Plant Physiol. (1998) 118: 827-834
Induction of Acclimative Proteolysis of the Light-Harvesting
Chlorophyll a/b Protein of Photosystem II
in Response to Elevated Light Intensities1
Dan-Hui Yang,
Jeanette Webster2,
Zach Adam,
Marika Lindahl3, and
Bertil Andersson*
Department of Biochemistry, Arrhenius Laboratories for Natural
Sciences, Stockholm University, S-106 91 Stockholm, Sweden (D.-H.Y.,
J.W., M.L., B.A.); and Department of Agricultural Botany, Hebrew
University of Jerusalem, Rehovot 76100, Israel (Z.A.)
 |
ABSTRACT |
Most
plants have the ability to respond to fluctuations in light to minimize
damage to the photosynthetic apparatus. A proteolytic activity has been
discovered that is involved in the degradation of the major
light-harvesting chlorophyll a/b-binding
protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine
or cysteine type and is associated with the outer membrane surface of
the stroma-exposed thylakoid regions. The identity of the protease is
not known, but it does not correspond to the recently identified
chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to
bacterial enzymes. The acclimative response shows a delay of 2 d
after transfer of the leaves to high light. This lag period was shown
to be attributed to expression or activation of the responsible
protease. Furthermore, the LHCII degradation was found to be regulated
at the substrate level. The degradation process involves lateral
migration of LHCII from the appressed to the nonappressed thylakoid
regions, which is the location for the responsible protease.
Phosphorylated LHCII was found to be a poor substrate for degradation
in comparison with the unphosphorylated form of the protein. The
relationship between LHCII degradation and other regulatory proteolytic
processes in the thylakoid membrane, such as D1-protein degradation, is discussed.
| |
INTRODUCTION |
The main focus of photosynthesis research has been the functional
and structural aspects behind the energy-transducing process. The
progress has been significant, and the photosynthetic protein complexes
can now be described at a very refined molecular level (Barber and
Andersson, 1994
). Conversely, relatively little is known about the
acclimating, regulatory, and protective processes that maintain high
photosynthetic efficiency during ever-fluctuating and even stressful
environmental conditions. The identification of such auxiliary
processes associated with thylakoid membranes and the characterization
of the enzymes involved are therefore central tasks of current
photosynthesis research. Examples of auxiliary enzymes or components
are kinases and phosphatases responsible for reversible protein
phosphorylation (Gal et al., 1997), desaturases controlling the lipid
composition and dynamics of thylakoid membranes (Wada et al., 1990),
stress-induced proteins such as the ELIPs (Adamska, 1997), and several
enzymatic activities involved in various forms of regulatory
proteolysis (Adam, 1996; Andersson and Aro, 1997). In the latter case,
D1-protein turnover during repair of damage caused by photoinhibition
is the most illustrative example (Prasil et al., 1992; Andersson and
Aro, 1997). The knowledge of chloroplast proteases involved in
regulatory proteolysis is still very limited. The presence of homologs
to bacterial proteases in chloroplasts, such as the Clp and FtsH
proteins, has provided new insights and possibilities (Gray et al.,
1990; Moore and Keegstra, 1993; Adam, 1996; Lindahl et al., 1996;
Ostersetzer et al., 1996).
In a previous study we discovered a regulatory proteolytic activity
involved in the degradation of LHCII during acclimation of spinach
(Spinacia oleracea) leaves from low- to high-intensity light
(Lindahl et al., 1995). LHCII is the most abundant protein in the
thylakoid membrane of higher plants (Jansson, 1994). It is not only a
key component for light harvesting, but is also essential for
regulation and distribution of excitation energy within the
photosynthetic apparatus and responds to both short- and long-term
fluctuations in light intensity and quality (Anderson and Andersson,
1988; Melis, 1991, 1996). This acclimation of the light-harvesting
apparatus is generally thought to optimize photosynthetic efficiency
and minimize light stress and photoinhibition to PSII (Andersson and
Barber, 1996; Melis, 1996; Park et al., 1997).
