From the Centro di Studio del CNR sulla Biologia
Cellulare e Molecolare delle Piante, Via Celoria 26, 20133 Milano,
Italy, the
Dipartimento di Biologia, Università degli
Studi di Padova, Via U. Bassi 58/B, 35100 Padova, Italy, and the
** Dipartimento di Scienze e Tecnologie Avanzate, Università del
Piemonte Orientale Amedeo Avogadro, Via Borsalino 54, 15100 Alessandria, Italy
Received for publication, December 18, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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The relationship between state transitions and
photoinhibition has been studied in Chlamydomonas
reinhardtii cells. In State 2, photosystem II activity was more
inhibited by light than in State 1. In State 2, however, the D1 subunit
was not degraded, whereas a substantial degradation was observed in
State 1. These results suggest that photoinhibition occurs via the
generation of an intermediate state in which photosystem II is inactive
but the D1 protein is still intact. The accumulation of this state is
enhanced in State 2, because in this State only cyclic photosynthetic electron transport is active, whereas there is no electron flow between
photosystem II and the cytochrome
b6f complex (Finazzi, G., Furia,
A., Barbagallo, R. P., and Forti, G. (1999) Biochim. Biophys. Acta 1413, 117-129). The activity of photosystem I and of cytochrome b6f as well as the coupling of
thylakoid membranes was not affected by illumination under the same
conditions. This allows repairing the damages to photosystem II thanks
to cell capacity to maintain a high rate of ATP synthesis (via
photosystem I-driven cyclic electron flow). This capacity might
represent an important physiological tool in protecting the
photosynthetic apparatus from excess of light as well as from other
a-biotic stress conditions.
The photochemical utilization of absorbed light is a critical step
in the photosynthetic process. Because harvesting of light, photochemistry, and electron transfer occur on widely different scales
of time, a correct balance among these different processes is required
to optimize the efficiency of CO2 fixation.
When light is absorbed in excess of what can actually be
utilized by photochemistry, damage to the photosynthetic apparatus may
be induced. Impairment of both photosystem I
(PSI)1 (1) and photosystem II
(PSII) (2) has been described, and this loss of activity has been
termed photoinhibition (3). It has been also shown that the degradation
of the PSII reaction center D1 subunit is a major consequence of
photoinhibition (2).
Some mechanisms contribute to protecting the photosynthetic apparatus
from an excess of light (4, 5). The first is the so-called
energy-dependent quenching, qE, i.e. the
increased thermal dissipation in the PSII antennae that follows the
generation of the electrochemical proton gradient across the thylakoid
membranes. It is supposed to protect the reaction center from the
consequences of a strong illumination by reducing the amount of energy
present in the antenna protein complexes (6).
The second one (6) is state transitions, a phenomenon that has been
discovered in Chlorella pyrenoidosa (7) and in
Porphyridium cruentum (8). It is a mechanism to balance
light utilization between the two photosystems that is based on the
reversible transfer of a fraction of the light-harvesting complex II
(LHCII) from PSII to PSI (reviewed in Refs 9-11). It is also supposed
to protect PSII from photoinhibition inasmuch as it can decrease the
size of its antenna.
The migration of LHCII to PSI (State 1-State 2 transition) results from
the phosphorylation of the former by a membrane-bound protein kinase,
which is activated under reducing conditions (reviewed in Refs. 9
and12). Under oxidizing conditions, the kinase is deactivated, and
LHCII is dephosphorylated by a thylakoid-bound phosphatase, which is
possibly regulated by the recently discovered immunophilin-like 40-kDa
lumenal TLP protein (13). After dephosphorylation, LHCII rebinds
to PSII (State 2-State 1 transition).
In higher plants, only a small fraction of the LHCII (15-20%,
reviewed in Ref. 10) migrates reversibly from PSII to PSI. In the green
alga Chlamydomonas reinhardtii, on the contrary, a much
larger fraction of the PSII antenna is transferred during State 1-State
2 transition (14), and a much larger decrease of PSII energy capture is
accordingly observed (15). In addition, cytochrome
b6f complexes accumulate in the
unstacked lamellae in State 2 (16). Therefore, it is unlikely that
state transitions serve the purpose of balancing the absorption of PSII
and PSI in Chlamydomonas. Instead, State 2 would represent a
structural condition where most of the excitation energy is utilized by
PSI photochemistry so that cyclic electron transport around PSI is likely to prevail over linear electron flow that involves both PSI and PSII.
