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Plant Physiol. (1998) 116: 755-764
Photosystem I Is an Early Target of Photoinhibition in Barley
Illuminated at Chilling Temperatures1
Staffan Erling Tjus*,
Birger Lindberg Møller, and
Henrik Vibe
Scheller
Plant Biochemistry Laboratory, Department of Plant Biology, Royal
Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871
Fredriksberg C, Copenhagen, Denmark
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ABSTRACT |
Light-induced damage to photosystem I
(PSI) was studied during low-light illumination of barley
(Hordeum vulgare L.) at chilling temperatures. A 4-h
illumination period induced a significant inactivation of PSI electron
transport activity. Flash-induced P700 absorption decay measurements
revealed progressive damage to (a) the iron-sulfur clusters
FA and FB, (b) the iron-sulfur clusters
FA, FB, and FX, and (c) the
phylloquinone A1 and the chlorophyll A0 or P700
of the PSI electron acceptor chain. Light-induced PSI damage was also
evidenced by partial degradation of the PSI-A and PSI-B proteins and
was correlated with the appearance of smaller proteins. Aggravated
photodamage was observed upon illumination of barley leaves infiltrated
with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate
peroxidase. This indicates that the photodamage of PSI in barley
observed during low-light illumination at chilling temperatures arises
because the defense against active oxygen species by active
oxygen-scavenging enzymes is insufficient at these specific conditions.
The data obtained demonstrate that photoinhibition of PSI at chilling
temperatures is an important phenomenon in a cold-tolerant plant
species.
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INTRODUCTION |
Light is the ultimate energy source for photosynthesis. However,
if the absorbed light energy that is transferred to the reaction centers exceeds the consumption, this may cause damage. Initially, mechanisms such as nonphotochemical thermal dissipation of excess energy (Demmig-Adams and Adams, 1992 ; Horton et al., 1994) and short-
and long-term dynamic regulation of the antenna size (Anderson and
Andersson, 1988; Aro et al., 1993) serve to protect the photosynthetic machinery. At later stages, when the protective capacities are exceeded, photoinhibition takes place. This damage results in a net
decrease in photosynthetic efficiency (Powles, 1984; Aro et al., 1993)
and has been estimated to cause losses of carbon assimilation of
approximately 10% in willow fields (Ögren and Rosenqvist, 1992).
A combination of high light with other stress factors such as chilling
or heat, drought, or low carbon dioxide supply greatly increases the
inhibition process (Powles, 1984; Demmig-Adams and Adams, 1992; Aro et
al., 1993).
PSII has long been considered the primary target for photoinhibition
(Andersson and Styring, 1991; Barber and Andersson, 1992; Prasil et
al., 1992; Aro et al., 1993) because PSI is more stable than PSII
during strong light treatments (Powles, 1984) and because its
inactivation has rarely been observed in vivo (Havaux and Eyletters,
1991). However, in isolated thylakoid membranes, PSI is as susceptible
as PSII to light stress (Satoh, 1970a; Inoue et al., 1986, 1989; Tjus,
1995). The primary target for photoinhibition of PSI upon illumination
of thylakoid membranes was shown to be FX,
FB, and FA (Inoue et al.,
1986). Also, it has been shown that oxygen is required for
light-induced inactivation of PSI to take place (Satoh, 1970a; Inoue et
al., 1989; Tjus and Andersson, 1993), which suggests that the damage is
caused by active oxygen species.
The increased sensitivity of PSI to light stress in isolated thylakoid
membranes compared with whole plants demonstrates that the PSI complex
is effectively protected by mechanisms residing within the chloroplast.
The site of superoxide production in chloroplasts is near to PSI (Asada
and Takahashi, 1987; Asada, 1994). A Cu,Zn-superoxide dismutase, which
converts superoxide to hydrogen peroxide, is present at a high
concentration in the chloroplast stroma, specifically around PSI (Ogawa
et al., 1995). Hydrogen peroxide is reduced to water by the action of
ascorbate peroxidase, which is bound to the stromal thylakoids near PSI
(Miyake and Asada, 1992). Most likely, the oxygen-scavenging enzymes of
the chloroplast may serve to protect PSI in vivo.
In the cold-sensitive plants cucumber and potato, light treatments at
low temperature implicate PSI, rather than PSII, as the primary target
for photoinhibition in vivo (Havaux and Davaud, 1994; Sonoike and
Terashima, 1994; Terashima et al., 1994; Sonoike, 1995, 1996a; Sonoike
et al., 1995; for review, see Sonoike, 1996b). The damage was suggested
to be caused by superoxide and/or singlet oxygen produced by PSI. It
was speculated that the low temperature caused a decrease in the rate
of carbon dioxide fixation that would result in an accumulation of
reducing power on the acceptor side of PSI. Furthermore, it was
proposed that the chilling temperature introduces lesions into the
lipid bilayer or inactivates the oxygen-scavenging enzymes such as
superoxide dismutase.