The identified proteolytic activity was associated with degradation of
the so-called "outer" or "peripheral" pool of LHCII enriched in
the rapidly phosphorylated 25-kD subunit (Larsson et al., 1987a, 1987b;
Anderson and Andersson, 1988), thereby reducing the antenna size of
PSII in spinach leaves by 20% to 30% in response to elevated light
intensities (Lindahl et al., 1995). The protease involved was found to
be of the Ser or Cys type and extrinsically bound at the outer surface
of the stroma-exposed regions of thylakoid membranes. Notably, the
proteolytic process did not occur in the absence of ATP; therefore, it
can be added to the list of the few known examples of ATP-dependent
proteolysis associated with the thylakoid membrane (Adam, 1996).
Another feature of the acclimative proteolysis of LHCII is that it
requires a relatively long period of induction; therefore, no change in
the size of the PSII antenna was seen during the first 2 d after
transfer of spinach plants to increased light intensities (Lindahl et
al., 1995).
To gain an understanding of the mechanism and regulation of the
acclimative reduction of the LHCII antenna, we studied the induction of
LHCII degradation and investigated the activation/deactivation pattern
of the proteolytic process after transfer of spinach plants from low-
to high-intensity light. The results revealed a light-dependent induction at the enzyme level and a regulatory control at the substrate
level. Evidence was also provided that the newly discovered chloroplast
ATP-dependent proteases Clp and FtsH were not responsible for the
acclimative LHCII degradation.
| |
MATERIALS AND METHODS |
Growth and Acclimation of Plants
Spinach (Spinacia oleracea) plants were grown for 4 weeks at an intensity of 30 µE m
2
s1 (low-intensity light). The light/dark cycle
was 10 h of light followed by 14 h of darkness. The mature,
low-light-grown spinach plants were transferred to an intensity of 600 to 800 µE m2 s1
(high-intensity light) for studies of the acclimative responses to high
light.
Isolation and Subfractionation of Thylakoid Membranes
Thylakoid membranes were isolated according to the method of
Andersson et al. (1976) from control plants grown at low light and from
acclimating plants after the indicated times at the high-light regime.
The isolated thylakoids were suspended in 15 mM
Tricine-NaOH, pH 7.8, 15 mM NaCl, 3 mM
MgCl2, 100 mM sorbitol, 1 mM ascorbate, and 0.1% (w/v) BSA (incubation buffer).
Chlorophyll concentration and the chlorophyll a/b
ratio were determined spectrophotometrically according to the method of
Lichtenthaler and Wellburn (1983).
Digitonin fractionation of isolated thylakoid membranes (Carlberg et
al., 1992) was performed at a chlorophyll concentration of 0.2 mg
mL1, and a final digitonin concentration of
0.1% (w/v) at room temperature for 30 s. The solubilization was
terminated by a 10-fold dilution with ice-cold incubation buffer.
Nonappressed thylakoid membranes (stromal lamellae vesicles) were
isolated by differential centrifugation. The supernatant remaining
after centrifugation at 40,000g for 30 min was centrifuged
at 100,000g for 1 h. The final pellet containing the
nonappressed membranes was resuspended in incubation buffer and stored
at 
80°C.
High-Salt Washings of Thylakoid Membranes
For studies of enzyme induction in vivo, thylakoid membranes were
isolated from control leaves and from leaves at various stages in the
acclimation process. Isolated thylakoid samples were washed three times
in incubation medium supplemented with 0.5 M NaCl to
release the extrinsically bound proteolytic activity from the membrane
(Lindahl et al., 1995). Control washings were made in incubation buffer
without the addition of extra NaCl. The wash supernatants were
concentrated, dialyzed against incubation buffer, and used for assays
of proteolytic activities and in reconstitution experiments. Protein
concentration was determined according to the method of Lowry et al.
(1951).
