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J Biol Chem, Vol. 274, Issue 51, 36446-36455, December 17, 1999
From the Lipid peroxidation was investigated in relation
with the hypersensitive reaction in cryptogein-elicited tobacco leaves.
A massive production of free polyunsaturated fatty acid (PUFA)
hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was
characterized during the development of leaf necrosis. The process
occurred after a lag phase of 12 h, was accompanied by the
concomitant increase of 9-LOX activity, and preceded by a transient
accumulation of LOX transcripts. Free radical-mediated lipid
peroxidation represented 10% of the process. Inhibition and activation
of the LOX pathway was shown to inhibit or to activate cell death, and
evidence was provided that fatty acid hydroperoxides are able to mimic
leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were
consumed, chloroplast lipids being the major source of PUFAs. The
results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free
PUFA substrates for LOX. The LOX-dependent peroxidative
pathway, responsible for tissue necrosis, appears as being one of the
features of hypersensitive programmed cell death.
In plant-pathogen interactions, a typical feature of plant
resistance is hypersensitive reaction
(HR),1 characterized by the
induction of rapid cell death at the site of an attempted attack by
either an avirulent strain of a pathogen or a non-pathogen. The
collapse of challenged cells, occurring during incompatible
interactions, was shown in most cases to be dependent on a gene for
gene plant pathogen interaction (1, 2). HR is accompanied by a battery
of defense mechanisms including de novo synthesis of
antimicrobial enzymes and metabolites, strengthening of the cell wall,
and the onset of systemic acquired resistance dependent on salicylic
acid accumulation (3, 4). HR often leads to dry lesions that are
supposed to limit pathogen growth. Other proposed roles is the release
in apoplasm of defense-related proteins and toxic metabolites, as well
as of signals that activate the defenses of both neighboring and
distant cells. Hypersensitive cell death appears to not be the result
of the direct action of released pathogenic factors but is rather under
the genetic control of the host. Indeed, several observations underline
that HR is an example of PCD in plants (1, 2). Furthermore,
hypersensitive cell death has morphological and molecular features
similar to the mammalian PCD, called apoptosis. These include cytoplasm
and chromatin condensation followed by their fragmentation, activation of calcium-dependent endonucleases (5-8) and of cysteine
proteases (9-11), and involvement of similar regulation factors (2). Some differences between HR and mammalian apoptosis were observed, however, such as changes in DNA laddering (5, 8) and the lack in HR of
the repressor role of Bcl-xL (12). One ultimate
characteristic of HR is the loss of membrane integrity, and thus HR is
often characterized by an associated electrolyte leakage (5, 13). This
feature is not encountered in mammalian apoptosis but is one
characteristic of the catastrophic cell death called necrosis, which is
not dependent on gene activation (14). In this way, the use of the term
"necrosis" assumes different meanings when referring to mammalian
cell death or to pathogen-associated plant cell death.
Membrane damage during HR is in close correlation with lipid peroxide
production and with AOS generation (12, 15-17). AOS can initiate lipid
peroxidation in membranes by fatty acid free radical production, and
the process can be propagated by autoxidation (18). The generation of
AOS during the oxidative burst is an important early event during the
course of plant-pathogen interactions and is well documented (19, 20).
In the HR, lipid peroxidation is often a late process occurring at the
same time as the appearance of necrosis. Since AOS production preceded
lipid peroxidation, it is generally admitted that AOS are implicated in
the initiation of membrane damage, and hence hypersensitive cell death.
Indeed, inhibition of oxidative burst by exogenous supplied enzymes,
scavengers, or inhibitors of AOS generator systems suppresses or delays
both lipid peroxidation and hypersensitive cell death (15, 21-23).
Lipid peroxidation might also be due to LOX (EC 1.13.11.12) activity
(24, 25). Initiation of HR membrane damage by LOXs has been suggested
as an alternative hypothesis to free radical action, and the process
might be propagated by autoxidation (26, 27). Indeed, induction of LOX
activity has been observed in several plants during incompatible
interaction and occurs after a lag phase of few hours (28). The
observation that an incompatible interaction can be suppressed in
transgenic tobacco plants expressing antisense LOX clearly demonstrates
the role of LOX in plant resistance to pathogens (29). Finally, since
(i) the LOX pathway leads to products, such as hydroperoxides,
alkenals, and aldehydes, that may kill plant cells and pathogen (30,
31) and (ii) HR triggering is an example of PCD, the induction of a LOX
pathway could be considered as an active process of membrane
degradation leading to plant cell death.
Thus, it is not clear at present whether lipid peroxidation during HR
is induced by AOS and free radicals or is the result of a LOX action.
Both mechanisms could operate in parallel or be exclusive. Furthermore,
the question of whether membrane lipid peroxidation induces cell death
or is the consequence of cell death is still open.
The HR induced in tobacco (Nicotiana tabacum) leaves by
cryptogein, a purified protein from the fungus Phytophthora
cryptogea (32), was investigated in this work. Cryptogein leads
also to defense gene activation (33) and systemic acquired resistance (34). Features of PCD were observed, as assessed by plasma membrane blebbing, cell shrinkage, and cytoplasmic condensation (6), and the
expression of hsr 203J, a gene proposed as the hallmark of
HR-inducing pathogens or elicitors (35). On tobacco cells cryptogein
induces an early oxidative burst (36) and late LOX activity (37). An
early AOS production was also characterized on leaves (38). The AOS
production and lipid peroxidation induced by cryptogein are in close
correlation with the intensity of necrosis (17). Applied to detached
leaves cryptogein causes total leaf necrosis, and this model appears
perfectly suited to biochemical analyses of necrotic associated
processes. Molecular insights on the peroxidation regiospecificity and
enantioselectivity of PUFAs were expected to discriminate between a
free radical mediated process, or a LOX pathway, i.e.
nonspecific versus specific peroxidation, respectively.
Thus, lipid peroxidation was analyzed in the present work using a
previously established hydroxy fatty acid HPLC assay (39) and was
further investigated by chiral phase HPLC (40). Our results
demonstrated the involvement of an induced 9-LOX-dependent lipid peroxidation pathway using free fatty acids as substrates. Evidence was provided that PUFA hydroperoxides are responsible for
tissue necrosis. The activation of the LOX pathway, leading to a
massive production of fatty acid hydroperoxides from membrane lipids,
appears as being an active process in plant-hypersensitive cell death.
The involvement of this pathway to pathogen growth limitation is also discussed.
Chemicals--
Cryptogein, prepared according to Bonnet et
al. (34) was provided by M. Ponchet (INRA, Antibes, France). PUFAs
and fatty acid standards were purchased from Fluka (Büchs) or
Sigma and MeJA from TCI (Interchim, Montluçon, France). The
hydroxy fatty acid chromatographic standards have been previously
described (41). In addition, 15-HEDE, used as an internal standard for HPLC quantification, was prepared from eicosadienoic acid, according to
the previously described procedure (42), and the chemical structure was
assessed by 1H and 13C NMR spectroscopy. An
enriched fraction containing 16:3 (16:3/18:3/18:2 composition 37/56/7)
was prepared from parsley leaf lipid extract, by TLC and galactolipid
hydrolysis, as described below.
