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Masumi Hirashima1, Ryouichi Tanaka and Ayumi Tanaka* Institute of Low Temperatue Science, Hokkaido University, N19 W8, Kita-ku, Sapporo, 060-0819 Japan Tetrapyrroles are well-known photosensitizers. In plants, various intermediate molecules of tetrapyrrole metabolism have been reported to induce cell death in a lightdependent manner. In contrast to these reports, we found that pheophorbide a, a key intermediate of chlorophyll catabolism, causes cell death in complete darkness in a transgenic Arabidopsis plant, As-ACD1. In this plant, expression of mRNA for pheophorbide a oxygenase was suppressed by expression of Acd1 antisense RNA; thus, AsACD1 accumulated an excessive amount of pheophorbide a when chlorophyll breakdown occurred. We observed that when senescence was induced by a continuous dark period, leaves of As-ACD1 plants became dehydrated. By measuring electrolyte leakage, we estimated that >50% of the leaf cells underwent cell death within a 5 d period of darkness. Light and electron microscopic observations indicated that the cellular structure had collapsed in a large population of cells. Partially covering a leaf with aluminum foil resulted in light-independent cell death in the covered region and induced bleaching in the uncovered regions. These results indicate that accumulation of pheophorbide a induces cell death under both darkness and illumination, but the mechanisms of cell death under these conditions may differ. We discuss the possible mechanism of light-independent cell death and the involvement of pheophorbide a in the signaling pathway for programmed cell death. Keywords: Arabidopsis • Cell death • Pheophorbide a • Senescence. Abbreviations: Acd1, Accelerated cell death 1; DAB, 3,3′diaminobenzidine; NCC, non-fluorescent chlorophyll catabolite; pFCC, primary fluorescent chlorophyll catabolite; PaO, pheophorbide a oxygenase; RCCR, red chlorophyll catabolite reductase; ROS, reactive oxygen species; WT, wild type. Introduction Numerous lesion-mimic mutants have been isolated in higher plants, and the genes responsible for lesion-mimic phenotypes have been identified. Some of these genes encode enzymes involved in chlorophyll metabolism, including Les22 encoding uroporphyrinogen decarboxylase (Hu et al. 1998) and Lin2 encoding coproporphyrinogen III oxidase (Ishikawa et al. 2001), both of which are involved in chlorophyll biosynthesis. Defects in the Accelerated cell death 1 (Acd1) and Accelerated cell death 2 (Acd2) genes also induce lesions in mutant leaves and both encode enzymes involved in chlorophyll breakdown; the Acd1 and Acd2 genes encode pheophorbide a oxygenase (Pruzinská et al. 2003, Tanaka et al. 2003, Yang et al. 2004) and red chlorophyll catabolite reductase (RCCR; Mach et al. 2001), respectively. The development of lesions through a defect in tetrapyrrole metabolism was originally described by Kruse et al. (1995), who analyzed transgenic tobacco plants expressing antisense mRNA for coproporphyrinogen oxidase. Later, antisense RNA suppression of other mRNA species, including those for uroporphyrinogen decarboxylase (Mock and Grimm 1997) and protoporphyrinogen oxidase (Molina et al. 1999, Lermontova and Grimm 2006), was described. Cell death is also induced by inhibiting chlorophyll biosynthetic enzymes by inhibitors such as diphenyl ether compounds, which are widely used as herbicides in agriculture (Witkowski and Special Issue – Regular Paper Light-Independent Cell Death Induced by Accumulation of Pheophorbide a in Arabidopsis thaliana 1Present address, National Institute of Floricultural Science, National Agriculture and Food Research Organization, 2-1 Fujimoto, Tsukuba 305-8519, Ibaraki, Japan. *Corresponding author: E-mail, [email protected]; Fax, +81-11-706-5493. Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 719 M. Hirashima et al. Halling 1988, Witkowski and Halling 1989). These reports indicate a close relationship between lesion formation and impairment of chlorophyll metabolism. The process of lesion formation by tetrapyrrole accumulation is not fully understood, but it is most likely that initiation of lesion formation is triggered by the generation of singlet oxygen, as a result of energy transfer from excited tetrapyrrole molecules. This hypothesis is consistent with the observation that lesion formation in tetrapyrrole metabolism mutants is light dependent (Mock and Grimm 1997, Meskauskiene et al. 2001, Gray et al. 2002, Yang et al. 2004). Lesion-mimic phenotypes of chlorophyll metabolic mutants are not due to their low chlorophyll synthesis activity, because inactivation of Mg-chelatase or Mg-protoporphyrin IX methylester cyclase does not cause lesion formation, although these mutants accumulate less chlorophyll (Mochizuki et al. 2001, Tottey et al. 2003, Rzeznicka et al. 2005). The reason why some mutants that are defective in chlorophyll metabolism develop lesions and others do not is unclear at the present time. It is likely that lesion formation is dependent on the level of tetrapyrrole intermediate molecules in the cell. Feedback mechanisms seem to prevent excessive accumulation of some intermediate molecules, such as Mgprotoporphyrin IX (Papenbrock et al. 2000), when a certain enzymatic step is blocked. In such cases, lesion formation is not observed. During senescence, chlorophyll is degraded to safe linear tetrapyrroles in a series of reactions catalyzed by chlorophyllase, Mg-dechelatase and pheophorbide a oxygenase (PaO). PaO is a Rieske-type