EPR detection of reactive oxygen in the
photosynthetic apparatus of higher plants under light stress
Éva Hideg and Imre Vass
Institute of Plant Biology, Biological
Research Center, Szeged, Hungary
INTRODUCTION
Plants performing oxygenic photosynthesis have developed a
balanced system of enzymatic and non-enzymatic defence against
reactive oxygen species (ROS), e.g.
singlet oxygen, hydrogen peroxide or various oxygen free
radicals. This way, although molecular oxygen and its highly
reactive forms are continuously produced in illuminated
chloroplasts, whose thylakoid membranes containing both highly
unsaturated fatty acids which can participate in free
radical cascades and excessive concentration of chlorophyll
a potential photosensitising dye , the antioxidant
system is usually sufficient to prevent damage under normal
metabolic conditions. However, this balance is frequently
disturbed in plants subject to unfavourable environmental
conditions and/or pollutants. Activated oxygen has been
implicated in the damage of plants upon numerous types of natural
and artificial stress conditions (Asada et al 1994, Demmig-Adams
and Adams 1994, Foyer et al 1994, Krause 1994, Hideg 1997).
Surprisingly, light the obligatory driving force of
higher plant photosynthesis is among these stress factors.
Under conditions when photochemically active radiation (PAR) is in excess, either due to unusually
high intensity irradiation or as a consequence of lowered photon
utilising capacity, reactive molecules capable of initiating
membrane, protein and pigment damage are photoproduced. The
complex set of these reactions is known as photoinhibition (PI,
Powles 1984). If stress conditions are not severe, plants may
prevent the above situation by facilitating energy utilisation,
e.g. down regulating the energy input or by dissipating the
excess energy (Allen 1992, Demmig-Adams and Adams 1992). Surplus
PAR, however, may exhaust the adaptation systems (reviewed by
Demmig-Adams 1992, Foyer and Harbison 1994, Krause 1994) and
result in the overexcitation of photosynthesis. PI results in a
net, in vivo decrease of photosynthetic activity. It is generally
accepted, that the primary target of damage is photosystem (PS) II.
PS II is a pigment protein complex with a reaction centre
consisting of a heterodimer of two membrane spanning proteins, D1
and D2. These either bind or contain the redox cofactors involved
in the electron transport (Namba and Satoh 1987). The bound
components are: the primary electron donor chlorophyll dimer
(P680), the primary electron acceptor pheophytin (Pheo), the
subsequent quinone electron acceptors QA and QB.
Electrons from the catalytic cleavage of water by a manganese
containing cluster bound to the lumenal side of D1/D2 are
transferred to P680 via TyrZ, a redox active residue
on D1. From P680, electrons are delivered to a mobile pool of
plastoquinone molecules by subsequent redox reactions via Pheo, QA
and QB (Scheme. 1).
Scheme 1
Photosynthetic electron transport in PS II of higher plants (see
explanation in text).
Oxygen evolving thylakoid membrane, PS II and other
sub-thylakoid membrane preparations provide good models for
studying light stress. These in vitro studies have revealed the
occurrence of two major routes, the so called acceptor side
induced and donor side induced photoinhibition (API and DPI,
respectively). Both API and DPI result in the impairment of PS II
electron transport followed by the selective degradation of the
D1 reaction centre protein (Mattoo et al 1984) and, to a lesser
extent, of the D2 protein (Schuster et al 1988). Prolonged PI
results in more general membrane damage, characterised by the
appearance of lipid peroxidation products (Hundal 1992, Hideg et
al 1994a) (Scheme 2). The two forms of PI are distinguished on
the basis of differences in the primary site of electron
transport malfunctioning, fragmentation pattern of the subsequent
D1 protein degradation, as well as in the light intensity and
oxygen requirement of the two process (for review see Aro et al
1993 and references therein). A third, alternative pathway of PI
has been suggested to operate under low light intensities. ROS
are also likely involved in this process, but their predicted
amount is below the dection level of methods available at present
(Keren et al 1995, 1997).
Both models of PI assume the formation of active oxygen (Aro et
al 1993, Telfer and Barber 1994 and references therein). In API,
which is caused by excess PAR in the presence of oxygen, singlet
oxygen production has been predicted as a result of increased
reaction centre chlorophyll triplet formation, which is a
consequence of the non-physiological over-reduction of the first
quinone electron acceptor in photosystem II (Vass and Styring
1992, Vass et al 1992, Aro et al 1993). DPI occurs when electron
flow from water to P680 is insufficient. There is a consensus
that the damage is triggered by the strong oxidants (P680+,
TyrZ+) created by primary charge separation and whose lifetime is
prolonged as a result of inoperative water splitting (Thompson
and Brudwig, 1988, Telfer and Barber 1989). In such case, both
electron transport and protein damage proceed in the absence of
oxygen even upon illumination with relatively lower intensities
of PAR (Jegerschöld and Styring, 1991).
