See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/278588263

4864

Data · June 2015

CITATIONS READS
0 95

2 authors:

Roberto Buonaurio Maurizio Servili

Università degli Studi di Perugia Università degli Studi di Perugia
87 PUBLICATIONS   968 CITATIONS    225 PUBLICATIONS   7,957 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

olive oil by products in research and development of new dairy products View project

VALOROLIO: La filiera degli oli di oliva di qualità: valutazioni economiche e di mercato delle innovazioni di processo.
View project

All content following this page was uploaded by Roberto Buonaurio on 17 June 2015.

The user has requested enhancement of the downloaded file.

Physiological and Molecular Plant Pathology (1999) 54, 155–169
Article No. pmpp.1998.0196, available online at http:\\www.idealibrary.com on

Involvement of lipoxygenase, lipoxygenase pathway

volatiles, and lipid peroxidation during the
hypersensitive reaction of pepper leaves to
Xanthomonas campestris pv. vesicatoria
R. B"* and M. S#
" Dipartimento di Arboricoltura e Protezione delle Piante, UniversitaZ di Perugia, Borgo XX Giugno, 74,
06121 Perugia, Italy and # Istituto di Industrie Agrarie, UniŠersitaZ di Perugia, Via. S. Costanzo, 06124 Perugia, Italy

(Accepted for publication December 1998 )

Lipoxygenase activity and protein, production of lipid-derived volatiles, and lipid peroxidation
levels were determined in pepper (Capsicum annuum L., cv. Early Calwonder-10R) leaves during
the hypersensitive reaction induced by avirulent race 2 of Xanthomonas campestris pv. Šesicatoria.
Lipoxygenase activity increased during the collapse phase of the hypersensitive reaction (8 to 12 h
after inoculation), and an increase in electrolyte leakage occurred. However, Western blot
analysis revealed that lipoxygenase proteins decreased during the same period. When only one
longitudinal half of a pepper leaf was inoculated with the avirulent bacterium race, a significant
increase in lipoxygenase activity was observed in both inoculated and noninoculated leaf halves,
10 h after inoculation. In addition, lipoxygenase protein decreased in inoculated leaf halves, but
remained unchanged in noninoculated ones. The evolution of some volatile compounds derived
from the lipoxygenase pathway [(E, E )-2,4-hexadienal, 1-hexanol, 3-hexen-1-ol, 2,4-hexadienal
and 2,4-eptadienal] and carotenoid degradation (α- and β-ionone) increased in the incompatible
interaction during the collapse phase of the hypersensitive reaction. The level of the oxidative
index (A \A ) of leaf lipid extracts, determined to estimate lipid peroxidation, significantly
#$& #!&
increased in the advanced stage of the hypersensitive reaction. Furthermore, determination of the
oxidative index in neutral lipid, glycolipid and phospholipid fractions showed that the oxidative
index was significantly increased only in the glycolipid fraction. Lipoxygenase activity and
protein, electrolyte leakage, volatiles and lipid peroxidation were not changed in pepper leaves
inoculated with the virulent race 1 of X. campestris pv. Šesicatoria during the time interval
considered (2–12 h after inoculations). The hypothesis that a lipoxygenase with chloroplastic
location is induced in the incompatible interaction, and which is responsible for the increase in
lipid peroxidation is advanced. # 1999 Academic Press

INTRODUCTION
The hypersensitive reaction (HR) induced in plants by phytopathogenic bacteria is
characterized by the rapid death of cells at the site of pathogen multiplication
and is associated with restriction of bacterial growth [20 ]. One of the earliest events
which occurs in the HR to pathogenic bacteria is irreversible membrane damage [20 ].
It has been suggested that lipoxygenase (LOX), an enzyme that catalyzes the oxidation
* To whom correspondence should be addressed.
Abbreviations used in text : ECW-10R, cv. Early Calwonder-10R, HR, hypersensitive reaction ; LOX,
lipoxygenase ; ROS, reactive oxygen species.

0885–5765\99\050155j15 $30.00\0 # 1999 Academic Press

156 R. Buonaurio and M. Servili
of polyunsaturated fatty acids with a (Z,Z)-1,4-pentadiene moiety to fatty acid
hydroperoxides [48 ], has a role in damaging plant membranes during the HR [49 ]. An
increase in LOX activity has been reported in several incompatible host-pathogen
interactions characterized by HR, including those provoked by bacteria [49 ]. The
strong involvement of this enzyme in establishing incompatibility has recently been
demonstrated in tobacco plants using genetically engineered tobacco lines with
antisense tobacco LOX cDNA constructs [44 ]. LOX could directly or indirectly
promote lipid peroxidation, a deterioration process of plant membranes, also activated
during HR [15, 27, 28 ]. This degradation process is directly initiated by LOX, as
reported by Maccarrone et al. [38 ] for soybean LOX-2, or indirectly by reactive oxygen
species (ROS) particularly singlet oxygen and superoxide radicals, generated during
the reaction catalyzed by LOX [24, 36 ]. ROS, which are thought to be involved in the
induction and\or execution of hypersensitive cell death [5, 34 ], can be generated in
HR by systems other than LOX, such as membrane bound NADPH oxidase and
peroxidase [7, 33 ]. They can also arise as a by-product of chloroplastic, mitocondrial,
peroxisomal and glyoxysomal redox systems (e.g. xanthine oxidase and glycolate
oxidase) [7 ]. Increases in certain isoforms of extracellular peroxidase, with NAD(P)H
oxidase activity, associated with apoplastic accumulation of hydrogen peroxide [6 ],
and stimulation of xanthine oxidase activity [11, 41 ] are reported in bacterially
induced HR.
What causes the restriction of bacterial growth in plants during HR is not clearly
established. Among the antimicrobial compounds which may be responsible for this
phenomenon, there are some volatiles derived from the LOX pathway, the degradation
process of linoleic and linolenic acids in which LOX is a key enzyme [19 ], and shown
to evolve early in bean leaves undergoing HR to pathogenic bacteria [14 ].
The present paper deals with changes in LOX activity and protein, volatiles derived
from the LOX pathway, and lipid peroxidation during HR induced in pepper leaves
by an avirulent race of Xanthomonas campestris pv. Šesicatoria (Doidge) Dye. Preliminary
results of this study were reported earlier [13 ].