Reconstitution of LHCII Proteolysis in Situ
In situ degradation of LHCII in low- and high-light-acclimating
thylakoid membranes was followed after high-salt washing and homologous
or heterologous reconstitution with an appropriate desalted
supernatant. The reaction mixture contained washed thylakoids (1 mg of
chlorophyll) resuspended in incubation buffer supplemented with 0.5 mM ATP. A concentrated and dialyzed wash supernatant obtained from thylakoids corresponding to 1 mg of chlorophyll was added
in darkness at 22°C. As a control, unwashed thylakoids were incubated
under the same conditions. Aliquots were frozen in liquid nitrogen and
kept at 80°C before analysis of LHCII degradation by SDS-PAGE and
western blotting.
Effect of Phosphorylation on LHCII Degradation
Thylakoids were incubated at 1 mg chlorophyll
mL1 in incubation buffer in the presence of
12.5 µCi [
-32P]ATP
mL1 and 0.5 mM unlabeled ATP.
Different extents of phosphorylation of thylakoid membranes were
induced in vitro by illumination at 160 µE m2
s1 at 25°C for various lengths of time.
Residual radioactive ATP was removed by centrifugation, and the
thylakoids were resuspended in fresh incubation buffer containing 0.5 mM ATP and 10 mM NaF to a concentration of 2 mg
chlorophyll mL1. Thylakoids were then
incubated in the dark at 25°C, and samples were removed after
0, 2, 4, and 6 h. LHCII degradation was analyzed by
two-dimensional gel electrophoresis (Larsson and Andersson, 1985;
Lindahl et al., 1995). The relative protein phosphorylation was
determined by autoradiography.
Assay of Proteolytic Activity Using Isolated LHCII or Fluorescein
Thiocarbamoyl-Casein as a Substrate
LHCII was isolated from high-light-grown spinach according to the
method of Burke et al. (1978). The purified LHCII was added to the wash
supernatants in incubation buffer in the presence of 0.1% (w/v) Triton
X-100. The ratio of LHCII protein to supernatant protein was 1:5. The
samples were incubated at 25°C for 2 h, and then assayed for
LHCII degradation by SDS-PAGE and western blotting. The antibody used
was raised against both the LHCII-27 and LHCII-25 subunits.
Proteolysis of fluorescein thiocarbamoyl-casein was monitored by
fluorescence emission spectra (500-600 nm) using an excitation wavelength of 495 nm (Twining, 1984).
Separation and Quantification of the LHCII-27 and LHCII-25
Polypeptides
Thylakoid proteins were separated either by two-dimensional
SDS-PAGE, as described previously (Larsson and Andersson, 1985; Lindahl
et al., 1995), or by SDS-PAGE according to the method of Laemmli (1970)
using a 12% to 22.5% acrylamide gradient. Quantification of the
LHCII-27 and LHCII-25 polypeptides was performed by Coomassie blue
staining of the second-dimension gel or western blot, followed by
laser-densitometry scanning.
Analyses of Clp and FtsH Proteins
Intact chloroplasts were isolated according to the method of
Bartlett et al. (1982) and thylakoid membranes were prepared from them.
The Clp protease was detected by western-blot analysis using antibodies
raised against the rice clpP and pea clpC gene products expressed in Escherichia coli (Ostersetzer and
Adam, 1996; Ostersetzer et al., 1996).
For FtsH analysis isolated thylakoid membranes were washed in either 2 M NaBr or 0.1 M NaOH; the suspensions were left
on ice for 30 min, and then centrifuged at 10,000g for 5 min. Membrane preparations and wash supernatants were assayed for the
presence of FtsH by western-blot analysis using an antibody raised
against the E. coli enzyme, as described by Lindahl et al.
(1996).
| |
RESULTS |
Regulation of LHCII Degradation at the Enzymatic Level
The acclimative reduction of the LHCII antenna is not an immediate
response after transfer of low-light-grown spinach plants to high light
(Lindahl et al., 1995). As illustrated in Figure 1A, no or very little decrease in the
LHCII antenna occurred during the first 2 d of the high-light
regime (including two night periods), as judged by the relatively
constant chlorophyll a/b ratio and the unchanged
proportion between LHCII and the PSII core (CPa). Only during d 3 to 4 was the reduction of the LHCII antenna complete, as revealed by a
decrease of the LHCII/CPa ratio from 6.0 to 3.5 (Fig. 1A) and by the
reduced level and changed proportion between the LHCII-27 and LHCII-25
subunits (Fig. 1A, inset). There was also an increase in the
chlorophyll a/b ratio from 2.7 to 3.1.
