Journal of Experimental Botany, Vol. 54, No. 388, pp. 1665±1673, July 2003
DOI: 10.1093/jxb/erg180
RESEARCH PAPER
Role of visible light in the recovery of photosystem II
structure and function from ultraviolet-B stress in higher
plants
Elena Bergo1, Anna Segalla1, Giorgio Mario Giacometti1, Delia Tarantino2, Carlo Soave2, Flora Andreucci3
and Roberto Barbato3,*
1
2
Dipartimento di Biologia, UniversitaÁ di Padova, Italy
Dipartimento di Biologia, Universita di Milano, Italy
3
Dipartimento di Scienze e Tecnologie Avanzate, UniversitaÁ del Piemonte Orientale, `Amedeo Avogadro',
Corso Borsalino 54, 15100 Alessandria, Italy
Received 22 October 2002; Accepted 19 March 2003
Abstract
Introduction
The effect of visible light on photosystem II reaction
centre D1 protein in plants treated with ultraviolet-B
light was studied. It was found that a 20 kDa
C-terminal fragment of D1 protein generated during
irradiation with ultraviolet-B light was stable when
plants were incubated in the dark, but was degraded
when plants were incubated in visible light. In this
condition the recovery of photosynthetic activity was
also observed. Even a low level of white light was
suf®cient to promote both further degradation of the
fragment and recovery of activity. During this phase,
the D1 protein is the main synthesized thylakoid
polypeptide, indicating that other photosystem II
proteins are recycled in the recovery process.
Although both degradation of the 20 kDa fragment
and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of
the 20 kDa fragment may occur even in the absence
of D1 synthesis. Comparing chemical and physical
factors affecting the formation of the fragment in
ultraviolet-B light and its degradation in white light, it
was concluded that the formation of the fragment in
ultraviolet-B light is a photochemical process,
whereas the degradation of the fragment in white
light is a protease-mediated process.
Although plants depend on light for their survival, in many
cases light can be harmful to them. An excess of light
brings about the inactivation of oxygenic photosynthesis, a
phenomenon known as photoinhibition (Powles, 1984).
The molecular target of photoinhibition is photosystem II
(PSII), a thylakoid multisubunit pigment±protein complex
capable of light-induced water splitting, with concomitant
reduction of plastoquinone molecules. The reaction centre
of PSII is composed of the D1 and D2 polypeptides to
which all redox cofactors, including P680, are bound
(Nanba and Satoh, 1989). An excess of light is thought to
impair electron transfer through PSII, causing oxidative
damage to the protein D1 (Vass et al., 1992). However, not
only the quantity but also the quality of light is important in
determining the actual extent of photoinhibition. It has
long been known that wavelengths in the ultraviolet-B
region of the spectrum (280±320 nm) are very effective in
inactivating photosynthesis, and that the molecular target
is again PSII (Jones and Kok, 1966). An increasing body
of evidence indicates that photoinactivation due to
ultraviolet-B light and that due to visible light are only
partially related phenomena, as inactivation occurs at
different sites of the photosynthetic electron transfer chain.
In visible light, inactivation of PSII takes place either at the
acceptor or donor side. In the ®rst case, over-reduction of
QA promotes the formation of the P680 triplet from which
singlet oxygen is formed, with the subsequent induction of
oxidative damage to the D1 protein (Vass et al., 1992).
Key words: D1 protein, photosynthesis, photosystem II,
ultraviolet-B.
* To whom correspondence should be addressed. Fax: +39 0131 254410. E-mail: [email protected]
Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; PAGE, polyacrylamide gel electrophoresis; PSII, photosystem II.
Journal of Experimental Botany, Vol. 54, No. 388, ã Society for Experimental Biology 2003; all rights reserved
1666 Bergo et al.
Proteins are then degraded by the recently discovered
DegP2 (Haussuhl et al., 2001) and FtsH proteases (Spetea
et al., 1999; Lindhal et al., 2000), hereafter referred to as
FtsH1; DegP2 initially cleaves the D1 protein in the loop
connecting the fourth and ®fth transmembrane segments,
originating the 23 kDa N-terminal fragment ®rst described
by Greenberg et al. (1987), whereas FtsH1 degrades this,
and possibly other fragments, further. In donor-side
photoinhibition, the accumulation of highly oxidizing
cations such as P680+ and/or TyrZ+ may provide the
driving force for direct photochemical cleavage of the
protein (Barber and Andersson, 1992) occurring between
the ®rst and second transmembrane segments (Barbato
et al., 1991), giving rise to a C-terminal fragment of about
24 kDa. In ultraviolet-B light, the main site of damage is
thought to be the Mn-cluster involved in the oxidation of
water (Melis et al., 1992; Vass et al., 1996), and its damage
prompts the breakdown of D1 protein, which is cleaved in
(or near) the second transmembrane segment by an as yet
unidenti®ed mechanism. This event originates 20 kDa
C-terminal and 10 kDa N-terminal fragments (Barbato
et al., 1995). Other sites of secondary damage inside PSII,
such as QA, QB, bound/unbound plastoquinone/plastoquinol, P680, and TyrZ have also been reported
(Greenberg et al., 1989; Renger et al., 1989; Melis et al.,
1992; Vass et al., 1992).