Plant Growth and Treatments--
Tobacco plants (N.
tabacum var. Petit Havana) were grown for 8 to 9 weeks
in a greenhouse, at 100-120 µmol/m2·s light radiance
(HQI-BT 400 watts-D OSRAM lamps, München, Germany), with a 14/10
h, 25/20 °C light/dark cycle and 60% relative humidity. Leaves
(about 5 g) were selected in the middle of the stem, detached, and
treated with 10 µl of an aqueous solution of cryptogein (0.2 µg/µl) or water for control, as described previously (17). For MeJA
treatment, tobacco plants were placed into an airtight 25-liter chamber
for 5 days, and MeJA (5 µl) was applied on a piece of filter paper
(43). Chemicals, in a 0.5% Tween 80 aqueous solution, were infiltrated
between two secondary leaf veins, applying the syringe tip to the
epidermis of excised leaves. Leaf petioles were then dipped into water
and the leaves kept in the dark at room temperature. Necrosis was
assessed from changes with time of leaf water content, and expressed as
% of initial FW.
Hydroxy Fatty Acid and Hydroperoxy Fatty Acid Analysis--
Free
and bonded hydroxy and hydroperoxy fatty acids were analyzed by HPLC as
free hydroxy fatty acids, after NaBH4 reduction and
hydrolysis. Tobacco leaves (2.5 g) were homogenized in 0.2 N NaOH, 5% (w/v) NaBH4, in the presence of the
internal reference 15-HEDE (100 nmol/g FW). The samples were frozen and
stored at
For the analysis of free hydroperoxide and hydroxy fatty acids, tobacco
leaf tissue (2.5 g) was ground at 4 °C in 6 ml of 100 mM
potassium phosphate buffer, pH 4.5, containing 1 mM both deferoxamine mesylate and ortho-phenanthroline, as
transition metal chelators, and 10 ml of 50/50 (v/v)
chloroform/methanol with 2 mM triphenyl phosphine, as
reducing agent, and in the presence of 15-HEDE (100 nmol/g FW). The
organic phase was recovered by centrifugation at 700 × g for 5 min, and the aqueous phase was further extracted
with 5 ml of chloroform. Both organic phases were pooled before vacuum
evaporation of the solvent without drying. Then, 2 × 10 ml of
70/30 (v/v) hexane/ethyl ether was added and evaporated again to
eliminate chloroform. The residue was applied onto a silica Sep-Pak
cartridge column (Millipore, St. Quentin-Yvelines, France), previously
equilibrated with 70/30 (v/v) hexane/ethyl ether. Product elution was
carried out successively with 1 ml of 70/30/0.1 (v/v/v) hexane/ethyl
ether/acetic acid and 10 ml of 70/30/1 (v/v/v) hexane/ethyl
ether/acetic acid. The first 1 ml was discarded and the next volume
recovered, evaporated to 800 µl, and analyzed by straight phase HPLC
as described above. In order to assess for free fatty acid
hydroperoxides, a second extraction of the same sample was carried out
in parallel in which triphenyl phosphine was omitted in the extraction
buffer. The extraction procedure and HPLC analysis was the same as
above and led to the separation of both free hydroperoxide and hydroxy
fatty acids.
Fatty Acid and Lipid Analysis--
The overall fatty acid
composition of tobacco leaf sample (80 mg FW) was determined according
to Miquel and Browse (44). Fatty acid methyl esters were analyzed by
gas phase chromatography (Hewlett-Packard 5890 series II, Les Ullis,
France) on a 15-meter × 0.53-mm Carbowax column (Alltech
Associates, Deerfield, IL) with flame ionization detection. The oven
temperature was programmed for 1 min at 160 °C, followed by a
20 °C/min ramp to 190 °C and a second ramp of 5 °C/min to
210 °C, and maintained at 210 °C for a further 5 min. The methyl
ester fatty acid peaks were quantified and identified by comparison of
their retention times with those of standards. Data are expressed in
nmol/mg DW.
For individual lipid analysis, tobacco leaves (0.5 g) were ground under
liquid nitrogen using a mortar and pestle. The tissue sample was
transferred into a screw-capped centrifuge tube with 6 ml of 10/10/1
(v/v/v) chloroform/methanol/formic acid and stored overnight at
LOX Activity Determination--
Frozen leaf tissue (2 g) was
ground in ice-cold 100 mM, pH 6, sodium phosphate buffer (4 ml), containing 2% (w/v) of polyvinyl polypyrrolidone, and a protease
inhibitor mixture (1/2-tablet for 4 ml of buffer, Roche
Molecular Biochemicals CompleteTM). The mixture was
centrifuged for 20 min at 16,000 × g, and the pellet
was discarded. This crude extract was used for the LOX assay and
protein quantification. The enzyme extract (0.5 ml) was incubated for
20 min at 25 °C, with 0.25 M sodium phosphate buffer, pH
7, at a final volume of 1.5 ml, and 5 µl of 18:2 (0.1 M)
in ethanol. The reaction was stopped by adding 200 µl of 1 N NaOH and 500 µl of 5% (w/v) NaBH4 in 0.2 N NaOH. After addition of the internal reference (100 nmol
of 15-HEDE), hydroxy fatty acids were extracted in 1.5 ml of 70/30
(v/v) hexane/diethyl ether and analyzed by straight phase HPLC and
chiral phase as above. Protein content was determined using Pierce BCA
protein assay reagent, following the enhanced protocol of the
manufacturer's instructions (Pierce), with bovine serum albumin as standard.
Preparation of the LOX-specific Probe and RNA
Analysis--
Total RNA fraction was extracted from control and
treated leaves at various times. RNA extraction was performed using the Plant RNA easy minikit, and poly(A)+ RNA extraction was
carried out using an Oligotex mRNA kit (both from Qiagen,
Courtaboeuf, France) following the manufacturer's instructions. The
LOX-specific probe was prepared by RACE amplification using the
MarathonTM cDNA Amplification Kit
(CLONTECH, Ozyme, St. Quentin-Yvelines, France)
with 1 µg of poly(A)+ RNA extracted from tobacco cells
treated with cryptogein for 60 min. The two primers used for the
5'-RACE reactions were a gene-specific primer deduced from the sequence
of the LOX1 of N. tabacum (45) (GenBankTM
accession number X84040), 5'-GAGGAGTAGCTGTTGAGGACTGGAGCTCCC-3' (30-mers), and a primer corresponding to the Marathon adapter 5'-CCATCCTAATACGACTCACTATAGGGC-3' (27-mers). Briefly,
poly(A)+ RNA were reverse-transcribed with Moloney murine
leukemia virus-reverse transcriptase using (T)30-NN as primer. The
second strand performed with a mixture of Escherichia coli
DNA polymerase I, RNase H, and E. coli DNA ligase was
monitored by addition of [32P]dCTP. Following the
creation of blunt ends with T4 DNA polymerase, the double strand
cDNA was ligated to the Marathon cDNA-Adapter. The 5'-RACE
reaction was performed on this cDNA population with the
ExpandTM Long Template PCR System (Roche Molecular
Biochemicals) for 30 cycles using the following steps: 94 °C for
30 s, 55 °C for 30 s, and 68 °C for 4 min. The PCR
products were analyzed by electrophoresis on 1.2% agarose gel in TAE
buffer. One unique band of almost 1 kilobase pair was extracted from
the gel and cloned in pGEM-Teasy vector (Promega, Charbonnieres,
France). Fluorescent sequencing was done by Genome Express S.A.
