PESTICIDE

Biochemistry & Physiology

Pesticide Biochemistry and Physiology 86 (2006) 714

www.elsevier.com/locate/ypest

On the mode of action of the phytotoxin (8R,16R)-()-pyrenophorin

Konstantinos A. Aliferis, Maria Chrysayi-Tokousbalides

Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece
Received 1 August 2005; accepted 14 November 2005
Available online 9 March 2006

Abstract
The response of Avena sterilis to the phytotoxic fungal metabolite (8R,16R)-()-pyrenophorin was studied under illumination. The
phytotoxicity of pyrenophorin at 70 lM was expressed as bleaching of leaf sections which was accompanied by increased electrolyte leakage, elevated superoxide dismutase activity, severe lipid peroxidation, rapid loss of photosynthetic pigments, and a decline in total protein. Proteolytic enzyme activity in the treated tissues was not aected and remained at the same low levels as in the untreated.
Pyrenophorin exerts its phytotoxicity through a mechanism operating under both light and dark as indicated by electrolyte leakage measurements. The presence of PSII inhibitor diuron (172 lM) did not reverse electrolyte leakage and bleaching caused by pyrenophorin.
The results indicate that the oxidative damage triggered in A. sterilis by the phytotoxin is not light dependent and is exerted through
a mechanism which involves electron misdirection and generation of reactive oxygen species.
 2006 Elsevier Inc. All rights reserved.
Keywords: Avena sterilis; Bleaching; Mode of action; Macrodiolides; Oxidative stress; Phytotoxin; Pyrenophorin; Superoxide dismutase

1. Introduction
Bioactive natural products have been of great value for
the development of new pesticides. The challenge lies not
only on the discovery of novel templates but also on the
elucidation of their modes of action at the subcellular level.
Although, there are numerous examples of natural products with bioactivity, research on the mode of action of
such compounds is relatively limited [19].
Regarding herbicides, major consideration has been
given to the investigation of phytotoxic substances of natural origin, such as phytotoxins produced by fungal pathogens [6,8,10]. Pyrenophorin (8,16-dimethyl-1,9-dioxacyclohexadeca-3,11-diene-2,5,10,13-tetraone) (Fig. 1) is a
fungal phytotoxin which belongs to macrodiolides, the
structure of which was proposed in 1965 [11]. This phytotoxin was rst isolated from Drechslera avenae, a pathogen
of cultivated oats [12] and later from Stemphylium radicinum [13] and Pyrenophora avenae Ito et Karib [14]. The
compound exists with two isomers and has also been
*

Corresponding author. Fax: +30 210 5294514.

E-mail address: mchrys@aua.gr (M. Chrysayi-Tokousbalides).

0048-3575/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.pestbp.2005.11.011

produced by total synthesis through many chemical

approaches [1523]. The isomer (8R,16R)-()-pyrenophorin has been isolated from cultures of a D. avenae pathotype with host specicity to Avena sterilis [24].
In respect to bioactivity, pyrenophorin is a secondary
metabolite with antimicrobial [12,25] and phytotoxic properties. It is a non-selective phytotoxin which not only causes chlorophyll retention in leaves but aects root growth of
graminaceous species as well [14,24]. The symptom of chlorophyll retention, known as green islands has been associated with fungal infections [2630] and phytotoxicity
expression of fungal metabolites, such as zinniol [31], and
gigantenone and petasol [32]. Interestingly, chlorophyll
retention has also been associated with bioactive compounds of variable chemistry and mode of action, such as
cycloheximide [3335], cytokinins [3638], EDTA [36,39],
paraquat [36], the polyamines spermidine and spermine
[30,40], and siderochromes [41].
Our approach to study, the mode of action of (8R,16R)()-pyrenophorin at the subcellular level was based on the
role and the signicance of the compound in nature. The
observation that pyrenophorin is produced rather early in
culture [24] indicates an involvement in host infection

K.A. Aliferis, M. Chrysayi-Tokousbalides / Pesticide Biochemistry and Physiology 86 (2006) 714

O
O
R

O
O
Fig. 1. Chemical structure of (8R,16R)-()-pyrenophorin.