MATERIALS AND METHODS

Plants, bacteria, inoculation and sampling
Pepper (Capsicum annuum L.) plants, cv. Early Calwonder-10R (ECW-10R), with the
Bs1 resistance gene to Xanthomonas campestris pv. Šesicatoria, were grown in sterilized
compost-enriched soil in a greenhouse at 22–28 mC, under natural light conditions.
About 40 days after sowing, plants were transplanted into pots and transferred to a
growth chamber at 28p2 mC, 60–70 % RH, 65 µE m−# s−" illumination and 14 h light
period.
Race 1 (strain Xcv 82-8) and race 2 (strain Xcv E3) of X. campestris pv. Šesicatoria,
respectively virulent and avirulent to pepper cv. ECW-10R, were used in all
experiments. Bacterial cultures were maintained as suspensions in 15 % glycerol at
k80 mC.
To prepare inocula, bacteria were grown on nutrient agar at 27p1 mC for 48 h,
suspended in deionized water and the suspension adjusted to 10) cfu ml−" from
absorbance measurements (A l 0n3).
'!!
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 157
The 3rd–6th leaves of pepper plants at the 8th true leaf stage were completely
infiltrated with the bacterial suspensions using a syringe with a 30 G needle.
Corresponding leaves infiltrated with water served as controls. Leaf samples collected
from 2 to 12 h after inoculation, were snap-frozen in liquid nitrogen and stored at
k80 mC before use.

Lipoxygenase extraction and actiŠity assay

Leaf samples (1–2 g fresh wt) were ground in a mortar with sand and 3–6 ml of
potassium phosphate buffer 0n1 , pH 7n0. The slurry was filtered through four layers
of nylon cloth and the fluid centrifuged for 30 min at 15,000 g. Lipoxygenase activity
was polarographically determined in the supernatant (crude extract) by the Grossman
and Zakut method [21 ], using the Rank Brothers (Bottisham, U.K.) oxygen electrode.
Desalting of the extracts was not necessary as no substantial differences in activity were
observed between desalted and non-desalted extracts. The assay mixture contained
0n1  citrate-phosphate buffer pH 5n5, 7n5 m linoleic acid and 0n025 % Tween 20 in
a total volume of 1n5 ml. The enzyme activity was inhibited at 50 % by 400 µ n-
propylgallate, a known LOX inhibitor [31 ]. Changes in LOX activity were also
evaluated using the spectrophotometric assay described by Axelrod et al. [3 ],
performed at pH 5n5 (citrate-phosphate buffer, 0n1 ), after desalting the crude extract
on Sephadex G-25 (PD-10 columns, Pharmacia). In both assays, autoxidation of the
substrate was negligible over the analysis time and no activity was detected in boiled
extracts.
The effect of different pH levels on LOX activities was evaluated polarographically
in pepper leaf extracts using linoleic acid solutions buffered with 0n1  citrate-
phosphate or 0n05  Tris–HCl buffers at different pH values (see Fig. 2 for details).

Western blot analysis

For Western blot analysis, leaf samples (about 1 g fresh wt) were ground in a mortar
with liquid nitrogen and then with buffer as described by Avdiushko et al. [1 ] : 0n44 
sucrose, 10 m KCl, 1n5 m EDTA, 0n1 m MgCl , 10 µ leupeptin, 150 m Tris–HCl,
#
pH 7n5. Forty micrograms of leaf extract were separated on 7n5 % SDS-PAGE [32 ] gels
and transferred to nitrocellulose paper overnight at 30 V and 4 mC. The blots were
incubated in TBS buffer (20 m Tris–HCl, 0n5  NaCl, pH 7n5) for 10 min and then
for 1 h in blocking solution (5 % skim milk in TBS buffer). The blots were soaked for
5 h in rabbit polyclonal antibodies raised against soybean leaf peak 3 LOX diluted
1 : 2,000 in antibody buffer (1 % skim milk in TBS buffer). Non-immune rabbit serum
was used as control. After washing with TBS buffer for 20 min, blots were treated with
Anti-rabbit IgG alkaline phosphatase conjugate (Sigma A-3687) diluted 1 : 30,000 in
antibody buffer for 2 h. After washing in TBS, bound antibody was visualized by
incubating the blot in BCIP\NBT (Sigma Fast B-5655). Images of the blots were
captured with a JVC videocamera and digitalized in a Macintosh computer using
Adobe Photoshop 2n5 software. Densitometry readings of the immunological detected
proteins were performed using the freeware program NIH image 1n58 provided by
National Institutes of Health, Research Services Branch, Bethesda, MD, U.S.A.
158 R. Buonaurio and M. Servili

Lipid peroxidation estimation

Lipid peroxidation of leaf tissues was evaluated by determining the oxidative index
(A \A ratio) of lipid extracts according to Maccarrone et al. [37 ]. Briefly, lipids
#$& #!&
were extracted from leaf samples (about 1 g fresh wt) by the procedure outlined by
Kates and Eberhardt [26 ] and dissolved in methanol. The absorbance at 235 nm
(detection of conjugated dienes) and 205 nm (detection of polyenoic fatty acids) were
recorded in lipid extracts diluted with methanol.
The oxidative index was also evaluated in lipid fractions separated from lipid
extracts on silicic acid columns by the Kates method [25 ]. Columns (1 cm in diameter)
filled with 1n2 g of BioSilA (200–400 mesh ; Bio-Rad laboratories) were loaded with
lipid extracts and eluted successively with 12 ml cloroform (neutral lipids), 48 ml
acetone (glycolipids) and 12 ml methanol (phospholipids).