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| Figure 1.
Induction of LHCII degradation and
activation/deactivation of the LHCII protease during acclimation of
spinach plants from low- to high-intensity light. Spinach plants grown
at low light (30 µE m2 s1) were
transferred to high-intensity light (600-800 µE m2
s1). The light/dark cycle was 10 h of light followed
by 14 h of darkness. A, Thylakoid membranes were isolated during
the acclimation period, and the reduction of the PSII antenna was
followed by measurements of the chlorophyll
a/b ratio ( ) and the LHCII/PSII core
(CPa) ratio ( ), as determined by mild SDS-PAGE. Each point is the
mean of four to six separate experiments. The arrow indicates the time
when the plants were transferred to high light. The inset shows the
relative amounts of the LHCII-27 and LHCII-25 polypeptides before (0 day) and after (4 days) high-light acclimation, as analyzed by
Coomassie blue-stained two-dimensional SDS-PAGE. B, Thylakoid membranes
isolated from leaves at various stages of acclimation were washed with
a high-salt buffer to release the protease extrinsically bound to the
outer membrane surface. The desalted and concentrated wash supernatants
were tested for proteolytic activity by addition to washed high-light
thylakoids. The degradation of LHCII was analyzed with SDS-PAGE and
western blotting. Results are the means of three independent
experiments + SE. Chl, Chlorophyll.
|
|
The lag period of the acclimative response suggests that the
proteolysis of LHCII requires some mechanism of induction. Such an
induction could be controlled at the enzyme level and/or at the
substrate level. To discriminate between these possibilities we took
advantage of the fact that the LHCII protease is extrinsically located
at the outer membrane surface of the stroma-exposed thylakoids and can
be readily washed off with high-salt buffers (Lindahl et al., 1995).
The wash supernatants were obtained from thylakoid membranes isolated
from control low-light leaves and from leaves at various stages of the
acclimation process, and used for different assays of proteolytic
activities.
As a first approach, washed thylakoid membranes isolated from
acclimating spinach leaves were used as a substrate. As illustrated in
Figure 1B, the different supernatants showed various abilities to
catalyze LHCII degradation. As judged by immunodetection, no proteolysis could be detected using the supernatant obtained from thylakoids derived from low-light-acclimated leaves. Only after 2 d (including dark periods) at the elevated light regime did the wash
supernatant possess a proteolytic activity toward LHCII, particularly
LHCII-25. The wash supernatant with maximal proteolytic activity (28%
degradation of LHCII-25) was obtained after 3 d of exposure to
high light (Fig. 1B).
After activation the proteolytic activity could either remain active or
be deactivated once the new reduced level of the LHCII antenna size had
been reached. The presence of proteolytic activity in the wash
supernatant was therefore investigated after the amount of LHCII had
reached a new stable value at the reduced level. The results revealed a
rapid reduction in proteolysis during d 4 (Fig. 1B), suggesting a
deactivation of the enzymatic activity.
These in situ degradations were complemented with experiments in which
isolated LHCII was used as a substrate for the various wash
supernatants. As judged by immunodetection, no or very little degradation occurred during the first 27 h after transfer of the dark-adapted leaves to the high-intensity light (not shown). Only after
approximately 2 d could a significant degradation be observed, particularly for LHCII-25. Using the artificial protease substrate fluorescein thiocarbamoyl-casein (Twining, 1984), a similar increase of
proteolytic activity was found for the various wash supernatants (not
shown).
These data imply that LHCII degradation is regulated at the enzymatic
level, which could involve expression and degradation of the protease
(turnover) or a posttranslational activation/deactivation process.