oxygenase that catalyzes the oxygenic ring opening of pheophorbide a between C4 and C5 (Hörtensteiner et al. 1998, Takamiya et al. 2000, Hörtensteiner 2006). The gene encoding PaO was initially identified as Acd1 from Arabidopsis and Lethal leaf spot 1 (Lls1) from maize (Pruzinská et al. 2003, Tanaka et al. 2003, Yang et al. 2004). In the maize lls1 mutant, necrotic spots are formed, which expand continuously over the entire leaf until eventually the whole plant dies. The lesion-mimic phenotypes of lls1 and acd1 mutants were reported to be light dependent, and the involvement of chloroplasts was postulated (Gray et al. 1997). Recent studies indicate that the substrate of PaO, pheophorbide a, accumulates in these mutants or transgenic plants expressing antisense PaO RNA (Pruzinská et al. 2003, Pruzinská et al. 2005, Tanaka et al. 2003). Considering that pheophorbide a is a powerful photosensitizer, excessive accumulation of pheophorbide a in these mutants would lead to the generation of reactive oxygen species (ROS) under light conditions, which ultimately causes cell death, as observed in other chlorophyll biosynthesis mutants (Mock and Grimm 1997, Meskauskiene et al. 2001). To explain the cell death process induced by tetrapyrroles, two mechanisms have been proposed. One is that ROS directly oxidize cellular components, such as lipids, proteins and DNA. The other is 720 that ROS function as signal molecules. It has been reported that in the flu mutant, in which an excessive amount of protochlorophyllide is accumulated, singlet oxygen acts as a signal molecule to regulate gene expression and induce growth retardation (op den Camp et al. 2003, Wagner et al. 2004, Danon et al. 2005). Recently, a new function of chlorophyll intermediate molecules has been proposed. Mg-protoporphyrin IX regulates gene expression in nuclei of Chlamydomonas and Arabidopsis, and it is suggested to be a chloroplast signal (Kropat et al. 1997, Kropat et al. 2000, Mochizuki et al. 2001, Strand et al. 2003, Vasileuskaya et al. 2005, Pontier et al. 2007). Pheophorbide a is reported to inhibit Mg-chelatase activity (Pöpperl et al. 1997). These results suggest the possibility that pheophorbide a functions as a signal molecule or an inhibitor of specific enzymes and thereby induces cell death. In this study, we examined the cell death process by pheophorbide a in detail and found that it induces cell death in Arabidopsis leaves in the dark. These results indicate that pheophorbide a induces cell death not by producing singlet oxygen, but by other mechanisms. Results Arabidopsis leaves wilted during senescence We reported previously that pheophorbide a accumulated in transgenic Arabidopsis plants expressing antisense PaO mRNA (As-ACD1) (Tanaka et al. 2003). As shown in Fig. 1A, the PaO protein level increased after the onset of leaf senescence in the dark in the wild type (WT), while it was not detectable in the leaves of As-ACD1 plants before or after dark incubation. These results indicate that PaO expression was almost completely suppressed in As-ACD1 leaves. During senescence, the chlorophyll content decreased and the leaves turned yellow–green in the WT plants (Fig. 1B, D). In contrast, the leaves appeared greener in As-ACD1 than in the WT. However, it was often observed that parts of the leaves, especially the tips, were crinkled (Fig. 1B, C). In addition, many small lesions were observed in the As-ACD1 plants during the 5 d period of dark incubation (Fig. 1E). After re-illumination of As-ACD1 leaves, they became bleached, as described in our previous report (Tanaka et al. 2003). The levels of chlorophylls and pheophorbide a per leaf fresh weight in the 4-week-old seedlings were determined by HPLC analysis (Fig. 1C and Table 1). In senescing WT leaves, the levels of total chlorophylls decreased with increasing leaf age. Pheophorbide a was not detectable in these leaves. In the apparently healthy leaves of As-ACD1, the chlorophyll levels and the Chl a/b ratios were very similar to those of the WT (As-ACD1 Nos. 1–4, 6 and 7 in Fig. 1C and Table 1). In contrast, in the crinkled leaves of As-ACD1, the levels of total chlorophylls increased considerably based on the leaf fresh Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. Light-independent cell death by pheophorbide a A WT 0d 5d As-ACD1 0d 5d Table 1 Levels of total chlorophylls and pheophorbide a in WT and As-ACD1 leaves 62.0 PaO Leaf No. 47.5 (kD) As-ACD1 1 2 3 4 5 6 7 8 9 10 11 C WT 1 2 3 4 5 6 7 8 Chl a/b Pheophorbide a Fresh weight (mg) (nmol/g FW) 1 3.13 3.05 ND 3.9 2 2.63 3.03 ND 5.9 3 2.49 2.96 ND 8.7 4 2.39 2.91 ND 9.6 5 1.96 2.79 ND 12.9 6 2.09 2.86 ND 9.5 7 1.78 2.97 ND 10.5 8 1.83 2.92 ND 7.4 9 1.24 2.72 ND 4.7 10 1.44 2.93 ND 3.6 11 1.54 2.91 ND 2.3 1 3.23 2.91 ND 2.4 2 3.51 2.93 1.6 3.2 3 3.00 2.93 ND 4.2 4 2.19 2.91 ND 12.5 5 3.45 2.73 25.3 3.0 6 2.16 2.99 ND 10.9 7 1.63 3.01 0.9 12.9 8 5.92 1.55 23.2 2.7 9 5.17 1.98 15.7 3.0 10 2.61 1.66 15.4 2.5 11 6.12 1.72 81.1 1.6 12 6.03 1.43 81.0 0.9 WT B WT Total Chls (µmol/g FW) 9 10 11 12 As-ACD1 As-ACD1 0d D 5d E WT WT As-ACD1 As-ACD1 Fig. 1 Phenotypes of As-ACD1 under light and dark conditions. (A) Immunoblot analysis of PaO protein in the WT and As-ACD1. Leaf extracts corresponding to 0.5 mg of rosette leaves were subjected to SDS–PAGE and electroblotted on a PVDF membrane. The membrane was incubated with an anti-PaO antibody as described in Materials and Methods. (B) WT and As-ACD1 plants were grown for 35 d under continuous light conditions. The leaves of As-ACD1 