TY - JOUR
AU - Kangasjärvi, Jaakko
AB - Abstract Short, high-concentration peaks of the atmospheric pollutant ozone (O3) cause the formation of cell death lesions on the leaves of sensitive plants. Numerous similarities between the plant responses to O3 and pathogens suggest that O3 triggers hypersensitive response-like programmed cell death (PCD). We examined O3 and superoxide-induced cell death in the O3-sensitive radical-induced cell death1 (rcd1) mutant. Dying cells in O3-exposed rcd1 exhibited several of the typical morphological characteristics of the hypersensitive response and PCD. Double-mutant analyses indicated a requirement for salicylic acid and the function of the cyclic nucleotide-gated ion channel AtCNGC2 in cell death. Furthermore, a requirement for ATPases, kinases, transcription, Ca2+ flux, caspase-like proteolytic activity, and also one or more phenylmethylsulfonyl fluoride-sensitive protease activities was shown for the development of cell death lesions in rcd1. Furthermore, mitogen-activated protein kinases showed differential activation patterns in rcd1 and Columbia. Taken together, these results directly demonstrate the induction of PCD by O3. Ozone (O3) is an atmospheric pollutant that is phytotoxic via its breakdown in the apoplast to form reactive oxygen species (ROS). Short, high-concentration peaks, so-called acute O3, cause visible damage in sensitive plants (Wohlgemuth et al., 2002). Although accumulating evidence has deepened our understanding of oxidative stress and antioxidant defenses in O3 responses (Kangasjärvi et al., 1994; Sandermann et al., 1998; Overmyer et al., 2003), the mechanisms involved in O3-induced cell death are still relatively unknown. Due to the strong chemical reactivity of O3, its toxicity has previously been attributed to an ability to form toxic ROS that directly damage membranes (for review, see Heath and Taylor, 1997). However, the view of O3 has recently shifted, where it is now regarded in many cases not as a toxin but rather as an elicitor of cell death (Sandermann et al., 1998). O3-induced plant responses resemble on several levels the hypersensitive response (HR), normally seen as the result of challenge by an avirulent pathogen (for review, see Rao and Davis, 2001; Langebartels and Kangasjärvi, 2004). Common to these two processes are the induction of a biphasic oxidative burst, salicylic acid (SA) accumulation, ion fluxes, the deposition of cell wall-strengthening phenolic compounds, induction of defense genes such as Phe ammonia lyase, pathogenesis-related protein-1 (PR-1), and glutathione S-transferase (GST), as well as induction of local and systemic pathogen resistance. This has led to the view that O3 misfires HR-like cell death and defense programs via mimicry of the oxidative burst induced by avirulent pathogens. The HR is genetically regulated, and a form of programmed cell death (PCD; Dangl et al., 1996), where ROS have a well-established role in the induction and propagation of cell death signals, both generally and in spontaneous lesion mimic mutants (Vranová et al., 2002). As has often been stated (Kangasjärvi et al., 1994; Pell et al., 1997; Sandermann et al., 1998; Rao et al., 2000a), the similarities between the plant responses during the HR and under O3 suggest that O3-induced cell death is executed by the same mechanisms as the HR. This makes O3 a convenient probe for the study of ROS-induced PCD and implies that radical hypersensitive mutants, such as radical-induced cell death1 (rcd1; Overmyer et al., 2000; Ahlfors et al., 2004a), represent a new class of lesion mimic mutants. Although it is generally stated that O3 can induce PCD (Rao et al., 2000a), the morphological and biochemical hallmarks of PCD in O3-exposed plants have only been described in tobacco (Nicotiana tabacum; Pasqualini et al., 2003). In this article, we have deepened the view with the use of various O3-sensitive and hormonal mutants of Arabidopsis (Arabidopsis thaliana), such as rcd1, jasmonate resistant1 (jar1), nonexpresser of PR proteins1 (npr1), and transgenic NahG plants, to study the mechanisms of O3-induced cell death. In addition, we have addressed in more detail the question of O3-induced PCD by characterizing morphological similarities to the HR and PCD and showing the requirement for active metabolism in the ROS-induced cell death in rcd1. RESULTS The Morphology of O3-Induced Cell Death Two alleles of rcd1 have been found in ROS-related mutant screens and have been introduced previously (Overmyer et al., 2000; Ahlfors et al., 2004a; Fujibe et al., 2004). rcd1 is sensitive to O3, which already can be measured as increased ion leakage at 2 h and maximum at 12 to 24 h after the beginning of an O3 exposure (Overmyer et al., 2000). The morphology of O3-induced cell death was further studied in rcd1 and parental wild-type Columbia (Col-0) 24 h after the beginning of an O3 exposure (250 nL L−1, 8 h), when the difference in cell death between these two genotypes is maximal. In Col-0 plants, no visible damage was apparent (Fig. 1A Figure 1. Open in new tabDownload slide O3-induced HR-like lesions exhibit PCD hallmarks. O3-exposed Col-0 (A–D) and rcd1 (E–H) leaves at 24 h (A, B, E, and F) and 8 h (C, D, G, and H) after the beginning of the exposure (250 nL L−1, 8 h). Leaves show the absence in Col-0 (A) and the presence in rcd1 (E) of O3-induced lesions. Autofluorescence micrographs visualize the deposition of phenolic compounds indicating the activation of HR-like cell death and defenses seen in a few individual cells (arrow) exhibiting microscopic HR-like cell death in Col-0 (B) and in large tissues of rcd1 (F, asterisk). DAPI stain (D and H) depicts all nuclei for comparison to TUNEL-stained sections (C and G), illustrating the absence in Col-0 (C) and the presence in rcd1 (yellow-green stain, G) of nuclei undergoing DNA fragmentation. Figure 1. Open in new tabDownload slide O3-induced HR-like lesions exhibit PCD hallmarks. O3-exposed Col-0 (A–D) and rcd1 (E–H) leaves at 24 h (A, B, E, and F) and 8 h (C, D, G, and H) after the beginning of the exposure (250 nL L−1, 8 h). Leaves show the absence in Col-0 (A) and the presence in rcd1 (E) of O3-induced lesions. Autofluorescence micrographs visualize the deposition of phenolic compounds indicating the activation of HR-like cell death and defenses seen in a few individual cells (arrow) exhibiting microscopic HR-like cell death in Col-0 (B) and in large tissues of rcd1 (F, asterisk). DAPI stain (D and H) depicts all nuclei for comparison to TUNEL-stained sections (C and G), illustrating the absence in Col-0 (C) and the presence in rcd1 (yellow-green stain, G) of nuclei undergoing DNA fragmentation. ) when rcd1 formed macroscopic cell death lesions (Fig. 1E). Lesions in rcd1 take the form of either defined HR-like foci or large confluent areas of dry, collapsed tissue that resemble disease symptoms. Although free of macroscopic lesions, O3-exposed Col-0 exhibited a small number (10–30/leaf) of microscopic lesions. These individual or small groups of cells accumulated autofluorescent phenolics (Fig. 1B, arrow) and were frequently in the vicinity of the vascular bundles. In contrast, O3-induced lesions in rcd1 exhibited a strong accumulation of autofluorescent phenolic compounds (Fig. 1F, asterisk). Lesion induction and autofluorescent phenolic accumulation were specific, O3-triggered events in these experiments, as all clean-air control samples did not display these symptoms. One of the biochemical characteristics of PCD is the controlled degradation of nuclear DNA into internucleosomal fragments (Dangl et al., 1996), which can be detected with the terminal-transferase dUTP nuclear end label (TUNEL) assay via labeling of the free 3′ ends liberated in nuclei undergoing