In agreement with this idea, Finazzi et al. (17) show that
although the cytochrome b6f turnover
was the same in State 1 and State 2, it was completely inhibited by the
addition of the PSII inhibitor DCMU in State 1, whereas no effect of
this inhibitor was observed in State 2. This result led Finazzi
et al. (17) to propose that in State 2 the reducing
equivalents involved in the reduction of the cytochrome
b6f are not produced at the level of
PSII but rather at the level of PSI. Under these conditions PSII is not
connected to the intersystem electron carriers but is still
photochemically active (17).
To investigate whether this lack of functional connection between PSII
and cytochrome b6f complex might
affect the sensitivity of the former to photoinhibition, we have
measured the effects of strong illumination on fluorescence emission,
O2 evolution, and cytochrome f reduction in
algae under State 1 or State 2 conditions. We have found that PSII is
more prone to photoinhibition in State 2. However, in this state the
loss of activity is not accompanied by a degradation of the D1 protein.
The effect on PSII seems to be rather specific as neither PSI nor
cytochrome b6f activities nor the
coupling of thylakoid membranes were affected by the treatment. Thus,
we suggest that state transitions in C. reinhardtii
represent a means to maintain a high ATP synthesis capacity, even when
damages to PSII are induced by illumination with extremely intense light.
Strains and Culture Conditions--
C. reinhardtii
wild type (from strain 137C) was kindly provided by the Laboratoire de
Physiologie Membranaire du Chloroplaste at the Institut de Biologie
Physico-Chimique of Paris (France). Cells were grown at 24 °C in
acetate-supplemented medium (18) under 60 µE
m State Transitions and Photoinhibitory Treatments--
State 1 was obtained through incubation of the cells in the dark under strong
agitation, whereas State 2 was obtained through dark incubation in
anaerobic conditions obtained by argon bubbling. Photoinhibition was
performed by illuminating the sample with white light on a thin layer
(~1 mm) of cells in a Petri dish ([chlorophyll] = 500 µg
ml Oxygen Evolution and Fluorescence Emission
Measurements--
Photosynthesis and respiration were measured as the
O2 exchange with a Clark-type electrode (Radiometer,
Denmark) at 24 °C. The actinic light was filtered through a heat
filter, and its intensity was 850 µE m Spectroscopic Measurements--
Spectroscopic measurements were
performed on whole cells at room temperature using a homemade
spectrophotometer as described by Joliot et al. (21).
In continuous light experiments, actinic light was provided by a
light-emitting diode array, placed on both sides of the cuvette. Its
intensity was 1500 µE m
In single turnover flash experiments, excitation was provided by a
xenon lamp (EG&G). Light was filtered through a Schott filter (RG 695)
and was of saturating intensity. Measurements were performed on algae
kept in State 2 to ensure dark reduction of the plastoquinone pool.
Repetitive (usually 10) illuminations were performed at the frequency
of 0.15 Hz. The transmembrane potential was estimated from the
amplitude of the electrochromic shift at 515 nm, which is known to give
a linear response with respect to the electric component of the
transmembrane potential (22). Under the conditions employed here, the
kinetics of the electrochromic signal exhibited two phases previously
characterized in Joliot and Delosme (23): a fast phase (phase a),
associated with PSI and PSII charge separation, and a slow phase, which
develops in the millisecond time scale and is associated with the
turnover of the cytochrome b6f
complex (phase b).
The kinetics of phase b was deconvoluted from membrane potential decay
assuming that the latter process exhibited first-order kinetics. Phase
b was then computed considering that the rate of membrane potential
decay between two consecutive acquisitions was linearly related to its
mean value in the same interval. Cytochrome f redox changes
were evaluated as the difference between the absorption at 554 nm and a
base line drawn between 545 and 573 nm. We have checked that this
procedure for deconvolution of cytochrome f signals was
reliable also in the case of continuous illumination (17).
Protein Analysis--
For protein analysis, algae were collected
at the indicated times, washed in 20 mM HEPES containing
protease inhibitors (200 µM phenylmethylsulfonyl
fluoride, 5 mM amino- To investigate the influence of state transitions on the
sensitivity of C. reinhardtii to photoinhibition, we have
performed experiments on cells placed either in State 1 or in State 2. We have measured fluorescence emission, photosynthetic activity (as O2 evolution), and the rate of cytochrome f
reduction after exposure to strong illumination. The measurements were
performed on the same batch of algae, collected either before starting
the light treatment or after different irradiation times.
Fluorescence Emission and Oxygen Evolution--
Illumination of
the algae with high light intensity largely modified their fluorescence
emission parameters; a large decrease of the maximal fluorescence
emission (Fm) was observed in State 1 cells (Fig.