wilted (black arrow) and finally became bleached (white arrow). (C) Rosette leaves of WT and As-ACD1 plants after dark incubation. The leaves were arranged from young to old (from No. 1 to No. 11 or 12). Lesions were observed in No. 5 and Nos. 8–12 of As-ACD1 leaves. (D) Three-weekold Arabidopsis were incubated for 5 d in complete darkness. Before dark incubation, WT and As-ACD1 leaves were indistinguishable (0 d). The leaves of the WT turned green–yellow after dark incubation, but those of As-ACD1 remained green (5 d). (E) The leaves of the WT and As-ACD1 after 5 d of dark incubation. The leaves of As-ACD1 wilted. weight (As-ACD1 Nos. 5 and 8–12 in Fig. 1C and Table 1). This increase was probably due to the dehydration of the leaves (see below). The level of pheophorbide a was higher in the crinkled leaves of As-ACD1 than in the apparently healthy leaves. These results indicate that accumulation of pheophorbide a led to the morphological changes in the leaves. Note that the leaves of a T-DNA insertion line, in which the Acd1/PaO gene was disrupted, also became Pigment contents were measured by HPLC in the leaves shown in Fig. 1C. Leaf numbers correspond to those in Fig. 1C. ND indicates that the pigment was not detectable. crinkled after the onset of senescence induced by dark incubation (data not shown). This suggests that the crinkling of the leaves in As-ACD1 was not due to a possible secondary mutation in this line. Light-independent cell death induced by pheophorbide a We examined the changes in the water content of the leaves during dark incubation (Fig. 2A). In the WT, there was no difference in the water content before and after the dark incubation. In contrast, the water content significantly decreased after 5 d of dark incubation in As-ACD1. These results are consistent with the observation that As-ACD1 leaves became crinkled after dark incubation, but WT leaves remained intact (Fig. 1E). Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 721 A Fresh weight / Dry weight M. Hirashima et al. 14 WT As-ACD1 12 10 8 6 4 2 0 B Electrolyte leakage (%) 0d C 80 70 60 50 40 30 20 10 0 0 5d WT As-ACD1 1 2 3 4 Dark incubation (days) 0d 5d 0d 5d 5 WT As-ACD1 D WT As-ACD1 Fig. 2 Analysis of cell death in As-ACD1 leaves under dark conditions. (A) Water content of the leaves of the WT and As-ACD1. The water content was estimated by dividing fresh weight by dry weight. There was no difference between the water content of WT and As-ACD1 leaves before dark incubation, but it decreased in As-ACD1 after dark incubation. Vertical bars represent the SD (n = 3). (B) Electrolyte leakage of the leaves of WT and As-ACD1 plants. Plants were incubated in complete darkness for 0, 1, 3 and 5 d. The leaves were harvested under dim green light and electrolyte leakage was measured. Vertical bars represent the SD (n = 6). (C) Trypan blue staining. Plants were incubated in complete darkness for 5 d and then leaves were stained with trypan blue, as described in Materials and Methods. (D) Leaves of WT and As-ACD1 plants were stained with DAB to monitor H2O2 accumulation after dark incubation. A small amount of H2O2 accumulated in As-ACD1 leaves after dark incubation. In order to investigate the integrity of the cells during dark-induced senescence, we measured the electrolyte leakage of the leaves (Fig. 2B). WT and As-ACD1 plants were grown for 3 weeks under continuous light and then 722 incubated in the dark for various periods, as indicated in Fig. 2B. Electrolyte leakage remained low during dark incubation in the WT. In contrast, a large increase in electrolyte leakage was observed in As-ACD1 leaves; >50% of electrolytes were leaked after 5 d of dark incubation. These results indicate that more than half of the As-ACD1 leaf cells lost membrane integrity during dark incubation. We also employed trypan blue staining to ascertain whether cell death was induced in As-ACD1 leaves during dark incubation (Fig. 2C). Before dark incubation, green leaves of both WT and As-ACD1 plants were not stained with trypan blue, indicating that cell death was not induced at this stage. After dark incubation, WT leaves were slightly stained with trypan blue (Fig. 2C), while As-ACD1 leaves were intensely stained. Taken together, the results of electrolyte leakage and trypan blue staining indicate that accumulation of pheophorbide a induced cell death in the dark. Prior to programmed cell death in plants, the generation of H2O2 is occasionally observed, which facilitates acute cell death. We therefore investigated whether the cell death in As-ACD1 under darkness is also accompanied by H2O2 production. For this purpose, we treated WT and As-ACD1 leaves with 3,3′-diaminobenzidine (DAB) before and after dark-induced senescence (Fig. 2D). Before dark incubation, the H2O2 levels were low in both WT and As-ACD1 plants. After 5 d of dark incubation, H2O2 was produced in the As-ACD1 leaves. Possible mechanisms of H2O2 generation are considered in the Discussion. Chloroplast degradation by pheophorbide a accumulation We further examined the cell death occurring in dark conditions in As-ACD1 plants by light and electron microscopy. Before dark incubation, the chloroplasts were evenly distributed at the periphery of cells, and they showed a normal shape in both WT and As-ACD1 cells (Fig. 3A, B). After 4 d of dark incubation, most of the chloroplasts seemed to have sedimented to the bottom of the cell in the WT (Fig. 3C). In apparently non-damaged cells of As-ACD1, cell shape and localization of the chloroplasts