Regulation at the Substrate Level
The fact that the initiation of the LHCII degradation involves a
regulation at the enzymatic level does not exclude activation at the
substrate level. For this reason, a series of reconstitution experiments was designed in which high-salt-wash supernatants from
low-light controls or high-light-acclimating leaves were homologously
or heterologously added to washed low- or high-light thylakoid
membranes (Fig. 2). After these
reconstitutions the proteolytic activity was determined in situ by
immunoblotting and compared with the LHCII degradation occurring in
unwashed thylakoid membranes. The wash supernatant derived from the
high-light thylakoids induced a pronounced degree of degradation when
it was homologously readded to the washed high-light thylakoids (Fig. 2), corroborating the high reconstitution ability of the proteolytic system (Lindahl et al., 1995). In contrast, when the wash supernatant derived from low-light thylakoids was heterologously reconstituted with
washed high-light thylakoids, a very poor degree of degradation was
obtained. Moreover, as expected, no degradation was obtained when the
low-light supernatant was added to its washed low-light thylakoid
counterpart. These observations are consistent with the requirement for
an enzymatic induction of the proteolytic process as discussed above. A
crucial observation was made in the fourth reconstitution experiment,
when the high-light-wash supernatant was added to the washed low-light
control thylakoids (Fig. 2). This heterologous reconstitution resulted
in only about 15% of the maximal LHCII degradation. Therefore, even
though the high-light-wash supernatant contained the active protease,
it could not degrade LHCII in the low-light thylakoids.
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| Figure 2.
Homologous and heterologous reconstitution of
LHCII proteolysis using high-light (HL)/low-light (LL) thylakoids
(Thy.) and their corresponding high-light/low-light wash supernatants
(Sup.). The degradation of LHCII was followed by SDS-PAGE and
western-blot analyses. Results are the means ± SE of four
independent experiments.
|
|
The homologous and heterologous reconstitution experiments provide
evidence that induction of the enzyme is not sufficient for the
acclimative LHCII degradation, but that some additional activation at
the substrate level is required. In the search for the molecular basis
of such a substrate activation, two previous observations with respect
to the long-term acclimation of LHCII have been considered. First,
there is a lateral segregation between the LHCII substrate and the
responsible protease along the stacked thylakoid membrane. The outer
pool of LHCII is normally associated with the PSII centers located in
the appressed thylakoid regions (Larsson et al., 1987a, 1987b;
Mäenpää and Andersson, 1989; Melis, 1991), whereas
the proteolytic activity is confined to the nonappressed thylakoid
regions (Lindahl et al., 1995). Second, the outer pool of LHCII is not
only a substrate for proteolysis during long-term acclimation (Lindahl
et al., 1995), but is also readily subjected to protein phosphorylation
(particularly the 25-kD subunit) and rapid lateral migration from the
appressed to the nonappressed thylakoid regions (Kyle et al., 1983;
Larsson and Andersson, 1985; Larsson et al., 1987b). The latter events are of significance for the so-called state transitions involved in the
short-term regulation of the photosynthetic antenna (Gal et al., 1997).
Consequently, the substrate activation could involve protein
phosphorylation and lateral migration of phospho-LHCII from its
original location in the appressed thylakoid regions to the
nonappressed thylakoid regions, where it would come in contact with the
protease.
The relative content of LHCII in the stroma-exposed regions of
thylakoids isolated from leaves at high light intensity was therefore
followed during the acclimation period in vivo (Fig. 3). A transient increase of the
steady-state level of LHCII in the nonappressed thylakoid regions was
observed during the acclimation period, as judged by SDS-PAGE analyses.
After 5 h in high light, there was almost a doubling of LHCII in
the stromal thylakoid fraction. This indicates a migration of LHCII
from the appressed to the nonappressed membrane regions similar to that
observed after thylakoid protein phosphorylation in vitro (Kyle et al., 1983; Larsson et al., 1987b). The relative amount of LHCII in the
stromal thylakoids increased further during the high-light conditions
and reached a maximal level after 1.5 d (including a dark period).
After 2.5 d, however, there was a decrease in the relative level
of LHCII (Fig. 3) and a concomitant overall decrease in the amount of
LHCII in the unfractionated thylakoids by approximately 30% (Fig. 1A).
We therefore interpret the transient appearance of LHCII in the stromal
thylakoids as a migration of LHCII during the lag period to the stromal
thylakoids, where proteolysis will occur once an activated protease is
present.