Irrespective of the underlying mechanism of damage,
recovery from photoinhibition depends on the synthesis of
new protein D1 and on the reassembly of repaired PSII
centres. Recovery of activity requires a low level of
visible light and is prevented by the presence of inhibitors
of plastidial protein synthesis, such as lincomycin.
Reactivation of PSII is a complex process, requiring
lateral migration of damaged complexes from grana
partitions to stroma-exposed regions of the thylakoid
membrane, their partial dismantling (Adir et al., 1990;
Barbato et al., 1992), reassembly with pigments and other
inorganic cofactors (van Wijk et al., 1994) and, lastly,
back-migration of the repaired centres to grana regions of
the thylakoid membrane (van Wjik et al., 1997; BaenaGonzalez et al., 1999). Additional phenomena such as
reversible phosphorylation of certain subunits and dimerization of repaired PSII centres may also be involved
(Baena-Gonzalez et al., 1999). Recovery from exposure to
ultraviolet-B light may be an even more complex
phenomenon, as PSII damaged by this radiation stay in
the grana and do not migrate to stroma-exposed lamellae,
so that the repair cycle is not activated (Barbato et al.,
1992).
As depletion of stratospheric ozone is causing renewed
concern about the increased level of ultraviolet-B radiation
reaching the Earth's surface (Smith et al., 1995), it is
important to understand how biological organisms cope, at
the molecular level, with this enhanced environmental
risk. This work investigated how the D1 protein and
photosystem II, affected by treatment with ultraviolet-B
light, behave during further incubation with visible light, a
condition known to allow recovery of photosynthetic
activity (Sass et al., 1997). Evidence is presented here for
the following: (i) the formation in ultraviolet-B light of the
well-de®ned 20 kDa C-terminal fragment of the D1 protein
is a photochemical process; (ii) subsequent incubation of
ultraviolet-B-treated plants in white light brings about the
proteolytic degradation of this photochemically generated
fragment; (iii) this occurs both in vitro and in vivo, even
when protein synthesis is blocked by the presence of
lincomycin; (iv) recovery of photosynthetic activity is
observed only when white light is present and absolutely
depends on protein synthesis.
Materials and methods
Plant material, thylakoid isolation and subfractionation
Growth of barley (Hordeum vulgare L.), and the fah1 mutant of
Arabidopsis thaliana L. (ferulic acid hydroxylase; Landry et al.,
1995), and the isolation of thylakoids were carried out as described
previously (Barbato et al., 2000; Casazza et al., 2001). For
ultraviolet-B irradiation of isolated thylakoids, 20% (v/v)
glycerol was added. Photosystem II membranes and octyl-bD-glucopyranoside-derived oxygen-evolving PSII cores were
obtained as described previously by Ghanotakis et al. (1989).
Irradiation with ultraviolet-B and visible light
A Vilbert-Lourmat 215M lamp was used as the source of
ultraviolet-B light. The ultraviolet-C component of the lamp was
screened out by two layers of 0.15 mm thick cellulose diacetate.
Irradiation with ultraviolet-B light was performed essentially as
described previously (Barbato et al., 2000). For irradiation of
thylakoids at different temperatures, a thermostatted beaker was
used. Irradiation at a low oxygen concentration (about 2 mM) was
carried out in a tightly stoppered quartz cuvette purged with
nitrogen. To minimize oxygen intake, samples were withdrawn
under a nitrogen stream. Low oxygen concentration was achieved by
a chemical trap consisting of 10 mM glucose, 0.2 mg ml±1 glucose
oxidase and 0.2 mg ml±1 catalase. Irradiation with white light was
performed with a slide projector giving a light intensity of
50 mmol m±2 s±1 at the surface of the sample. For experiments
with protease inhibitor, the CompleteÔ Mini Protease Inhibitors
cocktail (Roche) was used, which contains inhibitors to all ®ve main
classes of protease. For 3.5 ml of thylakoids at a chlorophyll
concentration of 100 mg ml±1, half a tablet was used.
In vivo labelling
For in vivo labelling, plants which had been treated for 3 h with
ultraviolet-B light, were incubated in white light (50 mmol m±2 s±1) in
the presence of 67 mCi ml±1 of [35S]methionine as described
previously (Barbato et al., 2000). At the desired time, leaves were
harvested and washed with distilled water several times, and the
thylakoids isolated as described above.
Protein analysis
SDS-PAGE in the presence of 6 M urea was performed in 12.5%
polyacrylamide gels (Barbato et al., 2000). After electroblotting of
proteins (Dunn, 1986) to poly(vinylidenedi¯uoride) membranes
(Gelman), D1 protein and its primary 20 kDa fragment, as well as
other PSII subunits, were detected with speci®c polyclonal antibodies. For immunodetection of D1 protein, two different antibodies
Photosystem II repair after ultraviolet-B light stress 1667
to D1 were used, one speci®c for the C-terminus of the protein
(Barbato et al., 1991), the second, a kind gift from Professor Aro
(University of Turku, Finland) raised to a synthetic peptide
corresponding to residues 234±242 of the D1 protein sequence.