in the cells were similar to those of the WT (Fig. 3D) after dark incubation. In contrast, in the crinkled leaves of As-ACD1, the chloroplasts were dispersed in the cells and plasmolysis was often observed (Fig. 3E). In the most severely damaged region, no intact chloroplasts were observed (Fig. 3F). These optical microscopic observations indicate that accumulation of pheophorbide a damaged the structural integrity of the cells. No significant differences were observed by electron microscopy in the structure of cells and chloroplasts between WT and As-ACD1 leaves before dark incubation (Fig. 4A, B). In the WT, after 5 d of dark incubation, the chloroplasts had become round, but the integrity of the membrane systems, including the chloroplast envelope, thylakoid membranes, Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. Light-independent cell death by pheophorbide a had disappeared (Fig. 4G). Finally, the entire cellular structure was disrupted (Fig. 4H). Interestingly, healthy living cells and damaged or dead cells were adjacent to each other (Fig. 4E, G). Dark incubation of part of As-ACD1 leaves induced cell death Fig. 3 Light microscopic observation of As-ACD1 leaves. Plants were incubated in complete darkness for 4 d and stained with toluidine blue. (A and B) Light micrograph of WT (A) and As-ACD1 (B) leaves before dark incubation. (C–F) Light micrograph of WT (C) and AsACD1 (D–F) leaves after dark incubation. In an apparently healthy leaf of As-ACD1 (D), the chloroplasts appear intact. In contrast, in crinkled leaves of As-ACD1 (E and F), the chloroplasts seemed collapsed. One such collapsed chloroplasts is indicated by the open arrow in (E). In addition, plasmolysis was often observed in the crinkled leaves (E). Plasma membranes that shrunk away from the cell wall are indicated by black arrows in (E). In the most severely damaged leaves (F), no intact chloroplasts were observed. Scale bar = 50 µm. tonoplast and plasma membranes, had not changed (Fig. 4C). However, drastic changes were observed when the As-ACD1 plants were incubated in the dark; in the apparently nondamaged region, all the membrane systems appeared intact and there were no differences between the WT and As-ACD1 in the observable cell structures (Fig. 4D). From the observations of cells with varying degrees of damage, the following stages of cell death were observed. In the first stage, the tonoplast was difficult to recognize, but the chloroplast envelopes and thylakoid membranes were intact and plasma membranes were appressed to the cell wall, as observed in WT cells (Fig. 4E). In the next stage, the chloroplast envelopes were fragmented, but the thylakoid membranes were intact and the stacked granum structures were well maintained (Fig. 4E, F). Furthermore, plasma membranes could not be detected. In the third stage, thylakoid membranes became less conspicuous and grana stacking In order to understand the mechanism of light-independent cell death, it is important to clarify whether cell death in As-ACD1 in darkness is propagative or not. Thus, we investigated whether light-independent cell death occurs only in the cells accumulating pheophorbide a, or whether it spreads to the neighboring cells in which cell death was not induced by dark incubation. We followed the method of Yang and co-workers (2004) who studied the mechanism of lightdependent cell death in the Arabidopsis acd1 mutant. We covered a part of the leaves with aluminum foil and kept them under illumination for 5 d (Fig. 5A–D, G–J). In the WT, chlorophyll breakdown was observed only in the covered regions (Fig. 5E). Neither light-dependent nor lightindependent cell death was observed (Fig. 5E) in the WT. In contrast, as reported by Yang et al. (2004), covered regions were greener than the uncovered areas in As-ACD1 leaves (Fig. 5F). However, the cells from both the covered and uncovered regions in As-ACD1 were heavily stained with trypan blue (Fig. 5K). These results indicate that cell death was induced in both a light-dependent and a light-independent manner. Interestingly, the covered regions of the leaves were occasionally more heavily stained with trypan blue than the uncovered regions (No. 6* in Fig. 5K), demonstrating that cell death was more severely induced in the covered regions. These data may indicate that cell death occurred in the covered regions first, and then propagated to the uncovered regions. We note that in As-ACD1, cell death in the covered regions affected the other leaves; the leaves that were older than the covered leaves often wilted and later became bleached (data not shown), which was consistent with the study of Yang et al. (2004). These results indicate that darkinduced cell death was not only propagated within the same leaf but also spread to other leaves. Discussion In this study, we found that cell death was induced in As-ACD1 plants in a light-independent manner (Fig. 1D, 1E). We confirmed cell death by measuring electrolyte leakage and staining with trypan blue (Fig. 2B, C). From observing As-ACD1 leaves using a light microscope and an electron microscope, we found that the cellular structure was disrupted during dark treatment (Figs. 3F, 4H). We also observed different states in two adjoining cells in As-ACD1 after dark incubation; one cell had lost the plasma membranes, the tonoplast and chloroplast envelope membranes, Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 723 M. Hirashima et al. Fig. 4 Electron microscopic observation of WT and As-ACD1 cells. (A, B) The