DNA degradation. The vast majority of O3-exposed Col-0 leaf sections examined were free of TUNEL staining (Fig. 1C). However, consistent with the low level of microscopic cell death observed by fluorescence microscopy in O3-exposed Col-0 leaves, a few TUNEL-positive nuclei were found especially in or around the vascular bundles in Col-0 (data not shown) when O3-exposed rcd1 had a high proportion of TUNEL-positive nuclei (Fig. 1G). All parallel clean-air control sections of both genotypes and controls lacking the terminal transferase were free of TUNEL-positive nuclei, indicating the specificity of the assay. At higher magnification (data not shown), the TUNEL-negative nuclei in rcd1 appeared larger and the 4′6-diamino-phenylindole (DAPI) stain more even and diffuse. In contrast, TUNEL-positive nuclei were smaller and contained brightly stained foci, indicative of nuclear shrinkage and chromatin condensation, two additional characteristics of PCD. The ultrastructure of rcd1 nuclei undergoing cell death was examined by transmission electron microscopy. Figure 2 Figure 2. Open in new tabDownload slide rcd1 nuclear morphology. Transmission electron micrographs of Col-0 (A and B) and rcd1 (C and D) nuclei. A and C, Nuclei from healthy leaves in the clean-air controls. B, Typical nucleus from O3-exposed Col-0. D, Nucleus in a rcd1 cell undergoing PCD from section through an O3-induced lesion. B and D, Taken from leaves sampled at 10 h after the beginning of a 6-h, 250 nL L−1 O3 exposure. Arrow in D indicates aggregates of condensed chromatin. v, Vesicles; cw, cell wall. Figure 2. Open in new tabDownload slide rcd1 nuclear morphology. Transmission electron micrographs of Col-0 (A and B) and rcd1 (C and D) nuclei. A and C, Nuclei from healthy leaves in the clean-air controls. B, Typical nucleus from O3-exposed Col-0. D, Nucleus in a rcd1 cell undergoing PCD from section through an O3-induced lesion. B and D, Taken from leaves sampled at 10 h after the beginning of a 6-h, 250 nL L−1 O3 exposure. Arrow in D indicates aggregates of condensed chromatin. v, Vesicles; cw, cell wall. shows typical Col-0 (A) and rcd1 (C) nuclei from healthy clean-air control sections and from leaves sampled at 10 h after the beginning of a 6-h O3 exposure (250 nL L−1; B and D). A nucleus from a dying cell within an O3-induced lesion of rcd1 was shrunken and generally more electron dense (Fig. 2D) and contained dark patches of condensed chromatin (arrows). Nuclei from O3-exposed Col-0 leaves had a normal morphology (Fig. 2B), indicating that the changes observed in rcd1 were specific to cell death lesions and not a general effect of O3 exposure. Healthy nuclei (A–C) were typically suspended in the cytoplasm, while nuclei from dying cells in O3-exposed rcd1 were frequently found adjacent to the plasma membrane, as shown in Figure 2D. Cells within the O3-induced lesions also frequently displayed vesiculation of the cytosol. In Figure 2D, a fully formed vesicle and a small vesicle in the vicinity of the plasma membrane can be seen. Based on these results, it can be concluded that the cell death in O3-exposed rcd1 exhibits nuclear DNA fragmentation, nuclear shrinkage, chromatin condensation, and cytosol vesiculation, all of which are characteristic of PCD (Dangl et al., 1996; Mittler et al., 1997). None of these features were seen in the O3-tolerant Col-0. Mutant Analysis of the Role of SA, Jasmonic Acid, and HR SA and jasmonic acid (JA) have been proposed to regulate plant PCD (Overmyer et al., 2003; Lam, 2004). To address the relationship between cell death and SA accumulation in rcd1, it was crossed with both SA-deficient transgenic NahG plants and the SA-insensitive npr1, and O3 sensitivity of the double mutants was evaluated 7 h after the beginning of an O3 exposure (250 nL L−1, 6 h). As shown in Figure 3A Figure 3. Open in new tabDownload slide O3 sensitivity of double mutants. Plants of the indicated genotypes were exposed to a single 6-h pulse of 250 nL L−1 of O3 (A) or 300 nL L−1 (B and C), and cell death was monitored as ion leakage at 7 h after the beginning of the exposure. NahG plants express a bacterial salicylate hydroxylase gene and thus are unable to accumulate SA. Mutant npr1 is SA insensitive and jar1-1 is JA insensitive. The dnd1 mutant does not develop HR as a response to avirulent Pseudomonas infection. Experiments have been replicated at least twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5–10). Bars labeled with a different letter differ significantly (P < 0.01) by Tukey's honestly significant difference post-hoc test. Figure 3. Open in new tabDownload slide O3 sensitivity of double mutants. Plants of the indicated genotypes were exposed to a single 6-h pulse of 250 nL L−1 of O3 (A) or 300 nL L−1 (B and C), and cell death was monitored as ion leakage at 7 h after the beginning of the exposure. NahG plants express a bacterial salicylate hydroxylase gene and thus are unable to accumulate SA. Mutant npr1 is SA insensitive and jar1-1 is JA insensitive. The dnd1 mutant does not develop HR as a response to avirulent Pseudomonas infection. Experiments have been replicated at least twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5–10). Bars labeled with a different letter differ significantly (P < 0.01) by Tukey's honestly significant difference post-hoc test. , both NahG and npr1 reduced, but did not abolish, O3 sensitivity of rcd1; the double mutants rcd1 NahG and rcd1 npr1 were significantly less O3 sensitive than rcd1, but significantly more sensitive than Col-0, npr1, or NahG. The JA-insensitive mutants jar1 and coronative insensitive 1 (coi1) have previously been shown to be O3 sensitive due to defective lesion containment (Overmyer et al., 2000; Rao et al., 2000b; Tuominen et al., 2004). To gain further insight into the role of JA, rcd1 was crossed with jar1 and the O3 sensitivity of the double mutant was evaluated 7 h after the beginning of an O3 exposure (300 nL L−1, 6 h; Fig. 3B). The rcd1 jar1 double mutant displayed substantially increased O3 damage when compared to the corresponding single mutants, further supporting the importance of JA in lesion containment. The absolute values of ion leakage differ to some extent in rcd1 between the experiments presented in Figure 3, A to C. This is a result of normal variation in O3 delivery kinetics in the growth chambers between different experiments. If the cell death in rcd1 is a result of PCD similar to the HR seen after infection with avirulent pathogens, it would be expected that a double mutant of rcd1 with a mutant blocked in the development of HR has a reduced number of lesions following O3 exposure. Thus, a mutant impaired in developing HR, defense, no death (dnd1), was analyzed. The dnd1 mutant fails to develop HR as a response to avirulent Pseudomonas infection (Clough et al., 2000). rcd1 was crossed with dnd1 and the double mutant was exposed to O3. In clean-air controls, dnd1 and rcd1 dnd1 sometimes displayed a slightly higher degree of cell death than Col-0 due to the spontaneous lesions (Clough et al., 2000) caused by the dnd1 mutation (data not shown). However, 7 h after the beginning of an O3 exposure (300 nL L−1, 6 h), the rcd1 dnd1 double mutant had the same level of damage as the clean-air controls, indicating that the dnd1 mutation blocks cell death in O3-exposed rcd1 (Fig. 3C). O3-Induced Accumulation of SA, JA, and Changes in Gene Expression Many lesion mimic mutants contain high amounts of SA under control growth conditions (Lorrain et al., 2003). To check the possibility that altered SA or JA biosynthesis was involved in the increased cell death of rcd1, the levels of SA (free and conjugated) and JA were analyzed in Col-0 and rcd1 in clean-air and O3-exposed samples. As previously reported (Rao et