Bioscience Reports, Vol. 23, Nos. 2 and 3, April and June 2003 ( 2003)
Participation of Chloroplasts in Plant Apoptosis
Vitaly D. Samuilov,1,2 Elena M. Lagunova,1 Dmitry B. Kiselevsky,1
Elena V. Dzyubinskaya,1 Yana V. Makarova,1 and Mikhail V. Gusev1
Receiûed January 23, 2003
Mitochondria are known to participate in the initiation of programmed cell death (PCD)
in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a
new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a
monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic
(epidermal cells). The peels from pea (Pisum satiûum L.) leaves were treated by cyanide as
an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplastcontaining guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN −-induced apoptosis of cells of both types in the dark and in the
light. On the other hand, methyl viologen and menadione known as ROS-generating
reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that
are not oxidized spontaneously by O2 were shown to prevent the CN −-induced nucleus
destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents,
and they had no influence on the CN − effect. The light-dependent activation of CN −induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by
a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors.
The above data suggest that apoptosis of guard cells is initiated upon a combined action
of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer
chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be
antiapoptotic for the guard cell.
KEY WORDS: Photosynthesis; respiration; apoptosis; chloroplasts; mitochondria;
reactive oxygen species; plastoquinone; cytochrome b6 f complex; Pisum.
ABBREVIATIONS: AO, alternative oxidase; BH, benzylhydroxamate; BQ, p-benzoquinone; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DAD, diaminodurene; DBT, 2,5di-tret-butyl-4-hydroxytoluene; DCMU, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; DNPINT, dinitrophenylether of iodonitrothymol; DPIP, 2,6-dichlorophenolindophenol; IA,
iodoacetamide; LHCII, light harvesting chlorophyll a兾b protein complex; MV, methyl viologen; NEM, N-ethylmaleimide; PCD, programmed cell death; PMSF, phenylmethylsulfonyl fluoride; PQ, plastoquinone; PS, Photosystem; ROS, reactive oxygen species; TMPD,
N,N,N′,N′-tetramethyl-p-phenylenediamine; UQ, ubiquinone.
INTRODUCTION
Mitochondria, the energy-transducing organelles of the chemotrophic eukaryotes,
play a central role in the apoptosis of mammalian cells. In response to death stimuli,
1
Department of Physiology of Microorganisms, Lomonosov Moscow State University, Moscow 119899,
Russia.
2
To whom correspondence should be addressed. E-mail: [email protected]; fax: +007-095-939
3807.
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0144-8463兾03兾0400-0103兾0  2003 Plenum Publishing Corporation
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they release soluble apoptogenic proteins into cytosol. These proteins include
cytochrome c, AIF (apoptosis-inducing factor) flavoprotein, procaspases 2, 3, and
9, the second mitochondrial activator of caspases (Smac), endonuclease G and serine
protease Omi兾HtA2 interacting with caspase inhibitor XIAP and inducing enhanced
caspase activity (reviewed by Van Loo et al., 2002).
Several lines of evidence indicate that programmed cell death (PCD) in animals
and plants has common mechanisms (Lam et al., 2001) that presumably developed
in prokaryotes for antiviral protection of cell population and for cell differentiation
(Hochman, 1997; Aravind et al., 1999; Samuilov et al., 2000a, Lewis, 2000). PCD
involving mitochondrial cytochrome c was shown to occur in plants (Sun et al.,
1999; Balk et al., 1999; Korthout et al., 2000).
Some indirect data show that chloroplasts responsible for the light energy transduction in phototrophic eukaryotes are implicated in plant apoptosis. Inhibition of
chloroplast biogenesis in Arabidopsis thaliana mutants (‘lesion mimic mutations’’)
prevents the damage caused by a process resembling hypersensitive response-associated apoptosis (Martienssen, 1997). A decrease in the chloroplast-localized protein
DS9 in tobacco mosaic virus-infected tobacco leaves was shown to accelerate cell
death (Seo et al., 2000).