Volatile compound determination

Ten discs cut with a 10-mm cork borer from pepper leaves (about 0n1 g fresh wt) were
put into 5 ml capped vials and stored at k80 mC until use. Before analysis, the vials
were incubated at 35 mC for 15 min and a solid-phase microextraction process was
used to sample volatile compounds using a fibre (65 µm Carbowax\divinylbenzene ;
Supelco, Inc., Bellefonte, PA, U.S.A.), exposed in the head space of the vials for 20 min
at 35 mC. Thermal desorbtion of volatile compounds was obtained by direct injection
of the fibre into the GC injector, set at 250 mC in splitless mode using a splitless inlet liner
of 0n75 mm ID for 10 min. A GC Varian 3600, equipped with a fused silica capillary
column DB-Wax, 50 m, 0n32 mm ID, 1 µm film thickness (J & W Scientific, Folsom,
CA, U.S.A.) and a split\splitless injector, coupled with a MS Varian Saturn 3 (Varian,
Walnut Creek, CA, U.S.A.) was used. The column was operated with helium at a
pressure of 15 psi with a flow rate of 2n2 ml min−" and a linear velocity of 30n7 cm s−" at
35 mC. The GC oven temperature was maintained at 35 mC for 8 min, then increased
to 45 mC at a rate of 1n5 mC min−", increased to 150 mC at a rate of 3 mC min−", increased
to 180 mC at a rate of 4 mC min−", increased to 210 mC at a rate of 3n6 mC min−", and held
there for 14n51 min. The total time of analysis was 80 min. The injector and transfer
line temperatures were maintained at 250 mC and 220 mC, respectively. The MS was
operated in the mass range of 10–350 a.m.u. at a scan rate of 1 s per scan and a manifold
temperature of 180 mC. The identification of volatile compounds was made by
comparing mass spectral data with those of some pure analytical standards and against
the NIST-92 library.

Other assays
Electrolyte leakage was measured as previously described [10 ] by conductivity
determinations on leaf discs.
Protein content was determined using the Bradford method [8 ].

RESULTS
Symptoms and electrolyte leakage changes
When pepper leaves were inoculated with avirulent race 2 of X. campestris pv. Šesicatoria
(incompatible interaction), they showed the first symptoms of macroscopic tissue
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 159
collapse 7–8 h after inoculation. At this time, a more intense green colour with respect
to the water-infiltrated leaves was visible on the lower leaf surface. Successively, large
areas of affected leaves turned greenish brown and then gradually withered and
coalesced until almost the entire infected leaf blade wilted and shrivelled (12 h after
inoculation). Electrolyte leakage increased significantly (twice the control value, P 
0n05) 8 h after inoculation and increased progressively, reaching 4n3 times the control
value at 12 h in the incompatible interaction (Fig. 1).

80

60
Electrolyte leakage (µS h–1)

40

20

0 2 4 6 8 10 12 14
Hours after inoculation
F. 1. Electrolyte leakage in pepper leaves inoculated with virulent race 1 (#) or avirulent
race 2 of Xanthomonas campestris pv. Šesicatoria (
) and in healthy pepper leaves (=). Each point
represents the mean of 3 independent experimentspstandard error.

Neither symptom appearance nor electrolyte leakage changes (Fig. 1) were detected
in pepper leaves inoculated with virulent race 1 of the bacterium (compatible
interaction), during the experimental period (2–12 h after inoculation). In this
interaction, the first symptoms (water soaking of leaf tissue) appeared 36 h after
inoculation.

Effect of bacterial infection on LOX actiŠity and protein

The pH profiles of leaf pepper LOX showed that enzymatic activity was detected over
a broad range of pH in both control and inoculated plants (Fig. 2). Although pH
profiles were very similar, a broader range of activity was observed in the incompatible
interaction [compare Fig. 2(C) with Fig. 2(A) and 2(B)].
LOX activity increased significantly up to 2n6–3n3 fold the control values (P  0n05)
from 8–12 h after inoculation in the incompatible interaction (Fig. 3). The increase
160 R. Buonaurio and M. Servili

(A) (B)
100
Lipoxygenase activity (%)

50

0
3 4 5 6 7 8 9 3 4 5 6 7 8 9
pH pH

(C)
100
Lipoxygenase activity (%)

50

0
3 4 5 6 7 8 9
pH

F. 2. pH activity profiles of pepper lipoxygenase extracted from healthy leaves (A), and leaves
inoculated with virulent race 1 (B) or avirulent race 2 of Xanthomonas campestris pv. Šesicatoria (C),
10 h after inoculation. Equal amounts of protein (1 mg protein) were added per assay and the
activity expressed relative to the maximum. The buffers used were : 0n1  citrate phosphate for pH
3n5 to 7n0 (#) ; 0n05  Tris–HCl for pH 7n0 to 9n0 ($).

started when tissue collapse was barely visible and electrolyte leakage began to
increase. Up to the end of the experiment in the compatible interaction, LOX activity
did not change significantly (Fig. 3).
Immunoblot analysis showed that LOX from pepper leaves cross react with
polyclonal antibodies raised against soybean leaf LOX and that only one band was
evident in the blots. In addition, based on prestained protein standards, the apparent
molecular mass of pepper LOX is 101p2 kDa. When non-immune rabbit serum was
used, no bands were observed. Immunological detection of LOX in bacterial
inoculated leaves showed a progressive decrease in LOX protein content from 8–12 h
after inoculation (35 and 55 % reduction, respectively) in the incompatible interaction,
and no change in the compatible one (Fig. 4).
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 161
80

Lipoxygenase activity (nmol O2 min–1 mg–1 protein)

60

40

20

0 2 4 6 8 10 12 14
Hours after inoculation
F. 3. Lipoxygenase activity in pepper leaves inoculated with virulent race 1 (#) or
avirulent race 2 of Xanthomonas campestris pv. Šesicatoria (
) and in healthy pepper leaves (=).
Each point represents the mean of 3 independent experimentspstandard error.