1A), whereas an increase of
the minimal one (Fo) occurred in State 2 (Fig. 1B). This
suggests that the consequences of illumination on the photosynthetic
apparatus of Chlamydomonas were not identical in the two
conditions. In both cases, however, the effect of illumination was to
reduce the Fv/Fm ratio (Fig. 1C), a parameter related to the
photochemical efficiency of PSII (25). This indicates that PSII was the
major target of photoinhibition in both State 1 and State 2. The Fv/Fm
decline was reversible in the dark, unless an inhibitor of protein
synthesis, lincomycin, was present in the medium (not shown). The
addition of this compound during illumination enhanced the
photoinhibition, and its effect was larger in State 2 (Fig.
1D).
Samples were also collected to measure the effects of illumination on
the photosynthetic O2 evolution. To this aim, State 1 was
re-established (by oxygenation in the dark) in algae preilluminated in
State 2 before O2 evolution was recorded. We have already
shown indeed that no oxygen is evolved by the algae in State 2 (17). During the State 2 to State 1 transition, no recovery of inhibition occurred; after the transition to State 1, the Fv/Fm of State 2-treated
algae was still largely inhibited if compared with an untreated (State
1) sample (not shown).
The oxygen evolution rates (measured before and after the
photoinhibitory treatment) are shown in Table
I. No decrease of oxygen evolution
was observed in both State 1- and State 2-illuminated samples in the
absence of lincomycin. In its presence, a loss of activity was
observed, which was again larger in State 2- than in State 1-treated
cells.
Electron Transport from PSII to Cytochrome b6f
Complex--
A more direct way to characterize the effects of
irradiation on PSII photochemical activity would be to measure directly
the rate of plastoquinone reduction. It is very difficult to measure this parameter in vivo, where the redox changes associated
to PQH2 formation (observed around 260 nm) are largely
masked by other absorption signals. However, it is possible to obtain
this information indirectly by measuring the rate of cytochrome
f reduction.
This rate can be expressed indeed as kred × [f·Fe3+S·bl+·bh] × [PQH2], where kred is the
second order rate constant for plastoquinol oxidation,
[f·Fe3+S·bl+·bh]
represents the concentration of active cytochrome
b6f complexes, and
[PQH2] expresses the concentration of plastoquinol.
Although [PQH2] is proportional to the fraction of
active PSII, at least in State 1 conditions (Ref. 17, see also
below), the product kred × [f·Fe3+S·bl+·bh]
depends on the catalytic efficiency of the cytochrome complex. Therefore, it is important first to check that the turnover rate of
cytochrome b6f complex per
se is not affected by the photoinhibitory treatment. Only in this
case, the product kred × [f·Fe3+S·bl+·bh]
can be taken as a constant, and the cytochrome f reduction rate can be used to obtain information on PSII activity.
The intrinsic cytochrome b6f activity
can be easily measured in State 2 conditions (i.e.
anaerobiosis) under a single turnover flash illumination regime of low
actinic light frequency. In these conditions indeed the PQ pool is
fully rereduced in the dark time between two consecutive illuminations
(26), and the catalytic properties of the complex can be studied
independently of the rate of PQ photoreduction. Thus, the kinetics of
cytochrome b6f under single flash illumination
was always measured in State 2: State 1 preilluminated cells were
dark-adapted to anaerobiosis before their cytochrome
b6f kinetics were measured.
Fig. 2 shows the kinetics of the
electrochromic shift (panels A and B) and of
cytochrome f redox changes (panel C and
D) measured before and after a photoinhibitory treatment of
C. reinhardtii. The slow phase of the electrochromic shift
(phase b, see "Materials and Methods") and cytochrome f
redox changes are representative of electron injection into the low and
high potential electron transfer chains of the cytochrome
b6f complex, respectively (23, 26).
Fig. 2 shows that all the electron transfer steps that follow
plastoquinol oxidation were not affected by the photoinhibitory illumination. No differences were observed between cells treated in
State 1 and State 2 (not shown).
During the measurements, PSII activity was inhibited with DCMU and
hydroxylamine (27). Their addition did not affect cytochrome b6f kinetics (as expected, since it
does not depend on PSII activity in State 2, see above) but
reduced the amplitude of the fast phase of the electrochromic signal
(phase a) because of the loss of PSII photochemistry. In the presence
of DCMU and hydroxylamine, phase a only depends on PSI-driven charge
separation. Its constancy before and after the photoinhibitory
treatment (Fig. 2, A and B) suggests that PSI was
not affected by photoinhibition in our conditions.
The treatment did not affect the permeability of the thylakoid membrane
either, as indicated by the finding that the addition of a ionophore
induced the same acceleration of cytochrome
b6f turnover in both untreated and
treated cells. The kinetic effect of ionophores is quantitatively
related to the magnitude of the electrochemical proton gradient
(reviewed in Ref. 28), i.e. to the permeability of membrane
to ions. This conclusion is in agreement with previous results with
isolated thylakoid membranes (29, 30).
