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PLANTS INTERACTING WITH OTHER ORGANISMS

Fatty Acid Hydroperoxides and H₂O₂ in the Execution of Hypersensitive Cell Death in Tobacco Leaves^1,[w]

Commissariat à l'Energie Atomique/Cadarache, Direction des Sciences du Vivant, Département d'Ecophysiologie Végétale et de Microbiologie, Laboratoire de Radiobiologie Végétale, F–13108 Saint-Paul-Lez-Durance cedex, France (J.-L.M., C.R., J.-P.A., C.B., C.T.); and Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B–9052 Ghent, Belgium (S.C., J.D., B.v.d.C., D.I., F.V.B.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H₂O₂-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H₂O₂ plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H₂O₂ and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H₂O₂ and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

The signaling cascades leading to the HR are starting to be elucidated. Several resistance genes have been cloned, and protein kinases, phosphatases, and GTP-binding proteins have all been implicated downstream of these recognition proteins (Martin et al., 2003; Rathjen and Moffett, 2003). Changes in ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS) and nitric oxide (NO) are among the earliest events following pathogen infection or elicitor treatment in cultured plant cells (Doke, 1997; Grant and Loake, 2000; Wendehenne et al., 2004). The production of ROS is biphasic, with a sustained second oxidative burst in response to an avirulent pathogen, and monophasic and transient with a virulent pathogen. This suggests that ROS production may be responsible for some of the defense-associated processes (Lamb and Dixon, 1997). Furthermore, ROS, and specifically H₂O₂, are key modulators of NO in triggering plant cell death (Delledonne et al., 2001; Neill et al., 2002; Wendehenne et al., 2004).

In order to obtain further insight into the role of H₂O₂ in plant signaling and cell death, we used transgenic tobacco (Nicotiana tabacum) plants (Cat1AS), which only express 10% of wild-type catalase activity. CAT1 is the dominant catalase isoform in leaves, where it serves in the removal of photorespiratory-derived H₂O₂ (Willekens et al., 1997). Cat1AS plants are phenotypically very similar to wild-type plants when grown under low-light (LL) conditions, and variable in planta H₂O₂ concentrations may be achieved by modulating light intensities (Dat et al., 2003). This experimental model was previously used to mimic a sustained oxidative burst, allowing the role of H₂O₂ in ethylene and salicylic acid production to be pinpointed, as well as pathogenesis-related protein accumulation (Chamnongpol et al., 1998; Dat et al., 2001). In addition, pathogen infection of Cat1AS plants or transgenic tobacco plants with reduced ascorbate peroxidase activity demonstrated the direct involvement of H₂O₂ in limiting pathogen spread and suggested its participation in HR cell death (Mittler et al., 1999). More recently, using Cat1AS tobacco plants and modulating light intensity, we showed evidence of the role of H₂O₂ in triggering cell death in tobacco (Dat et al., 2003). A genome-wide transcriptome analysis revealed that a sustained H₂O₂ stress in Cat1AS plants mimicked the molecular response reported for biotic and abiotic stresses, including cell death (Vandenabeele et al., 2003). In this study, we aimed to obtain additional insight into the role of H₂O₂ in the execution phase of plant cell death.

In addition to the involvement of oxidative stress, cytochrome c release, and the activation of nucleases and proteases during plant cell death execution, we have proposed additional executioners involved in leaf HR cell death, which find their origin in the catabolism of membranes (Rustérucci et al., 1999; Lam, 2004). We showed that a 9-lipoxygenase (LOX)-dependent massive production of free fatty acid hydroperoxides operated during cryptogein-induced HR in tobacco. This process involves both galactolipase and 9-LOX activities targeting chloroplastic lipids (Cacas et al., 2005). This active lipid peroxidation is sufficient to induce plant cell death. Using antisense constructs, 9-LOX was shown as being essential in preventing further pathogen invasion in tobacco (Rancé et al., 1998). The 9-LOX metabolism is rapidly activated during incompatible plant-pathogen interactions, whereas it is delayed and reduced in compatible interactions (Jalloul et al., 2002; Montillet et al., 2002; Vailleau et al., 2002). A LOX-dependent programmed cell death was also shown on lentil (Lens culinaris) root protoplasts treated with H₂O₂ (Maccarone et al., 2000) and application of fatty acid hydroperoxides to tomato (Lycopersicon esculentum) protoplasts leads to all features of programmed cell death (Knight et al., 2001).