C R1 R2
140
(A) (B)
Relative amount of LOX protein (%)

2
120
Hours after inoculation

4 100

80
6
60
8
40

10 20

12 0 2 4 6 8 10 12 14
Hours after inoculation

F. 4. (A) Changes in LOX protein in pepper leaves infected with Xanthomonas campestris pv.
Šesicatoria. Western-blot analysis of leaf extracts from control plants (C) and plants inoculated with
virulent race 1 (R1) and avirulent race 2 (R2) of the bacterium. Each lane was loaded with 40 µg
of proteins. (B) Densitometric analysis of western blot LOX bands in plants inoculated with
virulent race 1 (#) and avirulent race 2 (
) of the bacterium. Data, expressed as % of the control
values, are the means of 2 independent experimentspstandard error.
162 R. Buonaurio and M. Servili

Effect of half inoculation of leaŠes on LOX actiŠity and protein

When only one longitudinal half of pepper leaves was inoculated with the avirulent
race of X. campestris pv. Šesicatoria, a significant (P  0n01) enhancement in LOX
activity was detected 10 h after inoculation in both noninoculated and inoculated
halves compared to corresponding controls (Fig. 5). However, the increase was less
pronounced in the noninoculated portion (2 fold the control value) than in the
inoculated one (6 fold). Furthermore, LOX protein decreased in the inoculated leaf
half, but remained unchanged in the noninoculated one (Fig. 5).

700
c

600
Relative LOX activity and protein amounts

A B C D
500

400

300

b
200

a a
100

0
0 A B C D
F. 5. Lipoxygenase activity ( ) and protein (8) in leaves of pepper plants following
inoculation of a longitudinal half of the leaf with the avirulent race 2 of Xanthomonas campestris pv.
Šesicatoria, 10 h after inoculation. Activity and protein data, expressed as % of the control values,
are the mean of 3 independent and 1 experiment, respectively. Columns relative to the activity
or the protein levels with the same letters are not significantly different (P  0n01) by Duncan’s
multiple range test. A l water-infiltrated half ; B l noninfiltrated half ; C l bacterium-
inoculated half ; D l uninoculated half. Inset, SDS-PAGE immunoblot probed with soybean
anti-LOX antiserum.

Changes in Šolatile compounds

Inoculating pepper leaves with the avirulent race 2 of Xanthomonas campestris pv.
Šesicatoria caused a marked increase (3–42 times the control values) in the levels of (E,
E )-2,4-hexadienal, 1-hexanol, 3-hexen-1-ol, 2,4-hexadienal and 2,4-eptadienal at 8
and 12 h after inoculation and a marked reduction in hexanal, (Z)-2-hexenal and (E )-
2-hexenal at 12 h after inoculation (Table 1). In addition, 12 h after inoculation, there
 Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 163
T 1
Production of Šolatile compounds deriŠed from the LOX pathway in pepper leaŠes inoculated with Širulent race 1 (R1 ) or aŠirulent race 2 (R2 ) of Xanthomonas campestris
pŠ. vesicatoria and in leaŠes of pepper control plants (C )

Hours after inoculation

4 8 12

Compound C R1 R2 C R1 R2 C R1 R2

hexanal 855n5p38n6* 894n3p15n6 980n2p11n7 912n1p7n6 961n0p48n2 839n8p6n5 940n3p38n0 851n3p61n6 563n0p21n7
(Z)-2-hexenal 106n5p1n3 109n2p2n4 107n2p3n3 103n3p0n6 96n3p1n5 110n6p4n5 90n1p0n7 117n0p1n5 59n3p1n1
(E )-2-hexenal 27366n4p97n2 26217n3p323n7 27923n0p336n7 25599n0p279n0 26271n0p12n5 27364n8p335n7 24847n8p0n7 30241n4p230n0 12918n8p281n3
1-pentanol 3n0p0n2 3n3p0n3 1n6p0n3 3n0p0n4 3n7p0n1 4n3p0n08 3n3p0n1 2n4p0n2 15n0p3n9
(E, E )-2,4- 1n7p0n3 7n3p0n2 16n6p1n9 1n0p0n5 2n6p0n1 107n3p4n7 1n5p0n03 7n5p0n7 19n9p4n5
hexadienal
1-hexanol 171n7p1n5 344n4p17n7 332n5p28n6 152n8p19n5 273n6p14n0 466n0p49n2 236n2p11n8 167n6p14n5 1002n2p81n0
3-hexen-1-ol 6n5p0n7 16n2p1n4 13n7p1n3 6n1p0n8 11n1p0n6 44n1p6n8 9n0p0n5 6n5p1n2 345n2p54n2
3-hexen-1-ol (i) 940n9p83n7 2046n0p8n5 1846n3p164n6 888n6p123n4 1299n7p34n0 2881n8p267n3 1103n2p6n0 825n2p42n3 5223n8p684n0
2,4-hexadienal (i) 60n7p0n2 65n1p19n0 61n7p2n7 65n9p5n4 77n1p5n7 167n7p11n3 59n0p3n0 39n8p2n9 2514n1p339n3
2,4-eptadienal (i) 14n2p0n5 26n9p7n7 15n7p0n6 18n8p1n7 26n4p3n4 31n7p4n0 19n5p2n6 26n2p3n2 128n2p11n9
α-ionone 3n7p0n2 8n4p1n0 5n0p0n6 4n3p0n06 5n0p0n4 7n3p0n5 3n2p0n2 4n3p0n8 26n9p5n1
β-ionone 21n0p0n6 44n4p9n1 30n0p0n5 22n2p1n5 28n1p0n4 35n3p6n4 17n9p2n7 29n3p1n8 190n3p21n1