Having ascertained that the intrinsic activity of PSI and cytochrome
b6f complex was not affected by
photoinhibition, we have estimated the consequences of illumination on
PSII by measuring the turnover rate of cytochrome f under
steady state illumination conditions, as stated above.
Fig. 3 shows the results of such
measurements in the case of one representative experiment. Panels
A and B refer to measurements performed on dark-adapted
algae under both State 1 (A) and State 2 (B).
Similar cytochrome f kinetics was observed in both states; switching the light on generated an oxidation signal (absorption decrease) that rapidly attained a plateau level. After the light was
switched off, a reduction was observed that brought the signal to its
initial level. In State 1 and State 2 conditions, the extent of the
oxidation signal was equally sensitive to the addition of DBMIB, an
inhibitor of cytochrome f reduction by plastoquinol (31)
(Figs. 3, A and B, compare squares and
triangles). DCMU, which blocks plastoquinone reduction by
PSII (27), inhibited electron flow only in State 1 cells
(circles). This result confirms previous findings from
Finazzi et al. (17) that the transition from State 1 to
State 2 corresponds to a shift from a linear (involving PSII and PSI)
to a cyclic (involving only PSI) electron transport system.
Therefore, in State 2 it is not possible to measure PSII activity on
the basis of cytochrome f turnover. For this reason, the
consequences of preillumination on cytochrome f kinetics in the case of continuous illumination regime were measured in State 1, at
variance with single flash measurements. In State 2-preilluminated cells, State 1 was re-established by dark oxygenation of the cells. The
same treatment did not affect the rate of electron transfer in
dark-adapted cells (not shown). Panels C and D of
Fig. 3 present the results of such measurements. In the absence of
lincomycin, no differences were observed between preilluminated and
dark-adapted cells (squares). The steady state redox level
of cytochrome f was more oxidized, however, in
lincomycin-treated samples (asterisks). This suggests
that photoinhibition reduced the rate of plastoquinol generation by
PSII in the presence of the antibiotic, in agreement with the finding
that O2 evolution was inhibited (Table I). This conclusion
is also in agreement with previous results obtained under similar
experimental conditions in higher plant leaves (32). Again, the
consequences were more severe in the case of State 2- than State
1-treated cells (compare C and D, asterisks). In untreated
cells, lincomycin did not affect cytochrome f turnover (not
shown). The time courses of the decrease in PSII-driven
cytochrome f electron flow are shown in Table
II.
Stability of the PSII Reaction Center--
All the
measurements performed so far indicate that the activity of PSII is
decreased by photoinhibition both in State 1 and State 2 provided that
lincomycin is added to the cell suspension. This loss of activity is
generally associated to a damage of the D1 subunit of PSII, which is
subsequently rapidly degraded (see e.g. Refs. 2 and 33). To
verify if this was the case in our conditions, we have measured the
amount of D1 in both State 1- and State 2-treated samples using an
immunoblotting essay (Fig. 4). We found
remarkable differences between State 1 and State 2 cells; although the
amount of D1 was reduced upon photoinhibition in State 1-treated cells,
no substantial degradation was observed in State 2 despite a massive
loss of PSII activity (Fig. 4A).
It has previously been demonstrated that DCMU protects the D1 protein
from degradation (34-35), possibly by reducing the accessibility to
the protease(s) to the damaged PSII centers (34). Therefore, we have
repeated the photoinhibitory treatments in the presence of this
inhibitor. As shown in Fig. 4 (panel B), DCMU protected against D1 degradation in State 1-treated cells. At the same time, it
deeply affected the fluorescence parameters of State 1 cells, strongly
enhancing the Fv/Fm decrease (Fig. 5,
compare open and closed squares). In State 2, no
substantial effects of DCMU were observed (Fig. 5, circles).
This effect was not due to any overestimation of the Fo parameter due
to incomplete reoxidation of Qa Relationship between Fluorescence Emission, Oxygen Evolution,
Cytochrome f Reduction, and D1 Protein Levels during Photoinhibition of
C. reinhardtii Cells under State 1 and State 2 Conditions--
We
report here on the sensitivity of C. reinhardtii to
photoinhibition in State 1 and State 2 conditions. In both States, we
have observed that the PSI and cytochrome
b6f complex intrinsic activities were
not affected by a preillumination with very intense light (Fig. 2),
whereas the photochemical efficiency of PSII was reduced. This results
in a modification of several parameters, all related to PSII; the
fluorescence emission (Fig. 1), the rate of O2 evolution
(Table I) and of electron transport to cytochrome f (Fig. 3,
Table II), and the level of the D1 protein (Fig. 4) are reduced during
photoinhibitory treatments. A comparison of the effects of light on the
different parameters reveals that the Fv/Fm is decreasing in the
absence as well as in the presence of lincomycin (Fig. 1), whereas the
O2 evolution (Table I) and the cytochrome f
reduction rates (Fig. 3, Table II) are affected by the treatments only
in the presence of the inhibitor. Their sensitivity is lower than that
of the Fv/Fm, the cytochrome f reduction rate parameter
being nevertheless more affected. In addition, the loss of
photosynthetic activity largely precedes the degradation of the D1
protein (Fig. 4).