Antiserum to CP43 was raised in mouse; the properties of other
antibodies have been described previously (Barbato et al., 1992).
Blots were incubated in peroxidase-conjugated goat-anti mouse (or
anti-rabbit) IgG and immunoreaction visualized by an enhancedchemiluminescence kit (SuperSignal, Pierce). For quanti®cation of
proteins, 0.2 mg of chlorophyll was applied per gel lane; for detection
of breakdown fragments of D1 protein, a larger amount of
chlorophyll was applied (1±2 mg). For autoradiography, gels were
stained with Coomassie Blue R-250, destained in 7.5% acetic acid/
10% methanol, incubated with Amplify (Amersham), vacuum-dried
and exposed to X-ray ®lm at ±80 °C.
Lincomycin treatment
Whole rosette leaves from 20-d-old Arabidopsis plants were used.
They were cut at the petiole level, dipped and kept either in distilled
water or in a 1 mM lincomycin for 1.5 h in the dark at room
temperature. Plants were there transferred in the light or kept in the
dark further for the desired periods of time.
Fluorescence analysis
The ¯uorescence parameters of the fah1 mutant of A. thaliana leaves
treated or not with lincomycin, were recorded using a Photosynthetic
Ef®ciency Analyser (PEA, Hansatech) as described previously
(Tarantino et al., 1999).
Results
Degradation of thylakoid proteins in leaves induced by
ultraviolet-B light
The major effect of ultraviolet-B light on thylakoid
proteins is breakdown of the reaction centre D1 protein,
giving rise to a relatively stable fragment of about 20 kDa
(Trebst and Depka, 1990; Friso et al., 1994; Barbato et al.,
1995) which consists of the C-terminus of the protein
(Friso et al., 1994). When barley leaves were irradiated
with ultraviolet-B light in these experimental conditions
(Fig. 1A, UVB), this fragment was detected after only 1 h
exposure, peaked after 2±8 h, and decreased upon longer
exposure. After 24 h, pronounced loss of both the D1
protein and the 20 kDa fragment was observed, together
with the loss of other thylakoid subunits such as CP43 and
OEE1 (Fig. 1B), CP47 and D2 (Fig. 1C). However, the loss
of these proteins was not paralleled by the appearance of
any speci®c degradation product (see also Fig. 1 in Barbato
et al., 1995). No loss of either D1 protein or of any other
thylakoid polypeptides was observed when plants were
kept for 24 h either in the dark (Fig. 1, lane D) or in growth
light (data not shown).
Further degradation of the primary breakdown
fragment requires visible light
When the ultraviolet-B lamp was turned off, the fate of the
20 kDa breakdown fragment depended on whether visible
light was supplied to the system or not. Figure 2 shows
that, when plants previously irradiated for 3 h with
Fig. 1. D1 protein is speci®cally cleaved by ultraviolet-B light to an
immunodetectable 20 kDa fragment. Immunoblotting with polyclonal
antibodies to proteins D1 (A), CP43 and OEE1 (B), CP47 and D2 (C)
of thylakoids isolated from barley leaves irradiated with ultraviolet-B
(UVB) light or kept in the dark (D), for the indicated periods of time.
D1/D2, heterodimeric complex composed of D1 and D2 proteins;
20 kDa, stable breakdown fragment of D1 protein. Each gel lane
contained 1 mg of chylorophyll.
ultraviolet-B light were exposed to 50 mmol m±2 s±1 of
white light, the level of the fragment decreased with time
and, after 3 h, almost disappeared from the membrane
(Fig. 2, visible). By contrast, the amount of fragment did
not vary when plants were kept in the dark (Fig. 2, D). It
was found that even a very low level of white light was
effective in determining further decrease in the 20 kDa
breakdown fragment (data not shown). These results
suggest that the fate of this fragment is that of full
degradation, and that this process needs light.
Synthesis of D1 protein during degradation of the
20 kDa breakdown fragment
After 3 h exposure to ultraviolet-B light, barley leaves
were incubated in white light in the presence of
[35S]methionine, and the level of radioactivity incorporated into the D1 protein was evaluated by SDS-PAGE and
autoradiography (Fig. 3). In this experimental condition,
the D1 protein (as identi®ed by immunoblotting; see
1668 Bergo et al.
Fig. 2. The 20 kDa D1 breakdown fragment is further degraded in
white light. Barley plants were incubated for 3 h in ultraviolet-B light
to induce formation of 20 kDa fragment (UVB), and then moved to
either white light (visible) or dark (D) for the indicated periods of
time. Thylakoids were isolated and analysed by immunoblotting with
antibodies to D1 protein. Each gel lane contained 1 mg chylorophyll.
Barbato et al., 2000), was mainly labelled. Labelling of
other polypeptides was detected after longer exposure, but
their level was much lower than that of the D1 protein (see
Fig. 3).
Lincomycin prevents recovery from photoinactivation
but allows degradation of the 20 kDa fragment
Incubation with a low level of white light is usually
associated with the recovery of photochemical ef®ciency
from photoinactivation, a process requiring de novo
synthesis of D1 protein and reassembly of functional
PSII centres. Thus, a low level of visible light is required
for the repair cycle of PSII units and the 20 kDa fragment
of damaged D1 subunits to be further metabolized. The
aim of the following experiments was to determine
whether there is a link between these two phenomena.