The purpose of the present studies was to investigate the role of chloroplasts in
plant PCD. We developed a system of leaf epidermal peels that are monolayers
consisting of cells of two types characterized by the phototrophic (guard cells) and
the chemotrophic (epidermal cells) modes of life, respectively. This system is convenient for light microscopy and promises to be useful in studies on the role of
chloroplasts and mitochondria in PCD. Epidermal peels were earlier used in studies
of the plant pathogenesis response in epidermal cells (Allan and Fluhr, 1997). Using
cyanide as an inducer of PCD (Wang et al., 1996; Ryerson and Heath, 1996), we
established that plant apoptosis is stimulated by illumination. Here we show that
the apoptotic effect of illumination is mediated via the redox state of the chloroplast
photosynthetic electron transfer chain in the combination with ROS. Reduced
plastoquinone in thylakoid membranes is apparently a link of the system responsible
for the death signal transduction in guard cells. (For preliminary publications, see
Samuilov et al., 2000b; 2002.)
MATERIALS AND METHODS
A number of methods have recently been developed to record PCD. The ‘‘DNA
ladder’’ obtained upon electrophoretic separation of DNA internucleosomal fragments or the appearance of caspase activity are considered hallmarks of apoptosis.
However, PCD mediated by mitochondrial AIF does not result in the appearance
of the ‘‘ladder’’ and is caspase-independent (Ferri and Kroemer, 2001). Annexin V
(used for the determination of phosphatidylserine in the plasma membrane), antibodies against Bax, Bcl-2, cytochrome c, poly(ADP-ribose)polymerase and other
proteins are also used for PCD visualization, but they all do not yield unambiguous
results. In particular, annexin V binding cannot be used as an exclusive indicator of
cell surface phosphatidyl serine inasmuch as it also binds aldehyde (e.g., malondialdehyde)-modified lipids (Balasubramanian et al., 2001). As noted by Jones (2001),
Chloroplasts in Apoptosis
105
the above hallmarks of apoptotic death represent time points well beyond the
moment of death, they only define cell corpse processing and do not tell anything
about the execution of death.
We used a direct method of microscopy to obtain reliable information on the
state of the plant cell nucleus, the target of apoptotic processes. Apoptosis in animals
results in the disruption of the nucleus and in a complete disappearance of the cell
through phagocytosis. Apoptosis in plants leads to the disruption and disappearance
of the cell nucleus as well; however, the cell wall is retained or hydrolyzed at later
stages of cell corpse management. Thus, retention of the cell walls permits one to
record veracious PCD in plants.
The experiments were carried out using the lower epidermis of 7 to 15 day old
pea (Pisum satiûum L. cv Alpha) seedlings grown under conditions of round-theclock illumination at 20–24°C. Epidermal peels were separated with tweezers and
placed in distilled water. In order to ensure a quick entry of reagents into the cells
of the separated epidermis, we used the infiltration method based on incubation
under vacuum for 1–2 min. The specimens were placed on polystyrene plates and
incubated in distilled water with additions given in the figure captions at room
temperature in the dark or under a luminescent lamp at a light intensity of approx.
1000 lx. After the incubation, the specimens were placed in distilled water for 5 min
and then treated for 5 min with Battaglia fixative (containing chloroform, 96% ethanol, glacial acetic acid and 40% formalin at a ratio of 5:5:1:1). The specimens were
thereupon washed with ethanol for 10 min to remove the fixative, then incubated in
water for 5 min and stained with the nuclear stain Carazzi hematoxylin for 20 min.
The stained epidermal samples were washed with tap water to remove excess stain
and investigated in a light microscope. Two or, in most cases, three repeats of each
study were conducted. At least 300–500 cells were counted in each repeat. The percentage of cells with destructed nuclei plus nucleus-devoid cells was calculated.
Oxygen consumption and evolution by pea leaf slices were measured with a
Clark-type platinum electrode at 25°C. The solution in the experimental cell (1.5 ml)
contained a fresh mass of pea leaf slices (30 mg), 10 mM HEPES (pH 7.1) and
25 mM KCl. White light of the saturating intensity (∼100 W兾m2) was used in
experiments.
RESULTS
Cyanide produces multiple effects on the cell metabolism as an inhibitor of
heme catalase and peroxidases, mitochondrial cytochrome c oxidase and chloroplast
ribulose-1,5-bisphosphate carboxylase. In contrast to natural inducers of PCD, CN −
elicits synchronous apoptosis with a high yield of dead cells, which facilitates the
subsequent assessment of the data obtained. Figure 1 presents the results of light
microscopy of epidermal and guard cells in pea leaf epidermal peels preincubated
for 20 hr in the dark. Cells with sharp images of nuclei are clearly visible (Fig. 1A).