Similarly to PI by excess PAR, UV-B (280-320 nm) irradiation
causes a multitude of physiological and biochemical changes in
plants, although these two types of light stress are different at
several points. Increased doses of UV-B radiation reaching the
Earth's surface as a consequence of stratospheric ozone depletion
have increased interest in this form of light stress in the past
decade. It is well established that UV-B results in the rapid
inactivation of photosynthetic electron transport, altered
pigment composition, destruction of the membrane structure and it
may cause the dimerisation of thymine bases and lesions in DNA
(for reviews see Tevini and Teramura 1989, Vass 1997). The
increased synthesis of flavinoids (Bornman 1989) effective
quenchers of singlet oxygen, hydroxyl, superoxide and peroxy
radicals as well as the increased expression of genes for
flavinoid biosynthesis (Strid 1993) imply the involvement of ROS
in the process. In the thylakoid membrane, the primary target of
UV-B is PS II: damage by UV-B involves functional impairment of
PS II electron transport (Kulandaivelu and Noorudeen 1983, Renger
et al 1989, Hideg et al 1993) and degradation of PS II reaction
centre proteins, primarily D1 (Renger et al 1989, Greenberg et al
1989, Friso et al 1994a) and D2 (Friso et al 1994b).
Scheme 2
Scematic representation of the main events of light
stress in plants.
Our in vitro studies have confirmed production of ROS in the
above light stress conditions. The aim of the present work is to
review these studies and to show possible outlooks on in vivo
applications. Figures 4, 5 and 6 contain unpublished data, which
will be published in the printed, journal version of the First
Internet Conference on Photochemistry and Photobiology.
PRODUCTION OF ROS IN VITRO
The triplet chlorophyll singlet oxygen model of API was
evidenced by observation of reaction centre chlorophyll triplet
quenching by oxygen (Vass and Styring 1992, Vass et al 1992) and
direct detection of singlet oxygen (Hideg et al 1994b). Our
studies have shown that singlet oxygen production is a unique
characteristic of API among the three studied forms of light
stress (Hideg et al 1994a, 1994b, Hideg and Vass 1996) (Fig. 1)
and it originates in PS II (Hideg and Vass 1995) (Fig. 2).
Figure 1
EPR detection of singlet oxygen trapped by TEMP in
thylakoid membranes: untreated, exposed to 15 min PI (API),
or to 35 min UV-B irradiation and in Tris pre-treated
thylakoid membranes photoinhibited for 15 min (DPI). TEMPO
is detected as aN = 1.52 mT.
Singlet oxygen may trigger the subsequent D1 protein damage,
either directly or by promoting a special confirmational change
which makes the protein susceptible for proteolytical damage. The
possibility of a direct cleavage by singlet oxygen or by the
products of a singlet oxygen induced radical cascade has been
supported by the observation of D1 protein degradation without
the conditions of photoinhibition, indused by the chemical or
photodynamic generation of singlet oxygen (Mishra and Ghanotakis
1994).
Figure 2
EPR detection of singlet oxygen trapped by TEMP in PS
II and PS I thylakoid membrane sub-preparations upon exposure to
PI for 25 min.
Besides the role of this non-radical form of active oxygen in
API, the involvement of oxygen free radicals is feasible on the
basis of models on the events of both API and DPI pathways (for
review see Aro et al 1993 and references therein). The
possibility of radical formation has been supported by the
lessening of photoinhibition induced damage in samples containing
various free radical scavengers and/or antioxidant enzymes
(Sopory et al 1990, Richter et al 1990). PI induced free radical
production has been evidenced by spectrophotometry
in-non-oxygen-evolving PS II preparations (Chen at al. 1992,
1995) and also directly, by EPR spectroscopy in thylakoid
membranes (Hideg et al 1994a) and in Chlorella (Hirayama et al
1995). In DPI, mainly hydroxyl radicals (Fig. 3, Hideg et al
1994a) and superoxide radicals (Fig.5, Chen et al 1992) are
produced. API is dominated by carbon-centred radicals (Fig. 3,
Hideg et al 1994a), but hydroxyl radicals may also contribute in
the latter phase of damage (data not shown, Hideg et al 1994b,
Shiraishi et al 1994). In line with reports on photoinhibitory D1
protein damage in the absence of oxygen during DPI (Jegerschöld
and Styring 1991) but not in API (Hundal et al 1990), we found
that free radical production required the presence of oxygen
during API but not in DPI (Fig. 3), demonstrating that ROS
production and the propagation of PI are closely related.
