorogenic acid (Isman and
Duffey, 1982). Furthermore, we cannot rule
out possible synergistic interactions of
salicylates and chlorogenic acid on defense
against herbivores (see Nelson and Kursar,
1999).
The effects of other secondary
metabolites on O. brumata larvae are
thought to be minor in comparison to the
deleterious effects of salicylates (IV).
However, antioxidative flavonoids and also
tannins, which might lower the degradation
of salicylates, e.g. by inhibiting β-
glucosidase activity (Juntheikki and
Julkunen-Tiitto, 2000), may have
antagonistic effects together with salicylates
by mitigating the harmful nature of the
compounds.
O. brumata seems to be a very flexible
insect that may use chemically very
divergent plants such as Querqus spp.,
Prunus padus (e.g. Tikkanen, 2000) and
several willow species (Kirsten and Topp,
1991; Tikkanen et al., 1998, 1999, 2000) as
hosts. The leaves of Prunus padus contain
cyanogenic glycosides (Seigler, 1998) and a
low level of tannins (Julkunen-Tiitto,
personal communication), whereas Querqus
species are rich in tannic compounds
(Feeny, 1968, 1970).
The explanation for this adaptability can
be found in the low β-glucosidase activity in
the midgut of larvae. This is supported by
the low degradation efficiency of salicin in
the digestive tract of larvae and the
inefficiency of salicin in the artificial diet
experiment (IV). For example, the capability
of Papilio glaucus canadiensis to feed on a
salicylate-rich diet was assumed to be due to
low β-glucosidase activity of the midgut of
larvae (Lindroth, 1989).
The decreased feeding and growth of O.
brumata larvae on a salicylate-containing
diet may affect the survival and fecundity of
insects. According to the slow growth - high
mortality hypothesis, a slow larval growth
rate increases their mortality by natural
enemies since slow-growing larvae are
susceptible to predation for longer period of
time (Clancy and Price, 1987). For example,
Häggström and Larsson (1995) showed that
high predation on Galerucella lineola
(Coleoptera: Chrysomelidae) on suboptimal
willow species was the result of a longer
development time and an elevated daily
predation rate. However, Lill and Marquis
(2001) showed that plant quality affected the
survivorship and fecundity of Psilocorsis
quercicella (Lepidoptera: Oecophoridae)
directly rather than indirectly via increased
predation due to prolonged developmental
time.
The growth of highly salicylate-
specialized P. vitellinae (Coleoptera:
Chrysomelidae) larvae exhibited exactly the
opposite performance responses on these
same three willow species (Rank et al.,
1989) as compared to the generalist O.
brumata (IV). P. vitellinae larvae grew
fastest on S. myrsinifolia and S. pentandra
and slowest on the salicylate-free S.
phylicifolia. This was obviously due to the
capability of specialist larvae to use the
released glucose moiety in the degradation
of salicylates as an energy source (Pasteels
et al., 1983; Rowell-Rahier and Pasteels,
1986). The degradation of salicylates occurs
mainly in the exertile glands of larvae by β-
glucosidase activity and toxic
salicylaldehyde is secreted (Pasteels et al.,
1983; Rowell-Rahier and Pasteels, 1986).
Thus the midgut of larvae is not exposed to
the harmful effects of salicylates (Pasteels et
al., 1983).
Surprisingly, the feeding of P. vitellinae
did not correlate with the concentration of
salicylates in the tested S. myrsinifolia
plants (III) although the feeding of P.
vitellinae is known to be stimulated by
salicylates (Tahvanainen et al., 1985b). In
earlier studies, salicylate content correlated
27
positively with the feeding of specialist
Chrysomelidae leaf beetles (Smiley et al.,
1985; Rank, 1992). Apparently, within
salicylate-rich willow species, other factors
such as nutrient, sugar or water content or
toughness of leaves are more discriminatory
in the food selection of P. vitellinae. For
example, Matsuki and MacLean (1994)
showed that the effects of water content,
toughness and/or nitrogen content of willow
leaves were generally important for early-
season herbivores. There may also be a
minimum threshold level of salicylates for
P. vitellinae, above which feeding is not
further stimulated (Sipura, personal
communication).
The feeding of P. vitellinae was not
affected by the induction of salicylates
either (III). Earlier studies have shown that
the defoliation of plants decreases the
palatability of leaves for generalist
herbivores (Haukioja and Niemelä, 1979;
Bryant, 1987; Bolser and Hay, 1998), but
has no effect on, or even increases the
palatability of regrowth plant tissues for the
specialists (Pullin, 1987; Bolser and Hay,
1998). Therefore, it is assumed that the
induction of salicylates may reduce the
palatability of S. myrsinifolia leaves for
semi-specialist or generalist herbivores such
as O. brumata, which is able to tolerate only
low to moderate level of salicylates. For
example, the long-term induction of
salicylates in quaking aspen reduced the
quality of new flush foliage for a semi-
specialist moth, Choristoneura conflictana
(Clausen et al., 1991).