We have recently established an oxylipin profile of Arabidopsis (Arabidopsis thaliana) and shown that it is possible to discriminate 9-LOX-dependent, 13-LOX-dependent, and ROS-mediated lipid peroxidation (Montillet et al., 2004). Using such a profile, we assessed the modulation of the 9-oxylipin metabolism during tobacco HR and showed that, in cryptogein-elicited leaves, light inhibits both 9-LOX oxylipin metabolism and HR cell death (Cacas et al., 2005). In contrast, in Ralstonia-infected leaves, the HR is characterized by both 9-LOX-dependent and ROS-mediated lipid peroxidation. To date, ROS-mediated lipid peroxidation is assumed to reflect the production of H₂O₂; however, this has never been clearly assessed. Thus, we decided to directly assess the interplay between H₂O₂ and lipid hydroperoxide production and their relationship with plant cell death. Cat1AS tobacco plants were used as a model system (Dat et al., 2001) to compare pathogen- and elicitor-induced HR under variable H₂O₂ background levels. For this, cell death phenology and lipid peroxidation processes were monitored following sustained H₂O₂ stress, cryptogein application, or Pseudomonas syringae pv syringae infection. This allowed us to assess whether H₂O₂ is only involved in early signal transduction or is also involved in the execution of cell death during the HR. In addition, we address the importance of light in the development of HR cell death.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Oxidative Stress-Induced and HR Cell Death Phenology Is Different

We first compared the cell death phenology induced by a sustained H₂O₂ stress in Cat1AS tobacco plants with that provoked in plants infected with an avirulent pathogen. Therefore, all plants were pregrown in a 14/10-h day/night cycle at LL (100 µmol m^–2 s^–1 fluence rate), and both treatments were initiated at the start of the day. Cat1AS plants were exposed to high-light (HL) stress (350 µmol m^–2 s^–1 fluence rate), resulting in the increased production of photorespiratory H₂O₂ as described earlier (Willekens et al., 1997; Dat et al., 2003). In a parallel experiment, tobacco wild-type plants were inoculated with 10⁷ cfu mL^–1 of P. syringae pv syringae. The first signs of leaf injury appeared in HL-exposed Cat1AS plants within 12 h, although bleached lesions only became prominent after 36 h (Fig. 1B). By then, all palisade parenchyma cells had collapsed, while both the upper and lower epidermal cell layer and most of the spongy parenchyma cells remained macroscopically untouched (Fig. 1F). In contrast, in pathogen-infected tobacco leaves exposed to HL, all cell layers were destroyed within 24 h (Fig. 1, C and G). This was also reflected by the complete dehydration of the infected leaf segments. In the light, symptom development was also characterized by bleaching (Fig. 1C), a phenotype not observed for infection in the dark, however, which also leads to complete dehydration (Fig. 1D).

View larger version (80K): [in this window] [in a new window]   Figure 1. Leaf symptoms induced by HL in catalase-deficient tobacco (Cat1AS) and by pathogen infection or cryptogein application to wild-type tobacco leaves. All plants were grown at LL (100 µmol m–2 s–1 fluence rate) in a 14/10-h day/night cycle. A to G, Cat1AS tobacco plants were transferred to HL conditions (350 µmol m–2 s–1 fluence rate) at the beginning of the day; for comparison, Pseudomonas-inoculated wild types were transferred similarly to HL. A, Control leaf of Cat1AS plant. B, Leaf of Cat1AS plant exposed to HL for 31 h. C, Necrotic symptoms of Pseudomonas-inoculated wild-type leaf exposed to HL for 31 h. D, Same as in C, but incubation in complete darkness; Pseudomonas inoculation (107 cfu mL–1) was carried out between two secondary leaf veins on the left part of the leaf and mock inoculation on the right part. E to G, Microscopic photographs of leaf sections corresponding to leaves shown in A to C, respectively. While completely bleached (B), the Cat1AS leaf showed absence of tissue collapse (F), whereas the infected wild-type leaf (C and D) displayed complete tissue dehydration (G). H and I, Cryptogein (10 µL at 0.2 µg µL–1) was applied to the petiole of excised leaves at the end of the night period and then incubated in the dark for 22 h (H) or at HL in a day/night regime of 12/12 for 48 h (I). For I, the symptoms started appearing after 22 h (data not shown) and were fully developed within 48 h. H and I, Each photograph shows, from left to right, leaves from the same tobacco wild-type plant corresponding to the 7- to 10-leaf rank starting from the flower bud. The symptoms obtained in a night/day or continuous light regime were similar to those described in I. Photographs from one of two independent experiments are shown.

A very similar cell death phenology as in the Pseudomonas- and cryptogein-induced HR was also observed within 24 h after (1) the infiltration of 1 M H₂O₂ in Cat1AS leaves in a continuous dark or normal day/night regime (LL); (2) a transient intense light exposure of Cat1AS plants (7 h at 1,000 µmol m^–2 s^–1 fluence rate), followed by continuous dark or a normal day/night regime at LL; and (3) the infiltration of the NO donor, sodium nitroprusside (SNP; 1–2 mM), in leaf segments of wild-type plants and incubation either in the dark or after a transient HL exposure (6 h) and dark (data not shown).