al., 2000b), O3 exposure increased the levels of SA and JA in wild-type Col-0 (the latter is masked by the huge increase of JA in rcd1). In contrast to several other lesion mimic mutants, rcd1 had normal levels of SA and JA under control conditions (Fig. 4 Figure 4. Open in new tabDownload slide SA and JA levels in O3-exposed rcd1 and Col-0. O3 induced accumulation of SA and JA. Free SA (A), conjugated SA (B), and JA (C) were measured in whole rosettes of Col-0 and rcd1 in response to a single 6-h pulse O3 exposure of 300 nL L−1. The results represent means ± se (n = 5). The analysis was repeated twice with similar results for the different genotypes. Figure 4. Open in new tabDownload slide SA and JA levels in O3-exposed rcd1 and Col-0. O3 induced accumulation of SA and JA. Free SA (A), conjugated SA (B), and JA (C) were measured in whole rosettes of Col-0 and rcd1 in response to a single 6-h pulse O3 exposure of 300 nL L−1. The results represent means ± se (n = 5). The analysis was repeated twice with similar results for the different genotypes. ). However, in the O3-exposed rcd1 plants, the levels of both free and conjugated SA and JA were higher than in Col-0 (Fig. 4). SA and JA are known regulators of defense gene expression. Thus, defense gene expression was investigated in Col-0 and rcd1 8 h after the beginning of an O3 exposure (250 nL L−1, 6 h) using a customized macroarray (Table I Table I. Expression of selected stress- and defense-related genes in wild-type Col-0 and rcd1 The samples were harvested 8 h after beginning of a 6-h O3 exposure of 250 nL L−1 O3. The values depict the average ratios of mRNA abundance between O3-treated and clean-air-grown material from two biological repeats. The data from the macroarray were first normalized to the mean of mRNA abundance of actin genes ACT2 (At3g18780) and ACT8 (At1g49240), which were shown to be constitutively expressed by RNA gel blots. A complete list of the genes used can be seen at http://www.helsinki.fi/biosci/plantstress/contents/publications/macroarray.html. AGI Code . Annotation . Col-0 . rcd1 . At3g12500 Basic chitinase 7.8 20.3 At1g62380 ACC oxidase 2 2.2 5.4 At1g32210 Defender against death1 (dnd1) 4.3 3.4 At2g47730 glutathione S-transferase 6 (GST6) 2.0 3.9 At5g44420 Plant defensin (PDF1.2a) 7.8 3.1 At4g02520 Glutathione S-transferase, putative 26.8 63.9 At2g14610 Pathogenesis-related protein-1 (PR-1) 65.0 69.1 At3g04720 Hevein-like protein 10.3 30.8 At1g02930 Glutathione S-transferase, putative 18.1 24.8 At3g49120 Peroxidase, putative 7.2 5.6 At5g20230 Plastocyanin-like domain-containing protein 63.1 47.2 At5g39580 Peroxidase 5.1 4.4 At1g21250 Wall-associated kinase1 (WAK1) 2.9 7.7 At3g15350 Legume lectin family protein 10.2 15.3 At5g54810 Trp synthase β-subunit 3.7 5.1 AGI Code . Annotation . Col-0 . rcd1 . At3g12500 Basic chitinase 7.8 20.3 At1g62380 ACC oxidase 2 2.2 5.4 At1g32210 Defender against death1 (dnd1) 4.3 3.4 At2g47730 glutathione S-transferase 6 (GST6) 2.0 3.9 At5g44420 Plant defensin (PDF1.2a) 7.8 3.1 At4g02520 Glutathione S-transferase, putative 26.8 63.9 At2g14610 Pathogenesis-related protein-1 (PR-1) 65.0 69.1 At3g04720 Hevein-like protein 10.3 30.8 At1g02930 Glutathione S-transferase, putative 18.1 24.8 At3g49120 Peroxidase, putative 7.2 5.6 At5g20230 Plastocyanin-like domain-containing protein 63.1 47.2 At5g39580 Peroxidase 5.1 4.4 At1g21250 Wall-associated kinase1 (WAK1) 2.9 7.7 At3g15350 Legume lectin family protein 10.2 15.3 At5g54810 Trp synthase β-subunit 3.7 5.1 Open in new tab Table I. Expression of selected stress- and defense-related genes in wild-type Col-0 and rcd1 The samples were harvested 8 h after beginning of a 6-h O3 exposure of 250 nL L−1 O3. The values depict the average ratios of mRNA abundance between O3-treated and clean-air-grown material from two biological repeats. The data from the macroarray were first normalized to the mean of mRNA abundance of actin genes ACT2 (At3g18780) and ACT8 (At1g49240), which were shown to be constitutively expressed by RNA gel blots. A complete list of the genes used can be seen at http://www.helsinki.fi/biosci/plantstress/contents/publications/macroarray.html. AGI Code . Annotation . Col-0 . rcd1 . At3g12500 Basic chitinase 7.8 20.3 At1g62380 ACC oxidase 2 2.2 5.4 At1g32210 Defender against death1 (dnd1) 4.3 3.4 At2g47730 glutathione S-transferase 6 (GST6) 2.0 3.9 At5g44420 Plant defensin (PDF1.2a) 7.8 3.1 At4g02520 Glutathione S-transferase, putative 26.8 63.9 At2g14610 Pathogenesis-related protein-1 (PR-1) 65.0 69.1 At3g04720 Hevein-like protein 10.3 30.8 At1g02930 Glutathione S-transferase, putative 18.1 24.8 At3g49120 Peroxidase, putative 7.2 5.6 At5g20230 Plastocyanin-like domain-containing protein 63.1 47.2 At5g39580 Peroxidase 5.1 4.4 At1g21250 Wall-associated kinase1 (WAK1) 2.9 7.7 At3g15350 Legume lectin family protein 10.2 15.3 At5g54810 Trp synthase β-subunit 3.7 5.1 AGI Code . Annotation . Col-0 . rcd1 . At3g12500 Basic chitinase 7.8 20.3 At1g62380 ACC oxidase 2 2.2 5.4 At1g32210 Defender against death1 (dnd1) 4.3 3.4 At2g47730 glutathione S-transferase 6 (GST6) 2.0 3.9 At5g44420 Plant defensin (PDF1.2a) 7.8 3.1 At4g02520 Glutathione S-transferase, putative 26.8 63.9 At2g14610 Pathogenesis-related protein-1 (PR-1) 65.0 69.1 At3g04720 Hevein-like protein 10.3 30.8 At1g02930 Glutathione S-transferase, putative 18.1 24.8 At3g49120 Peroxidase, putative 7.2 5.6 At5g20230 Plastocyanin-like domain-containing protein 63.1 47.2 At5g39580 Peroxidase 5.1 4.4 At1g21250 Wall-associated kinase1 (WAK1) 2.9 7.7 At3g15350 Legume lectin family protein 10.2 15.3 At5g54810 Trp synthase β-subunit 3.7 5.1 Open in new tab ). In accordance with the increased levels of SA (Fig. 4A) and ethylene (Overmyer et al., 2000; Tuominen et al., 2004) during O3 exposure, ethylene and SA-regulated genes, such as wall-associated kinase1 (WAK1), PR-1, and GST (SA markers) and 1-aminocyclopropane-1 carboxylic acid (ACC) oxidase, hevein-like protein, and basic chitinase (ethylene markers), had substantially higher mRNA levels in the O3-exposed plants. Transcript levels for PDF1.2a, a combined ethylene/JA marker, also increased. For most genes, the differences in expression between rcd1 and Col-0 were rather limited, with a few exceptions. ACC oxidase, hevein-like protein, and basic chitinase gene expression were increased 2 to 3 times in rcd1 compared to Col-0. This likely reflected the higher ethylene emission (Overmyer et al., 2000) from rcd1 during O3 exposure. The Role of Proteases in ROS-Induced Cell Death of rcd1 Proteases have both degenerative and signaling roles during PCD. In mammals, caspases (Cys aspartic proteases) are central to the regulation of PCD. Plants do not possess classic mammalian caspases; instead, they use vacuolar processing enzymes (VPEs), proteases with caspase activity, as regulators of PCD (Hatsugai et al., 2004; Rojo et al., 2004). To study the role of various types of proteases, in vitro experiments were performed. Col-0 and rcd1 leaves were incubated with known protease inhibitors, summarized in Table II Table II. Inhibitors used in this study a Symbolb . Inhibitor/Reagentc . Target/Effectd . Infiltrate XXO . Spray O3 . μm e μm f Van Metavanadate ATPases 50,000 100,000 Lan LaCl3 Ca2+ channels 1,000 2,000 Ama α-Amanitin Transcription 2.2 4.4 Hba Herbimycin A Tyr kinases 1,000 2,000 K25 K252a Ser/Thr kinases 2.2 4.4 Pep Pepstatin Asp proteases 1.0 E-64 E-64 Cys proteases 1.0 VAD z-VAD-fmk Caspases 50 PMSF PMSF Ser proteases 500 Ion A23187 Ca2+ ionophore 10 Ca CaCl2 Increased Ca2+ 2,000 Mg MgCl2 Control 2,000 EGTA EGTA Decreased Ca2+ 1,000 Gd Gd(III)Cl3 Ca2+ channels 1,000 Symbolb . Inhibitor/Reagentc . Target/Effectd . Infiltrate XXO . Spray O3 . μm e μm f Van Metavanadate ATPases 50,000 100,000 Lan LaCl3 Ca2+ channels 1,000 