ultrastructure of WT (A) and As-ACD1 (B) cells before dark incubation. (C–H) The ultrastructure of WT (C) and As-ACD1 (D–H) cells after dark incubation. (G) An apparently healthy cell was adjacent to the collapsed cells in As-ACD1. (H) The cell structure was completely destroyed. (F) The envelope membranes of the chloroplast appeared to have collapsed but thylakoid membranes remained intact. The magnified image of the chloroplast (asterisk) is shown in F. Scale bar = 1 µm. and the other cell maintained membrane integrity (Fig. 4G). Therefore, it is likely that induction of cell death is cell autonomous in As-ACD1 in darkness. Light-independent cell death was estimated to occur at a frequency of approximately 50% in the As-ACD1 leaves that were kept in darkness for 5 d (see Fig. 2B). This frequency is much lower than that of light-dependent cell death, which was nearly 100% (see photographs in Tanaka et al. 2003, Pruzinska et al. 2003). The threshold of pheophorbide a accumulation for light-independent cell death is probably higher than that for light-dependent cell death. These results may indicate that the mechanisms for the induction of cell death differed between light and dark conditions. Based on this assumption, we hypothesize that the mechanism of cell death when a part of a leaf was covered with aluminum foil (see Fig. 5) was as follows: cell death was probably first induced in the covered region. The signal that cell death had occurred in part of the leaf might then be transmitted to neighboring cells or even to the entire leaf through a signaling pathway, which has not yet been determined. By receiving such information, the cells may activate their ‘emergency’ responses, which may resemble those for pathogen defense. Since programmed cell death in leaves nearly always accompanies chlorophyll breakdown, and at least one of the enzymes of chlorophyll catabolism is known to be induced upon pathogen infection (Kariola et al. 2005), it would be reasonable to assume that chlorophyll breakdown has started in these cells. In As-ACD1, chlorophyll breakdown 724 should result in accumulation of pheophorbide a, which would induce further cell death. Based on the abovementioned assumption, cells accumulating a relatively small amount of pheophorbide a may undergo cell death under illumination. Thus, no surviving cells would remain in the uncovered region of the leaf partly covered with aluminum foil for a certain period. This hypothesis is very similar to that of Yang et al. (2004). It differs in that the induction of cell death occurs in the covered region, where cell death is induced in a light-independent manner. During the chlorophyll degradation process, pheophorbide a is converted to the primary fluorescent chlorophyll catabolite (pFCC) in a two-step reaction by PaO and RCCR on chloroplast inner envelope membranes (Hörtensteiner 2006). pFCC undergoes several modifications and is finally converted to FCCs (Pruzinská et al. 2005). FCCs are then exported from the chloroplast to the vacuole, where they are converted to non-fluorescent chlorophyll catabolites (NCCs). This model predicts the presence of a chlorophyll catabolite transporter on chloroplast envelope membranes. Breast cancer resistance protein (BCRP) 1 is an ABC-type transporter, and has been reported to transport pheophorbide a in mice (Jonker et al. 2002). If transporters similar to BCRP1 are present on chloroplast envelope membranes, and if they are involved in the export of chlorophyll catabolites, they may also be involved in the excretion of pheophorbide a from the chloroplast. Therefore, it is possible that pheophorbide a has access to various organelles in the cell, Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. Light-independent cell death by pheophorbide a Fig. 5 Induction of leaf senescence in a limited area by covering parts of leaves with aluminum foil. Parts of the leaves of the WT (A, C, E, G, H) and As-ACD1 (B, D, F, I, J) were covered with aluminum foil to induce leaf senescence in limited areas. Plants were grown for 0 (A, B), 4 (C, D) and 9 (E, F) days after being covered with aluminum foil. As-ACD1 leaves with aluminum foil wilted (D) after 4 d. After 9 d, the uncovered region of As-ACD1 leaves was bleached, but the covered region remained green (F). (G–K) The leaves of the WT (G,H) and As-ACD1 (I,J) were harvested and stained with trypan blue (K) after 7 d. In (K) the numbers (1–10) correspond to (G–J); 1–5 are WT, and 6–10 are As-ACD1. Also, 1, 2, 4, 6, 7 and 9 are covered leaves, while 3, 5, 8 and 10 are uncovered leaves. Magnifications of 6 and 9 are indicated as 6* and 9*, respectively. such as the mitochondria, nucleus and tonoplast, and plasma membranes, where it may induce a range of deleterious effects. Only a proportion of the chlorophyll molecules are degraded to NCCs in the As-ACD1 leaves because chlorophyll degradation is interrupted at the level of pheophorbide a by suppression of PaO (Acd1) expression. It has been reported that the non-yellow coloring 1 (nyc1) mutant (Kusaba et al. 2007) and the stay green (sgr) mutant (Park et al. 2007) retained a substantial amount of chlorophyll under dark Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 725 M. Hirashima et al. conditions. Stacked granum structures were well maintained, and disruption of the cellular components was limited after dark incubation in these mutants compared with those of the WT. The phenotype of the As-ACD1 mutant was very different from those of the nyc1 and sgr mutants, indicating that