From these experiments, we can conclude that the cell death phenology provoked by a sustained H₂O₂ stress (phenotype I) and an avirulent pathogen infection, cryptogein elicitation, or H₂O₂ and NO pulses (phenotype II) are clearly different. In phenotype I, cell death is restricted to the palisade layer, whereas in phenotype II, all cell layers collapse, leading to a total dehydration of the leaf. In the latter case, subsequent bleaching takes place in the light.

H₂O₂ Stress Induces a Free Radical-Mediated Membrane Lipid Peroxidation Driven by Light

In order to investigate ROS-mediated lipid peroxidation in relation to H₂O₂ production, we took advantage of the Cat1AS plants and analyzed the effects of (1) increased production of photorespiratory H₂O₂ and (2) apoplastic infiltration of H₂O₂, both treatments leading to cell death symptoms (see above).

We assessed the kinetics of lipid peroxidation accumulation in both Cat1AS and wild-type plants transferred from LL to HL by HPLC analysis of the hydroxy fatty acids (HFAs), as previously described (Rustérucci et al., 1999). HFAs were hardly detectable in wild-type leaves but started to accumulate after 2 d of sustained H₂O₂ stress in Cat1AS leaves (Fig. 2A). Lipid peroxidation largely coincided with the appearance of bleaching, and HFA levels reached a plateau within 31 h (total hydroxy octadecatrienoic acids [HOTEs]; Fig. 2A, 1.3 µmol g^–1 dry weight [DW]; see Table I legend for HFA abbreviations). As the kinetics of accumulation of each positional HFA of both 18:2 (data not shown) and 18:3 (Fig. 2A) were identical and reached similar levels, this is most likely the result of a free radical-mediated lipid peroxidation instead of one catalyzed by an enzymatic process. Chiral phase HPLC analysis of each HFA showed that, in nonstressed wild-type and Cat1AS leaves as well as in stressed wild-type leaves, 13-hydroxy octadecadienoic acid (HODE) and 13-HOTE showed some S-specificity (Table I). This is indicative of a basal constitutive (13S)-LOX activity, as previously noticed (Rustérucci et al., 1999). In stressed Cat1AS leaves, all tested isomers were clearly racemic, confirming a free radical-mediated process (Table I). Additionally, we assessed membrane lipid peroxidation by analyzing free and esterified products of lipid peroxidation and the hydroperoxide formation using the volatile alkane assay (Degousée et al., 1995). We observed that at least 90% of HFAs are esterified and hydroperoxides accumulate at high levels, demonstrating membrane lipid peroxidation (see Supplemental Fig. 1). Lipid peroxidation was then investigated in leaves of wild-type and Cat1AS plants infiltrated with 1 M H₂O₂ and incubated either in the dark or in the light for 24 h. Both cell death and lipid peroxidation were absent on the infiltrated areas of wild-type leaves because endogenous catalase levels act as an efficient sink for the infiltrated H₂O₂ (data not shown; Willekens et al., 1997). In contrast, Cat1AS-infiltrated leaves developed cell death symptoms in the dark on about 40% of the infiltrated area together with lipid peroxidation (total HOTEs, 0.68 µmol g^–1 DW, as compared to 0.15 for controls). Moreover, in a day/night cycle at LL, this process was increased 10-fold (6.7 µmol g^–1 DW), with cell death symptoms covering the entire infiltrated area. Interestingly, the HOTE isomer distribution is randomized and similar to the distribution obtained following a sustained H₂O₂ stress in HL-treated Cat1AS plants (see Fig. 2B for comparison). Such randomized HOTE isomer and enantiomer distribution (Table I) is again a marker of a free radical-mediated lipid peroxidation process (Montillet et al., 2004).

View larger version (23K): [in this window] [in a new window]   Figure 2. Lipid peroxidation in leaves of catalase-deficient transgenic (Cat1AS) and wild-type (WT) tobacco plants after transfer to HL. A, Kinetics of lipid peroxidation given for each specific isomer of 18:3 for Cat1AS and wild-type plants transferred to HL conditions (350 µmol m–2 s–1 fluence rate) at the beginning of the day (day/night, 14/10 h). Lipid hydroperoxides of leaf extracts are analyzed by HPLC as HFAs obtained by the NaBH4 hydrolysis procedure. Results expressed as mean ± SD from three independent leaves. B, ROS-mediated isomer distribution for the HFAs of linolenic acid (18:3) in tobacco leaves. Inset, Peroxidation positions for 18:3. The isomer distribution for HL was obtained from the results described in A; results expressed as mean ± SD (n = 9). The distribution for H2O2 was obtained from Cat1AS leaves infiltrated with 1 M H2O2 and incubated in a normal day/night regime (LL); the oxylipin signature associated with the necrotic symptoms was characterized after 24 h. Results expressed as mean ± SD (n = 4). See Table I for abbreviations.