* Each data, expressed as peak areai10−$, is the mean of 3 replicatespstandard deviation ; i l isomer.
164 R. Buonaurio and M. Servili
0.5

0.4
Oxidative index (A235/A205)

0.3

0.2

0.1

0 2 4 6 8 10 12 14
Hours after inoculation
F. 6. Lipid peroxidation, estimated as oxidative index (A \A ), in pepper (cv. ECW 10R)
#$& #!&
leaves infected with virulent race 1 (#) or avirulent race 2 of Xanthomonas campestris pv. Šesicatoria
(
) and in healthy pepper leaves (=). Each point represents the mean of 2 independent
experiments. The low SE bars fit within the symbols.

T 2
Changes in oxidatiŠe index in different lipid classes in pepper leaŠes inoculated with aŠirulent race 2 of
Xanthomonas campestris pŠ. vesicatoria (12 h after inoculation)

Oxidative index
(A \A )
#$& #!&
Incompatible
Lipid class Control interaction t-test

Total lipids 0n304p0n002 0n373p0n02 *

(123n0)
Neutral lipids 0n662p0n044 0n581p0n025 n.s.
(88n0)
Glycolipids 0n084p0n005 0n133p0n005 **
(159n0)
Phospholipids 0n194p0n005 0n227p0n025 n.s.
(118n0)

Each value is the mean of 3 replicatespstandard error. According to the Student t-test : *,
differences significant at P  0n05 ; **, differences significant at P  0n01 ; ns, differences not
significant. Values in brackets are as percentage of the control.
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 165
was an increase in α-ionone and β-ionone, volatile compounds derived from carotenoid
degradation [23 ], a process in which LOX is involved [51 ].
Inoculating with the virulent race 1 of the bacterium did not induce any relevant
changes in levels of the volatile compounds considered, except for a strong increase in
(E, E )-2,4-hexadienal (Table 1).

Changes in lipid peroxidation

Lipid peroxidation, evaluated by determining the oxidative index (A \A ) in the
#$& #!&
total lipid extracts, increased significantly (P  0n01) only in the incompatible
interaction, at 10 h (1n07 times the control values) and 12 h (1n27 times the control)
after inoculation (Fig. 6). Lipid fractionation into lipid classes using silicic acid column
chromatography showed that the changes in the oxidative index in the incompatible
interaction 12 h after inoculation, were essentially due to peroxidation of glycolipids,
in which a significant increase (1n6 times the control values) was detected (Table 2).
No significant changes in the oxidative index were observed in the neutral lipid and
phospholipid fractions (Table 2).