Recovery of photosynthetic ef®ciency after ultraviolet-B
irradiation was measured by means of the Fv/Fm parameter. The experiment was performed on the fah1 mutant
of A. thaliana. This carryies a lesion in the phyenylpropanoid biosynthetic pathway, the leaves of which do not
contain ultraviolet-B-absorbing ¯avonoids. As a result,
this mutant is particularly sensitive to this radiation
(Landry et al., 1995; Booij-James et al., 2000) and a
relatively low level of ultraviolet-B light induces inactivation of photosynthesis without damage to DNA (C Soave,
unpublished results), making the mutant excellent material
for recovery experiments. The same result was obtained
with barley although with a slower recovery kinetics. The
result of a typical experiment is reported in Fig. 4A, in
which the loss and recovery of Fv/Fm are compared in the
presence or absence of lincomycin. In the experimental
conditions used here, which were similar to those used for
Fig. 3. D1 protein is speci®cally labelled during recovery from
ultraviolet-B stress. Plants were treated for 3 h with ultraviolet-B light
and then incubated in growth light in the presence of 67 mCi ml±1
[35S]methionine in visible light (50 mmol m±2 s±1). At the indicated
times, leaves were harvested and thylakoids were isolated and
subjected to SDS-PAGE and autoradiography.
barley, the presence of the antibiotic did not affect the
extent of PSII inactivation (loss of about 50% of activity
after 3 h of irradiation in both cases). However, when
plants were moved to visible light, the effect of lincomycin
became evident: recovery occurred, at least to some extent,
when lincomycin was absent, whereas no recovery was
observed in its presence. In the latter case, further loss of
activity was evident and, after 24 h, variable ¯uorescence
was no longer detectable. In the second part of the
experiment, thylakoids were isolated from both samples,
and the levels of D1 protein and breakdown fragments
were evaluated by immunoblotting with polyclonal antibodies. As shown in Fig. 4B, the D1 protein in A. thaliana
did not give a simple degradation pattern as in barley, but a
number of fragments were detected with apparent masses
between 25 and 16 kDa (Fig. 4B). With barley, a similar
degradation pattern was observed when a higher level of
ultraviolet-B light was used. As in the case of barley, the
amount of breakdown fragments decreased upon incubation in white light (Fig. 4B) and, after 24 h, they were
absent or barely detectable. When lincomycin was present
during incubation in white light (Fig. 4C), removal of
fragments still occurred, although to a slightly lower
extent. Thus, synthesis of new D1 protein is not necessary
for breakdown fragments to be further degraded.
Metabolism of D1 protein in vitro
Up to now it has been established that a low level of visible
light has two effects on leaves previously exposed to
ultraviolet-B radiation: recovery of photosynthetic activity
by de novo synthesis of D1 protein is activated, and further
Photosystem II repair after ultraviolet-B light stress 1669
Fig. 4. The presence of lincomycin does not prevent further
metabolism of the 20 kDa D1 fragment. Arabidopsis leaves were
treated with lincomycin in the dark, exposed to ultraviolet-B light for
3 h, and then incubated in visible light for up to 24 h. (A) Fv/Fm ratios
were measured in lincomycin-treated leaves (+Linco, circles) and
controls (±Linco, squares); (B) immunoblotting with antibody to D1protein (DE-loop) of thylakoids from leaves treated with ultraviolet-B
light in the absence of lincomycin for the indicated periods of time
(UVB). Samples treated for 3 h with ultraviolet-B light were moved to
white light (visible) for the indicated periods of time; (C) immunoblotting with antibody to D1-protein (DE-loop) of thylakoids from
leaves treated with ultraviolet-B light in the presence of lincomycin
for 3 h (UVB), and then moved to visible light (visible) for up to 24 h.
metabolism of the fragments produced by ultraviolet-B
light is promoted. In leaves and thylakoids, identical
degradation patterns were observed for the D1 protein
(Barbato et al., 1995); however, in isolated thylakoids,
recovery dependent on protein synthesis cannot occur.
Isolated thylakoids irradiated with ultraviolet-B light and
then incubated either at a low level of visible light or in the
dark, gave the results shown in Fig. 5A. The fragment
forming upon irradiation with ultraviolet-B light (Fig. 5A)
was stable in the dark (Fig. 5A, dark) but then further
degraded when thylakoids were incubated with a low level
(50 mmol m±2 s±1) of visible light (Fig. 5A, visible), in a
similar way as described for leaves. Even a very low level
Fig. 5. The 20 kDa D1 fragment is degraded in isolated thylakoids
but not in detergent-derived oxygen-evolving PSII particles.
