Journal of Chemical Ecology, Vol. 31, No.

4, April 2005 ( #2005)

DOI: 10.1007/s10886-005-3540-1

EFFECTS OF MECHANICAL WOUNDING

ON ESSENTIAL OIL COMPOSITION
AND EMISSION OF VOLATILES
FROM Minthostachys mollis

ERIKA BANCHIO,1,* JULIO ZYGADLO,2

and GRACIELA R. VALLADARES1
1
Centro de Investigaciones Entomológicas, FCEFYN, Universidad Nacional de Córdoba,
Av. Vélez Sársfield 299, 5000 Córdoba, Argentina
2
Cátedra de Quı́mica Orgánica, FCEFYN, Universidad Nacional de Córdoba, Av. Vélez
Sársfield 1600, 5016 Córdoba, Argentina

(Received May 27, 2004; revised August 23, 2004; accepted December 3, 2004)

Abstract—Plant tissues may show chemical changes following damage. This

possibility was analyzed for Minthostachys mollis, a Lamiaceae native to
Central Argentina with medicinal and aromatic uses in the region. Effects of
mechanical damage on its two dominant monoterpenes, pulegone and
menthone, were analyzed by perforating M. mollis leaves and then assessing
essential oil composition at 24, 48, and 120 hr; emission of volatiles was also
measured 24 and 48 hr after wounding. Mechanical damage resulted in an
increase of pulegone and menthone concentration in M. mollis essential oil
during the first 24 hr. These changes did not occur in the adjacent undamaged
leaves, suggesting a lack of systemic response. Postwounding changes in the
volatiles released from M. mollis damaged leaves were also detected, most
noticeably showing an increase in the emission of pulegone. Inducible
chemical changes in aromatic plants might be common and widespread,
affecting the specific compounds on which commercial exploitation is based.

Key WordsVPhytochemical induction, aromatic plants, Lamiaceae, mechan-

ical wounding, monoterpenes, pulegone, menthone, Minthostachys mollis.

INTRODUCTION

Plants respond to injuries or wounds from biotic or abiotic stresses by deploying

various biochemical defense mechanisms (Karban and Baldwin, 1997). These

* To whom correspondence should be addressed. E-mail: banchioe@com.uncor.edu

719
0098-0331/05/0400-0719/0 # 2005 Springer Science + Business Media, Inc.
720 BANCHIO ET AL.

defenses can be either direct, making the plant more resistant to further her-
bivory (e.g., toxic secondary metabolites, digestibility reducers) or indirect (e.g.,
volatiles released after attack). The latter facilitate Btop-down^ control of herbi-
vore populations by allowing pathogens, predators, and parasitoids to dis-
tinguish between infested and noninfested plants, thus aiding them in host or prey
location (Baldwin and Preston, 1999).
The distinction between both types of defenses is not clear-cut, since some
direct defenses, particularly those that slow herbivore growth by reducing their
digestive efficiency, may need the third trophic level to be effective (Jongsma
and Bolter, 1997; De Leo et al., 1998). Hence, for some direct defenses, we should
expect coordinated induction of both defense types (Baldwin and Preston, 1999).
Among the best studied examples of plant secondary metabolites with
defensive functions, monoterpenes (the C10 members of the terpenoid family of
natural products) occupy a prominent place. These colorless, lipophilic, volatile
substances represent the major constituent in plant essential oils and have been
implicated as defenses against a variety of insects and pathogens (Langenheim,
1994; Phillips et al., 1995; Isman, 2000; Ciccia et al., 2000; Bekele and
Hassanali, 2001; Harrewijn et al., 2001).
Induced chemical changes could also be important from a different point of
view, when aromatic or medicinal plants are considered. In this case, the change
per se would matter, particularly if the specific compounds giving economic
value to the plant, for fragrance, flavor, or pharmaceutical industries, are af-
fected (Valladares et al., 2002).
In the present paper, we analyze changes, induced by mechanical damage,
in the essential oil composition and emission of volatiles from Minthostachys
mollis (Kunth.) Griseb., a Lamiaceae native to Central Argentina with medicinal
and aromatic uses in the region. Damage-induced changes in essential oil com-
position and in the volatiles emitted have rarely been considered simultaneously
(e.g., Zabaras and Wyllie, 2001).
We have dealt with monoterpenoids, since the most important compounds
of M. mollis belong to this group (Valladares et al., 2002) and were given pre-
vious records of increased production of such compounds following herbivory
in various plant species (Karban and Baldwin, 1997; Tumlinson et al., 1999;
Valladares et al., 2002). Temporal and spatial (translocation) variations in the
plant response have been included in the analyses.