It has been shown in wheat that high levels of active oxygen, as
produced under severe stress conditions, directly inactivate and
degrade chloroplastic superoxide dismutase (Casano et al., 1997). When
the acceptor side of PSI is fully reduced, recombination between the
radical pairs
P700+/A0
or
P700+/A1
can generate the triplet state of P700 (Shuvalov et al., 1986; Golbeck,
1987; Golbeck and Bryant, 1991). Chlorophyll triplets can react with
molecular oxygen to create very toxic singlet oxygen that could cause
photoinhibitory damage to PSI.
One reason that photoinhibition of PSI has so far largely been
neglected is the use of relatively high light intensities in most
earlier investigations. High-light stress may actually protect PSI by
causing rapid photoinhibition of PSII and thereby depleting electron
donation to P700+. In agreement with this idea,
it is known that photoinhibition of PSI is prevented when isolated
thylakoids are illuminated in the presence of DCMU to block electron
transport from PSII (Satoh, 1970b). Under these conditions, P700 will
be oxidized into P700+, which is able to
dissipate excess excitation energy as heat (Nuijs et al., 1986) and
thereby to effectively quench deleterious effects otherwise caused by
excess light. Accordingly, it would be predicted that photoinhibition
of PSI in vivo may occur when the light intensity is sufficiently low
not to cause a direct photoinhibition of PSII. In the end, however,
inactivation of PSI will inevitably cause overreduction of the acceptor
side of PSII and, consequently, will induce damage to PSII.
In the present study, we demonstrated that low-light illumination at
chilling temperatures of barley (Hordeum vulgare L.), a
cold-tolerant plant, results in significant photoinhibition of PSI.
Furthermore, we show that the observed light-stress damage of barley
PSI is increased when superoxide dismutase and ascorbate peroxidase are
inhibited in the leaves prior to illumination.
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MATERIALS AND METHODS |
Barley (Hordeum vulgare L. cv Svalöfs Bonus) was
sown indoors in vermiculite. After 8 to 10 d of growth in
continuous low light (30 µmol photons m2
s1) at room temperature, the green parts of
primary leaves were cut off above the white base.
Uptake and Illumination Experiments
Detached leaves were kept at room temperature for 1 h to
reduce the turgor pressure, increasing their ability to absorb
treatment medium. The leaves were subsequently transferred to a beaker
containing 50 mm Tricine, pH 7.5, and 100 mm
sorbitol, which in some experiments was supplemented with 4 or 10 mm KCN, and were allowed 30 min to take up the medium and
regain their turgor. To further facilitate the uptake of medium, the
leaves were placed in an air flow supplied by a fan for 2 h to
increase the transpiration rate. These pretreatments were performed at
very low light intensities (approximately 2-5 µmol photons
m2 s1). At the end of
the uptake period (time 0 controls) and after different periods of
subsequent illumination, samples consisting of 50 leaves were taken
from each experiment. Thylakoid membranes were immediately prepared
from the leaves.
During illumination, the leaves were floated on the respective uptake
media contained in plastic trays. The illumination was performed at 2 to 4°C in a 600-L cold bench at a PPFD of 100 µmol photons
m2 s1 provided by a
sodium lamp (SON/T AGRO 400W, Philips, Eindhoven, The Netherlands). The
heat from the lamp was shielded by heat-absorbing glass plates covering
the cold bench, while the temperature inside was kept uniform by fans.
Control leaves (dark) were placed adjacent to the illuminated leaves
but were protected from the light.
Isolation of Thylakoid Membranes
Leaf samples (50 leaves, approximately 10 g) were homogenized
in a blender (Braun, Allentown, PA) equipped with exchangeable razor
blades (Kannangara et al., 1977) in 90 mL of 50 mm Tricine, pH 7.5, 100 mm Suc, and 5 mm
MgCl2. Chloroplasts were pelleted by
centrifugation at 1000g for 5 min, resuspended in the same buffer, and repelleted. Osmotic rupture of the chloroplasts was achieved by incubation in 5 mm MgCl2
for 5 min, after which the thylakoid membranes were obtained by
centrifugation at 2000g for 5 min. The thylakoid membranes
were washed twice in 10 mm Tricine, pH 7.5, repelleted as
above, and finally resuspended in 20 mm Tricine, pH 7.5, 400 mm Suc, 10 mm NaCl, and 5 mm
MgCl2. Aliquots were frozen in liquid nitrogen
and stored at  80°C until assayed for photosynthetic activities.
