Journal of Experimental Botany, Vol. 61, No. 13, pp. 3577–3587, 2010
doi:10.1093/jxb/erq171 Advance Access publication 1 July, 2010
RESEARCH PAPER
Production and diffusion of chloroplastic H2O2 and
its implication to signalling
Maria M. Mubarakshina1,2,3,*, Boris N. Ivanov2, Ilya A. Naydov2, Warwick Hillier3,
Murray R. Badger3 and Anja Krieger-Liszkay1
1
CEA Saclay, iBiTec-S, CNRS URA 2096, Service de Bioénergétique Biologie Structurales et Mécanismes,
F-91191 Gif-sur-Yvette Cedex, France
2
Institute of Basic Biological Problems RAS, 142290 Pushchino, Russia
3
RSB, Australian National University, ACT 0200 Canberra, Australia
* To whom correspondence should be addressed: E-mail: [email protected]
Received 21 January 2010; Revised 20 May 2010; Accepted 25 May 2010
Abstract
Hydrogen peroxide (H2O2) is recognized as an important signalling molecule. There are two important aspects to this
function: H2O2 production and its diffusion to its sites of action. The production of H2O2 by photosynthetic electron
transport and its ability to diffuse through the chloroplast envelope membranes has been investigated using spin
trapping electron paramagnetic resonance spectroscopy and H2O2-sensitive fluorescence dyes. It was found that,
even at low light intensity, a portion of H2O2 produced inside the chloroplasts can leave the chloroplasts thus
escaping the effective antioxidant systems located inside the chloroplast. The production of H2O2 by chloroplasts
and the appearance of H2O2 outside chloroplasts increased with increasing light intensity and time of illumination.
The amount of H2O2 that can be detected outside the chloroplasts has been shown to be up to 5% of the total H2O2
produced inside the chloroplasts at high light intensities. The fact that H2O2 produced by chloroplasts can be
detected outside these organelles is an important finding in terms of understanding how chloroplastic H2O2 can
serve as a signal molecule.
Key words: Diffusion, hydrogen peroxide, photosynthesis, reactive oxygen species.
Introduction
It is now widely accepted that the photochemical reactions
of photosynthesis are coupled directly to the generation of
reactive oxygen species (ROS) and their formation is
generally considered to be a damaging process. However,
ROS, and in particular hydrogen peroxide (H2O2), also
appear to play a major role in cellular signalling pathways
and the regulation of gene expression networks in plants
(Desikan et al., 2001; Vandenabeele et al., 2003; Apel and
Hirt, 2004; Laloi et al., 2004, 2007; Li et al., 2009). H2O2
can be generated by different sources within the cell: plasma
and apoplastic membranes, various NAD(P)H oxidases and
peroxidases as well as by organelles such as chloroplasts,
mitochondria, and peroxisomes. In addition, the amount of
H2O2 produced is tissue-specific (Mullineaux et al., 2006),
and this plays a very important role in developmental
processes (Swanson and Gilroy, 2010). Significantly, H2O2
has been recognized as the ROS inducing the largest
changes in the levels of gene expression in plants, and this
is probably due to its relative stability (Dat et al., 2000;
Bechtold et al., 2008; Fahnenstich et al., 2008; Foyer and
Noctor, 2009; Li et al., 2009). It has been shown that in
Arabidopsis thaliana up to one-third of the transcriptome is
changed upon exposure to H2O2 (Gadjev et al., 2006).
Many studies have demonstrated that chloroplast signals
initiate cellular responses during stress (Baier and Dietz,
2005). As an abiotic stress response signal H2O2 seems to
play the central role as an initiator of the response pathways
(Vanderauwera et al., 2005). To function as a signalling
molecule on a cellular level H2O2 has to be able to cross
organelle membranes. Alternatively, it could act as an
ª The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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3578 | Mubarakshina et al.
oxidant and change the redox state of cellular compounds
like glutathione and ascorbate. Furthermore, it could affect
the reduction state of the quinone pools in chloroplasts and/
or mitochondria, thereby exhibiting a signalling function
(for a recent review see Pogson et al., 2008).
Photosynthetic electron flow is regarded as the main
source of ROS in plants under high light stress. ROS that
can be produced are singlet oxygen, 1O2; superoxide, O:—
2 ;
hydrogen peroxide, H2O2, and the hydroxyl radical OH.