(A) Thylakoids were irradiated with ultraviolet-B (UVB) light for
30 min to induce formation of the 20 kDa fragment, and then
incubated in the dark (D), visible light (visible), and visible light in
the presence of 10 mM DCMU (visible+DCMU); each gel lane
contained 1 mg chlorophyll; (B) PSII membranes (PSII membranes)
and oxygen-evolving PSII complexes (PSII cores) were treated with
ultraviolet-B light for 0.5 h to induce the formation of the 20 kDa
fragment (UVB), and were then incubated in white light for the
indicated periods. Blots were probed with polyclonal antibodies
speci®c for C-terminus of D1 protein.
of visible light (5 mmol m±2 s±1) induced degradation of the
fragment (data not shown), suggesting that this phenomenon does not require electron transfer. Accordingly,
the presence of 10 mM DCMU during incubation in white
light did not affect the degradation of the 20 kDa fragment
(Fig. 5A, visible+DCMU). These results con®rm that
breakdown of the 20 kDa fragment does not require
concomitant D1 synthesis. When oxygen-evolving PSII
preparations such as PSII membranes (Fig. 5B, PSII
membranes) or highly resolved PSII cores (Fig. 5B, PSII
cores) were irradiated with ultraviolet-B light, formation of
the typical 20 kDa fragment was still observed, in the same
way as reported in leaves or isolated thylakoids (Barbato
et al., 1995). However, further incubation in white light of
these ultraviolet-B-treated preparations, at variance with
what occurred in leaves and thylakoids, did not promote
further degradation of the fragment (Fig. 5B). Therefore,
further metabolism of the fragment probably requires
factor(s) that are present in intact leaves or even in whole
1670 Bergo et al.
thylakoids, but not in simpler detergent-derived PSII
preparations.
In order to characterize the nature of the molecular
processes responsible for the generation of the 20 kDa
C-terminal fragment in ultraviolet-B light and that of its
degradation in visible light, the effects of temperature,
protease inhibitors and oxygen were further investigated.
As shown in Fig. 6A, the 20 kDa fragment is formed by
ultraviolet-B light in similar amounts at 0 and 20 °C. When
thylakoids containing the fragment were exposed to white
light, the fragment disappeared much faster at 20 °C
(Fig. 6B, 20 °C) than at 0 °C (Fig. 6B, 0 °C). When
irradiation with ultraviolet-B light was performed in the
presence of a cocktail of protease inhibitors, neither the
loss of D1 protein (Fig. 7A) nor the appearance of the
20 kDa fragment (Fig. 7B) was prevented. Instead, when
thylakoids containing the 20 kDa fragment were further
incubated in white light (Fig. 7C) in the presence of the
protease inhibitors, degradation of the fragment was
almost completely prevented. Lastly, in anaerobic conditions, the visible-light-induced degradation of the fragment (Fig. 8A) was much-less pronounced than in the
presence of oxygen (Fig. 8B).
Discussion
The reported loss of the D1 protein observed during
irradiation with ultraviolet-B light (Barbato et al., 1995) is
linked to an increased rate of degradation and a decreased
rate of synthesis. The D1 protein, when cleaved by
ultraviolet-B light, gives rise to a speci®c 20 kDa
C-terminal fragment (Friso et al., 1994; Barbato et al.,
1995). Three main observations must be considered in
relation to degradation and resynthesis of the D1 protein:
(i) the fate of the ultraviolet-B-generated 20 kDa D1
fragment is quite straightforwardÐboth in vitro and in vivo
further metabolism of this fragment needs light, but is
independent of concomitant synthesis of D1 protein; (ii) as
in the case of photoinhibition induced by visible light,
recovery from ultraviolet-B stress is dependent on the
synthesis of new D1 protein, a process requiring visible
light; recovery is in fact completely prevented by conditions in which synthesis of the D1 protein does not take
place; (iii) incubation with radioactive methionine during
recovery from ultraviolet-B light stress in a low level of
visible light leads to rather speci®c labelling of the D1
protein, radioactivity in other subunits being barely
detectable in our labelling and autoradiography conditions.
Some conclusions may be reached by bringing together
these observations. First, during recovery from
ultraviolet-B stress, low levels of visible light are required
for the synthesis of new D1 protein, and for the removal
from the membrane of the 20 kDa fragment generated by
ultraviolet-B light. Although these two processes are both
light-dependent, they do not seem to be closely related.
Fig. 6. Low temperature prevents metabolism of 20 kDa fragment in
visible light but not its generation by ultraviolet-B light. Immunoblotting with antibodies to D1-protein. (A) Thylakoids were exposed
to ultraviolet-B light at either 0 or 20 °C for the indicated periods;
(B) thylakoids were exposed to ultraviolet-B light for 30 min to
generate the 20 kDa D1 fragment (UVB) and then incubated in visible
light at either 20 °C or 0 °C for the indicated periods. Each gel lane
contained 1 mg chlorophyll.
New synthesis of D1 protein occurs continuously, independently of the damage induced by ultraviolet-B radiation. It is known that the synthesis and insertion of new
D1 protein depends on visible light at the level of
transcription, translation and elongation of the nascent
peptide (Zhang and Aro, 2002). Instead, ultraviolet-B
radiation simply decreases the rate of synthesis (Barbato
et al., 2000). Light-induced degradation of the 20 kDa
fragment occurs irrespective of whether D1 protein
synthesis is active or prevented. Secondly, it has previously been shown that the 20 kDa fragment is located in
grana membranes where it may be detected together with
other PSII proteins in structurally de®ned although not
functional centres (Barbato et al., 2000). During recovery
from ultraviolet-B stress, only the D1 protein is synthesised de novo (Fig. 3), suggesting the possibility that other
undamaged PSII subunits are recycled to reconstitute
functional PSII centres. Recovery of photosynthetic
activity needs the fragment to be removed from the
damaged centres, in order to make them available for
Photosystem II repair after ultraviolet-B light stress 1671
Fig. 8. Oxygen is involved in light-induced degradation of 20 kDa D1
breakdown fragment. Immunoblotting with antibodies to D1 protein.