and pathogen establishment. Studies on D. avenaeoat

interactions have shown that oxidative stress in leaf tissues
are associated with the development of leaf blotch disease
of oats caused by D. avenae [42].
Thus, the eect of the phytotoxin on physiological
parameters involved in oxidative leaf processes, such as
electrolyte leakage, lipid peroxidation, superoxide dismutase (SOD), photosynthetic pigments, and protein stability
was studied. Cytokinin bioassays were also included since
pyrenophorin has shown phytohormone-like action [24],
and cytokinins have been associated with Drechslera
spp.graminaceous plants interactions [29,43]. All tests
were performed by using leaf tissues of A. sterilis which
is the host of the pyrenophorin producing pathotype of
D. avenae.

no)ethanesulfonic acidMES, methionine, nitroblue

tetrazoliumNBT, riboavin, spermine, 2-thiobarbituric
acidTBA], Serva (bovine serum albuminBSA fraction
V, and Coomassie brilliant blue G-250), and ICN (4,6dioxoheptanoic acid). The solvents (acetonitrile, dichloromethane, diethyl ether, ethanol, and methanol) were of
analytical grade, purchased from Lab Scan Analytical
Reagents (Dublin, Ireland). Paraquat (1,1 0 -dimethyl-4,4 0 bipyridinium) of 33.5% purity and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of 99.6% purity were kindly
provided by Syngenta Hellas and Bayer CropScience
Germany, respectively.
2.3. Electrolyte leakage
Leaf segments of a total of 150 mg weight were placed
on the surface of the test solution (30 ml) in 9-cm diameter
Petri dishes and kept in a growth chamber at 22 1 C in
the dark for 20 h. Then they were exposed to continuous
illumination (90 lmol m2 s1) at a relative humidity of
85 5%. Additional experiments were conducted under
continuous light (90 lmol m2 s1) or dark. Conductivity
of solutions was measured with a conductivity instrument
(Hanna Instruments, HI9835).
2.4. Superoxide dismutase activity

Seedlings of A. sterilis were obtained from seeds collected in Votanikos, Athens, in July 2002. The seeds were
placed in a mixture of peatperlitesoil (2:1:1 v/v/v) after
glume removal. Seedlings were kept in a growth chamber
at 22 1 C, relative humidity of 85 5%, and a photoperiod of 16/8 h. The light source used was uorescent lamps
(Sylvania) providing illumination of approximately
90 lmol m2 s1.
Segments (3.0 0.5 cm) of the rst leaf of 9-day-old A.
sterilis seedlings were used in all tests. The ventral side of
each leaf was wounded with carborundum before cutting.
In chlorophyll induction synthesis experiments, seeds of
cucumber (Cucumis sativus Baboo F1) were used. The seeds
were put in a mixture of peatperlitesoil (2:1:1 v/v/v) in
the dark at 22 1 C and relative humidity of 85 5%
for 6 days.

Leaf segments of a total weight of 100 mg were placed

on the surface of the test solution (3 ml) in 5-cm diameter
Petri dishes and kept in a growth chamber at 22 1 C
and a relative humidity of 85 5% under continuous illumination (90 lmol m2 s1). Leaf segments were taken at
time intervals, quickly frozen in liquid nitrogen, and pulverized with a pestle in a mortar. Phosphate buer
50 mM, pH 7.8 (1.5 ml), was then added and the homogenate was centrifuged at 4 C for 15 min at 15,000g.
Total SOD (EC 1.15.1.1) activity of extracts was assayed
by monitoring the inhibition of photochemical reaction of
NBT according to the method of Beauchamp and Fridovich [44] with some modications. The reaction mixture
(3 ml) consisted of potassium phosphate buer (50 mM),
pH 7.8, methionine (13 mM), rivoavin (1.86 lM), EDTA
(79.5 lM), NBT (63 lM), and 200 lL of leaf extract. The
assay mixtures were illuminated for 5 min at a light intensity of 4.5 lmol m2 s1 at 22 C. One unit of SOD activity
was dened as the amount of enzyme required to cause a
50% inhibition of the reduction of NBT as monitored at
560 nm.