All epidermal cells lack nuclei after incubating epidermal peels with NaCN (Figs.
1B and 1C). Nuclei also disappear in most guard cells (Fig. 1C). Chromatin condensation occur in some of the guard cells; in other cells, disintegration of the nuclei
into several fragments of different size (Fig. 1B) was observed.
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Fig. 1. Light microscopy of epidermal and guard cells in epidermal peels from pea leaves preincubated
for 20 hr in the dark. The cells with destructed nuclei and nucleus-devoid cells were revealed after incubation with CN − A, control; B and C, 2.5 mM NaCN. Arrows show cells nuclei in Fig. 1A and nucleus
fragments in Fig. 1B.
Almost 100% of epidermal cells lost their nuclei within 1–2 hr of dark incubation with NaCN (Fig. 2A, cf. lines 1 and 3). Illumination did not exert any influence on CN −-induced destruction of nuclei in epidermal chloroplastless cells
(Fig. 2A).
The cyanide resistance of guard cells was significantly higher than that of epidermal cells (Fig. 2B, cf. lines 1 and 3). CN −-induced nucleus destruction in guard
cells was stimulated by illumination: 70% of the nuclei were destroyed in the presence of NaCN against the background of 17% in the dark after 24 hr of incubation
(Fig. 2B, cf. lines 3 and 4). The effect of CN − as an inducer of nucleus destruction
in guard cells manifested itself significantly later than in epidermal cells—only after
15–16 hr of incubation in the dark. It occurred earlier in the light, after 10 hr of
incubation.
Fig. 2. CN −-induced nucleus destruction in epidermal (A) and guard (B) cells of
epidermal peels from pea leaves in the dark (1, 3) and in the light (2, 4). 1 and 2,
control; 3 and 4, 2.5 mM NaCN.
Chloroplasts in Apoptosis
107
As an inhibitor of catalase and peroxidases, CN − can give rise to H2O2 accumulation in cells. Since CN −-induced nucleus destruction might be mediated by ROS,
we tested the influence of antioxidant traps for free radicals. α -Tocopherol, DBT
and mannitol (on antioxidant properties of mannitol see, for example, Shen et al.,
1997), all inhibited the CN −-induced destruction of nuclei in guard (Table 1) and
epidermal (Table 2) cells. Sorbitol that is not an antioxidant did not prevent CN −induced nucleus destruction. Anaerobiosis prevented to a considerable extent CN −induced nucleus destruction in the dark and in the light.
Illumination of native chloroplasts induces electron transport from H2O to
NADP + involving Photosystem (PS) II, cytochrome b6 f complex and PS I. NADPH
is then utilized in the CO2 assimilation reactions. In an effort to elucidate the
Table 1. CN −-Induced Destruction of the Guard Cell Nuclei in Pea
Leaf Epidermal Peels
Nucleus destruction (%)
Conditions
In the dark
In the light
Aerobiosis
Aerobiosis (48 hr)*
AerobiosisCα -tocopherol
AerobiosisCmannitol
AerobiosisCsorbitol
Anaerobiosis
AerobiosisCMV
AerobiosisCmenadione
AerobiosisCBQ
AerobiosisCDAD
AerobiosisCTMPD
AerobiosisCDPIP
AnaerobiosisCMV
AerobiosisCDCMU
AerobiosisCDNP-INT
AerobiosisCstigmatellin (1)
AerobiosisCstigmatellin (2)
AerobiosisCstaurosporine
AerobiosisCNEM
AerobiosisCIA
AerobiosisCPMSF
33.5J3.7
62.9J9.3
0
—
—
0
0
3.1J2.5
0*
0*
1.5J1.3*
12.4J1.5*
0
31.4J3.8
30.7J7.8
31.7J5.5
31.4J11.2
3.9J4.1
0
—
—
84.7J4.5
100
23.5J2.3
0
83.9J2.3
0
0
7.0J2.4
8.5J1.9
10.0J3.6
6.6J1.2
19.5J3.0
0
24.3J3.0
11.4J1.7
15.0J3.5
13.1J4.4
18.2J4.7
0
0
20.9J7.1
Additions: 2.5 mM NaCN or KCN, 0.1 mM α -tocopherol, 125 mM
mannitol or sorbitol, 5 mM MV, 0.1 mM menadione, BQ, DAD,
TMPD, or DPIP, 0.01 mM DCMU or DNP-INT, 0.005 mM stigmatellin (1), 0.02 mM stigmatellin (2), 0.0025 mM staurosporine, 1 mM
NEM, 10 mM IA, 0.5 mM PMSF. Anaerobic conditions were created
by addition of 50 mM glucose, 0.1 mg兾ml glucose oxidase, and
0.06 mg兾ml catalase; refined sunflower oil (5–6 mm in thickness) was
layered on top of the aqueous phase; the epidermal peels were preincubated for 1 hr under these conditions and then KCN was added. The
epidermal peels preincubated with staurosporine, NEM. IA or PMSF
for 1 hr before KCN addition. Incubation time with CN − : 21–23 hr
and 48 hr, if the asterisk marks off figures. The (—) mark shows that
the experiment was not carried out.