The differences observed in the Fv/Fm decrease between cells treated or
not with the antibiotic are consistent with the occurrence of a protein
synthesis-dependent recovery in both State 1- and State
2-treated cells (reviewed in Ref. 38). On the other hand, the
comparison between the fluorescence parameter on one side and the
electron transport measurements on the other suggests that this protein
synthesis-dependent recovery is able to minimize the
consequences of photoinhibition on electron transport by keeping the
loss of PSII activity within a level compatible with the functioning of
the overall photosynthetic process. This value can be estimated from
the traces of Fig. 1 and corresponds to a Fv/Fm decline of ~50%
(i.e. of the maximal decrease measured in the absence of the
antibiotic, Fig. 1D). Consistently with this idea, also in the presence of lincomycin a substantial inhibition of cytochrome f reduction rate and of O2 evolution can be
observed only between 30 and 60 min of treatment in State 1 (Fv/Fm
equal to 40% of the initial value) and after 20 min in State 2 (Fv/Fm = 25%). We believe that this apparent insensibility of
electron transport parameters to photoinhibition is a consequence of
the use of high light intensities to induce photosynthesis. Under these
conditions, the kinetic performance of the photosynthetic apparatus is
saturated, and the rate of oxygen evolution is limited by the rates of
the reactions occurring in the dark, most probably those of
CO2 assimilation by the Calvin Benson cycle, and not by the
light-driven electron flow. The latter has to be reduced beyond a
certain level (~50%) before becoming rate-limiting.
As stated before, the degradation and re-synthesis of the D1 subunit
are deeply involved in the loss of photosynthetic activity observed
here. This confirms previous findings suggesting that light affects the
stability of the D1 subunit (reviewed in Ref. 38). However, our data
indicate that the damage (and the subsequent repair) occurs in a time
scale of minutes, which is apparently considerably faster than the rate
of degradation of the D1 protein. This inconsistency is probably due
(at least to some extent, see below) to a misestimation of the D1
turnover rate, due to the presence of the protein synthesis inhibitor
in the reaction medium. It has been already reported that protein
synthesis inhibitors reduce the rate of D1 degradation (39), which is
otherwise very rapid (2, 34-35, 40-41). Their effect has been
interpreted as the consequence of a synchronization existing in
vivo between the synthesis and the degradation of this PSII
subunit, which is expected to reduce the rate of the synthesis when the
degradation is prevented. Alternatively, the involvement of non-nuclear
factors in the replacement of the newly synthesized proteins in the
membranes has also been proposed as an explanation for this phenomenon
(42-43). At present, our data do not allow discrimination between the
two possibilities.
The data presented here suggest that the effect of light on PSII
efficiency is likely due to a damage to its reaction center. This is
confirmed by the measurements of the amount of the protein D1, at least
in State 1 conditions (Fig. 4A, see under
"Results"), and by the effect of DCMU (Fig. 4B). It is
therefore a "classical" acceptor side photoinhibition,
i.e. it is due to the accumulation of reduced PSII acceptors
that induce the formation of P680 triplets. They may react with
O2, generating the oxidant species
1O2, which is responsible for the impairment of
PSII activity (44). In principle, the oxygen requirement of this
reaction could explain the differences observed between State 1- and
State 2-treated cells. However, we consider this possibility rather
unlikely, because oxygen entered the Petri dish during the treatment in State 2 despite the fact that argon was bubbled to maintain
anaerobiosis, and its concentration at the end of the treatment was
50-100 µM. This amount is largely sufficient to react
with 3P680.
Mechanism of Photoinhibition under State 1 and State 2 Conditions--
It was previously reported that the damage to the PSII
reaction center proceeds via the generation of an intermediate state (45-49), where charge separation in PSII is already perturbed (low Fv/Fm), but the D1 protein is still present in the reaction center. After its formation, degradation of the D1 protein occurs, giving rise
to an irreversible loss of activity (38, 45-49) that can be repaired
only by de novo protein synthesis (reviewed e.g.
in Ref. 38).