(Grenoble, France) using SP6 as the downstream primer and a
custom-designed primer as the upstream primer. Sequence analysis were
carried out with FASTA, NCBI, and the Wisconsin Sequence Analysis
Package (Genetics Computer Group, WI). The obtained sequence (998 base
pairs) showed 98% identity with the tobacco LOX1. This cDNA was
used as specific LOX probe for RNA analysis.
Northern blots were carried out according to standard protocols using
15 µg of total RNA per lane. After electrophoresis, RNA samples were
transferred and UV cross-linked to Hybond N+ filters
(Amersham Pharmacia Biotech). Hybridization was carried out with the
specific LOX cDNA, 32P-labeled by random priming
(rediprime, Amersham Pharmacia Biotech), at 42 °C
overnight as described previously (33). Filters were washed with 2 × SSC, 0.1% SDS at room temperature, 4 × 10 min, then with
0.2 × SSC, 0.1% SDS at 60 °C 2 × 10 min, and analyzed with a PhosphorImager (Molecular Dynamics, Les Bordes, France).
LOX-mediated Lipid Peroxidation in Cryptogein-induced
HR--
Lipid peroxidation was investigated in tobacco leaves using
straight phase HPLC analysis of hydroxy fatty acids obtained after NaBH4 reductive extraction and NaOH hydrolysis of total
lipids (39). Typical chromatograms of hydroxy fatty acids extracted from control leaves and cryptogein-treated leaves, when necrotic areas
appeared, are described in Fig.
1A. All positional isomers of
18:2 and 18:3 hydroxy fatty acids were separated and the
chromatographic peaks attributed. From our experiments, 16:3 represents
in tobacco leaves about 15% of total fatty acids (see below), and the
occurrence of corresponding hydroxy fatty acids was investigated. Two
isomers were enzymatically prepared and identified (see legend of Fig. 1), but they were not detected either in control or elicited leaf extracts. Therefore the 16:3 hydroxy fatty acids were not investigated further. In control leaves, the level of hydroxy fatty acids is low
with a preeminence for 13-HODE and 13-HOTE. In cryptogein-treated leaves, lipid peroxidation increased markedly, as observed on all 18:2
and 18:3 positional isomers, the major isomers being 9-HODE and 9-HOTE.
Since 9- or 13-LOX were characterized in plants, positional 9 and 13 isomers can arise from either LOX activity or autoxidation, whereas
12-HOTE and 16-HOTE can be considered to be specific of fatty acid
autoxidation. In addition, if peroxidation products are chiral, they
necessarily arise from LOX activity, whereas racemic products can be
obtained either by autoxidation or by a nonspecific LOX. Each
chromatographic peak was collected and submitted to chiral phase HPLC,
the example of (9R)- and (9S)-HOTE enantiomer
separation in control and treated samples being described in Fig. 1,
B and C. All the isomers were racemic mixtures in
control leaves, with the exception of 13-HODE and 13-HOTE which
exhibited an (S)/(R) enantiomeric ratio of 80/20
and 90/10, respectively (Table I). In
elicited leaves, as expected, 12- and 16-HOTE remained racemic, 13-HODE
and 13-HOTE (S)/(R) enantiomeric ratios were unchanged, whereas 9-HODE and 9-HOTE (S)/(R)
enantiomeric ratios reached values around 90/10 (Table I).
These data suggested that lipid peroxidation in elicited leaves is
dependent on (i) an intense LOX metabolism and (ii) a weak autoxidation
pathway, which was observed through the increased levels of the
non-enzymatic products 12- and 16-HOTE. In control leaves, metabolites
of the LOX pathway were observed at a low level and presented
13S-specificity. In elicited leaves, both 13- and 9-isomer
levels increased and presented S-enantioselectivity. Thus,
the time course of lipid peroxidation was analyzed in Fig. 2A in terms of
LOX-dependent peroxidation, illustrated by the evolution
level of the 13-isomers on the one hand and of the 9-isomers on the
other hand, and autoxidation, illustrated by the increase in the level
of 12-HOTE + 16-HOTE. Both types of enzymatic and non-enzymatic
peroxidation appeared to be induced simultaneously after a 12-h lag
phase. For the initial constitutive 13-specific LOX-dependent peroxidation, the metabolite level was shown
to increase, from 0.04 µmol/g FW reaching a plateau at 0.18 µmol/g FW after 15 h. The level of the metabolites at the 9-position increased markedly and continuously reaching 0.9 µmol/g FW after 30 h. The differences in kinetics of 13- and 9-isomer accumulation suggest the involvement of at least two different LOXs with
13S- and 9S-specificity, respectively. Globally,
LOX-dependent peroxidation appeared to be about 10 times
greater than autoxidation. As expected from a previous study that used
the thiobarbituric assay (17), the level of lipid peroxidation was
shown to be correlated to cryptogein-induced leaf necrosis, as measured
by tissue dehydration (Fig. 2B).
Time Course of LOX Induction in Cryptogein-elicited
Leaves--
The steady-state level of mRNA encoding for LOX was
analyzed using Northern blot experiments at different times following cryptogein application to tobacco leaves. A specific probe
corresponding to tobacco LOX 1, described by Véronési
et al. (45), was prepared by RACE-PCR and recognized a
2.9-kilobase pair mRNA band. The accumulation of LOX mRNAs
monitored in excised tobacco leaves infiltrated with cryptogein or with
water is described in Fig. 3,
A and B. A high transitory accumulation occurred
between 6 and 8 h after the treatment with cryptogein, whereas no
accumulation was observed in the water-treated leaves. Changes in 9-LOX
activity were analyzed in the same experiment and are described in Fig. 3C. In addition, in both elicited and control leaves,
attempts to characterize 13-LOX activity, as suggested by the previous metabolite analyses, were not successful (see below). In elicited leaves, the time course of 9-LOX activity level followed a similar transitory increase, reaching an apparent maximum (27 picokatals/mg protein) after 16 h after a lag phase of 8 h. An important
decrease was then observed (7 picokatals/mg protein after 28 h).
In control leaves the activity was at the limit of the detection level
(<2 picokatals/mg protein) for at least 72 h.