Singlet oxygen is primarily generated by charge recombination reactions within photosystem II (for a recent review see
Krieger-Liszkay et al., 2008). The production of hydrogen
peroxide may be detected at the donor site of photosystem
II during water splitting at a very low rate (Ananyev et al.,
1992; Hillier and Wydrzynski, 1993; Arato et al., 2004;
Clausen and Junge; 2004) but this process can probably be
largely neglected under physiological conditions. The reduction of O2 by a one-electron step results in the formation
of O:—
which then leads to the formation of H2O2. Two
2
potential pathways that lead to photosynthetic oxygen
reduction and hence H2O2 production under physiological
conditions have been discussed in the literature (Badger
et al., 2000). The first is linked to the photorespiratory cycle
and leads to H2O2 production in peroxisomes; the second is
associated with oxygen reduction in chloroplasts. The lightdependent reduction of O2 to H2O2 in chloroplasts was
discovered in 1951 by Mehler and was later called the
‘Mehler reaction’. It was shown that the primary product of
the oxygen reduction is O:—
2 (Allen and Hall, 1974; Asada
et al., 1974; Allen, 1977). Superoxide is mainly generated at
the acceptor side of photosystem I and to a very low level at
PSII and the plastoquinone pool (for a recent review see
Pospisil, 2009). Reduction of O:—
2 leads to the formation of
H2O2 that occurs either spontaneously or is catalysed by
superoxide dismutase. H2O2 can also be produced by
the reduction of O:—
2 by photosynthetic electron transport
chain components such as fully reduced plastoquinone
(Mubarakshina et al., 2006; Ivanov et al., 2007).
Many studies have focused on hydrogen peroxide as
a signalling molecule. In this context, the question arises as
to which organelles are the most important for the
generation of H2O2 and the increase in the cytosolic H2O2
concentration under light stress conditions, and to what
extent H2O2 is able to diffuse out of the compartments
where it is generated. Despite the fact that H2O2 is thought
to be able to diffuse through membranes, possibly through
aquaporins (Henzler and Steudle, 2000; Bienert et al., 2007),
a clear demonstration of this process is still missing from
the literature. There is no general agreement on the H2O2
production rate and concentration in different compartments of the cell (for a discussion see Veljovic-Jovanovic
et al., 2002; Queval et al., 2008).
In the present study, it was investigated whether H2O2
produced inside the chloroplasts can escape the chloroplast
in spite of the presence of powerful H2O2-detoxification
enzymes located in the stroma (Miyake and Asada, 1992;
Kieselbach et al., 2000; Yoshimura et al., 2002; Chew et al.,
2003). The generation of H2O2 in the light has been
investigated in isolated thylakoids and intact chloroplasts
indirectly by using spin trapping EPR spectroscopy and
directly by using the Amplex Red reagent, a H2O2-sensitive
dye. The amount of H2O2 that can be produced by
chloroplasts and diffuse out of them was estimated using
intact chloroplasts. In addition, the diffusion of H2O2 out
of chloroplasts was followed by using fluorescence microscopy. The Amplex Red reagent was used in the case of
chloroplasts and H2DCFDA, a ROS-sensitive dye, was used
in the case of protoplasts. Finally, the in vivo capacity for
light-induced O2 uptake in leaves was investigated by mass
spectrometry-based gas exchange.
Materials and methods
Plant material
Spinach leaves (Spinacia oleracea L.) were bought at the local
market. Arabidopsis plants (Arabidopsis thaliana) were grown in
a growth chamber, 8 h daylight period (light intensity 150 lmol
quanta m2 s1) at 21 C, 16 h night period at 19 C.
Thylakoids preparation
Thylakoids were isolated from spinach leaves as described by
Khorobrykh and Ivanov (2002), and were resuspended in a medium containing 0.4 M sorbitol, 20 mM NaCl, 5 mM MgCl2, and
25 mM HEPES (pH 7.6).
Chloroplasts preparation
Chloroplasts were isolated from spinach leaves according to
Laasch (1987) followed by centrifugation in a Percoll step gradient
(40–80% gradient) as described by Mullet and Chua (1983) with
some modifications. The medium for 40% Percoll contained
3.33 mM EDTA, 1.66 mM MgCl2, 83.3 mM HEPES (pH 7.6),
and 0.55 M sorbitol. The medium for 80% Percoll contained
10 mM EDTA, 5 mM MgCl2, 250 mM HEPES (pH 7.6), and
1.65 M sorbitol. Two green bands were separated after Percoll
gradient centrifigation. Only the inner part of the lower band that
corresponded to intact chloroplasts was used to ensure the
maximum intactness of chloroplasts. The chloroplasts were washed
in resuspension buffer without Percoll.
Protoplasts preparation
Arabidopsis leaves from 8–10-week-old plants were washed with
water and placed in media, containing 0.7 M sorbitol, 5 mM
CaCl2, 5 mM MES (pH 5.5), and polyvinylpyrrolidone (PVP)
5 mg ml1. Leaves were cut into 1 mm wide pieces with a razor
blade (submerged in the medium), washed with the same medium,
soaked with filter-paper, and placed into 5 ml of digestion
medium, containing in addition 1% Cellulase (Sigma) and 0.1%
Macerozyme R10 (Serva) for 2 h. Leaves were illuminated with
weak white light (65 lmol quanta m2 s1) during digestion. Then
the digestion medium was gently removed and replaced by
a medium, containing 0.7 M sorbitol, 5 mM CaCl2, and 5 mM
MES (pH 5.5). Leaf pieces were swirled gently to release the
protoplasts into the medium. The suspension was filtered through
80 lm nylon mesh and centrifuged at 30 g for 5 min. The pellet
was resuspended in a small amount of the medium (0.5–1 ml). All
operations were performed at room temperature.