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| Figure 3.
The relative amount of LHCII in the stroma-exposed
regions of thylakoid membranes isolated from spinach leaves during
their acclimation from low- to high-intensity light. Stromal lamellae
vesicles were isolated by digitonin incubation of thylakoid membranes
at different stages in the acclimation process, followed by
differential centrifugation. The relative amounts of LHCII were
determined by mild SDS-PAGE. The light/dark cycle was 10 h of
light followed by 14 h of darkness. The 100% value represents the
initial amount of LHCII in the stromal thylakoids before exposure of
plants to high light. Results are the means of three separate
experiments.
|
|
The requirement for lateral migration during the proteolysis of LHCII
could indicate a role for protein phosphorylation in the regulatory
process. This possibility was also considered in view of evidence for
the regulation of proteolysis via protein phosphorylation in several
biological systems (Molinari et al., 1995; King et al., 1996). In
chloroplasts this has been demonstrated for D1- and D2-protein
degradation during photoinhibitory conditions (Elich et al., 1992;
Koivuniemi et al., 1995). During the onset of light stress the two PSII
reaction center proteins become phosphorylated but the actual
proteolysis is preceded by dephosphorylation (Rintamäki et al.,
1996).
In view of these considerations and observations, thylakoid membranes
isolated from leaves in the acclimating phase were phosphorylated in
vitro to different extents in the presence of
[-32P]ATP. The extent of degradation of
LHCII-25 in the thylakoids was then monitored by two-dimensional gel
electrophoresis during a 6-h period. When the degradation of LHCII-25
was plotted against the relative extent of phosphorylation of this
polypeptide (Fig. 4), a clear correlation
could be seen. As the level of phosphorylation increased, the rate of
degradation of LHCII-25 decreased, and at about 70% phosphorylation no
degradation could be seen. Thus, it can be concluded that the protease
appears to be specific for the nonphosphorylated form of LHCII, so any
role for protein phosphorylation during the process of LHCII
degradation must be indirect (see ``Discussion'').
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| Figure 4.
Correlation between LHCII phosphorylation and
degradation. Thylakoid membranes were labeled using
[-32P]ATP. The maximal amount of phospho-LHCII
obtained at 160 µE m2 s1 for 15 min was
taken as 100% phosphorylation. The extent of degradation was
determined as a decrease in the LHCII-25/LHC-27 ratio during a 6-h
period. The analysis was with two-dimensional SDS-PAGE combined with
autoradiography and Coomassie blue staining. Results are from two
independent experiments (expt1 and expt2).
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Possible Relation between LHCII Degradation and the Clp and FtsH
Proteases
Chloroplast homologs to bacterial proteases have recently been
identified in higher plants (Adam, 1996). One of these proteins, Clp,
is an ATP-dependent Ser-type protease composed of two subunits, ClpA
and ClpP. Because the proteolytic activity degrading LHCII is both ATP
dependent and of the Ser type (Lindahl et al., 1995), this prompted us
to test whether Clp could be involved in this particular process. In
Figure 5A, the presence of the Clp
subunits in chloroplasts isolated from acclimating as well as
low-light control spinach leaves is shown by immunoblotting using
ClpA and ClpP antisera. Quantification revealed no changes in the
relative levels of the Clp subunits between the two chloroplast
samples. Furthermore, the two Clp subunits were absent from thylakoid
membranes isolated from both of these particular chloroplast samples
and were recovered in the soluble stromal fraction. This demonstrates that the Clp protease does not associate with the membrane under the
applied physiological conditions and that it cannot be responsible for
the acclimative proteolysis of LHCII, since this enzymatic activity
remained bound to the thylakoid membrane unless it was removed by
high-salt treatment.
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| Figure 5.
Analysis of the chloroplast proteases Clp and FtsH
in connection with LHCII degradation. A, The presence of ClpA and ClpP
in chloroplasts (lanes 1 and 2) and thylakoids (lanes 3 and 4) isolated
from high-light (lanes 2 and 4) and low-light (lanes 1 and 3) spinach
leaves was analyzed by SDS-PAGE and western blotting. B, Association of
FtsH with thylakoid membranes during acclimation was investigated by
washings with either NaBr or NaOH. Analysis was with SDS-PAGE and
western blotting. Lane 1, Unwashed thylakoids; lane 2, NaBr pellet;
lane 3, NaBr-wash supernatant; lane 4, NaOH pellet; lane 5, NaOH-wash
supernatant.