Product Specificity and Substrates of Elicited LOX--
Analyses
of LOX metabolites were conducted to assess free fatty acid
hydroperoxide formation by cryptogein action. Since the NaBH4/hydrolysis extraction procedure leads to the global
analysis of free and esterified hydroperoxide and hydroxy fatty acids, in a first type of experiment, reductive conditions were designed, in
which the hydrolytic step was omitted. Lipids were extracted from 24-h
cryptogein-treated leaves, by grinding at pH 4.5, in the presence of
iron chelators to avoid hydroperoxide degradation, and with triphenyl
phosphine in the chloroform/methanol mixture to reduce the
hydroperoxides under mild conditions (42). Although HPLC of these
extracts appeared to be more complex, as compared with the
NaBH4/hydrolysis procedure, free hydroxy fatty acids could,
however, be analyzed by straight phase HPLC, after a silica Sep-Pak
column enrichment. Under such experimental conditions, only the free
9-hydroxy fatty acid isomers were observed, and the 9-HODE/9-HOTE ratio
was 20/80 (results not shown). The 9-hydroxy fatty acids were
subsequently collected, submitted to chiral phase HPLC, and shown to
present the expected 90% S-enantioselectivity. The free
9-hydroxy fatty acids recovered under these conditions represented
60 ± 18% (n = 4) of the total 9-hydroxy fatty
acids obtained by the NaBH4/hydrolysis extraction
procedure. In a second type of experiment, the triphenyl phosphine was
not present in the extraction buffer. Free fatty acid hydroperoxides as
well as free hydroxy fatty acids could then be identified and
quantified by straight phase HPLC analysis. The HPLC analyses of free
fatty acid hydroperoxides described in Fig.
4 clearly show that, among the various
possible positional hydroperoxide isomers, only 9-HPODE and 9-HPOTE
were observed and, as above for free 9-hydroxy fatty acids, in a 20/80
ratio. By using the latter extraction procedure, the free
9-hydroperoxides represented 51 ± 7% (n = 2) of
the sum of free 9-hydroperoxide and 9-hydroxy fatty acids.
The preceding results suggested that free PUFAs should be the
substrates of cryptogein-induced LOX. Thus, LOX activity was measured
using 18:2 as a substrate and by HPLC to assess for 9/13 specificity.
The activity showed a maximum value at pH 7. Cryptogein-induced LOX was
shown as being regio-specific producing 98% of 9-HODE, and the
specificity was similar between pH 5 and 9. Substrate specificity was
analyzed by competition in a mixture of 16:3/18:3/18:2 (37/56/7,
respectively). All the PUFAs were substrates and shown to be
peroxidized with relative reaction rates of 16/79/5, for 16:3/18:3/18:2, respectively. In addition, since MeJA has been described to induce transcriptional activation of the 9-LOX gene and
accumulation of 9-LOX activity in tobacco (46), the MeJA-induced LOX
was analyzed similarly. Tobacco plants were treated with MeJA for 5 days, under the conditions described by Avdiushko et al. (43). The LOX activity analyzed on leaves was shown to be at the same
level as in leaves treated with cryptogein for 16 h and to exert
the same regio-specificity, 9-HODE representing over 96% of the
products. The enantioselectivity of MeJA- and cryptogein-induced LOXs
was determined on 9-HODE production, either in vitro, with an enzymatic extract, or in planta, after 18:2 substrate
infiltration into leaf and metabolite analysis (Table
II). Both enzymatic extracts possessed
the same 9S-enantioselectivity (around 92%). The 18:2 infiltration experiments led to similar increased levels of
(9S)-HODE and similar enantioselectivity (around 86%). In
both cases, although the induced LOX appeared very specific at the
9-position, the infiltration experiments led, in addition, to increased
levels of (13S)-HODE (around 85% enantioselectivity; Table
II). This result suggested again the presence of a
(13S)-LOX, which could not be characterized in
vitro in the present experiments.
PUFA Consumption in Cryptogein-elicited Leaves--
To investigate
the potential source of free fatty acids as substrates for the induced
LOX, global changes in the fatty acid composition of control and
elicited leaves were compared after treatment, when the symptoms were
not fully developed (18 h), and also compared with the composition of
control leaves at the beginning of the treatment (Fig.
5A). First, the fatty acid
composition of control leaves remained unchanged after leaf excision.
Second, the composition of all fatty acids did not change significantly in elicited leaves with the exception of 18:2 and 18:3, the major fatty
acid in leaves, which decreased to 63 ± 8% and 70 ± 9%
(n = 3) of initial levels, respectively. The different
classes of lipids were analyzed for their fatty acid composition, and
the decrease was confirmed for 18:2 and 18:3. As shown in Fig.
5B, decrease was observed only in phosphatidylcholine and
galactolipids. Proportional consumption of 18:2 and 18:3 was important
in phosphatidylcholine, reaching 41 ± 5 and 74 ± 10%
(n = 3) of initial level, respectively, and represented
a total of 4.6 µmol/g DW. Galactolipids appeared quantitatively as
the major source of PUFA consumption, since 18:2 and 18:3 reached
57 ± 11 and 61 ± 7% (n = 3) of initial
level, respectively, and represented a consumption of 1.2 and 20 µmol/g DW, respectively. An increase in the level of free 18:2 and
18:3 was also noticed, whereas the levels of other free fatty acids did
not change. Obviously, when symptoms were fully developed (24 h), the
total level of all fatty acids started to decrease significantly
(15-20% of consumption), whereas 18:2 and 18:3 reached 53 ± 8 and 54 ± 9% (n = 3) of initial levels,
respectively. In accordance with the observation that 16:3
hydroperoxides do not accumulate in elicited leaves, it is worth
mentioning that the decrease in 16:3 level was low, similar to fatty
acids that are not LOX substrates. With reference to the control, the
material balance calculation carried out on 18:3 indicated that,
24 h after cryptogein treatment, the steady-state level of
9-LOX-dependent peroxidation, as measured by 9-HOTE level
according to the NaBH4/hydrolysis procedure, represented
10 ± 6% (n = 3) of 18:3 consumption. Taken together, these results suggest the involvement of specific lipase(s) in the process and demonstrate the participation of the chloroplastic lipids.
Inhibition of Cryptogein-induced LOX Metabolism and Cell Death by
Limiting Oxygen Availability--
HR can be inhibited in the absence
of oxygen (12). In a first type of experiment, cryptogein-treated
leaves were kept under normal atmosphere for 1 h, allowing the
initiation of the oxidative burst, and then transferred at low oxygen
pressure (0.6%) for a further 25 h. As compared with leaves under
normal atmosphere, the leaves did not develop the necrotic symptoms.
LOX activity was shown to be induced at low oxygen pressure at
comparable level as in leaves under normal conditions, but metabolites
were not synthesized (results not shown). In a second type of
experiments, oxygen availability was limited by water-dipping
experiments. Cryptogein-treated and control leaves were kept under
normal atmosphere for 1 h and then half-dipped into water for the
next 23 h in order to compare symptom development on the same
leaf. As described in Fig. 6A,
necrotic symptoms developed in the upper aerial leaf part of
cryptogein-treated leaves but not in the lower immersed part. Although
LOX activity was induced in both parts at similar levels, metabolite
analysis showed increase of lipid peroxidation in the upper part but
not in the lower (Fig. 6B). Control leaves did not show any
symptom or activation of LOX metabolism. In both types of experiments,
the inhibition of HR was reversed, as soon as leaves were transferred
under normal oxygen conditions. These results showed that LOX activity
can be induced under low oxygen conditions. However, since oxygen is
the PUFA co-substrate of LOX, LOX-dependent peroxidation is
inhibited when oxygen is limiting, and consequently cell death.