To assess the nature of lipid peroxidation in the different cell death phenotypes described above, we used the ROS-mediated lipid peroxidation characteristics described in Figure 2B and the recently established oxylipin-profiling methodology, which is based on analysis of the HOTE isomer distribution (Montillet et al., 2004). This profiling method allows discrimination between LOX-dependent and ROS-mediated lipid peroxidation in plant tissues.

A 7-h intense light exposure (1,000 µmol m^–2 s^–1 fluence rate) leads to cell death in Cat1AS leaves within 24 h (see above). This cell death is not accompanied by an intense accumulation of HFAs (Fig. 3A) and similar results were also obtained by the infiltration of the NO donor SNP (Fig. 3B). In both cases, the lipid peroxidation level increased to maximum twice, as compared to controls, but never exceeded 0.5 µmol g^–1 DW. These levels are low compared to those observed in oxidative and HR cell death (see above and below). Oxylipin profiling was done similarly on cryptogein-elicited wild-type leaves following either a continuous light, a day/night (12/12), or a night/day (12/12) regime at HL and compared to the dark situation (see above; Fig. 1, H and I). Results from the two oldest leaves, showing marked death symptoms, revealed that, for all light conditions, total lipid peroxidation levels increased (at least to 1.1 µmol g^–1 DW; Fig. 3C), as compared to mock-treated leaves (0.2 µmol g^–1 DW); however, the increase is much less than that in the dark (3.7 µmol g^–1 DW). In the light, the ROS-mediated process was mainly observed, whereas 9-LOX-dependent lipid peroxidation prevailed during complete darkness (Fig. 3C). Chiral analyses of the individual HFAs were confirmatory; whereas the 9-isomers of cryptogein-treated leaves exhibited 90% (S)-enantiospecificity in the dark, they were only partly chiral with an (S)-enantiomer composition around 63% in the light (Table I).

View larger version (23K): [in this window] [in a new window]   Figure 3. Lipid peroxidation in tobacco leaves undergoing HR-like and HR symptoms induced by transient HL or NO and cryptogein (Cry). Lipid peroxidation was investigated according to the methodology previously described, showing total ROS-mediated, 13-LOX-, and 9-LOX-dependent lipid peroxidation (Montillet et al., 2004). A, Leaves from wild-type (WT) and Cat1AS plants were submitted at the beginning of the day period to a transient intense light (1,000 µmol m–2 s–1 fluence rate; 7 h), followed by a return to normal light conditions (LL; 100 µmol m–2 s–1 fluence rate; day/night, 14/10 h). The oxylipin signature was analyzed after 24 h of incubation. Wild type and AS1 T-HL represented leaves submitted to the transient intense light condition. For AS1 T-HL1 and AS1 T-HL2, the symptoms covered 20% to 40% and about 50% of the leaf surface, respectively. B, Leaves from wild-type plants were infiltrated with 1 to 2 mM SNP on the right side of the leaf, the left side being mock infiltrated. The infiltration was carried out 2 h after the beginning of the day and the leaves were incubated either in the dark for 24 h or at HL for 6 h (350 µmol m–2 s–1 fluence rate), followed by an additional 18 h in the dark. Under both conditions, necrotic areas represented at least 80% of the SNP-infiltrated leaf surface. Dark and Light indicate both conditions, Mock indicates water infiltration, and 1 to 2 mM represents SNP-infiltrated leaves at the indicated concentration. C, Lipid peroxidation was characterized from cryptogein-elicited wild-type leaves (rank 9–10; see Fig. 1I) incubated for 48 h at HL in a night/day (N/D) or a day/night (D/N) cycle (12/12), under continuous light and for 30 h in the dark. See Table I for abbreviations. Results expressed as mean ± SD. For A, n = 7; for B and C, n = 3.

Cryptogein Action on Leaves of Cat1AS Plants and Light Effects Reveal the Respective Roles of Fatty Acid Hydroperoxides and H₂O₂ in the Execution of HR Cell Death

In order to directly investigate the respective roles of 9-LOX metabolism and H₂O₂ in the execution of cell death, we compared the effect of cryptogein on cell death events under dark and HL conditions in wild-type and Cat1AS plants. In the light, we additionally compared the effects of ambient and high CO₂ concentration to modulate the H₂O₂ background. High CO₂ levels impair the accumulation of HL-induced photorespiratory H₂O₂ in Cat1AS plants (Willekens et al., 1997).