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Samuilov et al.
Table 2. CN −-Induced Destruction of the Epidermal Cell Nuclei in
Pea Leaf Epidermal Peels
Nucleus destruction (%)
Conditions
In the dark
In the light
Aerobiosis
AerobiosisCα -tocopherol
AerobiosisCDBT
Anaerobiosis
AerobiosisCMV
AerobiosisCmenadione
AerobiosisCBQ
AerobiosisCDAD
AerobiosisCTMPD
AerobiosisCDPIP
AerobiosisCDCMU
AerobiosisCDNP-INT
90.8J4.3
28.2J3.6
21.5J3.0
13.0J2.7
—
—
92.8J12.9
92.0J12.6
93.0J10.5
93.9J11.5
90.8J1.2
90.1J1.2
98.3J1.2
27.9J2.5
24.4J2.6
—
100
93.7J5.7
—
—
—
—
100
95.4J2.7
Conditions were as described for Table 1.
apoptosis-enhancing effect of illumination, we tested artificial electron acceptors.
The Hill reaction is light-induced and CO2-independent electron transport from H2O
to exogenous artificial acceptors (Hill reagents) in chloroplasts.
The efficient herbicide methyl viologen (MV) is reduced by PS I, preferentially
by FeS-center FB (Fujii et al., 1990). Mitochondrial bioenergetics is also affected by
MV (Palmeira et al., 1995). Menadione is reduced by PS II, cytochrome b6 f complex
and PS I of chloroplasts (Hauska, 1977; Samuilov et al., 1997) and by mitochondrial
NADH-ubiquinone reductase (Cadenas et al., 1977). The reduction products, methyl
viologen cation-radical and menadione anion-radical, are spontaneouly oxidized by
oxygen: O−2. is produced. Menadiol is also oxidized by O2 : H2O2 is produced (Cadenas et al., 1977; Yamashoji et al., 1991).
MV and menadione by themselves strengthened nucleus destruction (16J2%
and 19J4% against 5J1% in the control with no additions—data not shown) and
had no influence on the CN − effect in epidermal cells (Table 2). The menadioneinduced apoptosis was earlier shown in tobacco protoplasts (Sun et al., 1999). MV
and menadione by themselves exerted no influence on the nuclei of guard cells in
the dark or in the light, but they prevented CN −-induced nucleus destruction under
aerobic conditions (Table 1). A twofold drop in the CN − effect in guard cells incubated in the light occurred at MV and menadione concentrations of 2.5–3 mM and
approx. 0.01 mM, respectively (data not shown). Importantly, MV had no influence
on the anaerobic removal of the CN − effect.
Electron acceptors that are not oxidized spontaneously by O2 were tested in
further experiments. BQ, DAD, TMPD, and DPIP efficiently prevented the CN −induced nucleus degradation in guard cells incubated in the light and in the dark
(Table 1), but not in epidermal cells (Table 2). These compounds interacting with
PS II, cytochrome b6 f complex and PS I of chloroplasts (Saha et al., 1971; Hauska,
1977; Samuilov et al., 1997) give rise to the oxidation of the membrane PQ pool
and suppress NADP + photoreduction. They oxidize also the membrane UQ pool
Chloroplasts in Apoptosis
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in mitochondria via an interaction with NADH-UQ oxidoreductase, succinate-UQ
oxidoreductase and cytochrome bc1 complex of the respiratory chain.