DISCUSSION
Lipoxygenase activity is stimulated in plants infected by phytopathogenic bacteria
both in the incompatible interactions, characterized by HR, and in the compatible
interactions [49 ]. When compatible and incompatible plant-bacterium interactions
were compared, in general the activity increased more quickly and to a higher level in
resistant plants than in susceptible ones [49, 50 ]. We provide evidence here that LOX
activity is enhanced in pepper (cv. Early Calwonder-10R) leaves during the collapse
phase of the HR induced by the avirulent race 2 of X. campestris pv. Šesicatoria. No
increase was observed in LOX activity in the compatible interaction probably because
it was determined up to 12 h postinoculation, when symptoms were not yet visible.
Buonaurio and Umesh Kumar [9 ] demonstrated that LOX activity markedly
increased in a compatible pepper (cv. Early Calwonder) -X. campestris pv. Šesicatoria
combination, when fully expanded bacterial leaf spots were evident (12–15 days
postinoculation). Our data also show that the pH profile of LOX activity in the
incompatible interaction is slightly different from that observed in the compatible one
and in the control plants. The broader optimum pH range documented in the
incompatible interaction may be due to induction of membrane-associated LOX
caused by bacterial infection. Although it is not known at present in pepper plants,
membrane-associated LOX have been purified from tomato and cucumber plants and
have an optimum pH activity over a wide pH range [15, 18, 52 ].
Increased LOX activity was accompanied by a decrease in the level of LOX protein.
This finding is different from those obtained in other incompatible plant-bacterium
combinations. Increases in LOX activity paralleled increases in LOX protein in the
HR provoked by Pseudomonas syringae pv. syringae van Hall in tomato leaves [30 ],
whereas the protein remained practically constant when the activity increased in bean
leaves undergoing HR due to an avirulent race of P. syringae pv. phaseolicola [50 ]. There
are several possible explanations for our results. It is possible that increases in LOX
activity are due to changes in enzyme properties, i.e. increase in specific activity. Since
166 R. Buonaurio and M. Servili
induction and\or de noŠo synthesis of LOX isoforms have been reported in incompatible
plant-pathogen interactions [42, 53 ], it cannot be excluded that the antibodies used did
not react with the LOX form\s induced by the bacterium. Antibodies raised against
a rice LOX isozyme (Lox2 : Os : 1), which is strongly induced in an incompatible rice-
Magnaporthe grisea interaction, do not react with two other rice LOX isozymes (LOX
L-3 and LOX L-2), which are very slightly stimulated by the blast fungus (Shibata D.,
personal communication). Peng et al., [42 ] cloned the rice LOX gene (Lox2 : Os : 1 ) induced
by M. grisea infection and demonstrated that, similar to the Arabidopsis thaliana Lox 2
gene [4 ], homology with the other known LOX genes is relatively low and there is a
putative transit peptide sequence at the amino terminus suggesting that it is localized
in the chloroplasts [42, 46 ]. Based on these findings, it has been proposed that
Lox2 : Os : 1 rice and Lox2 A. thaliana genes should be classified in the Lox2 gene family
and the other known Lox genes in the Lox1 family [46 ]. It is worth noting that LOX
transcripts were rapidly down-regulated in bean leaves hypersensitively reacting to an
avirulent race of Pseudomonas syringae pv. phaseolicola [39 ], while LOX activity was
stimulated [15 ]. Similar to our hypothesis on LOX protein decline, Meier et al. [39 ]
have suggested that the probe they used did not necessarily detect the pathogen-
induced LOX transcripts.
Hydroperoxides generated by LOX activity are further metabolized in plants (LOX
pathway) by different enzymes including hydroperoxide lyase and allene oxide
synthase [19 ]. The LOX pathway, in many respects equivalent to the arachidonic
acid cascade in animals, generates a variety of compounds with interesting biological
activity, such as volatile aldehydes and alcohols (via hydroperoxide lyase) and the
growth regulators jasmonates (via allene oxide synthase) [45, 48 ]. The increased
evolution of some volatile compounds derived from the degradation of linoleic and
linolenic fatty acids was detected in the incompatible interaction concomitantly with
an increase in LOX activity, demonstrated that a LOX pathway is activated. Similar
to the data reported by Luning et al. [35 ] for pepper fruits, the majority of volatiles we
detected derived from 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. We
therefore suggest that 13-hydroperoxides are the major product of the bacterium-
induced LOX and that a 13-hydroperoxide lyase activity was induced during the HR.
Shibata et al. [47 ] purified a fatty acid hydroperoxide lyase from pepper fruits showing
maximum activity with 13-hydroperoxy-(9Z, 11E, 15Z )-linolenic acid as substrate. We
observed an increase in C -alcohols and a decrease in the corresponding C -aldehydes.
' '
This may indicate that an alcohol dehydrogenase is stimulated during the HR studied
[35 ]. Croft et al. [14 ] demonstrated that some volatiles derived by the LOX pathway
were evolved in bean leaves undergoing a HR to an avirulent race of Pseudomonas syringae
pv. phaseolicola. They also demonstrated that two volatiles, (E )-2-hexenal and (Z)-3-
hexenol, have antimicrobial activity against P. syringae pv. phaseolicola and that the
accumulation of these compounds correlates with the decrease in bacterial growth
observed during the HR [14 ]. Further research is necessary to determine whether
volatiles evolved in pepper plants undergoing a HR have antimicrobial activity against
X. campestris pv. Šesicatoria.
LOX activity can be stimulated in parts of plants far from the infection sites. A
systemic increase in LOX activity has been documented in immunized cucumber, rice
and tobacco plants [2, 12, 29 ]. We demonstrated that when only half a pepper leaf
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 167
was inoculated with an avirulent race of X. campestris pv. Šesicatoria, LOX activity
increased not only in the plant tissues invaded by the bacterium but also in non-
invaded tissues. Among the putative compounds which may act as signal molecules for
LOX activation in the uninvaded leaf portion, jasmonates could be considered, as they
stimulate LOX gene expression and LOX activity [40 ]. Since the increases in LOX
activity detected in both leaf halves were accompanied by a decrease in LOX protein
level in the inoculated half and an unchanged level in the adjacent noninoculated one,
it is possible that different LOX isoenzymes are involved : one bacterium-induced in
the invaded areas and another induced by signal compounds probably produced
during the HR.
The collapse phase of the bacterially induced HR is characterized by irreversible
membrane damage, as indicated by loss of electrolytes [27 ]. It has been suggested that
lipid peroxidation, mainly initiated by active oxygen species and\or LOX activity, is
responsible for membrane deterioration [15, 28 ]. Since the increase in LOX activity
found in this study precedes lipid peroxidation, we suggest that lipid peroxidation is,
at least in part, mediated by LOX. However, since electrolyte loss occurs before the
increase in lipid peroxidation, it seems possible that other degradative processes such
as lipase activation in leaf senescence [17 ] contribute to membrane damage. We
showed that glycolipids, the major lipid component of chloroplast membranes [22 ], are
the lipid fraction peroxidized most during the HR induced in pepper by X. campestris
pv. Šesicatoria. It is worth noting that chloroplasts from pepper leaves are strongly
disorganized and their membranes frequently undergo lysis during the collapse phase
of HR induced by the avirulent race 2 of X. campestris pv. Šesicatoria [43 ]. We suggest
that during the HR, there is induction of a LOX belonging to the Lox 2 gene
family and therefore probably localized in the chloroplasts. This bacterium-induced
LOX may initiate lipid peroxidation of chloroplast membranes. The recent hypothesis
that chloroplast lipids are the major sources of C -volatile formation [54 ], which in our
'
study were produced in great quantity, also supports the hypothesis of LOX pathway
activation in the chloroplast. Further studies are needed to purify the bacterial-
induced LOX in pepper and\or clone the relative LOX gene to determine whether the
enzyme is localized in the chloroplast or not.

The work was in part supported by a grant from M.U.R.S.T. 40 % (Progetto nazionale
‘ Ricerche epidemiologiche e fisiologiche sulle malattie crittogamiche delle piante ’) and
in part by a grant from M.U.R.S.T. 60 % to R. B.
We wish to thank Prof. M. Marte for critical discussion of the manuscript ; Prof. Dr
R. E. Stall, University of Gainsville, Florida for providing pepper seeds and Xanthomonas
campestris pv. Šesicatoria races ; Prof. D. Hildebrand, Departments of Agronomy,
Entomology and Horticulture, University of Kentucky, for rabbit polyclonal antibodies
raised against soybean leaf peak 3 LOX.