retardation of chlorophyll breakdown in darkness is not the major cause of the observed phenotype of As-ACD1. Singlet oxygen is probably not involved in the lightindependent cell death described in this study because, to the best of our knowledge, no strong and abundant molecules that could transfer their energy to pheophorbide a and convert it to its triplet state have been reported in the leaf cells of Arabidopsis in dark conditions. One could argue that a tetrapyrrole molecule may be able to perform a catalaselike reaction that possibly results in the production of H2O2 and singlet oxygen (Ghosh et al. 2008). However, it is unlikely that pheophorbide a is able to catalyze a catalase-like reaction, because pheophorbide a does not chelate a metal ion, which is thought to be essential in a catalase-like reaction (Ghosh et al. 2008). This assumption is apparently contradictory to our observation that pheophorbide a induces H2O2 production in leaves in the dark. We speculate that H2O2 production is not due to the excitation of pheophorbide a molecules, but it could be a result of a cellular response to the aberrant status of the cell. Such a response may be similar to that of the hypersensitive response in which H2O2 production is stimulated (Morel and Dangl 1997). We hypothesize that there are two possible mechanisms for the induction of light-independent cell death caused by accumulation of pheophorbide a. In the first hypothesis, we assume that pheophorbide a specifically inhibits the activity of channel proteins or other cellular components that are essential for membrane integrity. Such inhibition may cause dehydration (see Fig. 2A) or destruction of membrane systems (Figs. 3, 4) after accumulation of pheophorbide a by dark treatment. In the second hypothesis, we postulate that pheophorbide a functions as a signal molecule that regulates gene expression and induces programmed cell death. It has been recently established that Mg-protophorphyrin IX, the first chlorophyll intermediate after insertion of magnesium in the chlorophyll biosynthesis pathway, regulates gene expression in the nuclei as a chloroplast signal (Mochizuki et al. 2001, Pontier et al. 2007). In the chlorophyll biosynthesis pathway, Mg-protophorphyrin IX is the first precursor specific to chlorophyll biosynthesis. It would be reasonable to assume that the first precursor may be a better target of a feedback regulation than the later ones, if we consider that a rapid feedback is generally better than a slower one. In this sense, pheophorbide a is a good candidate for a signaling molecule in this pathway, as it is the first catabolite that is specific in the chlorophyll degradation pathway. 726 The question arises as to whether pheophorbide a plays a role in the induction of cell death under natural conditions in the WT. Fig. 1C and Table 1 show that the leaves that accumulated pheophorbide a retained a green color, but had aberrant Chl a/b ratios. The exact mechanism has not been determined, but we speculate that accumulation of pheophorbide a induces not simply oxidation of cellular components, but other types of cell death under light conditions, because the collapse of leaf cells preceded the bleaching of leaves. It has been reported that in Chlamydomonas reinhardtii, pheophorbide a accumulated under anaerobic conditions (Doi et al. 2001). Likewise, we occasionally observed the accumulation of pheophorbide a in Arabidopsis under high humidity or flooding conditions (R. Tanaka and A. Tanaka, unpublished results). Accumulation of pheophorbide a under anaerobic conditions is consistent with previous reports indicating that the conversion of pheophorbide a to RCCs requires molecular oxygen (Purzinská et al. 2003). If we consider that hypersensitive cell death upon pathogen infection must be induced irrespective of light irradiation, the cell death mechanism caused by pheophorbide a in darkness would be a likely strategy to accelerate cell death upon pathogen infection. In conclusion, the findings presented herein should prompt plant researchers to consider a new function for the tetrapyrrole molecules as well as an alternative mechanism for programmed cell death in plants. Materials and Methods Plant materials and growth conditions WT Arabidopsis thaliana (Columbia ecotype) and As-ACD1 plants expressing antisense Acd1 RNA were grown in a chamber equipped with white fluorescent lamps (FLR40SSW, NEC Co., Ltd., Tokyo, Japan) under continuous illumination at a light intensity of 80 µE m−2s−1 at 23°C. Production of As-ACD1 plants that overexpressed antisense RNA for the Acd1 gene was described in our previous report (Tanaka et al. 2003). HPLC analysis Three-week-old Arabidopsis leaves were harvested and ground in acetone. The samples were centrifuged at 10,000 × g for 10 min, and 80 µl of the supernatant was mixed with 20 µl of water. Following the method of Zapata et al. (2000), pigments were separated on a reversed phase column, Symmetry C8 (150 × 4.6 mm, Waters). Anti-PaO polyclonal antibody preparation The coding region of the Acd1 gene was amplified from cDNA. The amplicon was incorporated into the NdeI and XhoI sites of the pET43.1a vector (Novagen, Madison, WI, USA). The recombinant PaO protein containing the 6-histidine tag Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. Light-independent cell death by pheophorbide a was expressed in Escherichia coli and purified using a His-Bind Resin Chromatography Kit (Novagen) according to the manufacturer's instructions. The purified protein