Photosynthetic activity of chloroplasts and thylakoids
The photosynthetic activity of chloroplasts and thylakoids
was measured in a temperature-controlled (21 C) vessel with a
Hydrogen peroxide in chloroplasts | 3579
Clark-type O2-electrode. The reaction medium contained 0.4 M
sorbitol, 20 mM NaCl, 5 mM MgCl2, 25 mM HEPES (pH 7.6),
and 50 lg Chl ml1. Intact chloroplasts were shocked for 45 s in
5 mM MgCl2 and 25 mM HEPES (pH 7.6). 10 mM NH4Cl was
added to the shocked chloroplasts and thylakoids suspensions to
prevent a proton gradient. 1 mM K3[Fe(CN)6] was added as the
electron acceptor. The percentage of intactness of chloroplasts was
100% as tested by the ferricyanide reduction test (Heber and
Santarius, 1970) based on the ratio of light-driven O2 evolution
measured with intact and osmotically shocked chloroplasts. The
photosynthetic oxygen evolution in broken chloroplasts in the
presence of K3[Fe(CN)6] corresponded to 263632 lmol O2 mg1
Chl h1. The photosynthetic oxygen evolution in intact chloroplasts in the presence of 0.5 mM PGA and 4 mM NaHCO3
corresponded to 67612 lmol O2 mg1 Chl h1.
Electron paramagnetic resonance measurements
Electron paramagnetic resonance measurements were performed
with an ESR-300 X-band spectrometer from Bruker (Rheinstetten,
Germany). To detect H2O2-derived hydroxyl radicals the spin trap
a-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) was used. Addition of 50 lM FeEDTA produced OH from H2O2 via the socalled Fenton reaction. 4% ethanol was added to allow the
reaction between the spin trap and H2O2-derived hydroxyl
radicals. The reaction medium contained 0.4 M sorbitol, 20 mM
NaCl, 5 mM MgCl2, 25 mM HEPES (pH 7.6), 50 lg Chl ml1,
and 50 mM POBN. In the case of thylakoids 10 mM NH4Cl was
added to the suspension to prevent a proton gradient. Ethanol and
FeEDTA were added immediately after illumination to prevent
membrane degradation by ethanol during illumination. The time
of illumination was 3 min (if not otherwise specified) and the
duration of incubation with FeEDTA and ethanol was 3 min.
Spectra were recorded using a flat cell at room temperature,
9.72 GHz microwave frequency, 100 kHz modulation frequency,
1.005 G modulation amplitude, 62 mW microwave power, and
were the average of three scans.
Determination of H2O2 production by Amplex Red
H2O2 production by chloroplasts was measured by the AmplexRed
fluorescence assay. The Amplex Red reagent (10-acetyl-3,
7-dihydroxyphenoxazine) reacts with H2O2 in the presence of
horseradish peroxidase and forms resorufin, the fluorescent
product. Resorufin has a fluorescence emission maximum of
587 nm. The standard assay mixture contained 5 lM AmplexRed
(10 mM stock solution in DMSO), 10 U ml1 horseradish
peroxidase, 3 lg Chl ml1, and 4 mM NaHCO3 in the reaction
medium in a total volume of 2 ml (25 C). For fluorescence
emission spectra, the fluorescence was excited at 518 nm and
the spectra were recorded between 550–600 nm. The slit widths of
the excitation and the emission monochromator were set to 5 nm.
A calibration curve was recorded with known H2O2 concentrations. The sample was illuminated inside the fluorimeter using an
interference filter with a transmission from 245 nm to 490 nm. The
intensity of the actinic light was 200 lmol quanta m2 s1.
Observation of H2O2 using a confocal microscope
The Amplex Red reagent was used in order to show the
appearance of H2O2 outside chloroplasts. A 10 mM stock solution
of Amplex Red was prepared by dissolving in dimethyl sulphoxide
(DMSO). 3 ll drop of chloroplast suspension was mixed with
0.4 ll of Amplex Red, 10 U ml1 horseradish peroxidase, and
4 mM NaHCO3. Chloroplasts were scanned with a 488 nm laser
for resorufin excitation and a 635 nm laser for chlorophyll
excitation. Emission was detected from 540–600 nm for resorufin
fluorescence and from 640–750 nm for chlorophyll fluorescence.
Laser powers of microscope (Leica TCS SPE) were set to 5% and
10%, respectively, to avoid photobleaching and chlorophyll
degradation. The laser source acted as photosynthetically active
light, no additional light source was used during the experiment.
5% of 488 nm laser set corresponded to approximately 700–
800 lmol quanta m2 s1.
To follow H2O2 diffusion in protoplasts 5-(and-6)-carboxy-2#7#dichlorodihydrofluorescein diacetate (H2DCFDA) was used. For
each series of images a fresh sample was provided. The sample was
prepared on the slide from 4 ll drop of protoplast suspension
which was mixed with 4 ll of H2DCFDA to give a final
concentration of 50 lM. The drop was covered with a cover glass
and placed under the microscope. Protoplasts were scanned with
a 488 nm laser for DCF (dichlorofluorescein) excitation and
a 635 nm laser for chlorophyll excitation. Emission was detected
from 504–543 nm for DCF fluorescence and from 640–750 nm for
chlorophyll fluorescence. Laser powers were set to 10% and 20%,
respectively. The laser source acted as photosynthetically active
light, no additional light source was used during the experiment.