Chlorophyll content was determined according to the method of Arnon
(1949).
Electrophoresis
SDS-PAGE was carried out in 8 to 25% linear gradient gels
according to the method of Fling and Gregerson (1986) using
Mini-Protean electrophoresis chambers (Bio-Rad). To efficiently prevent
the deposition of protein aggregates in the sample wells, the samples were solubilized for 20 min at 75°C using 1.7% SDS, 34 mm DTT, and 34 mm
Na2CO3.
Immunoblot Analyses
To identify and quantify the PSI polypeptides, immunoblotting was
carried out essentially as described by Towbin et al. (1979). Proteins
were transferred by electrophoresis to nitrocellulose membranes in a
semidry blotting device. The membrane was incubated with
polyclonal antibodies raised in rabbits against the isolated barley PSI
complex. Subsequently, secondary antibodies ligated to alkaline
phosphatase were applied. Bromo-chloro-indolyl-phosphate and
tetrazolium blue were used for the coloring reaction. The developed
membranes were analyzed with a scanner (model JX330, Sharp Electronic,
Hamburg, Germany) combined with Image-Master software (Pharmacia).
Electron Transport Measurements
PSI electron transport was measured using a Clark-type oxygen
electrode and a 3-mL reaction mixture composed of 40 mm
Tricine, pH 7.5, 10 mm Suc, 167 µm MV, 0.1 mm DCIP, 1 mm sodium ascorbate, 10 mm NH4Cl, 10 µm DCMU, 5 mm sodium azide, and thylakoids corresponding to 15 µg of
chlorophyll. Similarly, PSII electron transport was assayed in a 3-mL
reaction mixture composed of 50 mm Tricine, pH 7.5, 20 mm NaCl, 5 mm MgCl2, 100 mm Suc, 1 mm phenyl-p-benzoquinone, and thylakoids corresponding to 15 µg of chlorophyll.
P700 Absorption Decay
Flash-induced P700 absorption changes were measured at 834 nm in a
300-µL reaction mixture containing thylakoid membranes corresponding
to 8 µg of chlorophyll dissolved in 20 mm Tricine, pH
7.5, 0.065% (w/v)
n-decyl- -d-maltopyranoside, 670 µm sodium ascorbate, and 20 µm DCIP. The
measuring beam and detection system were as previously described (Naver
et al., 1996). Actinic light pulses (532 nm, 6 ns) at full width of
half-maximum pulse amplitude were provided by a Nd:YAG laser (Quanta
Ray model GCR-100, Spectra Physics Lasers, Mountain View, CA) at a
frequency of 1 Hz. A total of 20 to 64 flash-induced decay curves were
collected and averaged for each sample. The recorded absorption changes
were resolved into exponential decay components by a
Levenberg-Marquardt nonlinear regression procedure (Press et al.,
1989).
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RESULTS |
Low-Light Illumination of Barley at Chilling Temperatures
Barley leaves were illuminated at 2 to 4°C with low light (100 µmol photons m2 s1).
Thylakoid membranes were isolated from the illuminated leaves, and
their photochemical activities were determined.
A small yet clear decrease in PSI activity (DCIP  MV) was induced by
the light treatment (Fig. 1). After
illumination for 8 h, the PSI activity was lowered by
approximately 20%, whereas the dark control was stable. At the same
time, PSII (H2O phenyl-p-benzoquinone) was slightly inactivated, but to the
same degree in both the light and dark, pointing toward a
light-independent effect of chilling. With 34 h of illumination,
PSI was not further inhibited, whereas the PSII activity had decreased
34%.
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| Figure 1.
Electron transport in thylakoid membranes isolated
from barley leaves subjected to illumination (100 µmol photons
m2 s1) at 4°C for 8 or 34 h.
Electron transport rates were assayed using an oxygen electrode. Error
bars indicate sds (n = 2-3). The 100%
activity was 229 and 178 µmol O2 (mg
chlorophyll)1 h1 for PSI and PSII,
respectively.
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To further corroborate photodamage of PSI and to specifically reveal
the lesion sites, flash-induced P700 absorption decay kinetics were
investigated. Photooxidation of P700 leads to an 834-nm absorption
change that rapidly relaxes. The total P700 absorption change,
similarly to the oxygraph measurements above, can be used to quantify
the remaining activity. In addition, the deconvolution of the decay
into different time components can reveal specific damages in the
electron acceptor chain. When isolated thylakoid membranes are used
without the addition of Fd, the terminal electron acceptors with an
intact PSI were FA and FB.