was used to raise polyclonal antibodies in rabbits. Immunoblot analysis Total protein was extracted from leaves by grinding with extraction buffer [50 mM Tris (pH 6.8), 10% (w/v) glycerol, 2% (w/v) SDS and 6% (v/v) 2-mercaptoethanol], and these samples were centrifuged at 10,000 × g for 10 min. The supernatants were separated by 10% SDS–PAGE, and the resolved proteins were transferred onto a Hybond-P membrane (GE Healthcare, Buckinghamshire, UK). The membrane was incubated with anti-Arabidopsis PaO (1 : 5,000). Anti-rabbit IgG linked to horseradish peroxidase was used as the secondary antibody. Chemiluminescent detection was performed using an ECL plus Western blotting detection system (GE Healthcare). The leaves were then placed in ethanol overnight to remove chlorophyll (Torres et al. 2002). Microscopy Dark-treated leaves were harvested and soaked in primary fixation buffer (2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4). They were then rinsed three times in 0.1 M cacodylate buffer, pH 7.4, and fixed with secondary fixation buffer (1% OsO4 in 0.1 M cacodylate buffer, pH 7.4) for 90 min. The samples were dehydrated in a graded series of ethanol dilutions (30, 50, 70, 90 and 100% ×3, v/v) for 15 min at each dilution and subsequently embedded in an epon resin mixture (TAAB Epon 812, TAAB Laboratories Equipment Ltd., Berkshire, UK). For light microscopic observation, specimens were stained with toluidine blue. For electron microscopic observation, ultra-thin sections were stained with 2% (w/v) aqueous uranyl acetate and lead citrate. Funding Measurement of the water content Six leaves were harvested under dim green light and wrapped in aluminum foil. After measuring the fresh weights, the samples were dried by incubation at 80°C for 13 h. Water content was estimated by dividing fresh weight by dry weight. Electrolyte leakage Plants were incubated in complete darkness for 0, 1, 3 and 5 d. The leaves were harvested under dim green light and then two leaves were soaked in 2 ml of distilled water in a test tube. Samples were shaken for 2 h and electrolyte leakage from the leaves was determined by measuring the conductivity of the solution using a TWIN compact meter (Horiba, Kyoto, Japan). Samples were boiled for 15min and shaken for 2 h. The conductivity was measured again in order to determine the total electrolyte content of the leaves. Trypan blue staining Arabidopsis leaves were harvested and stained with lactophenol–trypan blue solution (10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, 10 mg of trypan blue, dissolved in 10 ml of distilled water) (Koch and Slusarenko 1990). Leaves were boiled for 1 min in the solution and then decolorized overnight in chloral hydrate solution (75 g of chloral hydrate dissolved in 30 ml of distilled water). Detection of H2O2 DAB staining was performed on Arabidopsis leaves to visualize H2O2. Dark-treated leaves were harvested and vacuum infiltrated with DAB solution (10 mg ml–1 DAB–HCl, pH 3.8). Leaves were placed in a plastic box for 3 h and then fixed with a solution of 3 : 1 : 1 ethanol/lactic acid/glycerol for 15 min. The Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Creative Scientific Research No. 17GS0314 to A.T.; Grant-in-Aid for Scientific Research No. 68700307 to R.T.). Acknowledgments The authors thank Junko Kishimoto for her help with light and electron microscopy, and with preparation of the figures. We are also grateful to Sachiko Tanaka for her help in HPLC analysis. References Danon, A., Miersch, O., Felix, G., Camp, R.G. and Apel, K. (2005) Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana. Plant J. 41: 68–80. Doi, M., Inage, T. and Shioi, Y. (2001) Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis. Plant Cell Physiol. 42: 469–474. Ghosh, A., Mitchell, D.A., Chanda, A., Ryabov, A.D., Popescu, D.L., Upham, E.C., et al. (2008) Catalase–peroxidase activity of iron(III)TAML activators of hydrogen peroxide. J. Amer. Chem. Soc. 130: 15116–15126. Gray, J., Close, P.S., Briggs, S.P. and Johal, G.S. (1997) A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 89: 25–31. Gray, J., Janick-Buckner, D., Buckner, B., Close, P.S. and Johal, G.S. (2002) Light-dependent death of maize lls1 cells is mediated by mature chloroplasts. Plant Physiol. 130: 1894–1907. Hörtensteiner, S. (2006) Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57: 55–77. Hörtensteiner, S., Wuthrich, K.L., Matile, P., Ongania, K.H. and Kräutler, B. (1998) The key step in chlorophyll breakdown in higher plants. Cleavage of pheophorbide a macrocycle by a monooxygenase. J. Biol. Chem. 273: 15335–15339. Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 727 M. Hirashima et al. Hu, G., Yalpani, N., Briggs, S.P. and Johal, G.S. (1998) A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize. Plant Cell. 10: 1095–1105. Ishikawa, A., Okamoto, H., Iwasaki, Y. and Asahi, T. (2001) A deficiency of coproporphyrinogen III oxidase causes lesion formation in Arabidopsis. Plant J. 27: 89–99. Jonker, J.W., Buitelaar, M., Wagenaar, E., Van Der Valk, M.A., Scheffer, G.L., Scheper, R.J., et al. (2002) The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl Acad. Sci. USA 99: 15649–15654. Kariola, T., Brader, G., Li, J. and Palva, E.T. (2005) Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell 17: 282–294. Koch, E. and Slusarenko, A. (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell 2: 437–445. Kropat, J., Oster, U., Rudiger, W. and Beck, C.F. (1997) Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes. Proc. Natl