10% of 488 nm laser set corresponded to approximately 1500 lmol
quanta m2 s1. Series of images were taken over a period of 5 min
resulting in ;95 pairs of images of DCF and chlorophyll
fluorescence.
Mass spectrometry measurements
Mass spectrometry measurements were conducted on 1.3 cm2 leaf
discs obtained from 4–5-week-old Arabidopsis plants. The leaf
discs were placed into a sealed 1.6 ml closed leaf chamber and gas
sampling was performed via a membrane inlet mass spectrometry
(MIMS) system (Maxwell et al., 1998; Beckmann et al., 2009). The
leaf disc placed in the sealed leaf chamber was purged with
N2 before the addition of known volumes of 18O2 and CO2 to
provide concentrations of ;21% O2 and 3% CO2. Using MIMS
signals, samples were simultaneously recorded at m/z¼32, 36, and
44. Net CO2 assimilation was calculated as a decrease in m/z¼44
and net O2 evolution was obtained from gross O2 evolution via
increases at m/z¼32 and gross O2 uptake as decreases in m/z¼36.
Results
H2O2 production by thylakoids and chloroplasts
The generation of H2O2 by the photosynthetic electron
transport chain was measured indirectly in isolated spinach
thylakoids by using the spin trap a-(4-pyridyl-1-oxide)N-tert-butyl nitrone (4-POBN) and ethanol (Janzen et al.,
1978). 4-POBN/ethanol reacts with hydroxyl radicals (OH)
leading to the formation of a stable organic radical that can
be detected by EPR spectroscopy. The source of OH
radicals are from the so-called Fenton reaction between
H2O2 generated in the plant tissues and added ferrous
FeEDTA complex. Figure 1b shows the EPR signal of the
H2O2-derived hydroxyl radicals obtained in uncoupled
thylakoids after 3 min of illumination with white light
(1200 lmol quanta m2 s1). Ethanol and FeEDTA were
added immediately after illumination. No radical signals
from the spin trap were detectable in the dark (Fig. 1a).
Addition of catalase after illumination but prior to the
addition of ethanol and FeEDTA suppressed the EPR
signal completely, showing the specificity of the used spin
trap (Fig. 1c). In the absence of FeEDTA no signal was
obtained (Fig. 1d). These results show that OH radicals
were not generated directly in the light but originated from
H2O2 molecules accumulated during illumination. In the
presence of DCMU, a herbicide that binds to the
3580 | Mubarakshina et al.
Table 1. H2O2-derived hydroxyl radical production by spinach
thylakoids and chloroplasts measured by spin trapping EPR
spectroscopy
Samples (50 lg Chl ml1) were illuminated for 3 min at 1200 lmol
quanta m2 s1 in the presence of the spin trap 4-POBN (50 mM).
50 lM FeEDTA and 4% ethanol were added immediately after the
illumination. 10 mM NH4Cl was added in the case of thylakoids.
0.5 mM PGA and 4 mM NaHCO3 were added in the case of
chloroplasts. Where indicated 10 lM DCMU, 300 U ml1 catalase,
1 mM KCN, and 10 mM NH4Cl were added prior to the illumination.
100% is taken as the signal size obtained from illuminated thylakoids.
Four spectra of different preparations were used to calculate the
mean and the standard deviation.
EPR signal size (%)
Thylakoids dark
Thylakoids light
Thylakoids+DCMU light
Chloroplasts dark
Chloroplasts light
Chloroplast supernatant light
Chloroplast supernatant light (ethanol, FeEDTA,
and 4-POBN added to the supernatant)
Chloroplasts+catalase light
Chloroplasts+KCN light
Chloroplasts+KCN+NH4Cl light
Fig. 1. H2O2-derived hydroxyl radical production in spinach
thylakoids measured by spin trapping EPR spectroscopy. Thylakoids (50 lg Chl ml1) were illuminated for 3 min with 1200 lmol
quanta m2 s1 of white light in the presence of NH4Cl (10 mM)
and 4-POBN (50 mM). After illumination, FeEDTA and ethanol
were added and the sample was incubated for 3 min. Typical
EPR spectra of the 4-POBN/a-hydroxyethyl adduct are shown.
(a) Dark; (b) light; (c) light, in the presence of catalase (300 U ml1);
(d) light, in the absence of FeEDTA; (e) light, in the presence of
DCMU (10 lM). Spectra are shown on the same vertical scale,
but have been arbitrarily vertically offset for clarity. From the
spectra, the hyperfine splitting constants were determined to be
AN¼15.61 G and AH¼2.59 G, similar to values reported in the
literature (Finkelstein et al., 1982; Ramos et al., 1992).