2.2. Chemicals

2.5. Lipid peroxidation

(8R,16R)-()-Pyrenophorin was obtained from cultures

of a pathotype of D. avenae grown on oatmeal agar [24].
Reagents and other compounds used were purchased
from Merck (hydroquinone, b-mercaptoethanol, sucrose,
and trichloroacetic acidTCA), Sigma [azocasein, kinetin,
magnesium acetate, 2-mercaptoethanol, 2-(N-morpholi-

Leaf segments of a total weight of 100 mg were placed

on the surface of the test solution (3 ml) in 5-cm diameter Petri dishes and kept in a growth chamber at
22 1 C and a relative humidity of 85 5% under continuous illumination (90 lmol m2 s1). Leaf tissues were
then homogenized in ethanol:water (80:20 v/v) and

2. Materials and methods

2.1. Plant material

K.A. Aliferis, M. Chrysayi-Tokousbalides / Pesticide Biochemistry and Physiology 86 (2006) 714

centrifuged at 3000g for 10 min. The TBAmalondialdehyde (MDA) complex in A. sterilis tissues were estimated
as previously described [45]. Aliquots (1 ml) of appropriately diluted samples were used for determination of lipid
peroxidation. Pyrenophorin was used at a concentration
of 70 lM.
2.6. Analyses of photosynthetic pigments
Leaf segments of A. sterilis were placed on the surface of
the test solution (3 ml) in 5-cm diameter Petri dishes and
incubated as described above. Preparations were made
according to the method of Schoefs et al. [46].
After incubation leaf segments were taken at time intervals, quickly frozen in liquid nitrogen, and pulverized with
a pestle in a mortar. After the addition of methanol
(1.5 ml), leaf extracts were taken with a pipette and transferred to 2 ml Eppendorf tubes. Finally, extracts were claried by centrifugation at 2570g for 5 min at 4 C and the
supernatant was ltered through membrane lters (MN
Chromaphil GF-PET 45/25-Macherey-Nagel, Duren,
Germany). Extraction was performed at 04 C and under
weak light intensity to avoid pigment degradation.
Analysis of photosynthetic pigments was made using a
Shimanzhu HPLCMS (LCMS 2010 A). Absorbance of
solutions was measured using a Uvikon 922 spectrophotometer (Kontron Instruments). Pigment separation was
carried out by reversed phase HPLC as described by Darko
et al. [47] using a C18 column (particle size: 4.65 lm;
25 cm 34.6 mm i.d.) (Zorbax, DuPont, Wilmington,
DE, USA). The eluent consisted of acetonitrile, methanol,
and dichloromethane and the program was as follows: acetonitrilemethanol (85:15 v/v) was run isocratically from 0
to 10 min, followed by a 5 min linear gradient until the proportion
of
acetonitrilemethanoldichloromethane
(63:17:20 v/v/v) was reached. This composition was held
for 7 min. The nal dichloromethane proportion was
reached after a 3 min linear gradient and held until
30 min. During this step, the proportion of methanol was
kept at 18%. The column was then re-equilibrated for
20 min with the solvent mixture used initially. Absorbance
was read at 437 nm. All reversed phase HPLC analyses
were made at 25 2 C. Flow rate was 1 ml min1.
2.7. Extraction of soluble protein
Leaf segments of A. sterilis were placed on the surface of
the test solution (3 ml) in 5-cm diameter Petri dishes. After
incubation leaf segments were quickly frozen in liquid
nitrogen, pulverized with a pestle in a mortar and extracted
in 2.5 ml of extraction buer (pH 6.5) consisted of sucrose
(250 mM), ammonium sulfate (100 mM), magnesium
acetate (20 mM), 2-mercaptoethanol (5 mM), and Mes
(50 mM). Finally, the obtained extract was passed through
nylon sieve to remove plant debris [32]. Soluble protein was
determined by the method of Bradford [48], using BSA as
reference. All treatments were replicated three times. Data

subjected to ANOVA and means were compared by Students test at P 6 0.05.