The development of cell death on cryptogein-treated leaves kept in the dark, done by measuring solute leakage and water loss, was similar for wild-type and Cat1AS leaves (Fig. 4, A and B). Solute leakage precedes dehydration and all leaves were fully necrotic within 24 h. In parallel, total lipid peroxidation rose similarly in wild-type and Cat1AS leaves (Fig. 4C), with a massive accumulation of 9-LOX metabolites (80%–90% of total lipid peroxidation; data not shown). These results strongly suggest that an early and massive production of fatty acid hydroperoxides is enough to trigger HR cell death in the dark (Rustérucci et al., 1999). When the same infection was done with cryptogein under continuous HL conditions, either at ambient CO₂ (360 ppm) or at saturating CO₂ levels (3,000 ppm; to inhibit photorespiratory H₂O₂ production), cell death was delayed in wild-type leaves. Indeed necrotic symptoms only appeared after 48 h. Oxylipin profiles, characterized on each individual leaf, coincided with necrosis development (see Supplemental Fig. 2) and are indicative of a ROS-mediated process (Fig. 5). Although the average lipid peroxidation levels were 20% lower at high CO₂ levels, cell death phenology in wild type was similar under both CO₂ conditions. Contrary to wild type, the cryptogein-treated leaves from Cat1AS plants placed under 360 ppm CO₂ and under continuous HL were already completely necrotic within 22 h, while mock-treated Cat1AS leaves only showed tissue bleaching along the main veins due to the HL exposure (see also Dat et al., 2003). Under saturating CO₂, the timing of symptom development in the cryptogein-treated Cat1AS leaves was comparable to that observed under ambient CO₂ conditions, but the symptoms were much less developed (10%–20%; see Supplemental Fig. 2). In addition, the oxylipin profiles after 22 h of cryptogein-treated Cat1AS plants under high CO₂ levels revealed a 3-fold decrease of total HFA levels (Fig. 5). Taken together, these findings clearly show that, in Cat1AS leaves treated with cryptogein, the increased photorespiratory H₂O₂ levels are able to accelerate and/or aggravate the effect of the oxidative burst in the execution of cell death, in correlation with lipid peroxidation. In all cases, a ROS-mediated process is mainly observed (Fig. 5). Chiral analyses of individual HFAs confirmed this result (Table I) and showed that the 9-LOX-dependent process occurs at levels not exceeding 10% to 15% of total lipid peroxidation.

View larger version (17K): [in this window] [in a new window]   Figure 4. Comparison of cryptogein (Cry)-induced development of HR symptoms in the dark on detached leaves of wild-type (WT) and catalase-deficient transgenic (Cat1AS) plants. A, Solute leakage from leaf discs. B, Leaf dehydration. C, Total lipid peroxidation. See "Materials and Methods" for conditions. Results are expressed as mean ± SD from three independent experiments.

View larger version (19K): [in this window] [in a new window]   Figure 5. Lipid peroxidation of wild-type (WT) and catalase-deficient transgenic (Cat1AS) tobacco leaves treated with cryptogein and exposed to continuous HL under low and high CO2 conditions. Detached leaves from wild-type (A) and Cat1AS (B) plants were placed into a closed chamber under continuous HL (350 µmol m–2 s–1 fluence rate) under normal (360 ppm) or at high (3,000 ppm) CO2 conditions in order to inhibit photorespiration. The upstream oxylipin profile was determined for individual control leaves and elicited leaves undergoing HR symptoms, after 22 h for Cat1AS and 48 h for wild type (see Supplemental Fig. 2 for individual leaf analyses); see Figure 3 for lipid peroxidation characterization. Results are expressed as mean ± SD. For controls, n = 3; for cryptogein-elicited leaves, n = 6 to 9.

Both ROS- and 9-LOX-Mediated Lipid Peroxidation Occur in Response to the Avirulent Pathogen P. syringae pv syringae

In order to get further insight into the interplay between light and oxidative stress during the HR upon pathogen infection, we extended the cryptogein studies to an avirulent tobacco pathogen. Therefore, we infected wild-type and Cat1AS leaves of plants grown at LL with P. syringae pv syringae under either a day/night regime at HL or complete darkness.