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|
Another recently identified ATP-dependent protease of higher plant
chloroplasts homologous to a bacterial protein is the FtsH protease
(Lindahl et al., 1996). To find a relationship between this protease
and the LHCII-degrading activity, we subjected the isolated thylakoid
membranes to high-salt and alkaline washings, after which the presence
of FtsH in thylakoid pellets and wash supernatants was probed by
immunoblotting against a heterologous antiserum. As shown in Figure 5B,
FtsH could not be released from the membranes upon washing with either
NaBr or NaOH. Therefore, the firm association of FtsH with the
thylakoid membrane excludes any possible involvement of this
protease in the process of acclimative LHCII degradation.
| |
DISCUSSION |
The overall changes of the PSII antenna in response to variations
in the light environment for different plant species have been
thoroughly described (Osmond, 1994; Melis, 1996). Despite the wealth of
physiological information, the underlying molecular mechanisms for the
differences in antenna composition and the dynamic acclimations are
still not understood. The expression of the nuclear cab
genes encoding LHCII is light regulated at both the transcriptional and
translational levels (Green and Salter, 1996), and the hierarchy of Cab
protein assembly during biogenesis has been characterized (Melis,
1996). Aside from the recently discovered proteolytic process (Lindahl
et al., 1995; Lindahl, 1997), the process of reduction of the LHCII
antenna during acclimation to elevated light intensities is basically
unknown. An important feature of this acclimation is that it shows a
pronounced lag in its response, so a major aim of the present
investigation was to address the molecular control of this process.
It can be demonstrated that initiation of the acclimative LHCII
proteolysis requires regulation at both the enzyme and the substrate
levels. The lag period in the physiological response could be
attributed mainly to an expression or activation of the responsible
protease at high light. We have not been able to discriminate between
these two mechanistic alternatives. In the case of a posttranslational activation, redox control via plastoquinone or regulation via thiol
groups, as has been shown to occur for the enzymes involved in
reversible thylakoid protein phosphorylation (Gal et al., 1997; Vener
et al., 1998), are interesting possibilities for future experimental
investigations.
What is the mechanism that triggers LHCII degradation at the substrate
level? We conclude from the thylakoid-subfractionation experiments that the induction of degradation involves a lateral movement of the outer pool of LHCII, which is enriched in the rapidly
phosphorylated 25-kD subunit, from the appressed to the nonappressed
thylakoid regions where the protease is located (Lindahl et al., 1995).
It is known that phosphorylation of LHCII is a driving force behind its
lateral migration during the state-1 to state-2 transitions of the
short-term acclimation of light harvesting (Barber, 1982; Gal et al.,
1997). Therefore, it seems reasonable to assume that the lateral
migration of LHCII seen under the present long-term acclimation to
increased irradiance (Fig. 3) also involves protein phosphorylation. If
that is the case, a likely mechanism for the transformation of the
short-term, reversible reduction of the LHCII antenna (state
transition) to the long-term, irreversible reduction of the LHCII
antenna (degradation) would be the induction of proteolytic activity.
This enzyme induction will occur only after the plants are exposed for
a prolonged period to a high light intensity, as shown by the observed
lag phase in the physiological response (Fig. 1).
Such a simple mechanistic connection between short- and long-term
acclimation of the PSII antenna would be consistent with the fact that
both responses are targeted mainly to LHCII-25 (Larsson et al., 1987a,
1987b). However, it should be stressed that a role for protein
phosphorylation during LHCII degradation can only be indirect, because
it brings the protein substrate and enzyme together in the thylakoid
membrane system. We found that phospho-LHCII is a poor substrate for
the degradation compared with the unphosphorylated form (Fig. 4). LHCII
degradation, therefore, may be more similar to D1-protein degradation
during photoinhibitory conditions, in which the phosphorylated form of
the photodamaged protein is protected from proteolysis and no
degradation occurs in the absence of dephosphorylation (Koivuniemi et
al., 1995; Ebbert and Godde, 1996; Rintamäki et al., 1996).