CN −-induced nucleus destruction in illuminated guard cells was prevented by
DCMU, inhibiting electron transport between the primary (QA) and secondary (QB )
plastoquinones on the acceptor side of PS II in chloroplasts, and by DNP-INT,
inhibiting quinol oxidation by the Rieske FeS-center at the QO site in the chloroplast
cytochrome b6 f complex (Delosme et al., 1987; Barbagallo et al., 1999), but not in
the mitochondrial cytochrome bc1 complex (reviewed by Rich, 1984). The same
effect was produced by stigmatellin which blocks electron transfer at QO site in both
the b6 f and bc1 cytochrome complex (Iwata et al., 1998; Breyton, 2000). DCMU,
DNP-INT, and stigmatellin had no influence on CN −-induced nucleus destruction
in guard cells in the dark (Table 1) and in epidermal cells both in the dark and in
the light (Table 2).
Staurosporine, an inhibitor of protein kinase C, and cysteine protease inhibitors
NEM and IA suppressed CN −-induced nucleus destruction in guard cells both in
the dark and in the light (Table 1). PMSF, an inhibitor of serine proteases, caused
the same effect.
Figure 3 shows that CN −-inducd nucleus destruction in guard cells was intensified by exogenous H2O2 . The H2O2 effect was maximum at 0.1 mM of H2O2 , it did
Fig. 3. Effect of H2O2 , BH, pyruvate, and rotenone on CN −-induced nucleus destruction of the
guard cells in pea leaf epidermal peels. Additions: 2.5 mM KCN, 0.1 mM H2O2 , 5 mM sodium
pyruvate (Pyr), 0.05 mM rotenone (Rot). Epidermal peels were preincubated with BH, pyruvate
or rotenone for 30 min before KCN and H2O2 addition. Incubation time in the dark or in the
light: 20 hr.
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not change upon the increase in the H2O2 concentration up to 50 mM and was not
manifested at 50 mM of H2O2 even without CN − (data not shown). The destruction
of the guard cell nuclei was not induced by benzylhydroxamate (BH), an inhibitor
of alternative oxidase (ubiquinol oxidase) in plant mitochondria, nor by rotenone,
an inhibitor of NADH: ubiquinone reductase (on the respiratory chain of plant
mitochondria see reviews, Moore and Siedow, 1991; Siedown and Umbach, 1995;
2000; Affourtit et al., 2002), or the H2O2-rotenone combination (Fig. 3). The
destructive effect of CN − was decreased by BH. It was unaffected by rotenone. These
agents increased the destructive effect of the CN −–H2O2 combination to some extent.
Pyruvate that activates the AO of plant mitochondria via formation of a thiohemiacetal with the monomeric form of AO (Siedow and Umbach, 2000; Umbach et al.,
2002) induced nucleus destruction per se (in the absence of CN −). The action of
pyruvate was not affected by H2O2 . Pyruvate either had no influence or somewhat
decreased the CN − effect. It significantly diminished the CN − plus H2O2 effect
(Fig. 3).
Figure 4 (line 1) illustrates respiratory O2 uptake by pea leaf slices incubated in
the dark. Oxygen evolution occurred upon illumination. The steady-state rate of
photosynthetic O2 evolution was established after a lag phase. The nature of this lag
that is also observed in cyanobacterial cells (Barsky et al., 1984) and in isolated
spinach (Takahama et al., 1981) or pea chloroplasts (Allen, 1984) was discussed
earlier (Samuilov and Fedorenko, 1999). Light-induced O2 evolution was inhibited
by DCMU. Consecutive switching off the light had no influence on O2 consumption.
Fig. 4. O2 consumption and evolution by pea leaf slices (LS). Additions:
0.01 mM DCMU, 0.05 mM rotenone (Rot), 0.02 mM stigmatellin (Stigm),
2.5 mM KCN, 10 mM BH, 10 mM sodium pyruvate (Pyr), 0.005 mM
CCCP, 0.01 mM myxothiazol (Myx). On and Off, switching on and off
the light.