The time constant ( ) for charge recombination between
(FA/FB)
and P700+ is known to be >30 ms. With damage to
FA/FB, the charge
recombination will proceed from
FX with a time constant of
approximately 1 ms. With further damage to the electron transport
chain, the charge recombination from either
A1 or
A0, via the P700 triplet
state, takes place in 3 to 5 µs. Direct back-reaction from
A0 to
P700+, with a of approximately 30 ns, is
below the 1-µs time resolution of our experimental system (for
reviews of charge recombination, see Golbeck, 1987; Golbeck and Bryant,
1991). In this context it should be stressed that the decyl maltoside
concentration was carefully chosen to effectively dissolve the
thylakoid membranes while still retaining all of the bound antenna
pigments. This is important because excited free chlorophyll molecules
decay via chlorophyll triplet states with microsecond kinetics.
To achieve optimal deconvolution of the P700 decay kinetics, two time
windows were analyzed. On a wide, 10-ms time scale (Fig. 2B), the 1-ms back reaction from
FX, indicative of damaged
FB and FA centers, is
resolved (Golbeck 1987; Golbeck and Bryant 1991). In a narrow 200-µs
window (Fig. 2A), rapid, microsecond back reactions are unveiled,
representing damaged FX,
FB, and FA (Golbeck, 1987;
Golbeck and Bryant, 1991) and the total amplitude can be resolved. To
illustrate the results calculated from the decay curves, bar graphs
were constructed in which the amplitudes belonging to different decay
components within a sample are stacked in the same bar (Fig.
3).
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| Figure 2.
Flash-induced absorption change of P700 in
isolated thylakoid membranes. A and B represent 200-µs and 10-ms time
windows, respectively, of an illumination experiment carried out as
described in Figure 1. C and D represent 200-µs and 10-ms time
windows, respectively, of an illumination experiment carried out as
described in Figure 4 using leaves infiltrated with 10 mm
KCN. Arrows refer to different treatments in hours. D, Dark-treated; L,
light-treated.
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| Figure 3.
Resolution of flash-induced absorption changes of
P700 into decay components with different time constants. The data
shown in Figure 2, A and B, are a subset of the data used to produce this figure. A, B, and X refer to the Fe-S clusters FA,
FB and FX, respectively. The >30-ms time
component represents an intact PSI reaction center with recombination
from (FA/FB). The 1-ms time
component reflects recombination from FX
after loss of FA/FB activity. The 5-µs time
component represents recombination from either
A1 or A0 when
FA/FB and FX are all inactivated.
Error bars indicate sds for the total absorption amplitudes
(n = 2-8).
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In both windows, the 0-time controls and the 8- and 34-h dark samples
show decay kinetics (Figs. 2, A and B, and 3) calculated to be >30 ms,
demonstrating the presence of an intact PSI and antenna system. In the
200-µs window, thylakoids from the 8-h illuminated leaves
demonstrated (Figs. 2A and 3) a reduction to 91 ± 1% of the
total absorption amplitude and the appearance of a 5-µs decay
component, amounting to 15 ± 1% of the total control amplitude.
Furthermore, the 10-ms window (Figs. 2B and 3) resolved a 1-ms decay
component, representing 2.9 ± 0.8% of the 0 control amplitude,
and a main fraction showing intact PSI with an amplitude of 73 ± 1%. The 34-h illuminated sample showed the same pattern of damage as
at 8 h but was slightly more pronounced (Figs. 2, A and B, and 3).
Cu,Zn-superoxide dismutase and ascorbate peroxidase are effective
scavengers of active oxygen species. Both enzymes are inhibited by CN.
To test the importance of these two oxygen-scavenging systems in the
protection against light stress, leaves were infiltrated with 10 mm KCN prior to illumination. Otherwise, the light
treatment was as above. In the 8-h dark control, PSI activity as
determined with the oxygen electrode was decreased by approximately
10%. The 8-h light sample was inactivated by as much as 55% (Fig.
4). When illuminating for 34 h, the
dark control showed no additional decrease, whereas the light sample
was inactivated by approximately 63%. The same pattern was seen in
PSII, with 60 and 78% decreases of oxygen evolution at 8 and 34 h
of light treatment, respectively (Fig. 4). Accordingly, the prolonged
light treatment did not proportionally increase the damage to either
photosystem but, instead, the inactivation seemed to level out.
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| Figure 4.