Activation of Cell Death by LOX Substrates and
Metabolites--
Substrates and products of the LOX pathway were
tested in the induction of tobacco leaf tissue cell death. Although
PUFA 9-hydroperoxides could not be prepared with sufficient purity to
be tested (47), 13-HPODE can be conveniently prepared with soybean LOX
(42). The effects of 18:2 and 13-HPODE, at 1, 2, 5, and 10 mM concentration in 0.5% of a Tween 80 aqueous solution,
were first compared by infiltration between two secondary veins of
leaves. As shown in Fig. 7A,
after 4 h of incubation, 13-HPODE infiltration induced necrotic
areas above 2 mM concentration, whereas 18:2 infiltration was effective only at 10 mM (not shown). In a second phase,
necrosis induction by 5 mM of 13-HPODE was compared on the
same leaf with the effects of the corresponding alcohol (13-HODE),
tert-butyl hydroperoxide (tert-BuOOH) and again
of the free fatty acid (18:2), at the same concentrations. After 4 h, only 13-HPODE infiltration induced the necrotic areas (Fig.
7B). Surprisingly, tert-BuOOH was not effective
in developing necrotic areas, even at a concentration of 20 mM and after 20 h (results not shown). The symptoms
induced by 2 and 5 mM 13-HPODE infiltration were further
observed in water-dipping experiments (see above) indicating that
oxygen was then not necessary to induce the necrotic lesions (results
not shown). In leaves of MeJA-treated plants, for which 9-LOX activity
accumulated (see above), and of the corresponding control, necrotic
symptoms were not observed. Infiltration of 18:2 and 18:3 between two
secondary leaf veins was carried out at 1, 2, 5, and 10 mM
concentration. Leaf tissue was more sensitive to PUFA infiltration, as
compared with the preceding experiments. In control leaves necrotic
areas were induced, after 4 h, for 5 mM concentration
and above (results not shown). In the MeJA-treated material, necrotic
lesions appeared within 15 min in the 5-10 mM
18:3-infiltrated parts (results not shown), and within 4 h for all
concentrations of both PUFAs (see Fig. 7C for 18:2).
The above efficient hydroperoxide or PUFA concentrations appear
consistent with the decrease of the PUFA level observed in cryptogein-treated leaves. On the one hand, taking into account the
volume of compound infiltration (0.75 ml/g FW), a final level of 3.75 µmol/g of leaf initial FW can be calculated from a 5 mM product infiltration. On the other hand, assuming that 18:2 + 18:3
fatty acids represent 65% of total fatty acids in leaf lipids (17 ± 3 µmol/g FW; n = 3), the PUFA decrease in the
cryptogein-treated leaves (around 50%, after 24 h) can be
estimated to be around 5.5 µmol/g FW, which is a value close to the
preceding one. Taken together, these results confirmed that free PUFAs
are the LOX substrates in vivo and demonstrated that
application or in planta production of PUFA hydroperoxides,
at relevant physiological levels, induce leaf necrosis.
The involvement of a LOX pathway causing the development of tissue
necrosis during HR is shown for the first time in this work as follows:
(i) free fatty acid hydroperoxides are produced massively in tobacco
leaves, in response to cryptogein; (ii) the intensity of leaf necrosis
is correlated to the level of fatty acid peroxidation, dependent on
cryptogein-induced 9-LOX; (iii) since oxygen is the PUFA co-substrate
of LOX, LOX metabolism and cell death are both blocked when oxygen
availability is limited; (iv) finally, leaf necrosis can be induced
either by indirect production of hydroperoxides in planta,
infiltrating PUFAs into leaves where LOX activity has been induced by
MeJA, or by direct infiltration of physiological relevant levels of
fatty acid hydroperoxides.
The production of fatty acid hydroperoxides by a plant LOX was
initially described in potato-Phytophthora infestans
interaction, but arachidonate, the LOX substrate, was released from the
fungus (48). In our model, it is worth emphasizing that plant lipids are the precursors for hydroperoxide production. Thus, our results showed that cryptogein elicits a plant program to provide adequate substrates and enzymes for free fatty acid hydroperoxide production leading to cell death. Increasing LOX activity during the development of HR was described for many plants. Enzyme specificity appears to be,
however, dependent on the plant model. For instance, 13-LOX was
described in rice (49) and soybean (27), whereas 9-LOX was
characterized in tomato (50) and tobacco (Ref. 51 and this work). Since
it has been further shown that infiltration of 13-HPODE as well as
in planta production of 9-HPODE were able to induce tobacco
leaf necrosis, these data taken together suggest that, dependent on the
plant species, 13- and (or) 9-hydroperoxide isomers of fatty acids can
be produced as potent effectors of hypersensitive cell death.
Surprisingly, infiltration in the tobacco leaves of
tert-BuOOH up to 20 mM concentration did not
induce necrotic lesions. Thus, it can be proposed that, in addition to the hydroperoxides, fatty acid hydroperoxide metabolites and
degradation products would also play an important role in toxicity.
Autoxidation of esterified fatty acids was also observed and
tentatively attributed to membrane lipid peroxidation. This process,
representing around 10% of the total lipid peroxidation level, occurs
late after elicitation and is induced simultaneously with the enzymatic
process. Therefore, it can be proposed that this additional membrane
lipid peroxidation is likely initiated by free fatty acid
hydroperoxides produced massively via LOX action, rather than AOS
generated early during the oxidative burst (38). Anyway, the hypothesis
that massive lipid peroxidation occurring in cryptogein-induced HR is
the result of free radical production by the oxidative burst can be
ruled out. Additional experiments, including the study of other models, are needed, however, to demonstrate the systematic occurrence of the
LOX pathway in hypersensitive cell death, and the involvement of
AOS as effectors or (and) as signaling components in the process (22,
52, 53).
Analysis of changes in the fatty acid composition of cryptogein-treated
leaves was assessed for the importance of the peroxidative pathway.
During the first 18 h, whereas the level of the other fatty acids
did not change significantly, about 30% of the 18:2 and 18:3 were
consumed specifically, reaching 50% after 24 h (5.5 µmol/g FW).
Despite the high reactivity of hydroperoxides, the steady-state level
of PUFA hydroperoxides after 24 h was shown to represent still
10% of PUFA consumption. In addition, infiltration of 18:2 into
leaves, 16 h after cryptogein treatment, increased the 9-HODE
level, indicating that 9-LOX activity was not substrate-saturated in vivo. Overall, these results strongly suggest that
massive PUFA consumption occurs mainly via the LOX pathway. Among the lipid changes following tobacco elicitation, previous investigations described a late decrease in galactolipid levels (15). In the present
model, the analysis of the fatty acid composition of the lipid classes
showed further that PUFAs originated mainly from galactolipids. Thus,
chloroplasts appear as being the main source of PUFAs in the process.