2.8. Proteolytic enzyme assay
Proteolytic enzyme activity was determined using the
assay of Bunkers and Strobel [32]. The assay solution
was consisted of 0.2 ml K2PO4 (0.4 M), pH 6.0, 0.3 ml
azocasein (10 mg ml1), and 100 lg soluble protein. The
volume was made up to 1 ml with the extraction buer
mentioned above. Assay solutions were incubated for 2 h
in a water bath at 30 C. Cold TCA 12% (2 ml) was then
added to stop the reaction and to precipitate proteins.
The obtained mixtures were centrifuged at 1500g for
10 min, 0.5 ml of the supernatant was taken and brought
to a volume of 1.5 ml with distilled H2O. The absorbance
was read at 340 nm. Proteolytic enzyme activity was calculated according to Peterson and Huaker [49] and Bunkers
and Strobel [32]. One unit of azocasein activity was considered the amount of enzyme required for an absorbance
increase at a rate of 0.01 per minute. All treatments were
replicated three times. Data subjected to ANOVA and
means were compared by Students test at P 6 0.05.
2.9. Reversibility studies
Leaf segments of a total weight of 50 mg were placed on
the surface of the test solution (1.5 ml) in 5-cm diameter
Petri dishes and kept in a growth chamber at 22 1 C,
a relative humidity of 85 5% under continuous illumination (90 lmol m2 s1). Measurements of chlorophyll content were taken 36 h after treatment.
2.10. Induction of chlorophyll synthesis
Cotyledons were excised from 6-day-old cucumber seedlings etiolated under a UV lamp and placed in 5-cm diameter Petri dishes containing water solutions of kinetin,
pyrenophorin or sterilized distilled H2O. They were incubated at 25 C in the dark for 14 h and then were exposed
to light (90 lmol m2 s1) for 3 h [32]. The fresh weight
(fw) of the cotyledons was taken and the chlorophyll content was determined according to Hiscox and Israelstam
[50]. Absorbance of the extracts was read at 665 nm and
measurements were expressed as A665 g1 of fw.
3. Results and discussion
Electrolyte leakage was used as an indicator of membrane destabilization of A. sterilis tissues exposed to
various light conditions. Pyrenophorin (70 lM) induced
signicant electrolyte leakage from A. sterilis leaf tissues
during a 20-h dark period (Fig. 2). Subsequent exposure
of the tissues to 4-h illumination decelerated the eect
(Fig. 2). Electrolyte leakage caused by pyrenophorin was
expressed in a similar pattern under continuous light or
dark, in contrast to the rate, and intensity of conductivity

10

K.A. Aliferis, M. Chrysayi-Tokousbalides / Pesticide Biochemistry and Physiology 86 (2006) 714

Fig. 2. Electrolyte leakage measured as conductivity changes of medium containing Avena sterilis leaf tissues exposed to light after a 20-h incubation in the
dark. Maximal conductivity (DCmax = 117.8 ls cm1) obtained by boiled tissues was used as reference. Each point represents the mean of three replicates
and standard deviations (P 6 0.05) are plotted as vertical bars.

changes caused by the PSI electron diverter paraquat

(4 lM) which were higher under illumination than in
the dark (Fig. 3). Electrolyte leakage by pyrenophorin
remained unchanged in the presence of PSII inhibitor diuron (172 lM) which when applied alone had rather a slight
eect on the conductivity of the solution (Fig. 2). On the
other hand, diuron had a diminishing eect on paraquatinduced electrolyte leakage (Fig. 2). Based on the data
depicted in Figs. 2 and 3, pyrenophorin activity seems to
be not light dependent. The highest conductivity was measured in the solutions containing pyrenophorin and hydroquinone. Taking into account that both substances applied
separately aected conductivity in a similar manner
(Fig. 2), such conductivity enhancement indicates an inter-

action between pyrenophorin and hydroquinone under the

conditions set.
Since electrolyte leakage is often associated with
involvement of reactive oxygen species (ROS), the production of superoxide anion O2  was monitored in order to
test the triggering of a defence system mediated through
SOD. Treatment of A. sterilis leaf tissues with pyrenophorin at 70 lM caused a rapid increase in SOD activity reaching 92 and 135 U mg1 of protein 12 and 24 h after
treatment, respectively. The level of SOD in the untreated
tissues remained at low levels not exceeding the value of
3.5 U mg1 of protein throughout the experiment. The
increase in SOD activity in the treated tissues suggests formation of O2  molecules as a consequence of pyrenophorin

Fig. 3. Electrolyte leakage measured as conductivity changes of medium containing Avena sterilis tissues in the dark (closed symbols) or light (open
symbols). Each point represents the mean of three replicates and standard deviations (P 6 0.05) are plotted as vertical bars.