Electron transport in thylakoid membranes isolated
from barley leaves infiltrated with 10 mm KCN and subjected
to illumination (100 µmol photons m2 s1)
at 4°C for 8 and 34 h. Electron transport rates were assayed using an oxygen electrode. Error bars indicate sds
(n = 2-3). The 100% activity was 234 and 163 µmol O2 (mg chlorophyll)1 h1
for PSI and PSII, respectively.
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Flash-induced P700 decay measurements verified the PSI inactivation
(Figs. 2, C and D, and 5). After 8 h
of treatment, the dark control exhibited a minor microsecond decay with
an amplitude of about 3%. With 8 h of light, damage in PSI was
demonstrated in the 200-µs window (Figs. 2C and 5) as a decrease to
50 ± 4% in total absorption amplitude and in the appearance of a
5-µs decay component, contributing to 10 ± 2% of the
amplitude. Furthermore, the 10-ms window (Figs. 2D and 5) resolved a
1-ms decay component with 3.2 ± 0.4% of the P700 amplitude and
an intact fraction of 37 ± 1%. At 34 h, the dark control
showed reduction to 88% in total amplitude and demonstrated a
microsecond decay with an amplitude of 5.2%. The 34-h light sample was
heavily deteriorated and thylakoid isolation resulted in aggregated
material. The combination of aggregation and pronounced damage led to a
low signal-to-noise ratio that prevented the resolution of the decay
into time components. However, the total loss of amplitude was shown to
be approximately 78%.
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| Figure 5.
Resolution of flash-induced absorption changes of
P700 into decay components with different time constants. The data
shown in Figure 2, C and D, are a subset of the data used to produce this figure. Error bars indicate sds for the total
absorption amplitudes (n = 2-7). For further
explanation, see legend to Figure 3.
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Light-Stress Effects on the PSI Reaction Center Polypeptides
Thylakoid membranes isolated from the light-stressed barley leaves
were further analyzed by SDS-PAGE (Fig.
6A). The barley PSI-A and PSI-B proteins
are very difficult to resolve from each other during electrophoresis,
and we could not discriminate between them. In the material illuminated
without KCN, no pronounced changes in the polypeptide pattern were
detectable. However, the appearance of an additional protein band
migrating at 78 kD and slightly faster than the PSI-A/B band was
noticeable in the illuminated samples (Fig. 6A, lanes 4 and 5, marked
with an asterisk) but not in the dark controls (Fig. 6A, lanes 1-3).
In the illuminated and KCN-treated leaves, a marked decrease in the
content of the reaction center proteins was already evident at 8 h, and this reduction was even more pronounced at 34 h (Fig. 6A,
lanes 9 and 10). As observed in the samples without KCN, an additional,
faster-migrating band was observed at 78 kD. Quantitatively, the
appearance of the 78-kD band correlated with the decrease of PSI-A/B
and we therefore suggest that it is derived from degradation or
modification of the reaction center proteins. In addition, a
polypeptide migrating at 15 kD also appeared. It should be noted that
the dark controls (Fig. 6A, lanes 7 and 8) also showed minor amounts of
the 78-kD band. This correlates well with the observed nonspecific
inactivation of PSI induced in the 34-h dark sample of approximately
10% (Figs. 4 and 5).
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| Figure 6.
A, SDS-PAGE of thylakoid membranes isolated after
illumination of barley leaves with or without KCN, as specified in
Figures 1 and 4. Each sample corresponds to 8 µg of chlorophyll. Lane 1, 0 h; lane 2, 8 h of dark; lane 3, 34 h of dark; lane
4, 8 h of illumination; lane 5, 34 h of illumination; lane 6, KCN plus 0 h; lane 7, KCN plus 8 h dark; lane 8, KCN plus
34 h of dark; lane 9, KCN plus 8 h of illumination; and lane
10, KCN plus 34 h of illumination. B, Immunoblot of the same
samples in A, each corresponding to 0.5 µg of chlorophyll, using an
antibody directed against the PSI complex. The blot was
densitometrically scanned and the relative intensities of coloring are
specified above the separate lanes.