QB-binding pocket in photosystem II and inhibits the
photosynthetic electron transport, only a very small signal
was observed (Fig. 1e; Table 1). It is known that the
O2 reduction rate and thus H2O2 production by PSII is very
low (Zastrizhnaya et al., 1997; Khorobrykh et al., 2002;
Arato et al., 2004) compared with the Mehler reaction (for
a review see Badger et al., 2000).
When experiments with intact chloroplasts instead of
thylakoids were performed, a smaller EPR signal was
observed following illumination (Fig. 2b).The radical signal,
despite being much smaller than with thylakoids, exhibited
similar behaviour and was not detected in the dark
(Fig. 2a). The smaller radical signal is due to the presence
of chloroplast H2O2 detoxifying enzymes. In the presence of
KCN the size of the EPR signal was increased ;5 times
0
100
462.1
0
561.4
561.7
661.2
0
2565.8
75611.5
(Fig. 2c). The addition of KCN inhibits the ascorbate
peroxidase (APX) (Jablonski and Anderson, 1982; Dalton
et al. 1987), and Rubisco but the inhibitor has only a small
effect on overall photosynthetic electron transport activity
(10% inhibition of the linear electron transport in this study,
not shown; see also Ouitrakul and Izawa, 1973). The
addition of the uncoupler NH4Cl in the presence of KCN
further stimulated the production of H2O2 as seen by the 3fold increase of the signal size (Fig. 2d) relative to the signal
obtained in the presence of KCN only (Fig. 2c). Under this
condition the size of the signal in chloroplasts is comparable
to the one observed in thylakoids (Fig. 1b; Table 1).
The experiments performed with thylakoids and comparisons with intact organelles raises questions about the
accessibility and transport of small molecules. Indeed for
chloroplasts, is H2O2 able to diffuse outside the plastid?
It appears unlikely that 4-POBN is able to enter into
the chloroplast. Previous studies with intact roots show the
action of 4-POBN with ROS derived only from the
apoplast, and there was no interaction with the symplast
(Heyno et al., 2008). However, to demonstrate that only
external H2O2 outside chloroplasts was detected in Fig. 2b,
the chloroplast suspension was centrifuged after illumination. The 4-POBN assay was then conducted on the
chlorophyll-free supernatant. As can be seen in Fig. 2e,
signals of comparable sizes were measured in the supernatant and the chloroplast suspension. This indicates that
a significant fraction of H2O2, which was produced in
chloroplasts, was not detoxified by APXs or other detoxification enzymes in the stroma and was able to diffuse
out of the chloroplasts. To show that ethanol did not lead
to a perforation of the envelope membranes, a second set of
Hydrogen peroxide in chloroplasts | 3581
Fig. 3. Dependence of EPR signals of H2O2-derived hydroxyl
radicals on H2O2 concentration. Time of incubation of H2O2 with
FeEDTA and 4-POBN and ethanol was 3 min. The reaction buffer
was the same as in the case of measurements with thylakoids
or chloroplasts. The mean and standard deviations of four
measurements are shown.
Fig. 2. H2O2-derived hydroxyl radical production in spinach
chloroplasts measured by spin trapping EPR spectroscopy. Chloroplasts (50 lg Chl ml1) were illuminated for 3 min with 1200 lmol
quanta m2 s1 of white light in the presence of PGA (0.5 mM),
NaHCO3 (4 mM), and 4-POBN (50 mM). After illumination, FeEDTA
and ethanol were added and the sample was incubated for 3 min.
Typical EPR spectra of the 4-POBN/a-hydroxyethyl adduct are
shown. (a) dark; (b) light; (c) light, in the presence of KCN (1 mM);
(d) light, in the presence of KCN (1 mM) and NH4Cl (10 mM);
(e) signal measured in the supernatant after centrifugation of (b),
FeEDTA and ethanol were added prior to the centrifugation (time to
the start of the measurement was 3 min); (f) same as (d) but
ethanol, FeEDTA, and 4-POBN were added to the supernatant
after centrifugation. Then the sample was incubated for 3 min.
Spectra are shown on the same scale as on Fig. 1.
measurements was performed in which chloroplasts were
illuminated, separated into pellet and supernatant. All spin
trap reagents were only added to the supernatant (Fig. 2f).
Almost the same signal size was obtained under this
condition demonstrating that the diffusion of H2O2 out of
chloroplasts was independent of ethanol.
Estimation of H2O2 production in vitro
The question then arises as to the extent of H2O2 production
by chloroplasts, and then how much of this H2O2 can
diffuse out into the surrounding tissue. To estimate the
concentration of H2O2 in chloroplasts a calibration curve of
the dependence of the H2O2-derived EPR OH signal on
H2O2 concentration was performed (Fig. 3). As shown
in Fig. 3 the EPR signal size increases linearly with the
increase in H2O2 concentration. The detection limit for EPR
detection of H2O2 is <1 lM under the conditions used here.