The data reported here are in agreement with this idea, since the
decrease of the activity of PSII precedes the degradation of the
protein in State 1. The phenomenon is even more evident in State 2, where no degradation of the protein is observed, despite the fact that
PSII activity is more severely inhibited by the illumination (Figs. 1
and 5). Therefore, our results suggest that photoinhibition in State 2 induces a higher accumulation of the intermediate state. The large
increase of the Fo during photoinhibition in State 2 (Fig. 1) is also
consistent with this conclusion, as an increased Fo is a typical
signature of the formation of the intermediate state (49). The similar
consequences of photoinhibition in State 2 and in State 1 in the
presence of DCMU (enhanced Fv/Fm decrease and no D1 degradation) are
also consistent with this hypothesis, as this compound has been shown
to enhance the generation of the PSII intermediate state during
photoinhibition (49).
Thus, we suggest that the condition required to enhance photoinhibition
is the lack of electron transfer from Qa
Using Chlamydomonas mutants devoid of the cytochrome
b6f complex, it has been previously
reported that the presence of a plastoquinol molecule in the Qb site of
PSII exerts a protective role against the degradation of the D1 protein
by proteases by a mechanism resembling that of DCMU (48). The finding
that no degradation of D1 occurs in State 2 (Fig. 4) suggests that the
Qb site of PSII complexes is occupied by a PQH2 during the
photoinhibitory treatment under these conditions. This would stem from
the fact that PSII and the cytochrome
b6f complex are not connected
functionally (Ref. 17; see also Fig. 3, A and
B).
It has also been suggested that the PQ pool is not homogeneously
distributed in thylakoid membranes (51). The existence of PQ-diffusing
domains, the equilibration of which is very slow, have been already
reported (52-53). Thus, it is possible to think that the structural
rearrangements of the photosynthetic apparatus that follow State 2 transition (14-16) cause a physical separation between PSII and the
b6f complex by placing them in
different PQ domains. The cytochrome would be still connected to PSI
(explaining the shift to cyclic electron flow; see Ref. 17), whereas
photosystems II would be in separate domains, where plastoquinone would
be reduced very rapidly without being oxidized (at least, not in a fast
time scale). This would give rise to over-reduction of the acceptor of
PSII and to the loss of its photochemical capacity. At the same time,
the D1 protein would not be degraded because of the protecting role
played by the PQH2 present in the Qb site.
It is important to note, however, that the properties of PSII centers
in State 2 are different from those of the Qb nonreducing centers (54),
i.e. PSII, where PQ reduction in the Qb site is impaired. In
these centers, indeed, Qa
To summarize, we believe that the following events occur during
photoinhibition of State 2 cells. Transition to State 2 isolates PSII
from the b6f complex because of
physical constraints to PQH2 diffusion. Thus, PSII does not
participate to electron flow but is nevertheless able to reduce Qa and
the plastoquinone molecules that are still connected to it. During a
prolonged illumination, Qa is over-reduced, and all the PQ pool
connected to PSII is in the PQH2 form. This promotes loss
of photochemical activity while protecting the D1 subunit from degradation.
Physiological Consequences of Photoinhibition in State 1 and
State 2 Conditions--
We have previously demonstrated that cyclic
electron flow around PSI is induced by transition to State 2 in
C. reinhardtii cells (17). Here, we have demonstrated that
PSII is the major target of photoinhibition (Figs. 1, 3, and 4), in
agreement with previous results (e.g. Refs. 2 and 3),
whereas PSI activity is not affected by our illumination protocol (Fig.
2). For these reasons, we think that the consequences of
photoinhibition should be more serious on the overall photosynthetic
activity of cells placed in State 1, where PSII photochemistry
contributes to the electron flow, than in State 2, where it does not
(17).
In particular, in State 2 the capacity of light-induced ATP synthesis
should not be affected by photoinhibition despite the fact that PSII is
degraded. Indeed, in this condition ATP synthesis depends exclusively
on PSI-driven cyclic electron flow (Ref. 17; see also Refs. 55-57).
The ability to maintain a high level of ATP synthesis in photoinhibited
cells might have important consequences on several cellular processes.
In particular, the ability to recover from the photoinhibition itself
depends on the ATP-requiring protein synthesis (2, 38, 58). In
addition, the ability to re-establish linear electron flow (via State 2 to State 1 transition) is also influenced by the availability of ATP
(41).
Important consequences may then be envisaged in the case of
environmental stress. For example, a shortage of oxygen in the external
milieu would induce the reduction of the PQ pool and the transition to
State 2 (see e.g. Ref. 10). Illumination under these
conditions would promote acceptor side inhibition, reducing or
suppressing linear electron flow and lowering the ability of the cells
to generate the electrochemical proton gradient and, consequently, to
synthesize ATP. The activity of cyclic electron flow and the dependent
ATP synthesis would however protect the cells from severe energy crisis
and provide the conditions for recovery of photosynthetic activity. The
stronger inhibition of PSII taking place under these conditions,
therefore, would be largely compensated by the higher capacity to
recover from the damage.