Chloroplasts in Apoptosis
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The respiration of leaf slices was insensitive to rotenone (Fig. 4, line 2): plant mitochondria possess alternative pathways of intra- and extramitochondrial NADH and
NADPH oxidation (for review, see Siedow and Umbach, 1995). Stigmatellin hindered O2 uptake in the dark and O2 evolution in the light. CB − caused an additional
deceleration of O2 consumption: under these conditions, the mitochondrial respiration was maintained by an alternative (CN −-resistant) oxidase that was inhibited
by BH. The O2 uptake by intracellular mitochondria was insignificantly stimulated
by pyruvate and by the protonophore CCCP and suppressed by consecutive
additions of myxothiazol, an inhibitor of cytochrome bc1 complex of the respiratory
chain, BH, and CN − (Fig. 4, line 3).
In the final experiments, the effect of the glycolysis inhibitor 2-deoxy-D-glucose
(10 mM), the uncoupler of oxidative and photosynthetic phosphorylation CCCP
(0.005 mM), BH (10 mM), and the inhibitors of cytochrome bc1 complex myxothiazol (0.005 mM) and antimycin A (0.005 mM) was tested. All these agents added
separately or in combination caused the destruction of guard cell neither in the light
nor in the dark (data not shown). D-Mannose that inhibits glycolysis and induces
apoptosis in Arabidopsis roots and maize suspension-cultured cells (Stein and
Hansen, 1999) had no influence on pea guard cell nuclei at concentrations from
5 mM to 200 mM (data not shown).
DISCUSSION
Nucleus destruction in epidermal (chloroplastless and mitochondrion-containing) and guard (chloroplast- and mitochondrion-containing) cells occurred when
epidermal peels from the pea leaves were treated with CN −. The disappearance of
cell nuclei was preceded by their fragmentation (Fig. 1). We found that illumination
accelerated CN −-induced apoptosis of guard cells, but not of epidermal cells (Fig. 2).
Apoptosis of guard and epidermal cells was inhibited by antioxidants and anaerobiosis both in the light and in the dark (Tables 1 and 2), suggesting its mediation by
ROS.
As an inhibitor of catalase and peroxidases, CN − apparently leads of H2O2
accumulation in the cells. Hydrogen peroxide produces no destructive effect per se;
hydroxyl radicals, products of its one electron reduction, are dangerous oxidants:
the values of E′0 for H2O2兾OH • and OH •兾H2O are 0.32 and 2.31 V, respectively
(Koppenol, 1994). Because of its high E′0 value and radical nature, OH • indiscriminately oxidizes many organic compounds including DNA, proteins, and lipids and
initiates radical chain reactions.
It has long been known that photosynthetic electron transport (Fig. 5A) is
accompanied by the reduction of O2 to superoxide radical on the electron acceptor
side of PS I (the Mehler reaction). H2O2 is subsequently generated from O−2. in the
reaction catalyzed with superoxide dismutase. The electron flux to O2 constitutes
10% of the total electron flux from the water-oxidizing complex, even when the
supply of NADP + saturates (Asada and Takahashi, 1987). Besides, H2O2 is generated by the electron acceptor side of PS II (Ananyev et al., 1994) and by incomplete
oxidation of H2O on the electron donor side of PS II (for review, see Samuilov,
1997). Photorespiration produces H2O2 in peroxisomes through glycolate oxidase.
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Fig. 5. Photosynthetic and respiratory electron transfer chains in chloroplasts (A) and plant mitochondria (B). b6 f, cytochrome b6 f complex, c,
cytochrome c; DH, dehydrogenases; NADHin , NAD(P)Hin and
NAD(P)Hout , intramitochondrial and extramitochondrial nucleotides;
OS, oxidizable substrates; PC, plastocyanin; Q, plastoquinone for Fig.
5A and ubiquinone for Fig. 5B; WOC, water-oxidizing complex; I, II,
III, and IV, complexes of respiratory chain; for other designations, see
abbreviation list.
Moreover, the Mehler reaction activity should be increased by CN − that inactivates
chloroplast ribulose-1,5-bisphosphate carboxylase (Ishida et al., 1998) and thereby
enhances spontaneous oxidation of ferredoxin by O2 .
However, the apoptosis-enhancing effect of illumination cannot be accounted
for by assuming that chloroplasts are an extra source of ROS. Illumination and the
O−2. -generating reagents MV and menadione produced opposite effects on the CN −induced nucleus destruction in guard cells (Tables 1 and 2). The CN − effect increased
under illumination. Vice versa, it was weakened or prevented by MV and menadione. At the same time, apoptosis of epidermal cells was potentiated by these
reagents, and they had no influence on the CN − effect.