As can be seen in Fig. 4 the EPR signal of H2O2-derived
hydroxyl radicals from chloroplasts increased with increasing both light intensity and time of illumination. The light
dependency of the increase in signal size showed saturation
behaviour. The maximum signal size was reached at light
intensities of 1000–1500 lmol quanta m2 s1, where the
photosynthetic CO2 fixation in intact chloroplasts and the
rate of the electron transport in thylakoids also reach
saturation values (Walker and Osmond, 1986; Zhi-Fang
et al., 1999; Khorobrykh et al., 2004). The EPR signals
obtained from chloroplasts illuminated for 3 min with light
intensities from 400–1200 lmol quanta m2 s1 correspond
to a concentration of 5–9 lM H2O2 (Fig. 4A). It was
estimated that the rate of H2O2 appearance outside the
chloroplasts corresponds to 2 lmol H2O2 mg1 Chl h1 at
400 lmol quanta m2 s1 when a chlorophyll concentration
of 50 lg Chl ml1 was used. As shown in Fig. 2 and Table 1
the EPR signal of H2O2-derived hydroxyl radicals from
chloroplasts represents about 5% from the signal obtained
from thylakoids. If 5% is equal to 2 lmol H2O2 mg1 Chl
h1 at 400 lmol quanta m2 s1 then 100% (the signal from
thylakoids) would correspond to a rate of H2O2 production
of 40 lmol H2O2 mg1 Chl h1. This value is in a relatively
good agreement with previously published data obtained by
measurements using an oxygen electrode (Mano et al., 2001;
Khorobrykh et al., 2004; Mubarakshina et al., 2006).
The data presented in Figs 2 and 4 were obtained using
intact chloroplasts. No reduction of ferricyanide was seen in
the preparations of intact chloroplasts. A contamination
with broken chloroplasts increased the signal of H2O2derived hydroxyl radicals. The illumination of chloroplasts
with light intensities higher than 2000 lmol quanta m2 s1
led to a dramatic 2–3-fold increase of the H2O2-derived
hydroxyl radical generation. This is most likely due to
a destructive effect of light on the whole system: inhibition
of the antioxidant systems of the chloroplast (Mano et al.,
2001) and the destruction of the protein pigment complexes
3582 | Mubarakshina et al.
Fig. 5. H2O2 production in intact chloroplasts measured by the
resorufin fluorescence. (a) Chloroplasts (3 lg Chl ml1) were
illuminated with 200 lmol quanta m2 s1 of blue light in the
presence of PGA (0.5 mM) and NaHCO3 (4 mM); (b) in the
absence of chloroplasts.
Fig. 4. Influence of light intensity (A) and time of illumination (B) on
the EPR signal of H2O2-derived hydroxyl radicals from spinach
chloroplasts. PGA (0.5 mM) and NaHCO3 (4 mM) were added prior
to illumination. Chloroplasts (50 lg Chl ml1) were illuminated for
3 min with 1200 lmol quanta m2 s1 of white light in the case of
(A). The mean and standard deviations of four measurements are
shown.
which consequently led to a much higher level of ROS
generation.
In addition, H2O2 production by intact chloroplasts was
measured via the oxidation of Amplex Red to the fluorescent resorufin catalysed by horseradish peroxidase (Fig. 5).
Approximately 150 nM H2O2 was detected outside the
chloroplasts at a light intensity of 200 lmol quanta m2 s1
after 3 min of illumination of chloroplasts (3 lg Chl ml1).
This value is in good agreement with the data obtained using
the POBN assay (approximately 125 nM at the same light
intensity and time of illumination recalculated if 3 lg Chl
ml1 had been used instead of 50 lg Chl ml1 as in Fig. 4).
(Fig. 6C) indicating the specificity of the Amplex Red
reagent for H2O2.
To follow the diffusion of ROS inside a cell, protoplasts
from Arabidopsis thaliana and the ROS-sensitive dye
H2DCFDA were used (Fig. 7). Opposite to Amplex Red,
H2DCFDA can diffuse into chloroplasts. After diffusion
through membranes, it becomes de-esterified and can then
react with ROS to form the highly fluorescent product DCF
(dichlorofluorescein). H2DCFDA has recently been successfully applied to monitor H2O2 production in chloroplasts
and protoplasts (Kristiansen et al., 2009; Chen et al., 2010).
During illumination of the protoplasts a strong increase of
DCF fluorescence was observed (Fig. 7A). An overlay of
chlorophyll and DCF fluorescence shows that most of the
DCF fluorescence is localized inside the chloroplasts and
also some in the immediate neighbourhood of the chloroplasts after 5 min of illumination (Fig. 7A). One has to
keep in mind that inside protoplasts part of the H2O2 could
also have been produced by other organelles like peroxisomes and mitochondria and also by the plasma membrane. The increased production of H2O2 by each source
can lead to an increase of H2O2 in cytoplasm.
In the presence of DCMU, no DCF fluorescence was
detectable while the chlorophyll fluorescence was clearly
seen (Fig. 7B). This demonstrates that the increase in DCF
fluorescence in the absence of DCMU was caused by
photosynthetic electron flow.