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A more quantitative analysis of the PSI-A/B decrease (Fig. 6B) was
obtained by immunoblot analysis using antibodies raised against barley
PSI. The antibodies recognize two closely migrating bands in the
PSI-A/B region. Prolonged heat treatment during solubilization before
SDS-PAGE results in more protein migrating in the lower band, and,
accordingly, both bands are interpreted as representing PSI-A/B but
with different degrees of denaturation. The antibodies did not
recognize the new polypeptide at 78 kD, possibly because of lack of or
denaturation-induced changes of binding epitopes for the antibodies on
this tentative PSI-A/B product. A number of new polypeptide bands
appeared below 20 kD after photoinhibition (data not shown). However,
because the PSI antibodies also recognize the low-molecular-mass PSI
subunits, we were not able to determine the specific origin of the new
polypeptides. Illumination in the absence of KCN resulted in a small
but distinct PSI-A/B decrease of 4 and 8% in the 8- and 34-h light
samples, respectively (Fig. 6B, lanes 4 and 5). With KCN present during
illumination, the PSI-A/B was degraded by 52 and 72% in the 8- and
34-h light samples, respectively (Fig. 6B, lanes 9 and 10). The dark
control thylakoids showed no decrease in PSI-A/B except for the 34-h
KCN sample, in which an 11% decrease was observed.
Illumination at 4 mm KCN Specifying the Origin of the
Microsecond Decay
In the experiments described above, photodamage was demonstrated
after an 8-h period of light stress. In a series of experiments, the
period of light treatment at 4°C was reduced to 4 h, whereas the
KCN concentration was lowered to 4 mm to minimize the
non-light-induced damage from active oxygen and to accurately assess
PSI damage by flash-induced P700 decay kinetics (Fig.
7). Without KCN present during
illumination, the PSI activity after 4 h of illumination was
damaged, as shown by (a) a reduction to 89 ± 4% in total
amplitude; (b) the appearance of a 5-µs decay component with a
11 ± 2% amplitude, indicating damaged
FA/FB and
FX centers; and (c) a millisecond component with
3.4 ± 0.8% of the amplitude, representing destroyed FA/FB centers (Fig. 7). The
same pattern was observed upon illumination of leaves infiltrated with
KCN, but the damage was more pronounced (Fig. 7). The 4-h light sample
was decreased to 70 ± 2% in total amplitude, showed a 5-µs
component with 16 ± 1% amplitude, and a millisecond decay with
3.0 ± 0.4% amplitude.
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| Figure 7.
Flash-induced absorption changes of P700 in
thylakoid membranes isolated from barley leaves infiltrated with 4 mm KCN and subjected to illumination (100 µmol photons
m2 s1) at 4°C for 4 h.
 A834 was resolved into decay components
with different time constants. Error bars indicate sds for
the total absorption amplitudes (n = 4-8). For
further explanation, see legend to Figure 3.
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Independently of the presence or absence of KCN, the control samples
showed no microsecond decay component (Fig. 7), documenting that
neither the isolation procedure nor the presence of detergent resulted
in the generation of free chlorophyll molecules. Upon excitation by the
laser flashes, detached chlorophyll molecules would have decayed via
chlorophyll triplets with lifetimes in the microsecond range. However,
it can be argued that the light treatment could detach pigments and
that the 5-µs component observed after light stress would have a
contribution originating from such a pool of free chlorophyll. The P700
reaction center chlorophyll receives light energy from 200 to 300 antenna chlorophyll molecules, whereas a "free" chlorophyll
molecule serves only itself. Thus, absorption decay events originating
from the reaction center of PSI should have a much lower light
saturation level than decay reactions taking place in a free pigment
pool.
To substantiate that the observed microsecond decay does indeed
originate from the reaction center and not from free antenna chlorophyll molecules, a light saturation experiment regarding P700
absorption decay was performed using a sample treated with 4 h of
light and 4 mm KCN. Such thylakoids (Fig. 7) showed a
distinct damage that is also easily resolved from the noise level at
low excitation light intensities. The excitation laser flash was
initially set at saturating intensity to ensure quantitative excitation of all P700, and the intensity was then successively lowered with the
use of gray filters. At each light intensity the amplitude of P700
absorption change was monitored (Fig. 8A)
and resolved into constituting decay components (Fig. 8B). It is
evident from this experiment that the relative contribution from the
different decay components to the total absorption amplitude is stable
throughout the saturation curve. When the P700 absorption change
increases, the amplitudes of the decay components increase to the same
degree. Therefore, the contribution to
A834 from free pigment molecules is
concluded to be negligible.
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| Figure 8.
Light-saturation analysis of P700 decay
components. Isolated thylakoid membranes from 4 mm
KCN-infiltrated leaves illuminated for 4 h, as specified in Figure
7, were subjected to analysis of P700 absorption changes at different
excitation light intensities from 0 to the saturation level. A, Total
signal amplitude. B, The amplitude resolved into relative contribution
of three charge recombination components of >30 ms, 1 ms, and 5 µs.
For an explanation of the charge recombination times, see legend to
Figure 3.