K.A. Aliferis, M. Chrysayi-Tokousbalides / Pesticide Biochemistry and Physiology 86 (2006) 714

presence. Other phytotoxic substances such as the herbicide

paraquat and the phytotoxin cercosporin have been found
to aect the level of SODs [1,51]. The elevated activity of
SOD in A. sterilis leaves caused by pyrenophorin was the
response of leaf cells to O2  generated in the presence of
the phytotoxin. This led apparently to lipid peroxidation
which was evaluated as equivalents of MDA (Fig. 4). High
levels of MDA, which is formed through autooxidation
and enzymic degradation of polyunsaturated fatty acids,
were observed in tissues of A. sterilis 12 h after exposure
to pyrenophorin at 70 lM (Fig. 4). Lipid peroxidation
leading to loss of membrane integrity has been described
as a consequence of oxidative burst [5255], and directly
related to ROS production [53]. It has also been reported
that lipid peroxidation occurs in the early stages of leaf
blotch development after inoculation of Avena sativa with
D. avenae or Drechslera siccans [42]. Our results on the
eect of pyrenophorin on lipid peroxidation might also
be indicative of an involvement of the phytotoxin in disease
development in the system of D. avenaeA. sterilis.
Photosynthetic pigment analyses revealed that treatment
of A. sterilis leaf tissues with pyrenophorin resulted in a
rapid decrease in such plant constituents (Fig. 5). Such
drastic decrease in chlorophyll and carotenoid content
was expressed as intense bleaching. It was also observed
that the protein decline in bleached tissues due to the presence of pyrenophorin was faster than that in the untreated
samples in which protein remained at statistically signicantly higher levels (Fig. 6A). On the other hand, proteolytic enzyme activity was at the same low levels in both
treated and untreated tissues (Fig. 6B), indicating that protein breakdown was due to other processes but not to
proteolysis.
Tests on the interaction of pyrenophorin with other
bioactive substances on leaf tissues of A. sterilis under illumination gave additional clues on the mode of action of the
phytotoxin. Pyrenophorin caused bleaching of the leaf sections 36 h after treatment but untreated tissues aged normally and the onset of senescence was macroscopically

11

Fig. 5. Chlorophyll a, chlorophyll b, and b-carotene content in leaf tissues

of A. sterilis in the presence of pyrenophorin (70 lM) under illumination.
Each point represents the mean of three replicates and standard deviations
(P 6 0.05) are plotted as vertical bars.

Fig. 6. Protein content (A) and proteolytic enzyme activity (B) of Avena
sterilis leaf tissues in the absence (dotted line) or presence (solid line) of
pyrenophorin. Each point represents the mean of three replicates and
standard deviations (P 6 0.05) are plotted as vertical bars.

Fig. 4. Malondialdehyde (MDA) equivalents (nmoles ml1) in untreated

(dotted line) and treated with pyrenophorin (solid line) Avena sterilis leaf
sections. Each point represents the mean of three replicates and standard
deviations (P 6 0.05) are plotted as vertical bars.

apparent in 72 h (data not shown). The bleaching eect

of pyrenophorin in the light was signicantly reduced by
hydroquinone (Table 1). Such protective action of
hydroquinone was also observed, although to a lesser
degree, during the 4-h illumination phase shown in
Fig. 2. Quinones are central components of most photosynthetic and respiratory electron transfer chains due to their
ability to carry electrons [56]. These naturally occurring
benzene dihydroxyls have also the ability to scavenge free
radicals and quench triplet chlorophyll energy [57]. The

12

K.A. Aliferis, M. Chrysayi-Tokousbalides / Pesticide Biochemistry and Physiology 86 (2006) 714