Reversible protein phosphorylation involving protein kinases and phosphatases
regulates many aspects of cell physiology. Protein phosphorylation is involved in
short time chromatic adaptation known as state transitions of the photosynthetic
apparatus in plants and algae (for reviews, see Benneth, 1991; Allen, 1992; Vener et
al., 1998; Wollman, 2001). State transitions allow photosynthetic organisms to adapt
to changes in light quality and intensity via the phosphorylation and reversible
Chloroplasts in Apoptosis
113
migration of a fraction of the chloroplast PS II light-harvesting chlorophyll a兾b protein complex (LHCII) between PS II ad PS I. LHCII protein kinase activation is
redox-dependent and involves the reduction of the membrane PQ pool: an oxidized
PQ pool led to State 1 (LHCII distribution that favors PS II), whereas a reduced
pool led to State 2 (LHCII distribution that favors PS I). Plastoquinol at the quinoloxidation site (QO) of the cytochrome b6 f complex mediates kinase activation (Vener
et al., 1998; Wollman, 2001). Dynamic structural models of cytochrome b6 f complex
were proposed that relate the activation of the LHCII kinase to the occupancy of
the QO site and the movement of the Rieske protein (Vener et al., 1998; Wollman,
2001; Finazzi et al., 2001). The redox state of PQ controls transcription of chloroplast- and nuclear-encoded PS I and PS II genes (Pfannschmidt et al., 1999; 2001).
The results of this study show that the PQ redox state in plants is also involved
in controlling PCD. Indeed, inactivation of ribulose-1,5-bisphosphate carboxylase
by CN − should give rise to PQ reduction in the chloroplast photosynthetic electron
transfer chain. CN − should exert the same effect on mitochondrial ubiquinone in
the dark, because CN − inhibits mitochondrial cytochrome c oxidase. The sequence of
CN −-induced events can be depicted as follows: quinone reduction →protein kinase
activation→protein phosphorylation→PCD initiation. Menadione, BQ, DAD,
TMPD, or DPIP oxidizing quinol and a number of other intersystem electron carriers disturbed the sequence of these events and prevented CN −-induced destruction
of nuclei in guard cells (Tables 1 and 2). The same effect was produced by MV
interacting with the PS I electron acceptor complex and with the dehydrogenases of
the respiratory chain.
Light-induced activation of the thylakoid LHCII kinases was sensitive to
DCMU (Benneth, 1991; Allen, 1992). CN −-induced destruction of nuclei in guard
cells was also inhibited by DCMU (Table 1). Further support for the conclusion of
PCD control at the level of PQ comes from the data on DNP-INT and stigmatellin
sensitivity of the light-induced activation of the nucleus destruction (Table 1). DNPINT is a quinone analogue known to be a competitive inhibitor of the plastoquinol
oxidation at the QO site of the chloroplast cytochrome b6 f complex (Delosme et al.,
1987; Barbagallo et al., 1999). Inhibition of the QO site by another quinone analogue, stigmatellin, prevented LHCII kinase-dependent protein phosphorylation
(Finazzi et al., 2001) as well as guard cell nucleus destruction in the light (Table 1).
The CN −-induced nucleus destruction in guard cells was unaffected by MV
under anaerobic conditions (Table 1). MV, a PS I electron acceptor, should oxidize
plastoquinol and components of the cytochrome b6 f complex in the light.
Table 3 summarizes the conditions of the CN −-induced nucleus destruction in
guard cells. Taken as a whole, the data obtained show that apoptosis of chloroplastcontaining guard cells is initiated when the simultaneous availability of two factors—
ROS and reduced PQ in the photosynthetic electron transfer chain—is provided.
CN −-induced nucleus destruction in guard cells was suppressed by staurosporine suggesting its mediation via protein kinase C, and was sensitive to NEM or IA
and PMSF (Table 1) pointing to the involvement of cysteine and serine proteases.
It had been shown earlier that staurosporine and cysteine and serine protease inhibitors block apoptosis induction in tobacco epidermal cells (Allan and Fluhr, 1997),
in soybean (Levine et al., 1996), and tomato (De Jong et al., 2000) suspension cells.
114
Samuilov et al.