2,000 Ama α-Amanitin Transcription 2.2 4.4 Hba Herbimycin A Tyr kinases 1,000 2,000 K25 K252a Ser/Thr kinases 2.2 4.4 Pep Pepstatin Asp proteases 1.0 E-64 E-64 Cys proteases 1.0 VAD z-VAD-fmk Caspases 50 PMSF PMSF Ser proteases 500 Ion A23187 Ca2+ ionophore 10 Ca CaCl2 Increased Ca2+ 2,000 Mg MgCl2 Control 2,000 EGTA EGTA Decreased Ca2+ 1,000 Gd Gd(III)Cl3 Ca2+ channels 1,000 a Inhibitors used in this study with their respective abbreviations, concentrations used, and proposed targets. b Abbreviations used in the figures for each inhibitor. c Full name of all inhibitors and reagents used. d Proposed inhibitor target or the expected effect of the treatments. e Concentrations used for in vitro coinfiltration experiments with XXO as the radical source. f Concentrations used for pretreating plants by spraying intact plants with the inhibitor 1 h prior to O3 exposure. Open in new tab Table II. Inhibitors used in this study a Symbolb . Inhibitor/Reagentc . Target/Effectd . Infiltrate XXO . Spray O3 . μm e μm f Van Metavanadate ATPases 50,000 100,000 Lan LaCl3 Ca2+ channels 1,000 2,000 Ama α-Amanitin Transcription 2.2 4.4 Hba Herbimycin A Tyr kinases 1,000 2,000 K25 K252a Ser/Thr kinases 2.2 4.4 Pep Pepstatin Asp proteases 1.0 E-64 E-64 Cys proteases 1.0 VAD z-VAD-fmk Caspases 50 PMSF PMSF Ser proteases 500 Ion A23187 Ca2+ ionophore 10 Ca CaCl2 Increased Ca2+ 2,000 Mg MgCl2 Control 2,000 EGTA EGTA Decreased Ca2+ 1,000 Gd Gd(III)Cl3 Ca2+ channels 1,000 Symbolb . Inhibitor/Reagentc . Target/Effectd . Infiltrate XXO . Spray O3 . μm e μm f Van Metavanadate ATPases 50,000 100,000 Lan LaCl3 Ca2+ channels 1,000 2,000 Ama α-Amanitin Transcription 2.2 4.4 Hba Herbimycin A Tyr kinases 1,000 2,000 K25 K252a Ser/Thr kinases 2.2 4.4 Pep Pepstatin Asp proteases 1.0 E-64 E-64 Cys proteases 1.0 VAD z-VAD-fmk Caspases 50 PMSF PMSF Ser proteases 500 Ion A23187 Ca2+ ionophore 10 Ca CaCl2 Increased Ca2+ 2,000 Mg MgCl2 Control 2,000 EGTA EGTA Decreased Ca2+ 1,000 Gd Gd(III)Cl3 Ca2+ channels 1,000 a Inhibitors used in this study with their respective abbreviations, concentrations used, and proposed targets. b Abbreviations used in the figures for each inhibitor. c Full name of all inhibitors and reagents used. d Proposed inhibitor target or the expected effect of the treatments. e Concentrations used for in vitro coinfiltration experiments with XXO as the radical source. f Concentrations used for pretreating plants by spraying intact plants with the inhibitor 1 h prior to O3 exposure. Open in new tab , with and without the exogenous superoxide generating system, xanthine and xanthine oxidase (XXO; Jabs et al., 1996), which has previously been shown to have a similar effect on rcd1 as O3 (Overmyer et al., 2000). As seen in Figure 5 Figure 5. Open in new tabDownload slide Effect of protease inhibitors on superoxide-induced cell death. Col-0 and rcd1 plants were treated in vitro with a panel of protease inhibitors in the presence and absence of a XXO superoxide-generating system. Cell death was monitored as ion leakage. Inhibitors used, their abbreviations, and targets were as follows: Pep, pepstatin, aspartic proteases; E-64, Cys proteases; VAD, z-VAD-fmk (caspase inhibitor 1), caspases; PMSF, phenylmethylsulfonyl fluoride, Ser proteases. Inhibitor information with concentrations used is summarized in Table II. Inhibitor treatment of Col-0 plants resulted in no deviation from control and XXO-treated values (data not shown). Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars labeled with a different letter differ significantly (P < 0.01), according to Tukey's honestly significant difference post-hoc test. Figure 5. Open in new tabDownload slide Effect of protease inhibitors on superoxide-induced cell death. Col-0 and rcd1 plants were treated in vitro with a panel of protease inhibitors in the presence and absence of a XXO superoxide-generating system. Cell death was monitored as ion leakage. Inhibitors used, their abbreviations, and targets were as follows: Pep, pepstatin, aspartic proteases; E-64, Cys proteases; VAD, z-VAD-fmk (caspase inhibitor 1), caspases; PMSF, phenylmethylsulfonyl fluoride, Ser proteases. Inhibitor information with concentrations used is summarized in Table II. Inhibitor treatment of Col-0 plants resulted in no deviation from control and XXO-treated values (data not shown). Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars labeled with a different letter differ significantly (P < 0.01), according to Tukey's honestly significant difference post-hoc test. , both z-VAD-fmk (general caspase inhibitor 1; Garcia-Calvo et al., 1998) and phenylmethylsulfonyl fluoride (Ser-protease inhibitor) reduced the level of XXO-induced ion leakage in rcd1 to approximately the levels of the Col-0 plants. In contrast, pepstatin, an aspartic protease inhibitor, and E-64, a Cys-protease inhibitor, had no effect. In control experiments with XXO-treated Col-0, the same inhibitors had no effect (data not shown). Therefore, it can be concluded that Ser- and caspase-like protease activities were required for execution of the superoxide-induced cell death in rcd1. Cell Death Induced by O3 and ROS Requires Active Metabolism Inhibitors of active metabolism (Table II) were applied in planta by spraying intact rcd1 and Col-0 prior to a 250-nL L−1 O3 exposure. Cell death was monitored as ion leakage over a short time course at 0, 3, and 6 h. At these time points, ion leakage in plants that received the inhibitor treatments alone (in clean air) did not deviate from control values in Col-0 or rcd1 (data not shown), indicating that the inhibitors were nontoxic. As shown in Figure 6A Figure 6. Open in new tabDownload slide Effect of pharmacological inhibitors on O3-induced cell death. A, rcd1 and B, Col-0 plants were pretreated 1 h prior to exposure by spraying intact plants with inhibitor solutions followed by an exposure to 250 nL L−1 O3, and cell death was monitored as ion leakage from leaves collected at 3 and 6 h after the beginning of the 6-h exposure. Inhibitors used, their abbreviations, and targets were as follows: Hba, herbimycin A, Tyr-kinases; K25, K252a, Ser/Thr-kinases; Lan, lanthanum chloride, calcium channels; Ama, α-amanitin, transcription; Van, sodium metavanadate, ATPases. Inhibitor information with concentrations used is summarized in Table II. Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars marked with an asterisk (*) or double asterisks (**) were significantly different from the control at the P < 0.05 or P < 0.01 level, respectively, according to Tukey's honestly significant difference post-hoc test. Figure 6. Open in new tabDownload slide Effect of pharmacological inhibitors on O3-induced cell death. A, rcd1 and B, Col-0 plants were pretreated 1 h prior to exposure by spraying intact plants with inhibitor solutions followed by an exposure to 250 nL L−1 O3, and cell death was monitored as ion leakage from leaves collected at 3 and 6 h after the beginning of the 6-h exposure. Inhibitors used, their abbreviations, and targets were as follows: Hba, herbimycin A, Tyr-kinases; K25, K252a, Ser/Thr-kinases; Lan, lanthanum chloride, calcium channels; Ama, α-amanitin, transcription; Van, sodium metavanadate, ATPases. Inhibitor information with concentrations used is summarized in Table II. Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars marked with an asterisk (*) or double asterisks (**) were significantly different from the control at the P < 0.05 or P < 0.01 level, respectively, according to Tukey's honestly significant difference post-hoc test. , after 3 h of O3, the calcium channel blocker lanthanum, the transcriptional inhibitor α-amanitin, the Tyr kinase inhibitor herbimycin A, and the Ser/Thr kinase inhibitor K252a caused a statistically significant reduction (P < 0.05) in ion leakage in rcd1 as compared to O3 alone. Furthermore, at 6 h, herbimycin A, K252a, lanthanum, and α-amanitin pretreatments significantly diminished O3-induced ion leakage in rcd1. In Col-0, pretreatment with herbimycin A, K252a, lanthanum, α-amanitin, or metavanadate did not result in significant deviation from fumigation with O3 alone (Fig. 6B). In similar in vitro experiments using XXO instead of O3 as the death-inducing stimulus, comparable results were obtained with both Col-0 and rcd1 (data not shown). The fact that inhibition of protein kinases with K252a and herbimycin A reduced cell death in rcd1 prompted us to assess the effect of the protein phosphatase inhibitor calyculin A. Table III Table III. Induction of cell death by calyculin A . Controla . Calyculin Aa . Col-0 7.95 ± 1.46b 14.19 ± 3.80b rcd1 5.90 ± 1.13b 30.74 ± 10.49c . Controla . Calyculin Aa . Col-0 7.95 ± 1.46b 14.19 ± 3.80b rcd1 5.90 ± 1.13b 30.74 ± 10.49c a Values given are percent ion leakage ± sd (n = 5) induced by 100 μ m calyculin A measured at 18 h posttreatment. b,c Values followed by the same letter do not differ significantly for each other (P < 0.05), according to Tukey's honestly significant post-hoc test. Open in new tab Table III. Induction of cell death by calyculin A . Controla . Calyculin Aa . Col-0 7.95 ± 1.46b 14.19 ± 3.80b rcd1 5.90 ± 1.13b 30.74 ± 10.49c . Controla . Calyculin Aa . Col-0 7.95 ± 1.46b 14.19 ± 3.80b rcd1 5.90 ± 1.13b 30.74 ± 10.49c a Values given are percent ion leakage ± sd (n = 5) induced by 100 μ m calyculin A measured at 18 h posttreatment. b,c Values followed by the same letter do not differ significantly for each other (P < 0.05), according to Tukey's honestly significant post-hoc test. Open in new tab shows that treatment with calyculin A triggered a 5-fold increase in cell death in rcd1. In Col-0, calyculin A caused a slight, but statistically nonsignificant, increase in ion leakage. Calcium- and ROS-Induced Cell Death We have shown above that O3- and superoxide-induced cell death was attenuated by the calcium channel blocker lanthanum (Fig. 6A). To further elucidate the role of calcium, the effect of increased calcium flux was tested in XXO-challenged leaves with calcium ionophore A23187 and increased extracellular calcium levels (2 mm CaCl2). These treatments, or the control treatment with Mg2+, did not cause statistically significant changes in XXO-induced ion leakage in rcd1 (Fig. 7A Figure 7. Open in new tabDownload slide Effect of altered calcium flux on superoxide-induced cell death. A, rcd1 and B, Col-0 plants were treated in vitro with reagents to alter calcium flux in the presence and absence of a XXO superoxide-generating system. Cell death was monitored as ion leakage. Reagents used, their abbreviations, and targets were as follows: Ion, A23187, calcium ionophore; Ca, calcium chloride, increased extracellular calcium; Mg, magnesium chloride, divalent cation control; EGTA, chelator of extracellular calcium; Gd, gadolinium(III) chloride, calcium channel blocker; La, lanthanum chloride, calcium channel blocker. Inhibitor and reagent information and concentrations used are summarized in Table II. Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars marked with an asterisk (*) or double asterisks (**) were significantly different from the water control at the P < 0.05 or P < 0.01 level, respectively, according to Tukey's honestly significant difference post-hoc test. Figure 7. Open in new tabDownload slide Effect of altered calcium flux on superoxide-induced cell death. A, rcd1 and B, Col-0 plants were treated in vitro with reagents to alter calcium flux in the presence and absence of a XXO superoxide-generating system. Cell death was monitored as ion leakage. Reagents used, their abbreviations, and targets were as follows: Ion, A23187, calcium ionophore; Ca, calcium chloride, increased extracellular calcium; Mg, magnesium chloride, divalent cation control; EGTA, chelator of extracellular calcium; Gd, gadolinium(III) chloride, calcium channel blocker; La, lanthanum chloride, calcium channel blocker. Inhibitor and reagent information and concentrations used are summarized in Table II. Experiments have been replicated twice with similar results; one representative experiment is shown. All data points are mean ± sd (n = 5). Bars marked with an asterisk (*) or double asterisks (**) were significantly different from the water control at the P < 0.05 or P < 0.01 level, respectively, according to Tukey's honestly significant difference post-hoc test. ) or Col-0 (Fig. 7B) leaves. A reduction in calcium fluxes either by the chelation of extracellular calcium with EGTA or the use of calcium channel blockers lanthanum and gadolinium, however, caused a significant reduction in the XXO-induced ion leakage in rcd1 (Fig. 7A), suggesting that calcium influx from the apoplast is involved in the regulation of cell death in rcd1. In the ROS-tolerant Col-0, differences in cell death after the restriction of calcium flux were not statistically significant (Fig. 7B). O3 Induces Rapid Activation of Mitogen-Activated Protein Kinases Application of the Ser/Thr kinase inhibitor K252a decreased O3-induced cell death in rcd1 (Fig. 6). Since K252a acts as a competitive inhibitor of ATP for various kinases, including mitogen-activated protein (MAP) kinases, we assessed MAP kinase activation in rcd1 and Col-0 during O3 exposure. Protein extracts from O3-exposed rcd1 and Col-0 were analyzed with an antibody (anti-phospho-TEY) that detects the activated forms of both mammalian and plant MAP kinases where the Thr and Tyr residues in the activation loop are phosphorylated (Chang and Karin, 2001; Asai et al., 2002). Two putative MAP kinases of approximately 43 and 45 kD were activated in O3-treated tissue when compared to clean-air-grown plants (Fig. 8A Figure 8. Open in new tabDownload slide MAP kinase activity in rcd1 and Col-0. A, O3-induced MAP kinase activation in Col-0 and rcd1. Activated MAP kinases were detected in the wild-type Col-0 and rcd1 mutant by western blotting using an anti-phospho-TEY motif antibody, which recognizes the phosphorylated activation loop. Col-0 and rcd1 were exposed to 7 h of O3 (250 nL L−1) and samples collected at 0, 0.5, 1, 2, 4.5, and 8 h. B and C, Immunoprecipitation kinase assay with AtMPK6 (B) and AtMPK3 (C) antibodies. Protein samples from O3-exposed (7 h, 250 nL L−1) Col-0 and rcd1 plants were immunoprecipitated with AtMPK3 and AtMPK6 antisera. Leaf samples were collected at 0, 0.5, 1, 2, 4.5, and 8 h after the beginning of the exposure. Results are expressed as fold induction of myelin basic protein phosphorylating activity compared to myelin basic protein phosphorylation activity in leaf extracts from clean-air-grown plants. Figure 8. Open in new tabDownload slide MAP kinase activity in rcd1 and Col-0. A, O3-induced MAP kinase activation in Col-0 and rcd1. Activated MAP kinases were detected in the wild-type Col-0 and rcd1 mutant by western blotting using an anti-phospho-TEY motif antibody, which recognizes the phosphorylated activation loop. Col-0 and rcd1 were exposed to 7 h of O3 (250 nL L−1) and samples collected at 0, 0.5, 1, 2, 4.5, and 8 h. B and C, Immunoprecipitation kinase assay with AtMPK6 (B) and AtMPK3 (C) antibodies. Protein samples from O3-exposed (7 h, 250 nL L−1) Col-0 and rcd1 plants were immunoprecipitated with AtMPK3 and AtMPK6 antisera. Leaf samples were collected at 0, 0.5, 1, 2, 4.5, and 8 h after the beginning of the exposure. Results are expressed as fold induction of myelin basic protein phosphorylating activity compared to myelin basic protein phosphorylation activity in leaf extracts from clean-air-grown plants. ). These two kinases, which have been identified with specific antibodies as AtMPK3 