The key role of chloroplast lipids in synthesis of products derived
from fatty acid hydroperoxides, the so-called oxylipin pathway, was
first established using mutants of PUFA synthesis for freezing-induced
volatile aldehyde production (54). Furthermore, the metabolism site of
fatty acid hydroperoxides was shown to be located on the envelope
membranes of chloroplasts (55), and it appeared that some pathogen- or
MeJA-induced LOX genes are chloroplast-targeted (49, 56). In addition,
morphological changes on chloroplasts were previously observed in
various models of plant-pathogen interaction (6-8) and in
cryptogein-treated tobacco leaves (57). The present results highlighted
the major role of this organelle in the development of leaf HR.
A tobacco 9-LOX was previously purified and characterized on cultured
cells treated by an elicitor from the pathogen Phytophthora parasitica var. nicotianae (51). The 9/13-LOX
specificity (87/13 and 93/7 on 18:2 and 18:3 substrates, respectively)
is consistent with our results on cryptogein-induced
(9S)-LOX in leaves (98/2 on 18:2). The expression of the
gene was shown to occur in elicited cell cultures and, furthermore, in
plants upon infection (46). In the present work, a cryptogein-mediated
expression of the same gene was demonstrated. From the chiral analyses,
a low constitutive (13S)-LOX metabolism can be proposed in
control leaves. Upon cryptogein treatment, the metabolism is shifted
toward a massive production of (9S)-fatty acid
hydroperoxides. The initial low constitutive (13S)-LOX
activity could be sufficient for the synthesis of JA, an early plant
defense signaling compound considered to operate in plant pathogen
interactions (25, 30, 31). The enzymes required for JA synthesis have
been proposed as being expressed constitutively (58), and the 18:3
precursor might be provided by the induction of phospholipases, known
to occur rapidly after elicitation (19, 59). JA was mentioned as being
produced in elicited tobacco cells before 9-LOX activity induction and
shown to mediate 9-LOX gene expression (46). In the present work, cryptogein- and MeJA-induced LOX activities were shown to exhibit the
same 9S specificity. Thus, participation of JA in the
signaling cascade leading to cryptogein-induced 9-LOX gene expression
appears likely. In addition, early accumulation of JA was described in infected tobacco leaves undergoing HR (60). These results, taken together, suggest that at least two different LOXs, with 13 and 9 specificity, operating in signaling and in production of cell death
effectors, respectively, should be involved in tobacco leaf HR.
During the development of cryptogein-elicited leaf necrosis, free fatty
acid hydroperoxides were produced from membrane lipids, indicating the
involvement of hydrolase activity. Indeed, a late induction of acyl
hydrolase or lipase activities was observed in various models of plant
pathogen interactions (26, 61). In general, LOX substrates are free
fatty acids. However, in the early stages of seed germination, lipid
body-associated LOX led to lipid peroxidation prior to hydrolase action
(40). In addition, in elicited soybean seedlings, the induced LOX was
shown to act in vitro on phospholipids (27). Thus, in the
cryptogein-induced tobacco HR, the question whether LOX acts before or
after the hydrolase action was addressed. First, all hydroxy fatty acid isomers were characterized in esterified lipids whereas, consistent with the 9-LOX activity determined in vitro on free fatty
acids, only 9-positional isomers were analyzed as free fatty acid
hydroperoxides, suggesting that hydrolysis occurs before peroxidation.
Second, free 16:3 fatty acid was shown can be peroxidized by elicited 9-LOX in vitro but 16:3 not was consumed and apparently not
peroxidized in elicited leaves. Since 16:3 is exclusively located on
the sn-2 position of chloroplast lipids (62), this last
result could be explained assuming lipase(s) specific of the
sn-1 position act before 9-LOX. Finally, the observation
that MeJA-induced 9-LOX activity did not lead to either metabolite
accumulation or to HR symptoms, in the absence of PUFA infiltration,
also argues in favor of that hypothesis. This last result further
suggests that lipase(s) might be induced by a signaling pathway
different from JA. The characterization of elicited lipases, their
product specificity, and the signaling pathway leading to their
induction are currently under investigation.
The events leading to cryptogein-induced HR are tentatively summarized
in Fig. 8. The induction of LOX pathway
is an active process of membrane degradation leading to hypersensitive
cell death. Lipase activity appears to be involved upstream from 9-LOX action. JA was proposed as an early signal for 9-LOX gene induction. In
the light of our results, further peroxidation of membrane lipids was
considered as a consequence of massive production of fatty acid
hydroperoxides, rather than AOS production during the early oxidative
burst. The induction of the LOX peroxidative pathway was not described
in mammalian PCD (63) and can be considered as a characteristic of
plant HR-PCD. Apoptosis in mammalian cells and lipid peroxidation were
correlated, both induced by oxidative agents, and both inhibited in
transfected cells overexpressing the oncogene bcl-2 (64).
Apoptosis was shown to be induced by free fatty acid hydroperoxides but
was not repressed by the Bcl-2 protein, suggesting that the protein
acts before lipid peroxidation (65). In plants, HR induction appears
not dependent on the Bcl-2 family (12), and lipid peroxidation was
shown in this work as being an active process.
In many aspects, the production of free fatty acid hydroperoxides must
be considered as an important part of the plant defense response to
limit pathogen invasion. Products from the LOX pathway include hydroxy
fatty acids, observed in this work, which are substrates for cutin
biosynthesis, reinforcing tissue defenses (66). Lipid hydroperoxides
and derived products are toxic to microorganisms (25, 30, 31, 50, 67).
Finally, fatty acid hydroperoxides were also proposed as acting as
signaling compounds to neighboring cells (25) and eliciting phytoalexin synthesis (48, 66). A recent investigation (29), showing that tobacco
plants exhibiting HR and a resistance toward the pathogen P. parasitica displayed susceptibility when antisense 9-LOX plants
were infected, is in favor of that assumption.
The massive production of free fatty acid hydroperoxides observed in
this work was shown to induce plant cell death and can also be
considered as a defense mechanism against the invading pathogen. This
metabolism has been observed during the development of leaf HR, and
chloroplast lipids appear essential for the response. Among the
questions arising from these results, further investigations must be
focused to address the occurrence of this pathway in other plant-pathogen interactions, other tissues (i.e.
non-photosynthetic tissues), and in the cultured cell model. The active
process of membrane degradation described in this work, together with
the induction of nucleases (5-8) and proteases (9-11), can be
considered as one of the important features leading to the
hypersensitive programmed cell death.
We thank Dr. Michel Ponchet (from INRA,
France) for providing cryptogein and helpful discussions. We are also
grateful to Michel Péan and co-workers for greenhouse facilities
and to Christiane Richaud for help in preparing the manuscript.