For the day/night experiment, pathogens were infiltrated (5 x 10⁷ cfu mL^–1 in MgCl₂ 10 mM) at the start of the day period. In both wild-type and Cat1AS-infected leaves, cell death led to complete tissue dehydration within 24 and 48 h for Cat1AS and wild-type leaves, respectively. Assessment of the upstream oxylipin profile revealed that, in both wild-type and Cat1AS plants, total lipid peroxidation of 18:3 increased 5 to 7 times as compared to mock-treated plants within 24 h (Fig. 6A). This increase is due in part to 9-LOX-dependent lipid peroxidation, which represents 31% and 50% of total peroxidation for wild-type and Cat1AS leaves, respectively. After 48 h, however, ROS-mediated lipid peroxidation prevailed in the wild-type leaves. In the dark, symptoms were fully developed within 24 h, and Cat1AS leaves were fully desiccated, whereas dehydration in wild-type leaves was lower. Total lipid peroxidation of 18:3 increased 10-fold for wild-type and 30-fold for Cat1AS plants (Fig. 6B). The process shows an important 9-LOX-dependent lipid peroxidation, representing 65% to 75% of the increase in the wild-type and close to 90% in Cat1AS leaves. Further insight into 9-LOX involvement in HR development was obtained with the chiral phase HPLC analysis of the HFA isomers of wild-type leaves. Interestingly, both in the day/night cycle and in the dark, Pseudomonas infection led to the production of (9S)-HODE and (9S)-HOTE (Table II), in addition to the basal levels of (13S)-LOX activity in mock- and pathogen-infiltrated wild-type leaves. Consistent with our previous analysis, the enantioselectivity is bigger in the dark condition [88% for (9S)-HOTE] than during a day/night cycle [from 81% at 24 h to 66% at 48 h for (9S)-HOTE], thus demonstrating the early onset of a (9S)-LOX-dependent peroxidation pathway following Pseudomonas infection, together with a light-driven ROS-mediated process that prevails over time in the light.

View larger version (18K): [in this window] [in a new window]   Figure 6. Comparison of lipid peroxidation in P. syringae pv syringae-infected leaves of wild-type (WT) and catalase-deficient transgenic (Cat1AS) plants. Leaves from plants grown at LL (100 µmol m–2 s–1 fluence rate; day/night, 14/10 h) were infiltrated with P. syringae pv syringae (5 x 107 cfu mL–1 in 10 mM MgCl2) and incubated in (A) HL (350 µmol m–2 s–1 fluence rate; day/night, 14/10 h) or (B) the dark. The upstream oxylipin profile of Pseudomonas-inoculated leaves was determined after 24 h of incubation for wild type (WT PS 24 h) and Cat1AS (AS1 PS 24 h) plants and also after 48 h for wild-type (WT PS 48 h) plants; MgCl2-inoculated leaves were included as controls and analyzed after 24 h of incubation (WT Mock and AS1 Mock). See Figure 3 for lipid peroxidation characterization. Results expressed as mean ± SD (n = 3).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

To characterize in more detail the different cell death processes, we used a methodology that distinguishes between 9-LOX-, 13-LOX-, and ROS-mediated lipid peroxidation processes (Montillet et al., 2004). We showed previously that, in the dark, the HR in tobacco is characterized by the induction of the 9-oxylipin metabolism (Rustérucci et al., 1999) and that this process can be inhibited by light (Cacas et al., 2005). In this work, we show that, under various light conditions, the cryptogein-induced HR is (1) inhibited in young, fully developed leaves but not in the older leaves of the plant; (2) not dependent on the timing of the elicitation under a day/night cycle; and (3) mainly characterized by a ROS-mediated lipid peroxidation. Thus, by comparing the lipid peroxidation characteristics of cryptogein-induced HR in the dark and in the light, both in wild-type and Cat1AS plants, we were able to investigate the role of H₂O₂ in HR cell death. Under dark conditions, the development of cryptogein-induced HR was identical in both Cat1AS and wild-type leaves and the lipid peroxidation was characterized by the same massive production of the 9-LOX products. Thus, H₂O₂ detoxification by catalase is not essential for symptom development in the dark, and H₂O₂ is not involved as a signal in the induction of the 9-oxylipin pathway. Furthermore, our results also suggest that H₂O₂ produced by the oxidative burst in the dark does not play an important role in the execution of cell death, but that an early and massive production of fatty acid hydroperoxides has a more prominent role in this execution. Under continuous light conditions, we have observed that (1) the cell death symptoms (if not completely blocked in fully developed young leaves), which are correlated with the increase in lipid peroxidation levels, are delayed in comparison to the dark situation; (2) the main lipid peroxidation process is mediated by the production of H₂O₂, whereas 9-LOX metabolism is low; (3) when catalase activity is decreased (Cat1AS plants), the effect of H₂O₂ is amplified leading both to earlier development of symptoms and to increased ROS-mediated lipid peroxidation; and (4) under high CO₂, in Cat1AS leaves, both symptoms and ROS-mediated lipid peroxidation are decreased, showing that photorespiratory H₂O₂ participates here in cell death execution. Taken together, these results show that the production of both fatty acid hydroperoxides and H₂O₂ occurs in tobacco HR and can be considered as participants in the execution of HR cell death.