Interestingly, the phenotype reported here is similar to that already
reported in the case of other typical stress conditions as the nutrient
deficiency (see e.g. Refs. 59-61). It has been shown that
under phosphorous and sulfur deficiencies a decrease in the rate of
oxygen evolution is observed that correlates with a systematic
transition to State 2 and a loss of the ability to reduce the PQ pool
(60). Nitrogen starvation also induces a systematic transition to State
2 (61) due to over-reduction of the PQ pool and a loss of
photosynthetic activity (59). It seems therefore that the transition
from State 1 and State 2 is a common response to a-biotic stress, which
might protect the photosynthetic ability to perform ATP synthesis (see
also Ref. 60 for a further discussion), thus allowing the maintenance of vital processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 s
1 of continuous
white light. They were harvested during exponential growth and
resuspended at the required chlorophyll concentration in an high
salt minimal medium (19). The use of this medium prevented the
spontaneous transition to State 2 otherwise observed in the presence of
acetate (see Ref. 20).2
Chlorophyll concentration was measured as the absorbance at 680 nm of
the cell cultures in a spectrophotometer equipped with a scatter
attachment on the basis of a calibration curve constructed after
extraction of the chlorophyll with 80% acetone.
1) at room temperature. The light was
screened with a layer of water and infrared- and UV-absorbing filters.
The intensity of the light reaching the sample was 2300 µE
m
2 s
1. We ensured
that the layer of cells was sufficiently thin to minimize mutual
shadowing. When indicated, plastidial protein synthesis was inhibited
by adding lincomycin at the final concentration of 1 mM.
Samples were collected at the indicated times and used in the different
experiments at the required chlorophyll concentration.
2
s
1. Fluorescence was measured in the same
chamber used for O2 recordings using a PAM fluorometer
(Walz, Germany).
2
s
1. Measurements were performed on algae kept
under State 1 conditions obtained through a strong agitation in the
dark in air. Estimation of the rates of cytochrome f
turnover was done using a procedure previously employed (17). Briefly,
starting from the consideration that the rate of cytochrome
f oxidation and reduction is the same at steady state, the
latter can be expressed as
df
/dt = [f
] × kox × [PC+], where
[f
] is the fraction of reduced cytochrome f,
and kox × [PC+] represents the
product of the second order rate constant for cytochrome f
oxidation times the concentration of oxidized plastocyanin. Both
parameters can be easily calculated experimentally from the traces of
Fig. 3; [f
] is estimated comparing the plateau
absorption level measured in the absence and presence of DBMIB, whereas
kox × [PC+] is given by the
initial rate of cytochrome f oxidation provided that it is
measured when its reduction is inhibited, i.e. in the presence of DBMIB.
-caproic acid, and 1 mM
benzamidine) and 1 mM lincomycin, and resuspended in 100 mM dithiothreitol, 100 mM
Na2CO3. The algae were then solubilized in the
presence of 2% SDS and 20% (w/v) sucrose at 100 °C for 1 min.
Polypeptides were separated by denaturing SDS-polyacrylamide gel
electrophoresis in the presence of 6 M urea. Immunoblotting was performed with monospecific polyclonal antibodies against D1, as
described in Barbato et al. (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Photoinhibition of C. reinhardtii
cells in State 1 and State 2. Algae were collected during
the exponential growth phase and resuspended in the minimal high
salt medium (19). State 1 was obtained by incubating the algae
in the dark under vigorous agitation to prevent reduction of the PQ
pool. State 2 was obtained through dark incubation of the algae under
an argon atmosphere. Photoinhibitory treatments were performed
illuminating a thin layer of cells (~1 mm) with white light of 2300 µE m 2 s
1 at a
chlorophyll concentration of 500 µg ml
1.
Panel A, changes in the Fo (squares) and Fm
(circles) parameters in State 1-treated cells. Closed
symbols, control; open symbols, 1 mM
lincomycin. Panel B, State 2-treated cells. Panel
C, decrease of the Fv/Fm parameter upon photoinhibition in State 1 (upward triangles) and State 2 (downward
triangles). In panel D, the same traces as in
panel C are presented after normalization of the initial
value. The data represent the result of five independent experiments.
a.u., absorbance units.
Effects of photoinhibition on photosynthetic oxygen evolution in
Chlamydomonas cells
1 in
the presence of 5 mM NaHCO3. Photosynthetic oxygen
evolution rate (µmol mg
1 chlorophyll h
1) is
expressed as the sum of O2 evolution (in the light) and
consumption (in the dark). The latter was not affected by the
treatment. Other conditions are as in Fig. 1. Light intensity was 850 µE m
2 s
1. Data represent the result of five
different experiments.