METHODS AND MATERIALS

Plants. Healthy, pest-free, 6-month-old M. mollis plants, were grown in a

glasshouse without supplementary lighting. In each plant, a 5-cm-diam hole was
EFFECTS OF MECHANICAL WOUNDING 721

punched in the middle of the lamina of five leaves (of approximately the same
age and size, but from different nodes), thus removing 30Y40% of the leaf area.
Damaged leaves were cut off after either 24, 48, or 120 hr. The undamaged
leaves adjacent to wounded ones were collected at the same time and kept
separately. Five leaves similar in size and age to the damaged ones from an
undamaged plant were used as controls. All leaves were frozen until the
chemical analysis was carried out. Ten different plants (replications) were used
for each treatment.
Essential Oil Extraction. After weighing, the plant material was extracted
by hydrodistillation in a micro-Clevenger-like apparatus for 40 min. The
volatile fraction was collected in dichloromethane. Internal standard was added
(12 g of tymol in 2 l dichloromethane). Essential oils of M. mollis contain 50
different compounds, with two monoterpenes accounting for about 80% of their
volume: pulegone and menthone (Valladares et al., 2002). These two com-
pounds were used for the present study. The monoterpenes were quantified with
respect to thymol. The FID response factors for each compound generate an
equivalent area with a negligible error (<5%).
Collection of Plant Volatiles. The volatile collection system consisted of a
vacuum pump that created a constant air flow (300 ml/min) through a poly-
ethylene terephthalate (PET) chamber (1500 ml in volume) containing a plant;
the chamber was closed at one end with a cap predrilled to fit exactly the
collection trap. At the other end, a cap, with a hole through which the plant stem
passed, separated the bottom of the chamber from the plant pot ground. Air
exited the chamber through a reusable glass collection trap packet with 30 mg
Super Q absorbent (80Y100 mesh)(Alltech), which was rinsed (5Y10 ml
dichloromethane) prior to each volatile collection to remove impurities.
Volatiles were collected for 2 hr. Immediately after, the compounds were
eluted from the absorbent traps with 200 l dichloromethane and internal
standard was added (12 g of tymol in 2 l dichloromethane). Collected
volatiles were analyzed by gas chromatography (GC) as described below. Once
the volatiles were collected, the plant was cut and weighed.
For each plant, volatiles were collected 24, 48, and 96 hr after damage.
Volatiles were also collected from undamaged control plants. All plants were of
the same age and size, and collections done taken under similar conditions of
light, temperature (20Y24-C), and relative humidity (õ70%). Collections made
without plants, from an empty chamber, established that the background of
monoterpenes present was negligible. Each treatment was repeated at least five
times.
Chemical Analyses. Analyses were accomplished by using a Perkin-Elmer
Q-700 gas chromatograph equipped with a CBP-1 capillary column (30 m 
0.25 mm) and a mass-selective detector. Analytical conditions: injector and
detector temperatures 250 and 270-C, respectively; oven temperature pro-
722 BANCHIO ET AL.