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DISCUSSION |
Low-light illumination of barley leaves at chilling temperatures
induced a clear damage of PSI electron transport, quantitatively similar to that of PSII. The PSI damage appears to stabilize at a
degree of inhibition of approximately 15 to 20% (Fig. 1). This indicates that, after a first damaging hit to PSI, a level is subsequently reached where protection and potential repair mechanisms keep the inhibition at a relatively constant level. Contrary to this
stabilizing damage to PSI, the damage to PSII continues. In the context
of PSI inhibition, the progressing damage to PSII in the present study
can be viewed as a mechanism that serves to protect PSI from further
inhibition.
The combination of low temperature and low light used in this study is
typical of conditions experienced in the spring and autumn in temperate
climates such as in Nordic countries, where barley is an important
crop. Thus, it is likely that the observed photodamage of PSI would
also take place under field conditions and lower the overall
photosynthetic productivity of the plants.
The light-induced inactivation of PSI was relatively small when assayed
as oxygen consumption using MV as an electron acceptor. The site at
which MV accepts electrons may vary from one electron acceptor to the
other, depending on the concentration used (Sonoike and Terashima,
1994). With a concentration of 167 µm, as used in the
present study, MV would serve as an efficient electron acceptor only
from the iron-sulfur centers. A more clear-cut and diagnostic measure
of PSI damage was obtained by monitoring the flash-induced P700
absorption changes. These measurements provide quantification of the
activity decrease as well as specific identification of the lesion
sites (Figs. 2, A and B, and 3).
The damage sequence includes: (a) a small damage to the terminal
acceptors FA/FB represented
by the millisecond decay, (b) a larger fraction showing additional
destruction of FX that leads to the microsecond
decay, and (c) a fraction in which detectable absorption by P700 is
completely lost because of direct destruction of P700 or due to a very
rapid back-reaction from A0 as a result of
damaged A1. This sequential damage corroborates
the results of Sonoike and Terashima (1994), who concluded that lesions
are primarily localized to FX or
A1 in photoinhibited cucumber leaves. This was
evidenced by a close correlation between inactivation of
NADP+ reduction and loss of P700 absorption with
MV as the electron acceptor. In our studies the small contribution to
the total damage from the millisecond decay phase compared with a large
contribution from the microsecond decay phase indicates that once
FA/FB is damaged
Fx becomes unstable and quickly deteriorates.
Alternatively, FX could be the initial target.
However, since damage appears to involve active oxygen, we find it more
likely that FA or FB is the
initial target, because they are more exposed. FB
is known to be particularly sensitive to chemical inactivation (Golbeck and Warden, 1982; Malkin, 1984; Sakurai et al., 1991).
The PSI photodamage detected by activity measurements correlates well
with an actual decrease in the PSI-A/B reaction center protein content.
The concurrent appearance of a new polypeptide migrating slightly
faster than PSI-A/B and of several smaller polypeptides below 20 kD
possibly represents modified and/or degraded PSI-A/B. Terashima et al.
(1994) documented selective photoinhibition of PSI in cucumber leaves
under conditions similar to those used in the present study (for
review, see Sonoike, 1996b). Immunoblots using site-specific,
peptide-derived antibodies (Sonoike, 1996a) revealed selective
degradation of PSI-B in cucumber. In our study only 22% of the total
PSI-A/B content was retained in the most severely inhibited sample,
demonstrating that PSI-B as well as PSI-A were degraded. Additional
experiments will be necessary to determine the individual fates of the
PSI-A and PSI-B subunits during photoinhibition of barley.
In leaves illuminated in the absence of KCN, the remaining amount of
undegraded reaction center proteins was slightly higher than the
remaining electron transport activity, as measured both by oxygraph and
by flash photolysis. In the KCN-infiltrated leaves, there was no
significant difference between the remaining protein and activity
levels. Thus, the data suggest that protein degradation is not the
primary effect of photodamage in PSI but follows rapidly the initial
damage to the electron transport cofactors. A key topic of future
experiments will be whether the observed primary inhibition of the PSI
electron acceptors necessitates a subsequent degradation and
resynthesis of the PSI-A/B proteins in a fashion similar to the D1
protein after photoinhibition of PSII.
In thylakoid membranes, PSI is sensitive to photoinhibition even at
normal room temperature. This suggests that PSI itself is not resistant
to light damage, but effective protection mechanisms reside within the
chloroplast that are able to counteract the active oxygen species. The
higher resistance to photoinhibition at low temperatures seen in barley
compared with cucumber may be due to either a higher content of
oxygen-scavenging enzymes (e.g. superoxide dismutase, ascorbate
peroxidases, glutathione reductases, and peroxidases) or a higher
endurance of these enzymes at cold temperatures. The enzymes may be
present constitutively at high levels in barley or may be induced by
the cold-light treatment to circumvent or minimize radiation damage.