Acad. Sci. USA 94: 14168–14172. Kropat, J., Oster, U., Rudiger, W. and Beck, C.F. (2000) Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus. Plant J. 24: 523–531. Kruse, E., Mock, H.P. and Grimm, B. (1995) Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system. EMBO J. 14: 3712–3720. Kusaba, M., Ito, H., Morita, R., Iida, S., Sato, Y., Fujimoto, M., et al. (2007) Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence. Plant Cell 14: 1362–1375. Lermontova, I. and Grimm, B. (2006) Reduced activity of plastid protoporphyrinogen oxidase causes attenuated photodynamic damage during high-light compared to low-light exposure. Plant J. 48: 499–510. Mach, J.M., Castillo, A.R., Hoogstraten, R. and Greenberg, J.T. (2001) The Arabidopsis-accelerated cell death gene ACD2 encodes red chlorophyll catabolite reductase and suppresses the spread of disease symptoms. Proc. Natl Acad. Sci. USA 98: 771–776. Meskauskiene, R., Nater, M., Goslings, D., Kessler, F., Op den Camp, R. and Apel, K. (2001) FLU: a negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 98: 12826–12831. Mochizuki, N., Brusslan, J.A., Larkin, R., Nagatani, A. and Chory, J. (2001) Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc. Natl Acad. Sci. USA 98: 2053–2058. Mock, H.P. and Grimm, B. (1997) Reduction of uroporphyrinogen decarboxylase by antisense RNA expression affects activities of other enzymes involved in tetrapyrrole biosynthesis and leads to lightdependent necrosis. Plant Physiol. 113: 1101–1112. Molina, A., Volrath, S., Guyer, D., Maleck, K., Ryals, J. and Ward, E. (1999) Inhibition of protoporphyrinogen oxidase expression in Arabidopsis causes a lesion-mimic phenotype that induces systemic acquired resistance. Plant J. 17: 667–678. Morel, J.-B. and Dangl, J.L. (1997) The hypersensitive response and the induction of cell death in plants. Cell Death Differ. 4: 671–683. 728 op den Camp, R.G., Przybyla, D., Ochsenbein, C., Laloi, C., Kim, C., Danon, A., et al. (2003) Rapid induction of distinct stress responses after the release of singlet oxygen in Arabidopsis. Plant Cell 15: 2320–2332. Papenbrock, J., Mock, H.P., Tanaka, R., Kruse, E. and Grimm, B. (2000) Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol. 122: 1161–1169. Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., et al. (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19: 1649–64. Pontier, D., Albrieux, C., Joyard, J., Lagrange, T. and Block, M.A. (2007) Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling. J. Biol. Chem. 282: 2297–2304. Pöpperl, G., Oster, U., Blos, I. and Rüdiger, W. (1997) Magnesium chelatase of Hordeum vulgare L. is not activated by light but inhibited by pheophorbide. Z. Naturforsch., C, 52: 144–152. Pruzinská, A., Tanner, G., Anders, I., Roca, M. and Hörtensteiner, S. (2003) Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron–sulfur protein, encoded by the accelerated cell death 1 gene. Proc. Natl Acad. Sci. USA 100: 15259–15264. Pruzinská, A., Tanner, G., Aubry, S., Anders, I., Moser, S., Muller, T., et al. (2005) Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol. 139: 52–63. Rzeznicka, K., Walker, C.J., Westergren, T., Kannangara, C.G., von Wettstein, D., Merchant, S., et al. (2005) Xantha-l encodes a membrane subunit of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis. Proc. Natl Acad. Sci. USA 102: 5886–5891. Strand, A., Asami, T., Alonso, J., Ecker, J.R. and Chory, J. (2003) Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX. Nature 421: 79–83. Takamiya, K.I., Tsuchiya, T. and Ohta, H. (2000) Degradation pathway(s) of chlorophyll: what has gene cloning revealed? Trends Plant Sci. 5: 426–431. Tanaka, R., Hirashima, M., Satoh, S. and Tanaka, A. (2003) The Arabidopsis-accelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a: inhibition of the pheophorbide a oxygenase activity does not lead to the ‘stay-green’ phenotype in Arabidopsis. Plant Cell Physiol. 44: 1266–1274. Torres, M.A., Dangl, J.L. and Jones, J.D. (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl Acad. Sci. USA 99: 517–522. Tottey, S., Block, M.A., Allen, M., Westergren, T., Albrieux, C., Scheller, H.V., et al. (2003) Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proc. Natl Acad. Sci. USA 100: 16119–16124. Vasileuskaya, Z., Oster, U. and Beck, C.F. (2005) Mg-protoporphyrin IX and heme control HEMA, the gene encoding the first specific step of tetrapyrrole biosynthesis, in Chlamydomonas reinhardtii. Eukaryot. Cell 4: 1620–1628. Wagner, D., Przybyla, D., Op den Camp, R., Kim, C., Landgraf, F., Lee, K.P., et al. (2004) The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana. Science 306: 1183–1185. Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. Light-independent cell death by pheophorbide a Witkowski, D.A. and Halling, B.P. (1988) Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl. Plant Physiol. 87: 632–637. Witkowski, D.A. and Halling, B.P. (1989) Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl. Plant Physiol. 90: 1239–1242. Yang, M., Wardzala, E., Johal, G.S. and Gray, J. (2004) The woundinducible Lls1 gene from maize is an orthologue of the Arabidopsis Acd1 gene, and the LLS1 protein is present in non-photosynthetic tissues. Plant Mol. Biol. 54: 175–191. Zapata, M., Rodríguez, F. and Garrido, J.L. (2000) Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridinecontaining mobile phases. Mar. Ecol. Prog. Ser. 195: 29–45. (Received January 14, 2009; Accepted February 25, 2009) Plant Cell Physiol. 50(4): 719–729 (2009) doi:10.1093/pcp/pcp035 © The Author 2009. 729