*
This work was supported by European Community funding, as a
part of the "CAST" project within the 4th Framework Program in Biotechnology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
HR, hypersensitive reaction;
PCD, programmed cell death;
AOS, active oxygen
species;
LOX, lipoxygenase;
PUFA, polyunsaturated fatty acid;
HPLC, high pressure liquid chromatography;
JA, jasmonate;
MeJA, methyl
jasmonate;
15-HEDE, 15-hydroxy-11,13(Z,E)-eicosadienoic
acid;
FW, fresh weight;
DW, dry weight;
RACE, rapid amplification of
cDNA ends;
PCR, polymerase chain reaction;
16.0, palmitic acid;
16:1, palmitoleic acid;
t-16:1, trans-palmitoleic
acid;
16:2, hexadecadienoic acid;
16:3, hexadecatrienoic acid;
18:0, stearic acid;
18:1, oleic acid;
18:2, linoleic acid;
18:3, linolenic
acid;
9-HODE, 9-hydroxy-10,12(Z,E)-octadecadienoic acid;
t-9-HODE, 9-hydroxy-10,12 (E,E) octadecadienoic
acid;
13-HODE, 13-hydroxy-9,11(Z,E)-octadecadienoic acid;
t-13-HODE, 13-hydroxy-9,11(E,E) octadecadienoic
acid;
9-HOTE, 9-hydroxy-10,12,15(E,Z,Z)
octadecatrienoic acid;
12-HOTE, 12-hydroxy-9,13,15,(Z,E,Z)-octadecatrienoic acid;
13-HOTE, 13-hydroxy-9,11,15(Z,E,Z)-octadecatrienoic acid;
16-HOTE, 16-hydroxy-9,12,14,(Z,Z,E)-octadecatrienoic acid;
9-HPODE, 9-hydroperoxy-10,12(Z,E)-octadecadienoic acid;
13-HPODE, 13-hydroperoxy-9,11(Z,E)-octadecadienoic
acid;
9-HPOTE, 9-hydroperoxy-10,12,15(E,Z,Z)
octadecatrienoic acid;
13-HPOTE, 13-hydroperoxy-9,11,15(Z,E,Z)-octadecatrienoic acid;
tert-BuOOH, tert-butyl hydroperoxide.
Involvement of Lipoxygenase-dependent Production of
Fatty Acid Hydroperoxides in the Development of the Hypersensitive Cell
Death induced by Cryptogein on Tobacco Leaves*
,,,,,
CEA-Cadarache, Direction des Sciences du
Vivant, Département d'Ecophysiologie Végétale et de
Microbiologie, Laboratoire de Radiobiologie Végétale,
13108 Saint-Paul Lez Durance Cedex, § Laboratoire de
Biogénèse Membranaire, CNRS, UMR 5544, Université
Victor Ségalan Bordeaux II, 146 Rue Léo Saignant,
33076 Bordeaux Cedex, and ¶ Unité Associée
INRA-Université de Bourgogne 692, Laboratoire de Phytopharmacie
et Biochimie des Interactions Cellulaires,
BV 1540, Dijon Cedex, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Extraction was carried out, as described
previously (39). An aliquot of the extract (50 µl) was submitted to
straight phase HPLC (Waters, Millipore, St. Quentin-Yvelines, France)
using a Zorbax rx-SIL column (4.6 × 250 mm, 5 µm particle size,
Hewlett-Packard, Les Ullis, France), isocratic elution with 70/30/0.25
(v/v/v) hexane/diethyl ether/acetic acid at a flow rate of 1.5 ml/min, and UV detection at 234 nm. Hydroxy fatty acid isomers were identified using standards (41). Quantification was performed with reference to
15-HEDE, assuming that all hydroxy fatty acids have the same extinction
coefficient at 234 nm. For the enantiomer composition analysis,
chromatographic peaks were collected from straight phase HPLC and
submitted to chiral phase HPLC, using a Chiralcel OD column (250 × 4.6 mm, 5-µm particle size, Diacel Chemical Industries, Interchim,
France), with a mobile phase of 95/5/0.1 (v/v/v)
hexane/2-propanol/acetic acid at a flow rate of 1 ml/min, and UV
detection at 234 nm (40).
20 °C. After centrifugation (2,000 × g, 5 min), the supernatant was collected and the tissue pellet re-extracted with
2.2 ml of 5/5/1 (v/v/v) chloroform/methanol/water. Both extracts were
combined and washed with 3 ml of 0.2 M
H3PO4, 1 M KCl. Lipids were
recovered in the chloroform phase, dried under N2, and
dissolved in 0.5 ml of 2/1 (v/v) chloroform/methanol. Individual lipids were purified from the extracts by monodimensional TLC using either 25/25/25/10/9 (v/v/v/v/v) chloroform/methyl acetate/n-propyl
alcohol/0.25% aqueous KCl (w/v) for polar lipids or 90/15/2 (v/v/v)
hexane/diethyl ether/acetic acid for neutral lipids. Lipids were then
located by spraying the plates with a solution of 0.001% (w/v)
primuline in 80% acetone, followed by visualization under UV light.
The silica gel zones corresponding to individual lipids were scraped from the plates, and fatty acid methyl esters were prepared and analyzed as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
HPLC analysis of hydroxy fatty acids
extracted from control and cryptogein-treated leaves after
NaBH4 reduction and lipid hydrolysis. Control and
cryptogein-treated tobacco leaves were kept in the dark, and after
24 h, total hydroxy acids from free and bonded fatty acids were
extracted, using the NaBH4 reduction-hydrolysis procedure
and submitted to straight phase HPLC, as described under
"Experimental Procedures." A, HPLC traces of
cryptogein-treated leaf extract (lower trace) and of control
leaf extract (upper trace), using a Zorbax rx-SIL column.
The various hydroxy fatty acids isomers of 18:2 and 18:3 fatty acids
were identified, as described previously (41), and quantified with
reference to the internal standard 15-HEDE. In addition, among the four
isomers of 16:3, two were prepared from partly purified 16:3, using
soybean and tobacco LOX activities (13- and 9-specific on 18:2,
respectively), and were not detected in the chromatograms of both
control and elicited leaves (respective retention times 21.1 and 30.9 min). In the chromatogram of cryptogein-treated leaf extract, two early
eluting compounds appeared just before 15-HEDE, and were designated
X and Y. According to their UV spectra, both
compounds were not identified as hydroxy fatty acids. Hydroxy fatty
acids were collected from the preceding HPLC analyses and submitted to
chiral phase HPLC, using a Chiralcel OD column, as described under
"Experimental Procedures." B, separation of the
(9R)-HOTE and (9S)-HOTE enantiomers from control
leaf extract. C, separation of the (9R)-HOTE and
(9S)-HOTE enantiomers from cryptogein-treated leaf
extract.
Enantiomer composition of the hydroxy fatty acids obtained by the
NaBH4/hydrolysis procedure from control and cryptogein-treated
leaves
View larger version (25K):
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Fig. 2.
Time course of LOX-dependent
peroxidation and autoxidation in control and cryptogein-treated leaves;
relationship with tissue necrosis. Hydroxy fatty acids were
extracted from control and cryptogein-treated leaves at different times
following application, using the NaBH4 reduction-hydrolysis
procedure and analyzed by HPLC as described in Fig. 1. A,
open and closed symbols represented the level of
hydroxy fatty acids for water-treated and cryptogein-treated leaves,
respectively. 