Table 3. Conditions of the CN −-Induced Destruction of the Guard Cell Nuclei in Illuminated Pea Leaf Epidermal Peels
Conditions
Aerobiosis
AerobiosisCantioxidants
Anaerobiosis
AerobiosisCelectron acceptors
AnaerobiosisCMV
AerobiosisCDCMU
AerobiosisCDNP-INT
AerobiosisCstigmatellin
ROS
Presumable state
of PQ at the QO
site of b6 f complex
Nucleus
destruction
C
A
A
C
A
C
C
C
Reduced
Reduced
Reduced
Oxidized
Oxidized
Oxidized
Displaced
Displaced
C
A
A
A
A
A
A
A
Data on the nucleus destruction see Table 1.
This does not imply that protein kinase involved in PCD is identical with the protein
kinases controlling the excitation energy transfer between PS II and PS I. It is conceivable that there are different protein kinases controlled by the quinone redox
state.
Of particular interest is the role of the mitochondrial respiratory chain (Fig.
5B) in the apoptosis of guard cells. At first sight, the data on the CN −-induced
nucleus destruction in guard cells in the dark, on its prevention by antioxidants and
anaerobiosis, by exogenous electron acceptors as well as its resistance to DCMU,
stigmatellin and DNP-INT (Table 1) attest to the involvement of mitochondria in
PCD. However, the redox state of the photosynthetic chain is also modified under
these conditions. For example, the transition to State 2 (activation of LHCII kinases
and phosphorylation of LHCII polypeptides) in whole cells of Chlamydomonas reinhardtii was induced in the dark under anaerobic conditions established by adding
glucose and glucose oxidase (Wollman and Delepelaire, 1984; Finazzi et al., 2001).
The opposite effect—the transition to State 1—was achieved in the dark through
vigorous agitation of the cell suspension in air (Finazzi et al., 2001). Thus, the CN −induced nucleus destruction in guard cells both in the light and in the dark is apparently chloroplast-dependent.
Nevertheless, the respiratory chain state exerts influence on the nuclear apparatus of guard cells. Of special interest is the effect of pyruvate that brought about
an appreciable destruction of the guard cell nuclei in the absence of CN − (Fig. 3).
Pyruvate activates AO of plant mitochondria (Siedow and Umbach, 2000; Umbach
et al., 2002) and decreases the concentration of the reduced UQ (Millenaar et al.,
1998) that is an antioxidant in animal (Alleva et al., 2001) and plant mitochondria
(Maxwell et al., 1999; Popov et al., 2001). Reduced UQ blocked apoptosis induced
by biochemical agents (H2O2 or α -tocopheril succinate), but not receptor-induced
apoptosis in animal cells (Alleva et al., 2001). The action of pyruvate as an inducer
of apoptosis in guard cells (Fig. 3) is apparently due to decrease in the intramitochondrial reduced UQ content also. The destructive effect of the CN − plus H2O2
combination was considerably diminished by pyruvate (Fig. 3). Under these conditions, pyruvate was obviously a scavenger of exogenous and endogenous H2O2 .
Chloroplasts in Apoptosis
115
Nonenzymatic oxidative decarboxylation of α -ketoacids by H2O2 is well known
(Metzler, 1977): PyruvateCH2O2 →acetateCCO2CH2O.
The AO suppression by BH diminished considerably the destruction of guard
cell nuclei in response to CN −, but not CN − plus H2O2 (Fig. 3).
Thus, the role of chloroplasts and mitochondria in CN −-induced apoptosis of
guard cells is essentially different: reduced PQ in chloroplasts is apparently an
inducer and reduced UQ in mitochondria is apparently a suppressor of apoptosis.
The conditions for PCD manifestation in guard and epidermal cells were considerably different. The destruction of epidermal cell nuclei already reached its peak
after 1 hr of incubation with CN − (Fig. 2). Preliminary data obtained using phase
contrast videomicroscopy showed that CN −-induced disappearance of the epidermal
cell nuclei was preceded by the collapse of the cell vacuolar compartment and the
cessation of cytoplasmic streaming. Subsequently, an explosive dissolution of the
cell nucleus took place, aparently, via a mechanism based on autolysis by hydrolases
from the collapsed vacuole.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation for Basic Research (grant
01-04-48356). We are grateful to Dr. A. V. Oleskin for correcting the English version
of the manuscript.
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