and AtMPK6 (Ahlfors et al., 2004b), were activated transiently during O3 exposure with slight differences between Col-0 and rcd1; in Col-0, 45-kD kinase showed the highest phosphorylation level 1 h after the start of the exposure, whereas in rcd1, stronger phosphorylation of 45-kD kinase was already evident 0.5 h after the start of the exposure. Immunoprecipitation kinase assays with the specific antibodies against AtMPK3 and AtMPK6 revealed that, indeed, rcd1 had an earlier peak activity of AtMPK6 (at 0.5 h) and a slightly lower peak activity of AtMPK3 (at 1–2 h) when compared to Col-0. When the AtMPK3 and AtMPK6 protein accumulation was analyzed, no differences between rcd1 and Col-0 were found (data not shown). DISCUSSION Morphological Markers of PCD Are Induced by O3 The atmospheric pollutant O3 induces cell death in sensitive individuals of several species (Koch et al., 2000; Wohlgemuth et al., 2002; Pasqualini et al., 2003). Typically, Arabidopsis mutants, in which a component required for either antioxidative capacity or a process that controls cell death is deficient, are sensitive to O3 (Rao et al., 2000a; Overmyer et al., 2003; Langebartels and Kangasjärvi, 2004). One such Arabidopsis mutant, rcd1, was isolated based on the extensive cell death seen in middle-aged rosette leaves as a result of O3 exposure (Overmyer et al., 2000). RCD1 encodes a protein that most likely is involved in interactions between hormonal signaling cascades in abiotic stress (Ahlfors et al., 2004a). In this study, we used different experimental approaches to dissect processes involved in O3-induced PCD using the rcd1 mutant. O3-induced lesions in rcd1 were large, whereas the wild-type Col-0 had microscopic cell death. In both accessions, O3 caused an accumulation of autofluorescent phenolic compounds in and around the dying cells (Fig. 1). This response is also triggered by wounding or a resistance gene (R-gene) product upon recognition of an avirulent pathogen (Dietrich et al., 1994). In addition, the cell death in O3-exposed rcd1 exhibits nuclear DNA fragmentation, nuclear shrinkage, chromatin condensation, and cytosol vesiculation (Figs. 1 and 2), all of which are morphological and functional characteristics of PCD. PCD is an orderly and highly regulated disassembly of cellular functions. It requires active cellular processes, such as energy production, signal transduction, ion fluxes, transcription, and translation. Active cell death has been demonstrated with the use of pharmacological inhibitors in response to pathogens (He et al., 1993), H2O2 (Levine et al., 1996), high light in antisense catalase tobacco (Dat et al., 2003), and O3 (this study). The inhibitors used, however, are not generally specific for only one process and the unambiguous demonstration that a process is used (e.g. in PCD) requires additional ways of verification, such as mutant analysis. The Role of Hormones SA accumulation is a requirement for the execution of HR-like cell death and for the development of systemic acquired resistance (Durner et al., 1997). O3-exposed rcd1 had increased SA concentration when compared to Col-0 (Fig. 4A), and SA was required for O3 lesion formation in rcd1, since compromised SA signaling in rcd1 npr1 and rcd1 NahG double mutants diminished symptom development significantly, but not completely (Fig. 3A). This suggests that O3-induced cell death in rcd1 comprises both SA-dependent and SA-independent components. SA-dependent cell death may be taken as further evidence of O3-induced HR-like PCD. Cell death in several lesion mimic mutants is reduced in double mutants with compromised SA signaling, similarly suggesting a role for SA in lesion development (Lorrain et al., 2003). JA has a proposed role in lesion containment during O3 exposure (Overmyer et al., 2003; Tuominen et al., 2004). Treatment of O3-sensitive accessions (Arabidopsis mutant rcd1 and the ecotype Cape Verdi Islands (Cvi-0); tobacco Bel-W3) with methyl jasmonate reduced or abolished O3-induced cell death, and JA-insensitive or biosynthesis mutants (jar1, coi1, and fad3/7/8) displayed lesions following O3 exposure (Örvar et al., 1997; Overmyer et al., 2000; Rao et al., 2000b; Tuominen et al., 2004). JA levels increased dramatically in O3-exposed rcd1 (Fig. 4B). It has been proposed that the increase in JA accumulation in O3-exposed plants is a result of the cell death process itself, which causes a release of a substrate for JA biosynthesis from the membranes of the damaged cells (Vahala et al., 2003; Tuominen et al., 2004). This would form an autocatalytic containment mechanism for the lesion propagation where the magnitude of cell death would also determine the strength of signal for lesion containment by JA. By this mechanism, decreased JA sensitivity will also increase sensitivity to O3, which was apparent in the rcd1 jar1 double mutant that displayed higher lesion formation than either parent. A similar result was observed when the lesion mimic mutant hypersensitive response-like lesions1 (hrl1) was crossed with coi1; the resulting double mutant was unable to contain lesions and had exaggerated cell death (Devadas et al., 2002). Inhibitor Studies The inhibitor studies indicated a role for caspases and calcium in the induction of cell death by ROS in rcd1 (Figs. 5–7 6 7). Caspases are central to the regulation of PCD in mammals. Attempts to find similar proteins in plants have failed, although plants have been suggested to use the related protein family of metacaspases as regulators of cell death (Watanabe and Lam, 2004). However, measurements of metacaspase activity indicate that they are not directly responsible for earlier reported caspase-like activities in plants (Vercammen et al., 2004). Recently, Cys proteases belonging to the class of VPEs have been shown to be regulators of virus-induced PCD in tobacco (Hatsugai et al., 2004), and bacterial, fungal, and virus-induced PCD in Arabidopsis (Rojo et al., 2004). VPEs display caspase activity and are inhibited by caspase inhibitors (Hatsugai et al., 2004; Rojo et al., 2004). Superoxide-induced cell death in rcd1 was reduced by treatment with the caspase inhibitor z-VAD-fmk (Fig. 5), suggesting that an Arabidopsis VPE might be involved in the regulation of PCD in rcd1. O3 is a demonstrated inducer of calcium ion fluxes (Castillo and Heath, 1990; Clayton et al., 1999). Calcium is also required for activation of the NADPH oxidase (Bowler and Fluhr, 2000). Cell death in rcd1 was inhibited by diphenylene iodonium, suggesting an involvement of the NADPH oxidase in cell death activation (Overmyer et al., 2000). Thus, involvement in the activation of the NADPH oxidase presents one mechanism by which calcium influx could regulate cell death in rcd1. Chelation of extracellular calcium with EGTA has also been shown to release intracellular calcium in the cytosol (Cessna and Low, 2001). An alternative interpretation for the reduction of cell death in XXO + EGTA-treated rcd1 (Fig. 7A) could be that cytosolic calcium release leads to partial inhibition of PCD. The lack of O3-induced cell death in the rcd1 dnd1 double mutant further supports a requirement for cations, K+ or Ca2+, in the cell death in rcd1. The dnd1 mutant bears a mutation in a cyclic nucleotide-gated cation channel AtCNGC2 and fails to produce HR in response to pathogen infections (Clough et al., 2000). AtCNGC2 has previously been shown to be able to transport K+ or Ca2+ (Leng et al., 1999, 2002). Since the dnd1 mutant also has elevated SA levels and constitutively activated defenses (Clough et al., 2000), an alternative explanation for the lack of increased cell death in rcd1 dnd1 could be that the SA-dependent constitutive defenses are sufficient to protect the plant from O3 damage. However, the ecotype Cvi-0 