Thylakoids were irradiated for 60 min to induce formation of the
20 kDa fragment (UVB, A and B). (A) A chemical trap for oxygen
consisting of glucose, glucose oxidase and catalase was added to part
of a sample which, after 10 min in the dark, was exposed to white
light for the indicated periods; (B) in order to evaluate any side-effects
due to the presence of ultraviolet-B absorbing factors in enzymes used
to generate low-oxygen conditions, a sample containing glucose
oxidase and catalase (but not glucose) was used as a control.
Fig. 7. Proteolytic inhibitors prevent white light-induced degradation
of the 20 kDa fragment but not its formation by ultraviolet-B light.
Immunoblotting with antibodies to D1 protein. (A) Thylakoids were
incubated in ultraviolet-B light (UVB) either in the absence or
presence of a cocktail of protease inhibitors, for the indicated periods
of time. Control samples were kept in the dark, either in presence or
absence of inhibitors, as indicated. Last right lane shows data on a
sample kept in the dark throughout the experiment, and is taken as an
end-point control. Each gel lane contained 0.2 mg chlorophyll. (B) An
experiment similar to (A), in which thylakoids were irradiated with
ultraviolet-B light either in the presence or absence of protease
inhibitors for the indicated periods; D is a dark control sample. Each
gel lane contained 1 mg chlorophyll; (C) Thylakoids were irradiated
for 30 min with ultraviolet-B light to induce formation of 20 kDa
fragment (UVB) and were then incubated in white light, either in the
absence or presence of protease inhibitors for indicated periods. Each
gel lane contained 1 mg chlorophyll.
reassembly of new PSII units. In this context, visible lightdependent degradation of the 20 kDa fragment may be
seen as the ®rst step in repair from ultraviolet-B stress.
As for the mechanism by which the 20 kDa fragment is
produced, it must be noted that it is generated both in vivo
and in vitro, and that its production is blocked neither at
0 °C nor by the presence of protease inhibitors. This makes
enzymatic cleavage at the origin of the 20 kDa fragment
highly improbable, at variance with the case in which
breakdown of the D1 protein is induced by visible light
(Aro et al., 1990). In fact, primary cleavage in
ultraviolet-B and visible light occurs on different sides of
the thylakoid membrane, i.e. on the lumenal (Friso et al.,
1994; Barbato et al., 1995) or stromal sides (De Las Rivas
et al., 1992; Spetea et al., 1999), respectively. In visible
light, the primary cleavage of D1 protein is carried out by
the recently discovered DegP2 protease (Haussuhl et al.,
2001), an enzyme bound to the stroma-exposed surface of
thylakoids. As this enzyme does not have access to the
lumenal side of thylakoids, it cannot be responsible for
primary cleavage of D1 in ultraviolet-B light. Proteolytic
activity located on the lumenal side of the membrane is due
to DegP1 protease (Itzhaki et al., 1998). However, this
protein has not been directly involved in the metabolism of
the D1 protein. Although the activity of recently discovered plastidial proteases such as DegP2 and FtsH1 is
relatively independent of temperature, it is nevertheless
strongly inhibited at 0 °C (Haussuhl et al., 2001).
The visible-light induced degradation of D1 is a twostep process: a light-dependent (and temperatureindependent) triggering event is followed by lightindependent (and temperature-dependent) proteolysis;
and these two steps can be resolved by performing
irradiation at low temperatures (Aro et al., 1990; Spetea
et al., 1999). Instead, in ultraviolet-B light, the protein is
cleaved in just one photocleavage event, which is not
inhibited by low temperatures or protease inhibitors. Its
further degradation requires white light both in vitro and
in vivo. The light intensity required to degrade the 20 kDa
fragment is unable to induce degradation of the D1 protein
1672 Bergo et al.
in itself, not even in thylakoids previously inactivated on
their donor side and, therefore, extremely sensitive to
photoinhibition. Since anaerobic conditions slow down
degradation of the fragment, it is proposed that visible light
is required to prepare the photochemically generated
fragment for proteolytic degradation, possibly by the
action of singlet oxygen or other oxygen radicals. If this is
the case, the FtsH1 protease may be responsible for the
breakdown of the 20 kDa fragment, as in the case of the
23 kDa N-terminal fragment of D1 generated during
acceptor-side photoinhibition (Lindhal et al., 2000).
Accordingly, PSII preparations still able to photogenerate
the 20 kDa fragment in ultraviolet-B light are incapable of
further degradation of this fragment in visible light,
indicating that the proteolytic activity responsible for this
phenomenon was lost during the fractionation process: as
in the case of visible light, this ®nding may be taken as
evidence that the enzyme is not closely associated to the
reaction centre complex, but is stroma-located or
peripherically bound to the thylakoid surface.