Acclimation of spinach and Scots pine to cold (Schöner et al.,
1990; Krivosheeva et al., 1996) and of potato and wheat to high-light
stress (Martinez and Maestri, 1995; Mishra et al., 1995) induces higher
contents of oxygen-scavenging enzymes. The barley plants used in the
present study were nonhardened and grown in low light. Therefore, it is
possible that acclimated barley under natural field conditions may
display higher resistance of PSI to photoinhibition. In the present
study treatment of the barley leaves with KCN, which inhibits
Cu,Zn-superoxide dismutase and ascorbate peroxidase (Halliwell and
Gutteridge, 1989), resulted in dramatically increased photodamage to
PSI. This demonstrates the importance of these detoxifying enzymes for
the photoinhibitory protection. The CN-mediated pattern of
photoinactivation closely followed that seen without the addition of CN
but was more pronounced and thus more easily studied.
The inactivation of both photosystems seemed to level out after
prolonged illumination. This can be explained in terms of an interplay
between damaging and protective mechanisms. An initial damage to PSI
may subsequently cause acceptor-side-induced photodamage to PSII and
degradation of the D1 protein. The progressive damage to PSII reduces
its function as an electron donor and may thereby protect PSI by
increasing thermal dissipation of excess energy by
P700+. Under normal conditions, degraded D1 is
rapidly replaced by de novo synthesis (Aro et al., 1993). A similar
repair of PSI would appear to be much slower because of its lower rate
of turnover. However, under chilling conditions, proteolysis and de
novo protein synthesis are slowed down, leading to an accumulation of
damaged PSI and PSII reaction centers that may then act as energy
sinks. Low-temperature-induced photoinhibition has also been shown to produce photochemically down-regulated PSII reaction centers that are
able to convert excitation energy to heat, thus protecting other
reaction centers from further photodamage (Aro et al., 1990; Tyystjärvi and Aro, 1990; Ottander et al., 1993; Krause, 1994; Schnettger et al., 1994). Altogether, this complex regulatory scheme
acts to sequentially lower the photoinhibitory effects on both
photosystems. As a further shield against photoinhibition, new
protective proteins may be synthesized during the cold treatment.
In experiments in which KCN was present, the dark control showed a
slightly decreased PSI activity. Most probably this reflects a direct
damage to P700 induced by nonspecific active oxygen attack. Havaux and
Davaud (1994) reported an increased photodamage of PSI in potato leaves
infiltrated with 100 mm DDC, which is known to inhibit
Cu,Zn-superoxide dismutase. In barley, experiments with 100 mm DDC caused severe damage of the dark controls (data not
shown). When the DDC concentration was reduced to 25 mm,
results similar to those with 10 mm KCN were obtained,
although the damage of the dark control samples was increased (data not
shown). The damage induced by inhibiting superoxide dismutase and
ascorbate peroxidase would be expected to be found in PSI, where
superoxide is mainly produced. During light treatment using KCN,
however, PSII followed closely the photoinactivation curve of PSI. This could mean that the increased production of superoxide and other active
oxygen species from PSI also migrate and severely damage PSII.
The results obtained show that photoinhibition of PSI may be expected
to cause a loss in biomass yields, especially in winter cereals grown
in cold climates and in other species grown in climates with
fluctuating temperatures that touch their lower temperature-endurance limits. In the long term, detailed knowledge of the molecular mechanisms behind this photodamage may be a tool to transfer
light-stress tolerance traits between plant species or varieties using
genetic modification.
| |
FOOTNOTES |
1
This work was financed by a grant to S.E.T. from
the Energy Research Program of the Nordic Council of Ministers, and by
the Danish Center for Plant Biotechnology.
*
Corresponding author; e-mail set{at}kvl.dk; fax 45-35-28-3333.
Received July 15, 1997;
accepted November 5, 1997.
| |
ABBREVIATIONS |
Abbreviations:
A0 and A1, primary and
secondary electron acceptors of PSI.
DCIP, dichlorophenol indophenol.
DDC, diethyl dithiocarbamate.
FA, FB and
FX, [4Fe-4S] iron-sulfur clusters of the PSI electron
acceptor chain.
MV, methyl viologen.
| |
ACKNOWLEDGMENTS |
We thank Prof. Bertil Andersson and Harry Teicher for valuable
discussions.
| |
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Top
Abstract
Introduction