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, 138 (3), 1516-26

Fatty Acid Hydroperoxides and H2O2 in the Execution of Hypersensitive Cell Death in Tobacco Leaves

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Fatty Acid Hydroperoxides and H2O2 in the Execution of Hypersensitive Cell Death in Tobacco Leaves

Jean-Luc Montillet et al. Plant Physiol.

Abstract

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 H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.
Figure 5.
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.
Figure 6.
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 × 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).

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