grammed from 60-C (3 min) to 240-C at 4-/min; carrier gas helium at a constant
flow of 0.9 ml/min; and source 70 ev. Oil components were identified by a
combination of mass spectral and retention time data, which were compared both
with those of authentic compounds and with those published in Zygadlo et al.
(1996). GC analyses were performed with a Shimadzu GC-RIA gas chromato-
graph, fitted with a 30 m  0.25 mm fused silica capillary column coated with a
Supelcowax 10. The GC operating conditions were: oven temperature
programmed from 60-C (3 min) to 240-C at 4-/min; injector and detector
temperatures 250-C; detector FID; carrier gas nitrogen at a constant flow of
0.9 ml/min.
Volatile Emission Rate. An estimation of the amount of monoterpenes
emitted in relation to their content in the plant tissues was obtained by
calculating, for each treatment, the quotient between the concentration values of
menthone or pulegone in the headspace and their respective concentration in the
essential oil (headspace/essential oil).
Statistical Analyses. Differences in menthone and pulegone content be-
tween treatments were tested for statistical significance using MannYWhitney
test, since data were not normally distributed.

RESULTS

Oil Concentration. Menthone and pulegone concentration in the essential

oil of wounded leaves (Figure 1) increased six and four times, respectively, in
comparison to control plants (P < 0.05, Umenthone = 239.0, Upulegone = 373.0),

FIG. 1. Changes in oil concentration (means T SE) of a menthone and b pulegone from
Minthostachys mollis leaves as a result of mechanical damage. *Values significantly
different from controls (P < 0.05, MannYWhitney test).
EFFECTS OF MECHANICAL WOUNDING 723

TABLE 1. VARIATIONS IN THE MAIN VOLATILES RELEASED FROM Minthostachys mollis

PLANTS AS A RESULT OF MECHANICAL DAMAGE

Compound Control 24-hr Postwounding 48-hr Postwounding

Menthone 1.13 T 0.14 0.85 T 0.39 0.88 T 0.37

Pulegone 0.21 T 0.03 0.85 T 0.27* 0.23 T 0.07

Values are expressed in nanograms per milligram fresh weight (means T SE).
*Values significantly different from controls (P < 0.05, MannYWhitney test).

during the first 24 postwounding hours. After that time, concentration of both
compounds returned to its prewounding level.
Undamaged leaves adjacent to wounded leaves did not differ significantly
from controls regarding pulegone and menthone concentration after the first 24
hr (P > 0.05, Umenthone = 48.0, Upulegona = 241.5), despite the dramatic increase
observed in their opposite (damaged) leaves (Figure 1). However, and in
coincidence with the damaged leaves, after 120 hr, their concentration of
pulegone decreased below the level of control leaves (P < 0.05, U = 56.0).
Plant Volatiles. Headspace experiments showed a change in concentration
of the compounds emitted by damaged plants compared with undamaged ones.
Emission of menthone was similar in both damaged and undamaged plants (P >
0.05, U24 hr = 46, U48 hr = 73.5) and did not change over time (Table 1). Instead,
emission of pulegone increased dramatically upon wounding (P < 0.05, U24 hr =
105) and then decreased back to prewounding levels (P > 0.05, U48 hr = 73.5)
(Table 1).
Emission Rate of Volatiles. The emission rate of pulegone with regard to its
concentration in the essential oil did not show important changes following

FIG. 2. Volatile emission rates of menthone and pulegone from Minthostachys mollis
plants, 24 and 48 hr after being mechanically wounded, presented as differences from the
corresponding rates in undamaged plants.
724 BANCHIO ET AL.

mechanical wounding, whereas the emission rate of menthone was noticeably

lower at 24 hr and higher at 48 hr than that of control plants (Figure 2).