REFERENCES
1. Avdiushko S, Croft KPC, Brown GC, Jackson DM, Hamilton Kemp TR, Hildebrand D. 1995.
Effect of volatile methyl jasmonate on the oxylipin pathway in tobacco, cucumber, and Arabidopsis.
Plant Physiology 109 : 1227–1230.
2. Avdiushko SA, Ye XS, Hildebrand DF, Kuc J. 1993. Induction of lipoxygenase activity in
immunized cucumber plants. Physiological and Molecular Plant Pathology 42 : 83–95.
168 R. Buonaurio and M. Servili
3. Axelrod B, Cheesbrough TM, Laakso S. 1981. Lipoxygenase from soybeans. Methods in Enzymology
71 : 441–451.
4. Bell E, Mullet JE. 1993. Characterization of an Arabidopsis-lipoxygenase gene responsive to methyl
jasmonate and wounding. Plant Physiology 103 : 1133–1137.
5. Bestwick CS, Brown IR, Bennett M, Mansfield JW. 1997. Localization of hydrogen peroxide
accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv. phaseolicola.
Plant Cell 9 : 209–221.
6. Bestwick CS, Brown IR, Mansfield JW. 1998. Localized changes in peroxidase activity accompany
hydrogen peroxide generation during the development of a nonhost hypersensitive reaction in lettuce.
Plant Physiology 118 : 1067–1078.
7. Bolwell GP, Wojtaszek P. 1997. Mechanisms for the generation of reactive oxygen species in plant
defence—A broad perspective. Physiological and Molecular Plant Pathology 51 : 347–366.
8. Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of
protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 : 248–254.
9. Buonaurio R, Umesh Kumar NN. 1995. Active oxygen-producing and -scavenging enzymes in
pepper leaves infected with Xanthomonas campestris pv. Šesicatoria. Journal of Phytopathology 143 :
165–168.
10. Buonaurio R, Montalbini P. 1991. Changes in superoxide dismutase, peroxidase and catalase
activities during the hypersensitive reaction caused by Pseudomonas syringae pv. syringae in bean leaves.
In : 4th International Working Group on Pseudomonas syringae pathoŠars. Florence, Italy, 10–13 June 1991.
Florence : Stamperia Granducale, 138–148.
11. Buonaurio R., Montalbini P. 1994. Xanthine oxidase activity in bean leaves infected by Pseudomonas
syringae pv. phaseolicola. In : Proceedings of the 8th International Conference on Plant Pathogenic Bacteria.
Versailles, France 9–12 June, 1992. Paris : INRA Editions (Les Colloques, no 66), 633–639.
12. Buonaurio R, Schiappa T, Polverari A, Pezzotti M. 1995. Induction of lipoxygenase activity is
associated with systemic acquired resistance of tobacco to Erysiphe cichoracearum. In : Aspects of Applied
Biology 42, Physiological responses of plants to pathogens. Warwick : The Association of Applied Biologists,
327–330.
13. Buonaurio R, Umesh Kumar NN, Cappelli C. 1993. Involvement of lipoxygenase activity in the
hypersensitive reaction caused by Xanthomonas campestris pv. Šesicatoria (Doidge) Dye in pepper
(Capsicum annuum) leaves. In : 6th International Congress of Plant Pathology. Montre! al, Canada, July
28–August 6, 1993 (poster n. 12.3.11).
14. Croft KPC, Juttner F, Slusarenko AJ. 1993. Volatile products of the lipoxygenase pathway evolved
from Phaseolus Šulgaris (L) leaves inoculated with Pseudomonas syringae pv. phaseolicola. Plant Physiology
101 : 13–24.
15. Croft KPC, Voisey CR, Slusarenko AJ. 1990. Mechanism of hypersensitive cell collapse : correlation
of increased lipoxygenase activity with membrane damage in leaves of Phaseolus Šulgaris (L.) cv. Red
Mexican inoculated with avirulent race 1 of Pseudomonas syringae pv. phaseolicola. Physiological and
Molecular Plant Pathology 36 : 49–62.
16. Droillard MJ, Rouetmayer MA, Bureau JM, Lauriere C. 1993. Membrane-associated and soluble
lipoxygenase isoforms in tomato pericarp-characterization and involvement in membrane alterations.
Plant Physiology 103 : 1211–1219.
17. Ferguson CHR, Simon EW. 1973. Membrane lipids in senescing green tissues. Journal of Experimental
Botany 24 : 307–316.
18. Feussner I, Kindl H. 1994. Particulate and soluble lipoxygenase isoenzymes–comparison of molecular
and enzymatic properties. Planta 194 : 22–28.
19. Gardner HW. 1991. Recent investigations into the lipoxygenase pathway of plants. Biochimica et
Biophysica Acta 1084 : 221–239.
20. Goodman RN, Novacky AJ. 1994. The hypersensitiŠe reaction in plants to pathogens. St. Paul : APS Press.
21. Grossman S, Zakut R. 1979. Determination of the activity of lipoxygenase (lipoxidase). Methods of
Biochemical Analysis 25 : 303–329.
22. Hiller RG, Goodchild DJ. 1981. Thylakoid membrane and pigment organization. In : Hatch MD,
Boardman NK eds. ; The biochemistry of plants., Vol. 8, Photosynthesis. New York : Academic Press, 1–49.
23. Isoe S, Hyeon SB, Sakan T. 1969. Photooxygenation of carotenoids. I. The formation of
dihydroactinidiolide and β-ionone from β-carotene. Tetrahedron Letters 22 : 279–281.
24. Kanofsky JR, Axelrod B. 1986. Singlet oxygen production by soybean lipoxygenase isozymes. The
Journal of Biological Chemistry 261 : 1099–1104.
25. Kates M. 1986. Techniques of lipidology. Isolation, analysis and identification of lipids. Amsterdam : Elsevier.
26. Kates M, Eberhardt FM. 1957. Isolation and fractionation of leaf phosphatides. Canadian Journal of
Botany 35 : 895–905.