also has elevated SA levels and is nevertheless O3 sensitive, and depletion of SA in crosses with NahG reduced cell death in both Cvi-0 and rcd1 (Fig. 3A; Rao et al., 2000b). Thus, elevated SA levels in dnd1 would be predicted to increase cell death and not reduce it. Therefore, it is likely that the lack of O3-induced cell death in dnd1 and rcd1 dnd1 is explained by the lack of a functional HR due to the deficient function of the cation channel rather than through increased SA. K+ transport is also involved in stomatal regulation, which is a crucial step in determination of O3 sensitivity (Kollist et al., 2000). Although there are no indications in the literature that AtCNGC2 could have a role in stomatal regulation, and thus that the dnd1 mutation could affect stomatal conductance, this remains a possibility and requires further study. O3-Induced MAP Kinase Activation Plant MAP kinase cascades are activated by a large number of stimuli, including pathogen infection and O3 (Zhang and Klessig, 2001; Ahlfors et al., 2004b). Constitutive MAP kinase activation has been shown to lead to HR-like cell death, suggesting that MAP kinase signaling is a part of the ROS-induced PCD (Ren et al., 2002). Inhibition of Ser/Thr kinases (including MAP kinases) with K252a suppressed cell death and phosphatase inhibitors increased cell death in rcd1 (Fig. 6; Table III), indicating that kinase activation is needed for the early phases of cell death in rcd1. However, when the timing and magnitude of cell death in rcd1 and Col-0 (Overmyer et al., 2000) are compared with the AtMPK6 and AtMPK3 activation (Fig. 8), it is likely that cell death and kinase activation are not directly linked; Col-0 had a high induction of AtMPK3 and AtMPK6 activity but little cell death when compared to rcd1. Furthermore, the O3-sensitive jar1 has similar MAP kinase activity compared to Col-0 (Ahlfors et al., 2004b). However, it is possible that ROS production and AtMPK6 activation could be linked. The more open stomata of rcd1 (Ahlfors et al., 2004a) allow more O3 to enter the plant leaf and to react with the components of the cell wall and plasma membranes, creating more ROS directly from O3 degradation. This higher oxidative load could also cause the earlier AtMPK6 peak activation in O3-exposed rcd1. The protein phosphatase inhibitor calyculin A, which increased cell death in rcd1 (Table III), has previously been shown to increase ethylene evolution and ACC synthase activity in tomato significantly without an inductive treatment (Spanu et al., 1994; Tuomainen et al., 1997). In O3-exposed plants, ethylene is required for the active ROS production responsible for lesion propagation (Overmyer et al., 2000; Moeder et al., 2002). In tobacco, the induction of ethylene biosynthesis takes place through SIPK, the tobacco homolog of Arabidopsis AtMPK6 (Kim et al., 2003), and in Arabidopsis, AtMPK6 directly activates ethylene synthesis by phosphorylating the ACC synthases AtACS6 and AtACS2 (Liu and Zhang, 2004). Thus, the fast and high induction of ethylene biosynthesis involved in the formation of O3 lesions in rcd1 (Overmyer et al., 2000) is most likely affected by the earlier peak activity of AtMPK6, since AtACS6 was also specifically activated by O3 in rcd1 (Overmyer et al., 2000). Whether the AtMPK3/6 activation is a result of the increased cell death, or vice versa, requires further study. Could Multiple Modes of Cell Death Occur in rcd1? The inhibitor treatments reduced, but did not fully block, cell death in ROS-treated rcd1. This could be due to the efficacy of the inhibitor treatments, since the action of a given inhibitor is unlikely to completely block its target pathway(s). Another interpretation is that both PCD and necrotic cell death may take place. It has been suggested that both death by rampant oxidation and PCD may occur, depending on the magnitude of O3-induced oxidative stress (Pell et al., 1997). Furthermore, Rao and Davis (1999) have presented evidence of both O3-induced necrotic and HR-like cell death, where the mechanism was dependent on genotype. Both rcd1, and to a smaller extent Col-0, displayed TUNEL-positive nuclei (Fig. 1), but since the TUNEL assay does not discriminate between random and programmed DNA fragmentation (Collins et al., 1992; Dangl et al., 1996; Pasqualini et al., 2003), it is possible that mosaics of apoptotic and necrotic cells can occur in the same O3-exposed tissue. Mixtures of cells bearing signs of different modes of death within the same tissue have been described in the study of cell death in mammals (Levin et al., 1999) and have recently been proposed to take place also in plants (Greenberg and Yao, 2004). It could be that signals emanating from the few cells undergoing necrotic cell death by rampant oxidation by O3-derived ROS may trigger surrounding cells to die by PCD, resulting in large areas of affected tissue and lesion propagation. This is analogous to the penumbra of secondary cell death seen at the periphery of acute hypoxic or traumatic lesions, which has been described extensively in mammals (Jacobson et al., 1997). A mixture of necrotic and PCD provides a model for some of the results seen in this study and for the mechanism by which O3 lesion initiation could take place in the oxidative cell death cycle (Overmyer et al., 2003). The higher initial stomatal conductance and the delay in the O3-induced stomatal closure in rcd1 (Ahlfors et al., 2004a) allow more O3 to enter the leaf during the initial phase of the exposure. This could cause more or larger lesion initiations by the direct action of the O3-derived ROS. However, the presence of characteristic biochemical and morphological markers of PCD in O3-exposed rcd1 suggest that the lesion propagation takes placed by hormone-controlled PCD, where the increased activation of MAP kinases is involved in the regulation of hormone biosynthesis. Thus, only lesion propagation would be dependent on active metabolism, ion fluxes, and SA signaling (as indicated by the inhibitor and double-mutant experiments), while the primary cell death observed would be necrotic, caused by the ROS from O3 degradation. This hypothesis requires further study, especially the role of stomatal function during the early phases of O3 challenge. MATERIALS AND METHODS Plants and Growth Conditions Arabidopsis (Arabidopsis thaliana) was grown on a 1:1 mixture of peat (Type B2; Kekkilä, Tuusula, Finland) and vermiculite at 22°C/18°C (day/night) with 70%/90% relative humidity under short-day (12 h, 250 μmol m−2 s−1) irradiance with 5 plants/8 × 8-cm pot. The isolation and genetics of the rcd1-1 allele used throughout this study has been described previously (Overmyer et al., 2000; Ahlfors et al., 2004a). Twenty-one- to 27-d-old plants (prior to bolting) were used for all experiments. Plants for O3 exposure and clean-air controls were grown side by side under identical conditions and were selected at random for separation into the two experimental groups. Mutant seeds were obtained from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org/abrc/) and the Nottingham Arabidopsis Stock Centre (NASC; http://nasc.nott.ac.uk). Seeds of the transgenic NahG (line B15) in the Col-0 background were a kind gift from Syngenta (Research Triangle Park, NC). rcd1 NahG, rcd1 npr1, rcd1 jar1, and rcd1 dnd1 double-mutant lines were created by crossing the respective plants with a glabrous rcd1 line as the pollen acceptor. F1 generation plants were confirmed as true crosses by the presence of trichomes and allowed to self-pollinate. Appropriate homozygous lines were identified from the F2 populations by PCR screening with dCAPS markers for rcd1-1, jar1-1, dnd1, and npr1-1, and by kanamycin resistance screening on plates for the presence of the NahG transgene. All