Alternatively, light may play a regulatory role: the
activity involved in the degradation of the 20 kDa fragment
may be due to a light-activated protease, and therefore
different from the FtsH1 protease, unless FtsH1 itself is
light-activated. Evidence for the presence of plastidial
light-activated protease(s) has been reported (Ostersetzer
and Adam, 1997) during import studies with in vitro
synthesized Rieske protein; in that case, FtsH1 was
involved. Given the low level of light required, together
with the lack of any effect of DCMU, it seems that electron
transport is not involved in the phenomenon.
From the results reported in this paper, it seems clear
that the metabolism of D1 protein in light is a very
complex issue, depending both on qualitative and quantitative aspects of light. In visible light, the metabolism of
D1 is a two-step phenomenon, in which the protein is
oxidatively damaged and removed by the combined action
of DegP2 and FtsH1 proteases. Shifting towards
ultraviolet-B, the primary degradative process may be
photolytic in nature, and photocleaved D1 generates the
20 kDa C-terminus. This then becomes the substrate of a
protease the activity of which, directly or indirectly, is
regulated by visible light. In natural conditions, in which
ultraviolet and visible light are mixed, a rather complex
picture emerges, in which turnover of D1 protein is
differently affected by the two kinds of light.
Acknowledgements
This work was supported by the Italian Ministry of University
and Scienti®c and Technological Research Grant-Co®n (CIP
MM5153929; RB, CS), National Program for Antarctic Research
(PNRA), and CNR Target Project on Biotechnology. Financial
support by the Dipartimento di Scienze e Tecnologie Avanzate to
RB is acknowledged.
References
Adir N, Shochat S, Ohad I. 1990. Light-dependent D1 protein
synthesis and translocation is regulated by reaction center II.
Reaction center II serves as an acceptor for the D1 precursor.
Journal of Biological Chemistry 265, 12563±12568.
Aro EM, Hundal T, Carlberg I, Andersson B. 1990. In vitro
studies on light-induced inhibition of photosystem II and D1protein degradation at low temperatures. Biochimica et
Biophysica Acta 1019, 269±275.
Baena-Gonzalez E, Barbato R, Aro EM. 1999. Role of
phosphorylation in the repair cycle and oligomeric structure of
photosystem II. Planta 208, 196±204.
Barbato R, Bergo E, SzaboÁ I, Dalla Vecchia F, Giacometti GM.
2000. Ultraviolet B exposure of whole leaves of barley afffects
structure and functional organization of photosystem II. Journal
of Biological Chemistry 275, 10976±10982.
Barbato R, Friso G, Rigoni F, Dalla Vecchia F, Giacometti GM.
1992. Structural changes and lateral redistribution of photosystem
II during donor side photoinhibition of thylakoids. Journal of Cell
Biology 119, 325±335.
Barbato R, Frizzo A, Friso G, Rigoni F, Giacometti GM. 1995.
Degradation of the D1 protein of photosystem II reaction centre
by ultraviolet-B light requires the presence of functional
manganese on the donor side. European Journal of
Biochemistry 227, 723±729.
Barbato R, Shipton CA, Giacometti GM, Barber J. 1991. New
evidence suggests that the initial cleavage of the D1-protein may
not occur near the PEST sequence. FEBS Letters 290, 162±166.
Barber J, Andersson B. 1992. Too much of a good thing: light can
be bad for photosynthesis. Trends in Biochemical Sciences 17,
61±66.
Booij-James IS, Dube SK, Jansen, MAK, Edelman M, Mattoo
AK. 2000. Ultraviolet-B radiation impacts light-mediated
turnover of the photosystem II reaction center heterodimer in
Arabidopsis mutants altered in phenolic metabolism. Plant
Physiology 124, 1275±1283.
Casazza P, Tarantino D, Soave C. 2001. Preparation and
functional characterization of thylakoids from Arabidopsis
thaliana. Photosynthesis Research 68, 175±180.
De Las Rivas J, Andersson B, Barber J. 1992. Two sites of
primary degradation of the D1 protein induced by acceptor or
donor side photoinhibition in photosystem II core complexes.
FEBS Letters 301, 246±252.
Dunn SB. 1986. Effects of the modi®cation of transfer buffer
composition on the renaturation of proteins in gels and on the
recognition of proteins on western blots by monoclonal
antibodies. Analytical Biochemistry 157, 144±153.
Friso G, Spetea C, Giacometti GM, Vass I, Barbato R. 1994.
Degradation of the photosystem II reaction center D1-protein
induced by UVB radiation in isolated thylakoids. Identi®cation
and characterization of C- and N-terminal breakdown products.
Biochimica et Biophysica Acta 1184, 78±84.
Ghanotakis DF, Demetriou F, Yocum CF. 1989. Isolation and
characterization of an oxygen-evolving photosystem II reaction
center core preparation and a 28 kDa chl a-binding protein.
Biochimica et Biophysica Acta 891, 15±21.