It should also be considered that high light intensities can lead to
kinase inactivation (Schuster et al., 1986; Horton and Hague, 1988;
Walters and Horton, 1991), which could complicate a potential
physiological role of protein phosphorylation during long-term
acclimation to high light. However, at the rather moderate nonphotoinhibitory irradiance applied in our present acclimation studies (600 µE m2
s1), no reduction in the kinase activity took
place (not shown). Kinase inactivation readily occurred at higher light
intensities (>1000 µE m2
s1), in accordance with previous studies
(Schuster et al., 1986; Horton and Hague, 1988; Walters and Horton,
1991).
Degradation of LHCII should potentially release chlorophyll molecules
that would be highly toxic to the photosynthetic apparatus if they were
not rapidly degraded or quenched by some other mechanism. ELIPs may be
involved in the binding of liberated chlorophyll molecules, thereby
providing quenching of any toxic effects during LHCII degradation. This
suggestion would be in accordance with the function proposed for ELIPs
as "transient pigment-binding proteins" (Adamska, 1997) and with
the experimental finding that ELIPs bind both chlorophyll and lutein
(I. Adamska, unpublished data). ELIPs in spinach leaves were recently
shown to have unusually high expression under conditions of LHCII
degradation (Lindahl et al., 1997).
In our efforts to identify the LHCII protease, we have at least ruled
out that it could be related to the chloroplast Clp or FtsH proteases.
On the other hand, it has previously been shown that a
detergent-activated proteolytic activity against LHCII is present in
isolated bean thylakoids (Anastassiou and Argyroudi-Akoyunoglou, 1995).
A comparison of the proteases responsible for acclimative proteolysis
of LHCII and the detergent-induced degradation reveals similarities as
well as discrepancies. Both enzymes are of the Ser type and probably
contain sulfhydryl groups essential for the catalytic activity. The
detergent-induced proteolytic activity of LHCII is, in contrast to the
acclimative degradation of LHCII, independent of ATP. The relationship
between the two proteases is still uncertain, because additional
information concerning the localization and the nature of the
association with the thylakoid membrane of the detergent-activated
protease is lacking.
Recently, a proteolytic activity involved in low-light-induced
degradation of ELIPs, which are homologous to the Cab proteins, has
been characterized in vivo and in vitro and the protease has been
partially purified (Adamska et al., 1996). This ELIP protease was also
found to be of the Ser type and extrinsically located at the
stroma-exposed thylakoid surfaces. However, the fact that its activity
does not require ATP argues against an involvement of this protease in
the acclimative degradation of LHCII. The identity of the LHCII
protease, therefore, remains unknown, and its identification and
characterization provide a central challenge for gaining further
knowledge of the light acclimation of PSII and its light-harvesting
antenna.
| |
FOOTNOTES |
1
This work was supported by the Swedish Natural
Science Research Council, the Carl Trygger Foundation, and a network
grant from the European Commission Human, Capital, and Mobility program (contract no. ERB CHRX CT 940619).
2
Present address: Department of Medical
Nutrition, Karolinska Institute, Huddinge Hospital, Novum, S-141 86 Huddinge, Sweden.
3
Present address: Department of Agricultural
Botany, Hebrew University of Jerusalem, Rehovot 76100, Israel.
*
Corresponding author; e-mail bertil.andersson{at}biokemi.su.se; fax
46-8-15-36-79.
Received March 31, 1998;
accepted August 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ELIP, early light-inducible protein.
LHCII, major light-harvesting chlorophyll
a/b-binding protein of PSII.
LHCII-25 and
LHCII-27, 25- and 27-kD subunits, respectively, of LHCII.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge Drs. I. Ohad and I. Carlberg for their
constructive suggestions and discussions of this work, as well as K. Svennersjö for her help typing the manuscript.
| |
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Abstract
Introduction