Oxylipin metabolism in the dark involves the induction of galactolipase and 9-LOX activities, respectively (Cacas et al., 2005). This metabolism can be induced early after pathogen assault, operates within 24 h, and is sufficient for the development of HR symptoms. During cryptogein elicitation in the light, this oxylipin pathway is inhibited at the transcriptional level. Under those conditions, the light-driven increase in H₂O₂ levels leads to ROS-mediated peroxidation of the membranes in a later phase. Since this ROS-mediated lipid peroxidation is closely correlated to cell death symptoms, and since these two processes occur earlier, are more pronounced, and are also inhibited partly under high CO₂ in cryptogein-elicited Cat1AS leaves, it can be argued that H₂O₂ plays a direct role in the execution of cell death. The fact that H₂O₂-detoxifying enzyme activities, catalase, and cytosolic ascorbate peroxidase decline during the development of HR in leaves (Dorey et al., 1998; Mittler et al., 1998) consolidates our observations. Interestingly, in the infection by the incompatible pathogen P. syringae pv syringae, our results indicate that 9-LOX metabolism and H₂O₂ production are both involved in the HR process.

H₂O₂ is known to play different roles in the HR (Doke, 1997; Lamb and Dixon, 1997; Grant and Loake, 2000). It contributes to cell wall reinforcement, limiting pathogen spread, and it can also directly participate in pathogen killing. For example, in tobacco, the modulation of catalase activity by antisense technology leads to more resistant plants against pathogen attack (Mittler et al., 1999), whereas overproduction leads to more sensitive plants (Polidoros et al., 2001; Talarczyk et al., 2002). H₂O₂ was shown to initiate a cell death program in Arabidopsis plants and suspension cultures (Desikan et al., 1998; Vandenabeele et al., 2004), in tobacco BY-2 cells (Houot et al., 2001), and in Cat1AS tobacco plants exposed to HL to activate a cell death process that involves the activation of an oxidative burst (Dat et al., 2003). Here, we show that H₂O₂ can also be involved in late execution during HR programmed cell death via light-dependent membrane lipid peroxidation. To explain the role of light, it might be argued that the Fenton reaction, leading to HO^· production from H₂O₂ and thus to lipid peroxidation, needs a cyclic reduction of Fe³⁺ into Fe²⁺ (Halliwell and Gutteridge, 2000). This process can be achieved by a light-driven recycling of redox compounds such as ascorbate. Additionally, the free fatty acid hydroperoxides that are produced enzymatically can also be substrates of Fenton-like reactions, leading, similarly, to the production of alkoxy radicals (Halliwell and Gutteridge, 2000) and thus enhancing the free radical-mediated lipid peroxidation in the light.

Investigation of the light effect in plant pathogen interactions is only in its infancy (Karpinski et al., 2003). In Arabidopsis, a light-signaling pathway was shown to interact with the salicylic acid-mediated signal transduction pathway and necessary to trigger the HR response to pathogens (Genoud et al., 2002; Zeier et al., 2004). Additionally, in the lsd1 mutant, the runaway cell death, but not the initial HR after Pseudomonas parasitica inoculation, was shown to be dependent on light and to involve photorespiratory H₂O₂ production (Mateo et al., 2004). More specifically, light-mediated leaf cell redox homeostasis and antioxidant cross-talk between mitochondria and the other organelles was shown to be essential in plant resistance to tobacco mosaic virus (Dutilleul et al., 2003). In tobacco cells, the involvement of redox status change of ascorbate and glutathione was proposed as part of the signaling pathways leading to programmed cell death (de Pinto et al., 2002). Redox regulation of transcription factors in eukaryotes was initially demonstrated in yeast (Saccharomyces cerevisiae; Delaunay et al., 2002), while NPR1, a key transcriptional regulator of plant systemic acquired resistance, is under redox control (Mou et al., 2003). In cryptogein-elicited leaves, the light effect on the inhibition of the 9-oxylipin pathway might be related to such redox regulation. However, if in the elicited material this metabolism can be switched off, keeping a place for H₂O₂-involved HR cell death, in the case of pathogen infection the response is composite. Indeed, in Pseudomonas-infected leaves, under normal light conditions, the ROS-mediated lipid peroxidation appears as the main process but, in addition, the activation of the 9-oxylipin pathway also leads to a massive accumulation of (9S) fatty acid hydroperoxides (30%–50% of total lipid peroxidation). Such a production might be at the origin of signaling and death processes involving reactive electrophile species (Farmer et al., 2003). Many aspects of the light response remain to be elucidated and, in this way, the cryptogein model might help in the characterization of the key factors involved in the induction/regulation of the 9-oxylipin pathway leading to the massive production of fatty acid hydroperoxides.