genotypes were confirmed in the F3 generation. Superoxide and O3 Treatments Extracellular superoxide (XXO in sodium phosphate buffer, 10 mm, pH 7.0) was applied by vacuum infiltration into the apoplast of detached leaves as described (Jabs et al., 1996; Overmyer et al., 2000). Three completely expanded middle-aged leaves from each plant were used. Treatments lasted 18 to 20 h at 22°C in closed 50-mL tubes. The following reagents were included: xanthine (100 μ m), xanthine oxidase (0.05 units mL−1), plus inhibitors or other reagents at the concentrations given in Table I. Standard O3 exposure, 250 nL L−1 or 300 nL L−1 O3 for 6 h, unless otherwise noted, with parallel clean-air controls at all time points was as described (Overmyer et al., 2000). At the times indicated, cell death was measured by ion leakage of 2 detached leaves into 5 mL, or whole rosettes into 1 mL, milli-Q water for 1 h, followed by quantification with a conductivity meter (Mettler Toledo, Greifensee, Switzerland). Data are expressed as a percentage of total ions (determined after killing plants by boiling) and are the means of 5 to 10 replicates. Histological Procedures For detection of autofluorescent phenolic deposits, plants were cleared by boiling 3 min in alcoholic lactophenol (2:1, 95% ethanol:lactophenol), rinsed in 50% ethanol, and then rinsed twice in water. Cleared leaves were mounted and viewed as by Dietrich et al. (1994). Control samples were microscopically free of autofluorescence in all experiments. For the TUNEL assay, samples were vacuum infiltrated and fixed overnight in 4% paraformaldehyde in phosphate-buffered saline and cryoprotected for 24 h each at 4°C in 15% and then 30% Suc in phosphate-buffered saline. Leaf samples were then submerged in Tissue-Tek OTC Compound 4583 (Miles, Elkhart, IN) and snap frozen in liquid nitrogen prior to cryosectioning (Leitz Kryostat 1720; Ernst Leitz Wetzlar, Wetzlar, Germany). Sections were mounted on Superfrost Plus slides (Menzel-Gläser, Erie Scientific Company, Portsmouth, NH) and stained with the In Situ Cell Death Detection kit (Roche Applied Science, Espoo, Finland), according to the manufacturer's instructions, and counterstained with DAPI. Sections were examined by epifluorescent microscopy (Olympus Provis AX70; Olympus Optical, Tokyo) with standard DAPI and fluorescent filter sets. Electron Microscopy O3-exposed and control leaf samples for electron microscopy were fixed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.0, by vacuum infiltration and then overnight at 4°C. Samples were washed and stored at 4°C until further analysis. Samples were then postfixed in 1% osmium tetroxide (EMS, Washington Park, PA), dehydrated through an ethanol series, and embedded in Epon XL 112 (Ladd Research Industries, Williston, VT) and polymerized. Blocks were sectioned on a Reichert ultracut microtome using a diamond knife (Diatome, Bienne, Switzerland) and mounted on copper grid slots. Sections were examined with a transmission electron microscope (JEOL JEM-1200EX; JEOL, Tokyo) at an accelerating voltage of 60 kV. Hormone Measurements JA and SA were extracted and quantified with [1,2-13C]JA and [13C1]SA as internal standards as described by Baldwin et al. (1997), with the modifications described by Vahala et al. (2003). Gene Expression Profiling Expression of 127 defense-related genes was studied by a cDNA macroarray analysis using cDNA clones and expressed sequence tag clones from the ABRC (Columbus, Ohio). All clones were resequenced. Seventy-five nanograms of each PCR-amplified sample were blotted onto Hybond N+ membranes with a 96-well dot-blot device; 75 ng of oligo(dT)21 and pSPORT and pBS plasmids provided the negative controls. The 2 constitutively expressed genes, ACT2 (At3g18780) and ACT8 (At1g49240), were each applied to the membrane 4 times. Hybridization and detection were according to Overmyer et al. (2000), except that 33P-dCTP was used for probe labeling. RNA for the analysis was extracted from plants 8 h after the beginning of a 6-h, 250-nL L−1 O3 exposure. The results were normalized by reference to the mean hybridization signals for ACT2 and ACT8. Genes with expression levels below a numerical value of 0.001 in any of the samples were excluded from this analysis. Hybridizations were performed at least twice, and the results represent the mean of the duplicate signals. Inhibitor Treatments Inhibitors were used at the concentrations stated in Table I. In XXO experiments, inhibitors were coinfiltrated with the radical-generating system. In O3 experiments, plants were pretreated 1 h prior to exposure by spraying intact plants with inhibitor solutions. All inhibitor solutions for spraying were dissolved in water with 0.05% Tween 20 to mediate surface wetting. The solvent for stock solutions for K252a, herbimycin A, pepstatin, z-VAD-fmk, and A23187 was DMSO; for E-64 and PMSF, it was ethanol; and the remaining inhibitors were in aqueous solutions. Where appropriate, controls were conducted by adding solvent and Tween 20 to the spray solution or adding solvent to incubation media at the concentrations resulting from dilution of stocks into working solutions. All reagents were from Sigma Aldrich Chemicals (St. Louis), except K252a, E-64, and z-VAD-fmk, which were from Calbiochem (San Diego). One-way ANOVA tests were performed with two-sided Dunnet's or Tukey's honestly significant difference post-hoc tests as indicated using SPSS 8.0. MAP Kinase Activity Measurements Protein extractions, TEY westerns, and immunoprecipitation kinase assays were performed as described previously (Kroj et al., 2003; Ahlfors et al., 2004b). ACKNOWLEDGMENTS We are grateful to Prof. Ian Baldwin and Dr. Günter Brader for supplying JA and SA standards, respectively, and to Mika Korva for plant care. We gratefully acknowledge the Department of Ecology and Environmental Sciences at the University of Kuopio, where this work was initiated. We would like to thank the Electron Microscopy Unit of the Institute of Biotechnology at the University of Helsinki for providing laboratory facilities. LITERATURE CITED Ahlfors R, Lång S, Overmyer K, Jaspers P, Brosché M, Tauriainen A, Kollist H, Tuominen H, Belles-Boix E, Piippo M, et al ( 2004 a) Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses. 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Trends Plant Sci 6 : 520 –527 Author notes 1 This work was supported by the Academy of Finland (grant nos. 43671 and 37995), by the Finnish Centre of Excellence Programme (2000–2005), and by an Academy of Finland/German Academic Exchange Service grant (SA10256/313–SF–PPP–pz). R.P. was supported by the Finnish Graduate program in Environmental Physiology, Molecular Biology, and Ecotechnology, the University of Kuopio, and The Finnish Graduate School in Environmental Science and Technology, Åbo Akademi. 2 Present address: Biology Department, CB No. 3280, University of North Carolina, Chapel Hill, NC 27599–3280. 3 Present address: A.I. Virtanen Institute, University of Kuopio, FIN–70211 Kuopio, Finland. 4 Present address: Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE–90187 Umeå, Sweden. 5 Present address: Department of Biology, University of Joensuu, PO Box 111, FIN–80101 Joensuu, Finland. * Corresponding author; e-mail jaakko.kangasjarvi@helsinki.fi; fax 358–9–191–59552. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055681. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Ozone-Induced Programmed Cell Death in the Arabidopsis radical-induced cell death1 Mutant
JF - Plant Physiology
DO - 10.1104/pp.104.055681
DA - 2005-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ozone-induced-programmed-cell-death-in-the-arabidopsis-radical-induced-pvb00m08wx
SP - 1092
EP - 1104
VL - 137
IS - 3
DP - DeepDyve
ER -