27. Keppler LD, Novacky A. 1986. Involvement of membrane lipid peroxidation in the development of
a bacterially induced hypersensitive reaction. Phytopathology 76 : 104–108.
Involvement of lipoxygenase, lipoxygenase pathway volatiles, and lipid peroxidation 169
28. Keppler LD, Novacky A. 1987. The initiation of membrane lipid peroxidation during bacteria-
induced hypersensitive reaction. Physiological and Molecular Plant Pathology 30 : 233–245.
29. Kessmann H, Staub T, Ligon J, Oostendorp M, Ryals J. 1994. Activation of systemic acquired
disease resistance in plants. European Journal of Plant Pathology 100 : 359–369.
30. Koch E, Meier BM, Eiben HG, Slusarenko A. 1992. A lipoxygenase from leaves of tomato
(Lycopersicon esculentum Mill) is induced in response to plant pathogenic Pseudomonads. Plant Physiology
99 : 571–576.
31. Kristie D, Thompson JE. 1989. Inhibition of lipoxygenase activity : a cautionary note. Phytochemistry
28 : 2577–2581.
32. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature 227 : 680–685.
33. Lamb C, Dixon RA. 1997. The oxidative burst in plant disease resistance. Annual ReŠiew of Plant
Physiology and Plant Molecular Biology 48 : 251–275.
34. Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H O from the oxidative burst orchestrates the
# #
plant hypersensitive disease resistance response. Cell 79 : 583–593.
35. Luning PA, Carey AT, Roozen JP, Wichers HJ. 1995. Characterization and occurrence of
lipoxygenase in bell peppers at different ripening stages in relation to the formation of volatile flavor
compounds. Journal of Agricultural and Food Chemistry 43 : 1493–1500.
36. Lynch DV, Thompson JE. 1984. Lipoxygenase-mediated production of superoxide anion in senescing
plant tissue. FEBS Letters 173 : 251–254.
37. Maccarrone M, Rosato N, Finazzi-Agro' A. 1995. Electroporation enhances cell membrane
peroxidation and luminescence. Biochemical and Biophysical Research Communications 206 : 238–245.
38. Maccarrone M, van Aarle PGM, Veldink GA, Vliegenthart JFG. 1993. In Šitro oxygenation of
soybean biomembranes by lipoxygenase-2. Biochimica et Biophysica Acta 1190 : 164–169.
39. Meier BM, Shaw N, Slusarenko AJ. 1993. Spatial and temporal accumulation of defense gene
transcripts in bean (Phaseolus Šulgaris) leaves in relation to bacteria-induced hypersensitive cell death.
Molecular Plant—Microbe Interactions 6 : 453–466.
40. Melan MA, Dong XN, Endara ME, Davis KR, Ausubel FM, Peterman TK. 1993. An Arabidopsis
thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant
Physiology 101 : 441–450.
41. Milosevic N, Slusarenko AJ. 1996. Active oxygen metabolism and lignification in the hypersensitive
response in bean. Physiological and Molecular Plant Pathology 49 : 143–158.
42. Peng Y-L, Shirano Y, Ohta H, Hibino T, Tanaka K, Shibata D. 1994. A novel lipoxygenase from
rice. Primary structure and specific expression upon incompatible infection with rice blast fungus. The
Journal of Biological Chemistry 269 : 3755–3761.
43. Polverari A, Buonaurio R, Marte M. 1994. Aspetti ultrastrutturali della reazione di ipersensibilita'
indotta in peperone da Xanthomonas campestris pv. Šesicatoria. Petria 4 : 319–320.
44. Rance I, Fournier J, Esquerre! Tugaye! MT. 1998. The incompatible interaction between Phytophthora
parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense
lipoxygenase sequences. Proceedings of the National Academy of Sciences of the United States of America 95 :
6554–6559.
45. Rosahl S. 1996. Lipoxygenases in plants—their role in development and stress response. Zeitschrift fur
Naturforschung 51c : 123–138.
46. Shibata D, Axelrod B. 1995. Plant lipoxygenases. Journal of Lipid Mediators and Cell Signalling 12 :
213–228.
47. Shibata Y, Matsui K, Kajiwara T, Hatanaka A. 1995. Purification and properties of fatty acid
hydroperoxide lyase from green bell pepper fruits. Plant and Cell Physiology 36 : 147–156.
48. Siedow JN. 1991. Plant lipoxygenase : structure and function. Annual ReŠiew of Plant Physiology and Plant
Molecular Biology 42 : 145–188.
49. Slusarenko AJ. 1996. The role of lipoxygenase in plant resistance to infection. In : Piazza GJ, ed.
Lipoxygenase and lipoxygenase pathway enzymes. Champaign : AOCS Press, 176–197.
50. Slusarenko AJ, Croft KP, Voisey CR. 1991. Biochemical and molecular events in the hypersensitive
response of bean to Pseudomonas syringae pv. phaseolicola. In : Smith CJ, ed. Biochemistry and Molecular
Biology of Host-Pathogen Interactions. Oxford : Clarendon Press, 126–143.
51. Sumner JB, Sumner RJ. 1940. The coupled oxidation of carotene and fat by carotene oxidase. The
Journal of Biological Chemistry 134 : 531–533.
52. Todd JF, Paliyath G, Thompson JE. 1990. Characteristics of a membrane-associated lipoxygenase in
tomato fruit. Plant Physiology 94 : 1225–1232.
53. Yamamoto H, Tani T. 1986. Possible involvement of lipoxygenase in the mechanism of resistance of
oats to Puccinia coronata avenae. Journal of Phytopathology 116 : 329–337.
54. Zhuang H, Hamilton Kemp TR, Andersen RA, Hildebrand DF. 1996. The impact of alteration
of polyunsaturated fatty acid levels on C-6-aldehyde formation of Arabidopsis thaliana leaves. Plant
Physiology 111 : 805–812.

View publication stats