, total hydroxy fatty acids; 
, 9-HOTE + 9-HODE
representative of 9-LOX activity; 
, 13-HODE + 13-HOTE representative
of 13-LOX activity; inset, 
, 12-HOTE + 16-HOTE,
representative of PUFA autoxidation. B, relationship between
leaf necrosis and total lipid peroxidation; tissue necrosis was
evaluated by measurement of leaf dehydration and total lipid
peroxidation by total hydroxy fatty acid level. Mean and S.D. from
three independent experiments are given.
View larger version (52K):
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Fig. 3.
Time course of the 9-LOX gene expression and
of 9-LOX activity in control and cryptogein-treated leaves.
Tobacco leaves were harvested after various times and frozen in liquid
nitrogen. Total RNA or protein extraction was carried out as described
under "Experimental Procedures." A, Northern blot
analysis of LOX transcripts accumulating in cryptogein-treated tobacco
leaves (15 µg of total RNA per lane); upper panel,
hybridization with the tobacco LOX probe; lower panel,
ethidium bromide staining of rRNA, as loading control. B,
Northern blot analysis of LOX transcripts from water-treated leaves;
conditions as in A. C, evolution of 9-LOX
activity analyzed in the same preceding experiment, with 18:2 as
substrate; open and closed symbols represented
the level of 9-LOX activity for water-treated and cryptogein-treated
leaves, respectively. Mean and S.D. from analyses carried out on three
independent extracts.
View larger version (20K):
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Fig. 4.
HPLC characterization of free hydroperoxy
fatty acids extracted from cryptogein-treated leaves. Total lipids
of 24 h cryptogein-treated leaves were extracted, omitting
reduction, by the methanol/chloroform procedure in the presence of
iron-chelating compounds, as described under "Experimental
Procedures." Hydroperoxides and hydroxy fatty acids, as well, were
collected from a silica Sep-Pak column and subjected to straight phase
HPLC, using a Zorbax rx-SIL column. Trace A, partial
chromatogram depicting hydroperoxide separation from cryptogein-treated
leaf extract. Trace B, reference hydroperoxides were
prepared from an equimolar mixture of 18:2 and 18:3 (1 mM),
with soybean LOX (Fluka; 1 mg/ml) in a pH 7 tetraborate buffer (10 mM), leading to a nonspecific peroxidation (42); after 20 min reaction at 25 °C, hydroperoxides were extracted with
hexane/ethyl ether (70/30) and submitted to HPLC, as above; the 13- and
9-isomers, mentioned in trace B, were collected, reduced
with NaBH4, and identified as hydroxy fatty acids.
Enantioselective peroxidation of 18:2, either by LOX extracts or in
planta; comparison of cryptogein- and MeJA-treated leaves
View larger version (24K):
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Fig. 5.
Changes in fatty acid composition of tobacco
leaves after cryptogein treatment. Total lipids were extracted
from control leaves before treatment and 18 h after water or
cryptogein application. Fatty acid composition of total lipids, or of
the various classes of lipids, separated by TLC, was evaluated by GPC,
as described under "Experimental Procedures." A, total
fatty acid composition. B, respective composition in 18:3
and 18:2 fatty acids, of galactolipids (GL),
phosphatidylcholine (PC), phosphatidylglycerol and
ethanolamine (PG-PE), neutral lipids (NL), and
free fatty acids (FA). Cross-hatched bars,
control, time 0; white bars, control, time 18 h;
black bars, cryptogein treatment, time 18 h. In order
to compare, results are expressed in µmol/g DW. Mean and S.D. from
three analyses of the same experiment are given, in which fatty acid
composition of total lipids (A) and of the various classes
of lipids (B) were analyzed simultaneously.
View larger version (36K):
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Fig. 6.
Effect of water dipping on leaf necrosis and
on the peroxidative metabolism of lipids. Cryptogein-treated and
control leaves were half-dipped into water 1 h after the treatment
and left to observe the development of the symptoms. The upper and
lower parts of leaves were analyzed at different times to determine the
evolution of the 9-LOX activity level and of 9-hydroperoxide metabolite
level by the NaBH4 reduction-hydrolysis procedure, as
described under "Experimental Procedures." A, symptoms
after 24 h of cryptogein-treated leaf (leaf upper face, left
panel; leaf lower face, right panel); leaf necrosis
appearing only in the upper aerial part. B, evolution of
9-LOX activity level and of 9-hydroxy fatty acid level; in order to
compare upper and lower part of the leaves, metabolite analysis results
were expressed in µmol/g DW. B, open and
closed symbols represented the levels for control and
cryptogein-treated leaves, respectively. , 9-LOX activity and
9-hydroxy fatty acid level of the upper aerial leaf part; 
, 9-LOX
activity and 9-hydroxy fatty acids level of the lower dipped leaf part.
Mean and S.D. of three independent experiments are given.
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Fig. 7.
LOX substrate and product infiltration on the
development of tobacco leaf necrosis. Leaves from control plants
were infiltrated between 2 secondary leaf veins with various
concentrations of compounds in 0.5% Tween 80. A,
infiltration of increased concentrations of 13-hydroperoxide of 18:2
(0, 1, 2, 5, and 10 mM).
B, comparison of 5 mM substrate reactivity was
carried out on the same leaf by infiltration of 18:2 free fatty acid
(18:2), tert-butyl hydroperoxide (tert-BuOOH),
13-hydroperoxide of 18:2 (HPODE), and the corresponding 13-hydroxy 18:2
fatty acid (HODE). C, leaves from 5 days MeJA-treated
tobacco plants, as described under "Experimental Procedures",
infiltrated with 1, 2, 5, or 10 mM 18:2 fatty acid. The
symptoms presented in A-C were those observed after 4 h of incubation. Typical photographs from triplicate experiments
leading to the same results were shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (125K):
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Fig. 8.
Schematic overview of the hypothetical
mechanism for free fatty acid hydroperoxide production induced by
cryptogein on tobacco leaves and leading to cell death. Massive
free PUFA hydroperoxide formation from membrane PUFAs was shown to
induce HR necrotic symptoms. The production was attributed to the
conjunction of an increase in LOX and putative lipase activities.
Transient accumulation of LOX mRNA transcripts was described, and
it was proposed that JA is likely involved in the regulation of this
process. Membrane autoxidation, being a minor and late process, was
likely attributed to the effects of PUFA hydroperoxides, rather than to
AOS, produced early during the oxidative burst.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: CEA, Laboratoire
de Radiobiologie Végétale, Dépt. d'Ecophysiologie
Végétale et de Microbiologie, Centre d'Etudes de
Cadarache, 13108 Saint-Paul Lez Durance Cedex, France. Tel.: 33 (0)4 42 25 64 86; Fax: 33 (0)4 42 25 62 86; E-mail:
ctriantaphylid@cea.fr.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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