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Fig. 2.
Effects of preillumination on the kinetics of
the electrochromism signal (A and B)
and cytochrome f (cyt f; C
and D) redox changes. Panels A
and C, untreated algae; panels B and
D, preilluminated algae. Open symbols, control
samples; closed symbols, 1 µM carbonyl cyanide
p-trifluoromethoxyphenylhydrazone. The cells (50 µg
of chlorophyll ml 1) were illuminated with red
flashes at the frequency of 0.15 Hz. DCMU and hydroxylamine were added
at the concentrations of 10 µM and 1 mM,
respectively, to block PSII activity. Treated algae were illuminated
for 90 min under the same conditions as in Fig. 1.
View larger version (30K):
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Fig. 3.
Effects of photoinhibition on redox changes
of cytochrome b6f
(cyt f) complex observed under
continuous illumination. Panel A, State 1, untreated.
Panel B, State 2, untreated. Panel C, State 1, 90 min of illumination. Panel D, State 2, 90 min of
illumination. Squares, no additions; circles, 10 µM DCMU; triangles, 2 µM DBMIB;
asterisks, 1 mM lincomycin (added during
preillumination). Note that in panel D, State 1 was
re-established before measuring cytochrome f kinetics. Other
conditions are as in Fig. 1. Upward arrow, actinic light on.
Downward arrow, actinic light off. Light intensity was 1500 µE m 2 s
1.
[Chlorophyll] = 50 µg of chlorophyll
ml
1.
Effect of photoinhibition on the electron flow through cytochrome
b6f
s
1. Data represent the
result of five independent experiments.
View larger version (42K):
[in a new window]
Fig. 4.
Light-induced degradation of the D1 protein
in State 1 and State 2 photoinhibited C. reinhardtii
cells. State 1 and State 2 cells were incubated in light as
in Fig. 1. Samples were collected after the indicated minutes,
thylakoid membranes were isolated, and their content of D1 subunit was
assayed by Western blotting. Panel A, control; panel
B, DCMU-treated samples.
. We checked indeed that
it was rapidly oxidized in the dark (not shown), in agreement with
previous work (36). Very similar kinetics of Qa
relaxation was also observed in State 1 and State 2 cells (not shown).
This suggests that the Qa-Qb equilibration rate (37) is not affected by
the state transitions, in agreement with our previous findings that
indicate a full photochemical competence of PSII in State 2 (17).
View larger version (22K):
[in a new window]
Fig. 5.
Effect of DCMU on the fluorescence parameters
of C. reinhardtii cells in State 1 and State 2 conditions. Same conditions as in Fig. 1. Squares,
State 1 cells. Circles, State 2 cells. Closed
symbols, control. Open symbols, 10 µM
DCMU. In panel B the same traces as in panel A
are presented after normalization of the initial value. The data
represent the result of five independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
to the
intersystem chain, a condition achieved in State 2 (17) or in State 1 in the presence of DCMU. This induces over-reduction of the Qa quinone
acceptor, promoting acceptor side photoinhibition (33-35, 38, 41,
50).
is oxidized at a rate that is
~1000 times slower, whereas we have observed that the reoxidation of
Qa
(measured as the fluorescence decline after an
illumination) is rapid in both in State 1 and State 2 cells (see
"Results").
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ACKNOWLEDGEMENTS |
---|
We thank Gianluca Elli and Stefano Santabarbara (Milan) for valuable discussions.
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FOOTNOTES |
---|
* This work was supported by the Consiglio Nazionale delle Ricerche Target Project on Biotechnology, by Ministero Universitá e Ricerca Scientifica e Tecnologica Special Project mm05153928, Fotosintesi Stress e Protezione, and by the Università del Piemonte Orientale (to R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a doctoral fellowship from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica.
§ To whom correspondence should be addressed. Tel.: 39 02 26604423; Fax: 39 02 26604399; E-mail: giovanni.finazzi@unimi.it.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M011376200
2 F.-A. Wollman, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
PS, photosystem;
DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone;
DCMU, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea;
Fo, minimum value of
fluorescence emission measured at open reaction centers;
Fm, maximal
value of fluorescence emission measured at closed reaction centers;
Fv, variable fluorescence (Fm Fo);
1O2, singlet oxygen species;
PC, plastocyanin;
PQ, plastoquinone;
PQH2, plastoquinol;
Qa, primary quinone acceptor of
photosystem II;
Qb, secondary quinone acceptor of photosystem II;
LHCII, light-harvesting complex II.
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REFERENCES |
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