Figure 3
EPR detection of free radicals by DMPO in thylakoid membranes or
Tris pre-treated thylakoid membranes upon exposure to PI for 30
min (realisation of API and DPI, respectively). Experiments were
carried out both in the presence (no label) and
absence(anaerobic") of oxygen. EPR spectra were best
interpreted with the following hyperfine splitting constants: aN = abetaH = 1.47 mT
for DMPO-OH ( * ) and aN = 1.65 mT,
abetaH = 2.32 mT
for DMPO-CH3 ( o ).
UV-B irradiation also results in free radical (Fig. 4) but not
in singlet oxygen production (Fig 1). The latter observation
demonstrates that the primary site of UV-B induced electron
transport impairment is different from that of PI by excess PAR.
In thylakoid membranes, UV-B irradiation results in parallel
production of several, mainly hydroxyl and carbon centred free
radicals (Hideg and Vass, 1996). Comparing a series of samples
with increasing PS II purity thylakoid membranes, PS II
and PS II core complex preparations suggests that the
primary event is hydroxyl radical production in PS II, the other
observed free radicals are probably produced in reactions
initiated by these primary products.
Figure 4
EPR detection of free radicals by DMPO in thylakoid
membranes and two sub-thylakoid preparations upon UV-B
irradiation for 30 min. Hyperfine splitting constants: aN = abetaH = 1.47 mT
for DMPO-OH ( * ) and aN = 1.65 mT,
abetaH = 2.32 mT
for DMPO-CH3 ( o ).
Superoxide anion radicals are formed as by-products during the
operation of photosynthetic electron transport (Mehler, 1951).
Their production site is at PS I, where molecular oxygen provides
an alternative sink for electrons in illuminated thylakoid
membranes in the absence of NADP (Takahashi and Asada 1988, Asada
1992). Accordingly, superoxide radicals can be trapped in
isolated thylakoid membranes with Tiron (Fig. 5, Hideg et al
1995). This signal is inhibited by DCMU which blocks the
electron transport between PS II and PS I , increased by
the addition of methyl-viologen enhancer of the Mehler
reaction (Takahashi and Katoh 1984, Shiraishi et al 1992)
(data not shown, Hideg et al 1995), and no superoxide trapping
was observed from the PS II core complex, even under conditions
of API (Fig. 5). These results demonstrate that the source of the
observed EPR signal is indeed the trapping of superoxide radicals
from PS I by Tiron. Also, API only slightly enhanced the EPR
signal in thylakoid membranes, indicating that superoxide
radicals are not the main promoters of API (Fig. 5, Hideg et al
1995). On the other hand, DPI resulted in intense superoxide
production (Fig. 5) in PS II core complexes, in accordance with
previous suggestions (Chen et al 1992, 1995). Superoxide radicals
are also produced in thylakoid membranes and PS II preparations
exposed to UV-B irradiation (data not shown), but sensitivity of
Tiron itself to UV-B irradiation does not allow precise
conclusions.
Figure 5
EPR detection of superoxide radicals trapped with
Tiron in thylakoid membranes (untreated and 30 min exposure
to API) and in PS II core complex preparations exposed to
either API or DPI for 30 min.
Conclusions
Table 1 summarises results of our in vitro ROS detection
studies. As it is shown here, singlet oxygen production appears
as a unique characteristic of API, providing a possibility for in
vivo identification of the process (see below). Although hydroxyl
radical production is a common characteristic of DPI and stress
by UV-B irradiation, further details of the two light stress
suggest distinct mechanism.
EPR detection of singlet oxygen is in line with the model
which has been suggested earlier (Vass et al 1992, Vass and
Styring 1992), that in API, the singlet oxygen molecules produced
in impaired photosynthetic reaction centres participate in the
initiation of the subsequent D1 protein damage. However, there is
still no consensus on the role of free radicals in light stress.
It is not yet clear whether the free radicals observed are
degradation by-products, initiators or promoters of the protein
and membrane lipid damage.