Table 1
Eect of pyrenophorin and other bioactive substances on bleaching of
Avena sterilis leaf segments expressed as chlorophyll content (A665 g1 fw)
36 h after treatment under illumination
Treatment (lM)

Chlorophyll content
(A665 g1 fw)

Control
Pyrenophorin (70)
Pyrenophorin + hydroquinone (70 + 454)
Hydroquinone (454)
Pyrenophorin + spermine (70 + 861)
Spermine (861)
Pyrenophorin + 4,6-dioxoheptanoic acid (70 + 632)
Dioxoheptanoic acid (632)
Pyrenophorin + diuron (70 + 172)
Paraquat (4)
Paraquat + diuron (4 + 172)
Diuron (172)

15.44a
2.37b
8.52c
13.59e
8.68c
12.37e
1.58b,d
6.02g
2.19b
0.29d
14.26a,e
10.43f

Means followed by the same letter are not signicantly dierent

(P 6 0.05).

antagonism of pyrenophorin activity which was exhibited

by the polyamine spermine (Table 1) also implies a free
radical interference since polyamines can act as ROS scavengers and membrane stabilizers [5861]. On the other
hand, diuron alleviated the eect of paraquat but it did
not prevent bleaching caused by pyrenophorin (Table 1).
This nding is in accordance with the results from electrolyte leakage experiments (Fig. 2) despite of the fact that the
light conditions are dierent. The above observations indicate that PSII electron chain after diuron inhibition site is
not important for pyrenophorin action and that the activity of the phytotoxin is not directly related to PSI electron
transport which is the target of paraquat. Furthermore,
4,6-dioxoheptanoic acid did not prevent tissues from
bleaching (Table 1). Since the compound is related to chlorophyll formation by inhibiting porphyrin biosynthesis [62]
and prevents damage caused by diphenyl ether herbicides
[63], it can be suggested that pyrenophorin phytotoxicity
is due to a dierent mechanism than inhibition of protoporphyrinogen oxidase.
Taking into consideration that (8R,16R)-()-pyrenophorin not only causes chlorophyll retention on detached
leaves in the dark but also aects root growth of monocots
with phytohormone-like activity [24], the association of
pyrenophorin with cytokinins was studied. It has been
shown that cytokinins have the ability to induce chlorophyll biosynthesis in etiolated cucumber cotyledons [64].
Therefore, a cucumber cotyledon bioassay was employed
to examine the possible interference of pyrenophorin with
induction of chlorophyll synthesis. Data in Fig. 7 show that
chlorophyll content in cucumber cotyledons treated with
70 lM kinetin was statistically signicantly higher than in
samples treated with pyrenophorin at the same concentration or in the untreated samples indicating that pyrenophorin activity is independent of cytokinin induction.
The results presented above indicate a correlation
between ROS production, lipid peroxidation and electrolyte
leakage in A. sterilis leaf tissues caused by pyrenophorin.This

Fig. 7. Eect of pyrenophorin (70 lM) and kinetin (70 lM) on chlorophyll content of cucumber cotyledons expressed as A665 g1 (fw). Each
column depicts the mean of three experiments with three replications each.
Standard deviations are plotted as vertical bars and same letters designate
no signicant dierences (P 6 0.05).

suggests that oxidative damage seems to be the mechanism

responsible for membrane alteration leading to abnormal
breakdown of proteins and photosynthetic pigments.
Oxidative processes have also been associated with the
action of the phytotoxins cercosporin [1,65], tentoxin [66],
rubellin [9], and interactions of Drechslera spp. and A. sativa [42]. The observation that pyrenophorin phytotoxicity
symptoms on A. sterilis leaves resemble disease symptoms
at the early stage of development combined with the fact
that this metabolite is produced in D. avenae cultures rather early [24] indicate a possible role of the compound with
D. avenaeA. sterilis interaction. Work is in progress to
study the eects of pyrenophorin in relation to structure
and functions of cell organelles of A. sterilis.
Acknowledgments
This work has been partially funded by the European
Union and the Ministry of National Education and
Religious Aairs of Greece.
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