Besides both types of hydroperoxide production, many other parallel processes, such as the activation of nucleases and proteases, are involved in HR cell death execution. The fact that different effectors of cell death can operate independently also provides the plant with a diverse scala of strategies to execute different and specific types of cell death (e.g. during pathogen and developmentally driven cell death processes; Heath, 2000).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Treatments

Cat1AS is a transgenic line of tobacco (Nicotiana tabacum) cv Petit Havana SR1 with approximately 10% of wild-type catalase activity (Chamnongpol et al., 1996). Cat1AS and control wild-type plants (SR1) were precultivated under LL (100 ± 20 µmol m^–2 s^–1 fluence rate; day/night, 14/10 h) at 25°C and 70% relative humidity. Mature preflowering plants (6–8 weeks old) were used for all experiments. Otherwise stated, HL treatment consisted of 350 ± 50 µmol m^–2 s^–1 fluence rate, with the selected lightning cycle. H₂O₂ (1 M) or SNP (1 or 2 mM) treatments were carried out by infiltration, applying the syringe tip to the epidermis of the leaves.

For the elicitation experiments, leaves were detached from the plants and treated at the petiole with 10 µL of an aqueous solution of cryptogein (0.2 µg µL^–1), followed after absorption by 3 x 10 µL of water. Control leaves were treated similarly with water. The leaves were then placed horizontally into a couple of closed chambers and watered by the petiole. A couple of chambers allow controlled lighting, humidity (70%), and the capacity to simultaneously carry out the experiments under low and high CO₂ atmosphere (360 or 3,000 ppm).

Plants were maintained at LL (day/night, 14/10 h) before pathogen infection. For induction of the HR, 5 x 10⁷ cfu mL^–1 of Pseudomonas syringae pv syringae in 10 mM MgCl₂ was inoculated by leaf infiltration (Chamnongpol et al., 1996), 50% to 75% of the leaf surface being infiltrated with the bacteria. The Pseudomonas-infected wild-type plants were maintained either in the dark or at HL (day/night, 14/10 h). Mock-infiltrated plants (10 mM MgCl₂) were used for controls.

Lipid Peroxidation Analyses

Total free and esterified hydroxy and hydroperoxy fatty acids were analyzed by HPLC as free HFAs, after NaBH₄ reduction and hydrolysis, as described previously (Rustérucci et al., 1999; Montillet et al., 2004).

Solute leakage was determined by measuring the conductivity change of pure water (5 mL; 18 M) after 2-h incubation of five leaf discs (diameter 9 mm), as referred to the conductivity at equilibrium.

Microscopic Analysis of Phenotypes

For sectioning, fresh plant material was fixed in formaldehyde (5%), acetic acid (5%), and alcohol (45%), and passed over a graded ethanol series. Infiltration and embedding in Historesin (7022–18500 Leica Historesin embedding kit; Leica Microsystems, Heidelberg) were performed according to the manufacturer's instructions. Sections (5–10 µm) were cut on a rotary microtome (Minot-Milerotonn 1212; Leitz, Wetzlar, Germany) with disposable Superlab Knifes (Adamas Instrumenten, Leersum, The Netherlands). Morphological sections of leaves were stained with 0.05% toluidine blue for 10 min before examination under a light microscope.

	ACKNOWLEDGMENTS

 
We thank the GRAP team (CEA/Cadarache, DSV-DEVM) for technical support in plant culture and experiments in closed chambers, Stijn Morsa for excellent technical assistance, and Céline Davoine for reading and editing the manuscript.

Received January 19, 2005; returned for revision April 13, 2005; accepted April 25, 2005.

	FOOTNOTES

 
¹ This work was supported by the Research Fund of the Ghent University (Geconcerteerde Onderzoeksacties; grant no. 12051403) and by an individual Marie Curie postdoctoral fellowship of the European Union (to J.D.).

² Present address: Panomics, Inc., 2003 East Bayshore Road, Redwood City, CA 94063.

³ Present address: Laboratoire de Génomique Fonctionnelle des Plantes, Faculté des Sciences–Ilot des Poulies, 33 Rue St. Leu, F–80039 Amiens cedex, France.

⁴ Present address: Laboratoire de Biologie Environnementale EA3184-usc, Institut National de la Recherche Agronomique, Université de Franche-Comté, Place Leclerc, F–25030 Besançon, France.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059907.

^* Corresponding author; e-mail ctriantaphylid{at}cea.fr; fax 33–4–42–25–26–25.

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