ROS IN VIVO
The above results indicate that ROS probably also play an
important role in light stress in vivo. However, direct extension
of in vitro spin trapping techniques to in vivo studies meets
experimental difficulties. As it is shown in Fig. 6, infiltration
of the trap 2,2,5,5-tetramethyl-pyrrolidine into intact leaves
indicates the occurrence of singlet oxygen production. However,
the nitroxide radical PROXYL is rapidly metabolised in the leaf
and its conversion hampers in vivo singlet oxygen detection
studies, especially quantitative ones. Extraction of the
hydroxylamine product of such reactions into an organic solvent
and its conversion back to the EPR active PROXYL may solve this
problem (data not shown) but makes quantitative in vivo studies
difficult.
Figure 6
EPR absorption spectra of
2,2,5,5-tetramethyl-pyrrolidine infiltrated broad bean leaf
segments after exposure to API by 2000 micromol m-2 s-1
PAR for times indicated in the figure. PROXYL radicals (downward
arrows) were observed with aN = 1.51 mT
hyperfine spliting.
Free radical detection is even more obstructed in vivo.
Ascorbate, which is present at high concentrations in leaves as
well as other reducing agents react with DMPO spin adducts
yielding EPR silent compounds. Rapid post-stress isolation of
thylakoid membranes in the presence of the spin trap helps this
situation, as it is illustrated by the example of detecting free
radicals in UV-B exposed leaves (Fig. 7). Nevertheless, it is
important to note, that while this post-stress detection
technique provides evidence for the occurrence of oxidative
stress, the trapped free radicals are very likely not the primary
in vivo products of such damage. On the other hand, increased
quantities of ascorbate (monodehydroascorbate) radicals in the
supernatant fraction after UV-B stress (Fig. 7) reveal the
possibility of utilising these radicals as natural, built-in
stress markers (Hideg et al 1997). In vivo, in situ detection of
ROS is necessary in order to understand the mechanism of light
stress as well as that of other plant stress situations.
Application of the spin trapping EPR spectroscopy technique,
which has been proved useful in in vitro experiments, offers an
insight into such studies already. Improvement of spin traps and
ROS sensors will provide an opportunity to judge whether pathways
identified in in vitro are relevant to field conditions and might
help finding more stress tolerant biotypes among agricultural
plants.
Figure 7
EPR spectra of pellet (thylakoid) and supernatant
fractions of crude leaf extracts from untreated and UV-B treated
broad bean leaves prepared in the presence of DMPO. Two line EPR
spectra in the supernatant fraction are from
monodehydro-ascorbate radicals. The spectrum observed in the
pellet fraction was best interpreted as a sum of spectra from
various DMPO radical adducts with the following hyperfine
splitting constants: aN = abetaH = 1.47 mT
for DMPO-OH ( * ), aN = 1.65 mT,
abetaH = 2.32 mT
for DMPO-CH3 ( o ) and aN = 1.65 mT,
abetaH = 1.07 mT,
agammaH = 0.14 mT
for the peroxy radical adduct DMPO-OR ( # , two
lines of the 6-line spectrum are hidden).
ACKNOWLEDGEMENTS
Our experimental studies concerning the topic of this review
have been supported by research grants from the Hungarian
Scientific Research Fund, OTKA F-6241 (1993-1995) and T-17455
(1994-1998).
EXPERIMENTAL PROCEDURES
Samples
Thylakoid membranes were prepared from market spinach
according to the method of Takahashi and Asada (1982),
re-suspended in a buffer containing 40 mM Hepes
(pH 7.2), 0.4 M sucrose, 15 mM NaCl and 5 mM
MgCl2, and kept on ice in the dark until use.
Tris-washing, which removes Mn and the water soluble subunits of
the water splitting enzyme was performed according to Wydrzynski
et al (1985). Tris-treated thylakoids were pelleted by
centrifugation, re-suspended under dim light in the above Hepes
buffer containing 20 mM NaHCO3 and stored on ice
in the dark until use. As a result of this treatment, the oxygen
evolving ability of the samples was decreased by more than 85%.
PS II enriched (BBY-type) preparations were made according to a
slightly modified version of the method described by Berthold et
al (1981), stored and measured in 50 mM Mes (pH 6.0),
0.4 M sucrose, 10 mM NaCl and 5 mM MgCl2.
PS II core complexes were prepared according to Ghanotakis et al
(1987) and kept in a buffer containing 20 mM Mes (pH 6.0),
0.4 M sucrose, 10 mM NaCl, 5 mM CaCl2
and 2 mM dodecyl-maltoside until use.
PS I enriched membrane preparations (PSI-110-type particles)
were made from thylakoid membranes according to the method of
Mullet et al (1980), resuspended and measured in a buffer
containing 0.1 M sucrose, 25 mM Hepes (pH 7.8),
10 mM NaCl and 5 mM MgCl2.