Imaging of H2O2 outside of chloroplasts
To determine the site of the Amplex Red reaction with
H2O2 in our conditions the fluorescence of resorufin was
followed by confocal microscopy. As shown in Fig. 6A
resorufin fluorescence is observed outside chloroplasts after
3 min of illumination and no such fluorescence is detected
inside chloroplasts. In the presence of DCMU, no resorufin
fluorescence was seen (Fig. 6B) demonstrating that the
appearance of H2O2 outside chloroplasts is the result of
photosynthetic electron transport. Almost no resorufin
fluorescence was detected in the presence of catalase
Mass spectrometry measurements of oxygen;
estimation of H2O2 in vivo
To assess the rates of O2 uptake via a putative Mehler
pathway reaction in vivo, the rate of 18O2 uptake in leaf
discs from Arabidopsis was measured with a membrane inlet
mass spectrometer. To suppress the oxygenase activity of
Rubisco the leaf was measured with saturating CO2
concentration since the affinity of the Rubisco oxygenase to
O2 is low compared with the Mehler reaction (Canvin et al.,
1980). Without suppression of photorespiration the
Hydrogen peroxide in chloroplasts | 3583
peroxisomal glycolate oxidase activity will produce H2O2.
Both, rates of gas exchange in the light and in the dark were
measured. There are conflicting results reported in the
literature concerning the rate of respiration in the light. It
has been suggested that dark respiration in the light occurs
at the same rates as in darkness (Sharp et al., 1984) or at an
even lower rate (Villar et al., 1994). For our experiments,
the respiration in the dark measured as 18O2 uptake was
subtracted from the net 18O uptake in the light. The
remaining light-stimulated O2 uptake was then assigned as
a contribution due to the Mehler reaction. Figure 8 shows
that the putative Mehler O2 uptake in leaf discs estimated in
this way, corresponded to rates of 0.36, 1.1, and 2.2 lmol
O2 m2 s1 at light intensities of 100, 650, and 1200 lmol
quanta m2 s1 of white light. These values accounted for
on average ;4% of the total electron transport flux
(measured from the net 16O2 evolution rate) at a light
intensity of 100 lmol quanta m2 s1, a ;7% flux at 650,
and a ;11% flux at 1200 lmol quanta m2 s1, respectively.
The data were obtained by serial application of different
light intensities during the measurement of oxygen uptake
in one leaf disc. This approach was used in order to be able
to detect the very small differences in oxygen uptake at
different light intensities due to the Mehler reaction.
The average chlorophyll content in C3 plants is 0.5 g m2
according to Lawlor (1987). Using Arabidopsis leaves, an
average chlorophyll content of 0.26 g m2 was obtained.
Using this value, the data obtained by mass spectrometry
were calculated as the rate of oxygen uptake per mg1
Chl h1 and 4.9, 15.2, and 30.4 lmol O2 mg1 Chl h1 was
obtained at 100, 650, and 1200 lmol quanta m2 s1.
Taking into account the stoichiometry of 18O2 linkage into
the hydrogen peroxide molecule as 1:1, the data obtained by
mass spectrometry would be equal to the rate of H2O2
production. If up to 5% of the H2O2 produced can leave the
chloroplasts (Table 1) the H2O2 concentration outside the
chloroplasts will be in the nM range in vivo.
Discussion
Fig. 6. Confocal imaging of the resorufin fluorescence in
chloroplast suspension. Horseradish peroxidase (10 U ml1) and
NaHCO3 (4 mM) were added. Magenta colour, chlorophyll
fluorescence; green colour, resorufin fluorescence; and overlay
are shown. (A) Dark (0 min) and after 3 min of illumination;
(B) in the presence of 10 lM DCMU; (C) in the presence of
catalase (1000 U ml1). The laser at 488 nm was used for
excitation of resorufin and as an actinic light (approximate light
intensity 700–800 lmol quanta m2 s1).
In the present article, it was investigated whether and how
much chloroplastic H2O2 can diffuse out of chloroplasts
under physiological conditions. It is known that while the
photosynthetic oxygen uptake can achieve up to 30% from
total photosynthetic electron transport, less than 10%
corresponded to the Mehler reaction in C3 plants (for
a review see Badger et al., 2000) under saturating CO2
concentrations. Photorespiratory H2O2 was recognized as
one of the most important signal molecules for maintaining
cell homoeostasis during photosynthesis (Foyer and
Noctor, 2005). The fact that chloroplastic H2O2 production
is much less than peroxisomal H2O2 production and the
existence of powerful antioxidant systems in chloroplasts
may question the ability of chloroplast-generated H2O2 to
diffuse over a relatively long distance out of chloroplasts to
trigger the expression of nucleus-encoded genes. The
possibility for H2O2 to diffuse out of chloroplasts has
3584 | Mubarakshina et al.
Fig. 7. Distribution of H2O2-mediated fluorescence of DCF in Arabidopsis protoplasts. Magenta colour, chlorophyll fluorescence; green
colour, DCF fluorescence, and overlay are shown. Fluorescence before illumination and after illumination (5 min) is shown. In the case of
DCMU only the images after illumination (5 min) are given. The laser at 488 nm was used for excitation of DCF and as an actinic light
(approximate light intensity 1500 lmol quanta m2 s1).