Greenberg BM, Gaba V, Canaani O, Malkin S, Mattoo AK,
Edelman M. 1989. Separate photosensitizers mediate
degradation of the 32 kDa photosystem II reaction center
protein in the visible and UV spectral regions. Proceedings of
the National Acadedmy of Sciences, USA 86, 6617±6620.
Greenberg BM, Gaba V, Mattoo AK, Edelman M. 1987.
Identi®cation of a primary in vivo degradation product of the
rapidly turning over 32 kd protein of photosystem II. EMBO
Journal 6, 2865±2869.
Photosystem II repair after ultraviolet-B light stress 1673
Haussuhl K, Anderssson B, Adamska I. 2001. A chloroplast
DegP2 protease performs the primary cleavage of the
photodamaged D1 protein in plant photosystem II. EMBO
Journal 20, 713±722.
Itzhaki H, Naveh L, Lindhal M, Cook M, Adam Z. 1998.
Identi®cation and characterization of DegP, a serine protease
associated with the luminal side of the thylakoid membrane.
Journal of Biological Chemistry 273, 7094±7098.
Jones LW, Kok B. 1966. Photoinhibition of chloroplast reactions.
I. Kinetics and action spectra. Plant Physiology 41, 1037±1043.
Landry LG, Chapple CC. Last RL. 1995. Arabidopsis mutants
lacking phenolic sunscreen exhibit enhanced ultraviolet-B injury
and oxidative damage. Plant Physiology 109, 1159±1166.
Lindhal M, Spetea C, Hundal T, Oppenheim AB, Adam Z,
Andersson B. 2000. The thylakoid FtsH protease plays a role in
the light induced turnover of the photosystem II D1 protein. The
Plant Cell 12, 419±431.
Melis A, Nemson JA, Harrison M. 1992. Damage to functional
components and partial degradation of photosystem II reaction
center proteins upon chloroplast exposure to ultraviolet-B
radiation. Biochimica et Biophysica Acta 1100, 312±320.
Nanba O, Satoh K. 1989. Isolation of a photosystem II reaction
center consisting of D-1 and D-2 polypeptides and cytochrome b559. Proceedings of the National Academy of Sciences, USA 84,
109±112.
Ostersetzer O, Adam Z. 1997. Light-stimulated degradation of an
unassembled Rieske FeS protein by a thylakoid-bound protease:
the possible role of the FtsH protease. The Plant Cell 9, 957±965.
Powles SB. 1984. Photoinhibition of photosynthesis induced by
visible light. Annual Reviews of Plant Physiology 35, 15±44.
Renger G, Volker M, Eckert HJ, Fromme R, Hohm-Veit S,
Graber P. 1989. On the mechanism of photosystem II
deterioration by UV-B radiation. Photochemistry and
Photobiology 49, 97±105.
Smith RC, Prezelin BB, Baker KS, et al. 1995. Ozone depletion:
ultraviolet radiation and phytoplankton biology in antarctic
waters. Science 255, 952±959.
Sass L, Spetea C, Mate F, Nagy F, Vass I. 1997. Repair of UV-B
induced damage of photosystem II via de novo synthesis of the
D1 and D2 reaction center subunits in Synechocystis sp. PCC
6803. Photosynthesis Research 54, 55±62.
Spetea C, Hundal T, Lohmann F, Andersson B. 1999. GTP
bound to chloroplast thylakoid membranes is required for lightinduced, multienzyme degradation of the photosystem II D1
protein. Proceedings of the National Academy of Sciences, USA
98, 6547±6552.
Tarantino D, Vianelli A, Carraro L, Soave C. 1999. A nuclear
mutant of Arabidopsis thaliana selected for enhanced sensitivity
to light-chill stress is altered in PSII electron transport.
Physiologia Plantarum 107, 361±372.
Trebst A, Depka B. 1990. Degradation of the D1 protein subunit of
photosystem II in isolated thylakoids by UV light. Zeitschrift fuÈr
Naturforschung 45c, 742±750.
Vass I, Sass L, Spetea C, Bakou A, Ghanotakis DF, Petrouleas
V. 1996. UV-B induced inhibition of photosystem II electron
transport studied by EPR and chlorophyll ¯uorescence.
Impaiment of donor and acceptor side components.
Biochemistry 35, 8964±8973.
Vass I, Styring S, Hundal T, Koivuniemi A, Aro EM, Andersson
B. 1992. Reversible and irreversible intermediate during
photoinhibition of photosystem II: stable reduced QA species
promote chlorophyll triplet formation. Proceedings of the
National Academy of Sciences, USA 89, 1408±1412.
van Wijk KJ, Nilsson LO, Styring S. 1994. Synthesis of reaction
center proteins and reactivation of redox components during
repair of photosystem II after light-induced inactivation. Journal
of Biological Chemistry 269, 28382±28392.
van Wijk KJ, Roobol-Boza M, Kettunen R, Andersson B, Aro
EM. 1997. Synthesis and assembly of the D1 protein into
photosystem II: processing of the C-terminus and identi®cation of
the initial assembly partners and complexes during photosyetm II
repair. Biochemistry 36, 6178±6186.
Zhang L, Aro EM. 2002. Synthesis, membrane insertion and
assembly of the chloroplast-encoded D1 protein into photosystem
II. FEBS Letters 512, 13±18.