Anaerobic conditions were achieved by bubbling the samples with
argon. Chlorophyll content was determined according to Arnon
(1949).
Light stress
In order to achieve the conditions of API, thylakoid
membranes, PS II or PS II core complex preparations
were diluted to 100 microgram chlorophyll / mL
with the corresponding buffer, without any external electron
acceptor and illuminated by 1000-1500 micromol m-2
s-1 PAR from a KL-1500 (DMP, Switzerland) lamp through
an optical fiber guide in a 1 cm cuvette from the side. DPI
was studied either in PS II core complex preparations at
pH 8.0 (Tris-HCl buffer instead of Mes) in the presence of
0.2 mM DBMIB as electron acceptor or in Tris-washed
thylakoids at pH 7.2 (Hepes thylakoid buffer) under the same
light conditions as above. UV-B irradiation was provided by a
VL-215M lamp (Vilbert Lourmat, France) with maximal emission at
312 nm and 20 micromol m-2 s-1
total UV-B intensity, as measured with a Cole-Parmer 97503-00
radiometer (Cole Parmer, USA). Samples were irradiated from above
through a cellulose acetate film (Courtaulds Chemicals, UK) to
remove UV-C radiation. 200 microL volumes of isolated
photosynthetic membranes were irradiated in corresponding buffer
solutions in a 1 cm cuvette from above. In in vivo experiments 6
weeks broad bean (Vicia faba L.) leaves were used. For
both PI and UV-B irradiation experiments, detached leaves were
placed on wet tissue paper with their adaxial sides up, under
moderate air flow from a fan. This method assured that leaves
were neither dried nor heated during the treatment. When
indicated, leaves were infiltrated with a 60 mM aqueous
solution of 2,2,5,5-tetramethyl-pyrrolidine. Leaf segments
(2 x 10 mm) were cut from the middle of part of
the leaf (avoiding the midrib) immediately after cessation of
strong illumination and put in the EPR spectroscope in a quartz
tube.
ROS detection
Free radicals contrary to the majority of biomolecules
are paramagnetic. This offers EPR spectroscopy an ideal
tool for their detection. However, since free radicals are
short-lived and present at very low concentrations, spin traps
are applied to facilitate their detection. Spin trapping EPR
spectroscopy is based on the reaction between a diamagnetic
compound (the spin trap) and the free radical yielding a
paramagnetic and relatively stable product (Scheme 3). Free
radicals were trapped with 67 mM DMPO.
DMPO is the most useful trap for the study of oxygen-centred free
radicals due to its many adducts with distinct EPR spectra
(Janzen and Liu 1973). However, DMPO trapping may underestimate
the amount of superoxide radicals in the sample, due to the
conversion of DMPO-OOH into DMPO-OH (Finkelstein et al 1979,
1980). The use of Tiron, a quinone
compound yielding a stable quinone radical upon reacting with
superoxide may provide a better solution for studying superoxide
production (Greenstock and Miller 1975, Miller and MacDowall
1975).
Scheme 3
Principle of spin trapping (see details in text).
Similarly to free radical trapping, singlet oxygen, the
non-radical form of reactive oxygen was detected utilising TEMP, which forms the nitroxide radical TEMPO with singlet oxygen (Lion et al 1976,
1980). Samples contained 10 mM TEMP. Singlet oxygen
detection with TEMP may be jeopardised by the conversion of the
produced TEMPO into EPR silent hydroxylamine by the reducing
compounds formed in the thylakoid membrane. In order to avoid
this situation, samples were extracted after the light stress
treatment into ethylacetate and aired in the presence of
catalytic amounts of PbO2 before EPR spectroscopy, as
described earlier (Hideg and Vass, 1993). Conversion of
2,2,5,5-tetramethyl-pyrrolidine into the nitroxide PROXYL upon reaction with singlet oxygen
was utilised in leaves similarly to that of TEMP in in vitro
studies.
EPR spectra were measured with a Bruker ECS-106 spectrometer.
X-band spectra were recorded at room temperature with
9.45 GHz microwave frequency, 16 mW power, 100 kHz
modulation frequency and 0.2 mT modulation amplitude, as
described earlier (Hideg and Vass, 1993, Hideg et al 1994a,
1994b). In order to insure comparative EPR quantitation, all
spectra of spin traps in samples were measured under identical
experimental conditions, at uniform 15 microL volumes in
uniform glass capillaries. EPR gain parameters were kept uniform
in experiments illustrated on the same figure.
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