According to Karban et al. (1997),
defensive chemicals have a dosage-
dependent effect on herbivores. A chemical
may have little detrimental effect on
herbivores when it is present at a low or
moderate concentration but has a dramatic
effect at higher concentrations (Karban et
al., 1997), as was shown in the case of
salicylates (IV).
To summarize, salicylates seem to greatly
influence the feeding and growth of a
variety of herbivores by stimulating or
inhibiting their feeding, through toxic
effects or by reducing the nutritive value of
leaves. Salicylates may also interact with
other traits of leaves, e.g. nitrogen content,
or other chemicals such as chlorogenic acid.
For example, the deleterious effects of
phenolic glycosides of Populus tremuloides
on the performance of Malacosoma disstria
were pronounced at low protein levels
(Lindroth and Bloomer, 1991).
The salicylate content of S. myrsinifolia
was shown to be highly variable (II, III).
The variability may impair herbivory
performance if herbivores have to choose
among different plants and plant tissues
(Karban et al., 1997). Induced defense
increases the variability and in this way may
also benefit plants under herbivore pressure
(Karban et al., 1997). Variability may be
important in evolutionary terms, since it
slows down the evolution of herbivore
adaptation to plant resistance (Shelton,
2000). Patchy feeding by herbivores due to
plant variability can be important for the
population structure of plants if it reduces
damage to certain plant individuals and
leads to higher herbivory on the neighboring
plants (Shelton, 2000).
4. CONCLUSIONS
Salicylates were found to be stable
compounds in intact willows and thus
maintenance costs of the salicylate
dependent defenses are relatively low.
However, the synthesis of high levels of
salicylates exploits plant resources to a
marked degree, which was observed as a
trade-off between the growth of plants and
synthesis of salicylates. Phe was suggested
to be a common and limiting factor for the
growth and synthesis of phenolics, but when
the Phe-demand was satisfied, other factors
began to limit the growth and synthesis of
salicylates.
Apparently, there is a delicate balance in
the allocation of Phe between proteins and
phenolics, and the majority of Phe is used
for the synthesis of phenolics. However,
priority in allocation is determined by
genotypic factors such as the inherent
growth rates of plants and also extrinsic
factors such as herbivore pressure or the
nutrient status of the environment.
28
Salicylates acted as strong anti-feeding
deterrents against a generalist moth, O.
brumata. Degradation of salicylates by
foliar and larval enzymes activates these
compounds by releasing deterrent, toxic and
nutritive quality lowering moieties. On the
other hand, the feeding choice of a highly
specialized leaf beetle, P. vitellinae, was not
altered by systemic induction of salicylates.
However, the induction may impair the
feeding of less specialized herbivores such
as O. brumata and increase the variability of
foliage, which may in turn subsequently
decrease the herbivory load.
ACKNOWLEDGEMENTS
I wish to express my warmest gratitude to
my supervisors, Riitta Julkunen-Tiitto,
Jorma Tahvanainen and Tuomas Sopanen
who have patiently guided me in my work
and studies, and when necessary also in my
personal life. I also thank my dear collagues
Olli-Pekka Tikkanen and Mika Sipura for
their inspiring co-work; these two guys
opened a window for me on the fascinating
life of herbivores and infected me with their
own enthusiasm for ecological study. I want
to thank Professor Pirjo Vainiotalo for her
fruitful co-operation and for her valuable
advice on the metabolism of salicylates. My
sincere thanks also go to our laboratory
technicians Outi Nousianen and Sinikka
Sorsa for their aid in lab work and to Matti
Savinainen for his help with computers and
other mysterious devices.
I will certainly remember for the rest of
my life the members of the “Discussing and
Critical Science Community”, who provided
the most enjoyable working atmosphere, and
other doctoral students for the happy and
unforgettable moments we spent around the
coffee table in cafeteria Kuutti. I especially
want to express my gratitude to Susanna
Nuutinen for guiding me in the use of the
computer programs and Tommi Nyman for
helping me untie some knots of the English
language. I will warmly remember Riitta
Tegelberg who patiently shared our working
space in spite of my messy working habits
and the constant smell of horses. I also want
to thank the head of the Department of
Biology, Heikki Hyvärinen, for providing
excellent working facilities and also other
members of our department for creating a
warm and friendly working atmosphere. I
thank my friend, Mari Penttinen for offering
me a helping hand whenever I needed it and
also other members of “Oiva’s stallion club”
for enjoyable moments spent in my leisure
time. Finally, I thank my family for their
support during my endless studies and all
my life. The Finnish Academy and the
Faculty of Natural Sciences of the
University of Joensuu supported my work
financially.
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