Fig. 8. Mass spectrometry measurements of light oxygen uptake
by chloroplasts in vivo. Leave discs of Arabidopsis were used.The
oxygen uptake was measured as the rate of 18O2 exchange in the
light. The respiration oxygen uptake in the dark was subtracted
from the light oxygen uptake. The averages of three experiments
are shown.
already been discussed previously by Ivanov (2000) and
Mano et al. (2001) on the basis of experiments in which
chloroplasts were illuminated in the presence of methylviologen. Here, using intact chloroplasts and spin trapping EPR
spectroscopy, it was possible to show that H2O2 produced
in chloroplasts under physiological conditions can leave the
chloroplasts even at relatively low light and short illumination. This was confirmed by using fluorescence microscopy
(Figs 6, 7). The rate of H2O2 appearance outside the
chloroplasts increased with an increase in light intensity
and time of illumination (Fig. 4) and corresponded to
approximately 2 lmol H2O2 mg1 Chl h1 at 400 lmol
quanta m2 s1. H2O2 production of the same order of
magnitude was obtained using the Amplex Red reagent
(Fig. 5). It is unlikely that H2O2 can pass through the
chloroplast envelope membrane simply by diffusion and it
seems more likely that it passes through aquaporins
(Henzler and Steudle, 2000; Bienert et al., 2007).
The appearance of H2O2 in cytoplasm may also reflect an
inactivation of detoxification enzymes in different organelles
during illumination. The concentration of H2O2 in cytoplasm dramatically increased when APX activity is decreased
(Polle, 2001; Pnueli et al., 2003). It has been reported in
the literature (Mano et al., 2001; Miyake et al., 2006;
Kitajima et al., 2007) that APX activity is lost upon high
light illumination. Mano et al. (2001) reported that illumination of leaves for 20 min in the presence of methylviologen
leads to a strong inactivation of tAPX, sAPX, and cAPX
(see also Miyake et al., 2006; Kitajima et al., 2007). In the
experimental conditions used in the present study, the
time of illumination was short and no methylviolgen was
added. The biggest part of chloroplast-generated H2O2 was
detoxified inside the chloroplasts as can be seen by
comparing the spectra b and d in Fig. 2. The inhibition of
APXs by KCN resulted in much higher amount of H2O2
Hydrogen peroxide in chloroplasts | 3585
outside of chloroplasts. Thus if, under severe stress conditions, APXs were completely inhibited, the appearance of
H2O2 in cytoplasm could be very significant and could
achieve up to 75% of the H2O2 produced via photosynthetic
electron transport (see spectrum d in Fig. 2; Table 1).
It is probable that H2O2 produced inside chloroplasts can
serve as a signal molecule for the regulation of expression of
responsive chloroplast-encoded genes (Casano et al., 2001).
Great attention has been given to the expression of cAPX
(the nuclear-encoded gene of cytoplasmatic APX). It was
shown that the level of cAPX was much lower in a mutant
that expressed catalase in chloroplasts and had a lower
H2O2 concentration in these organelles than in the wild type
(Yabuta et al., 2004). In addition, it was reported that the
expression of cAPX is induced in high light (Karpinski
et al., 1999; Chang et al., 2004) and that, in the presence of
methylviologen, the level of cAPX expression is correlated
with the level of H2O2 (Yabuta et al., 2004). It was
suggested that chloroplastic H2O2 might be a redox signal
that activates the expression of cAPX (Karpinski et al.,
1999). It was proposed later that this function could be
assigned to the extracellular H2O2 (produced, for example,
by plasma membrane or apoplast) or to abscisic acid that
perhaps works together with H2O2 (Fryer et al., 2003;
Yabuta et al., 2004; Bechtold et al., 2008). It has been
shown recently that abscisic acid secreted from vascular
cells regulates the expression level of high light responsive
genes by integration of H2O2- and redox-mediated retrograde signalling (Galvez-Valdivieso et al., 2009). Since
cAPX is the nuclear-encoded gene it is possible that the
total H2O2 appearance in the cytoplasm may be a signal
for cAPX regulation independently from which organelle
(or other sources) H2O2 came from. Except for cAPX,
400 H2O2-responsive protein families were found in Arabidopsis thaliana (Vandenbroucke et al., 2008).
In the experiments presented here, it is demonstrated that
a significant part of the chloroplast-derived H2O2 diffuses
out of chloroplasts and this may be sufficient to trigger
signalling processes in the cytoplasm or nucleus and to lead
to a change in the expression level of responsive genes. Our
findings could give a new background for the understanding
of the regulatory effect of H2O2 as a signal molecule in the
whole cell.
Acknowledgements
We would like to thank Sun Un and Bill Rutherford
(CEA Saclay) for fruitful discussions and Prue Kell
(ANU) for growing Arabidopsis. This work is supported
by an Incoming Marie Curie Fellowship (Incoming
International Fellowships (IIF) Call: FP7-PEOPLE-20074-2-IIF), the Joint French-Australian Science and
Technology Programme (Australian DEST grant number
FR07001, French FAST grant number16139QE), the
Russian Foundation for Basic Research (Grant number
08-04-0141), and by the Agence Nationale de Recherche
reference ANR-09-BLAN-0005-01.
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