DISCUSSION

Oil Concentration. M. mollis plants responded to mechanical damage by

dramatically increasing, after 24 hr, the concentration of the two most abundant
monoterpenes (menthone and pulegone) in their essential oil. Zabaras and
Wyllie (2001) found a comparable behavior in another aromatic plant, Ocimum
minimum, whose main compound linalool increased 24 hr after suffering
mechanical wounding.
In the present work, leaves adjacent to damaged ones remained unaltered in
their chemical composition at least regarding menthone and pulegone concen-
tration. In other systems, the defensive reaction has been recorded not only at or
near the site of damage, but also throughout the plant, as a result of signaling
molecules enabling communication among different plant tissues (Mc Auslane
et al., 1997; Constabel and Ryan, 1998; Baldwin and Preston, 1999; Gatehouse,
2002). An elicitor may be required for a systemic response to be induced
(Tumlinson et al., 1999).
In M. mollis, most of the essential oil transformations after injury occurred
during the first 24 postwounding hours, after which the damaged leaves started
to return to their prewounding metabolic state. From an ecological perspective,
rapid chemical changes may be interpreted as an attempt to minimize sub-
sequent predation (Clausen et al., 1991).
Monoterpenes are known to be induced by herbivore feeding in M. mollis
(Valladares et al., 2002) and in other plant species (e.g., Mc Auslane et al.,
1997; Paré and Tumlinson, 1999). Therefore, their role during the wounding
response is likely to be involved in protecting damaged leaves from further
attack. This function could be attributed mainly to pulegone, considering that its
toxic (Fournet et al., 1996; Franzios et al., 1997; Ellis and Baxendale, 1997;
Lamiri et al., 2001; Harrewijn et al., 2001), growth-inhibiting (Hummelbrunner
and Isman, 2001), repellent, and oviposition-deterring (Harrewijn et al., 1994)
properties have been demonstrated on various insect species. However, men-
thone has also showed insecticidal (Lee et al., 2001) and genotoxic activities
(Franzios et al., 1997).
Plant Volatiles. In agreement with other studies (e.g., Turlings et al., 1990;
Paré and Tumlinson, 1997), our results show that artificial damage can induce
the release of volatile terpenoids. Although both major constituents of M. mollis
essential oil increased after wounding, only the emission of pulegone changed.
Zabaras et al. (2002) also found a dramatic increase of volatile emission in
EFFECTS OF MECHANICAL WOUNDING 725

Melaleuca alternifolia leaves after mechanical damage, but in that case, the
compounds involved remained unchanged in the essential oil.
The increase in pulegone emission rate 24 hr after damage may be inter-
preted as an attempt to minimize subsequent predation (Harrewijn et al., 2001) or
as a potential cue for natural enemies of herbivores (De Moraes et al., 1998).
Emission Rate of Volatiles. The amount of monoterpenes emitted as vola-
tiles represented only a small fraction of the total monoterpenes present in the
plant. Extrapolation over a typical 6-month growing plant suggests that <1% of
total monoterpenes would be released to the atmosphere. A low rate of vola-
tilization is consistent with observations in other Lamiaceae (e.g., Tyson et al.,
1974; Gershenzon et al., 2000).
Even in undamaged plants, the relative concentration of various mono-
terpenes emitted from M. mollis was quite different from that found in the
essential oil within the plant. Emitted monoterpenes contained higher pro-
portions of menthone (60.2% in the headspace vs. 10% in the oil) and lower
amounts of pulegone (11.1 vs. 85%). Studies on other plant species have also
shown that the composition of emitted monoterpenes may differ from their
presence in the plant oil (Werker, 1993; Guillet et al., 1998; Gershenzon et al.,
2000). Membranes of storage compartments might be selectively more
permeable to particular monoterpenes, or the emitted monoterpenes may be
associated with entirely different secretory compartments than the stored
compounds (Gershenzon et al., 2000).
It is likely that changes in both oil concentration and headspace serve
important ecological roles during the leaf postwounding period. Moreover, from
a commercial point of view, the effects of wounding on leaf oil composition
could also have an important impact.

AcknowledgmentVThis research was supported by a grant from the Consejo Nacional de

Investigaciones Cientı́ficas y Tecnológicas to E. Banchio.

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