Download Light-Independent Cell Death Induced by

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.
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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.
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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.
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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.
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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.
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