From the
The resistance of maize plants to cold stress has been
associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling
treatment in the light. The cold-induced protein has been isolated,
characterized by amino acid sequencing, and pulse labeled with
radioactive precursors, showing that it is the product of
post-translational modification by phosphorylation of the minor
chlorophyll a/b protein CP29 rather than the product of a
cold-regulated gene or an unprocessed CP29 precursor. We show here that
the CP29 kinase activity displays unique characteristics differing from
previously described thylakoid kinases and is regulated by the redox
state of a quinonic site. Finally, we show that maize plants unable to
perform phosphorylation have enhanced sensitivity to cold-induced
photoinhibition.
In the photosynthetic apparatus of higher plants, the efficiency
of the electron transport and light-harvesting functions is regulated
in response to environmental and metabolic conditions. The earliest
described mechanism is the state I-state II transition and consists of
the phosphorylation-induced redistribution of light-harvesting complex
II (LHCII)
Recently, a new
regulatory process has been reported that is elicited by strongly
photoinhibitory conditions such as those produced by illumination of
maize plants at low temperature. Under these conditions, a new
thylakoid protein appears in cold-resistant cultivars and is absent
from cold-sensitive plants. While the protein has been isolated and
characterized as a chlorophyll a/b protein located close to
the PSII reaction center (RC), its origin is still unclear.
This is the
first report of CP29 phosphorylation and suggests that the reversible
modification of membrane-intrinsic, chlorophyll a/b binding
proteins can be a mechanism for the modulation of light harvesting and
excitation energy transfer to the reaction center.
The 34-kDa polypeptide was purified
from thylakoid membranes of cold-treated maize leaves (6 h at 4 °C
in the presence of high light). Since several thylakoid polypeptides
have molecular masses in the 28-35-kDa range, we isolated the
protein by two consecutive preparative SDS-PAGE systems to avoid the
presence of minor contaminations. The same procedure was applied to
CP29.
The polypeptides were then blotted onto polyvinylidene
difluoride filters and submitted to automated Edman degradation. As
they were N-terminally blocked, peptide fragments were generated by
limited proteolysis using trypsin. Following digestion, two of the
resulting peptides were purified by HPLC and sequenced in the case of
CP34, and three peptides were purified and sequenced in the case of
CP29. The results of this analysis are shown in Fig. 2. Fragments I and
II of both CP29 and CP34 gave sequences of 32 and 20 amino acids and
were identical in the two apoproteins. They matched almost exactly with
segments of the protein sequence deduced from the lhcb4 cDNA
clone of barley encoding the CAB protein CP29
(20) . The two
stretches of residues span, respectively, amino acids 102-131 and
212-231 of the barley apoprotein precursor. Only few differences
could be observed for fragments I and II, as shown in Fig. 2.
Fragment III, sequenced only for CP29, differed more from the
corresponding segment of its barley counterpart, especially at its
N-terminal side (see Fig. 2).
To test these possibilities, we
examined the patterns of thylakoid proteins synthesized at 25 and 4
°C in the presence of light. We labeled plastid proteins in
vivo by feeding [
Fig. 3, a and
b, show the results of this analysis. In particular, it was
clear that the chilling treatment did not cause de novo synthesis of the CP34 protein, since the labeling of this
polypeptide was as low as that of CP29 on a protein quantity basis, as
determined by densitometry of gels and relative autoradiographs. The
low [
The
autoradiographic pattern was very similar in chilled and control
samples. However, incorporation in all of the polypeptides was lower in
the cold-exposed plants as a result of the effect of low temperature on
protein synthesis
(26, 27) . The D1 protein is an
exception to this rule; its labeling was higher in the cold-treated
membranes, consistent with the photoinhibitory conditions applied
(28, 29) .
When we performed the above experiment with maize
lines, which produce very little or any CP34 upon cold stress in the
light, essentially no differences were observed in the phosphorylation
pattern apart from the lower labeling of the CP34 band (not shown).
To clarify
this point, we treated thylakoid membranes containing CP34 with
phosphatase enzymes in vitro. Both acidic and alkaline
phosphatases were used, and the former was less active than the latter
at neutral pH (not shown). When treated with alkaline phosphatase at pH
7.00, the CP34 band, detected by immunoblotting, disappears, indicating
that the change in mobility is only due to phosphorylation (Fig.
5 A). Surprisingly, the dephosphorylation was shown to be
slower at higher pH, 8.5 versus 7.0, closer to the alkaline
phosphatase optimum. Moreover, the addition of the mild detergent DM
increases the dephosphorylation rate at both pH values, thus suggesting
that external (stromal) pH changes may induce differences in the
accessibility of the phosphorylation site. We designed an experiment to
ascertain if the domain of the antenna complex involved in
phosphorylation-dephosphorylation events was exposed on the stromal or
lumenal site of the membrane. Thylakoids and PSII membranes (BBY
particles), exposing, respectively, the stromal and the lumenal domains
of the polypeptide to the solvent, were used. When thylakoids were
treated with alkaline phosphatase, dephosphorylation was more effective
if membranes were previously destacked by EDTA washing to increase the
accessibility to the stromal surface of grana membranes.
Dephosphorylation was more efficient when alkaline phosphatase was
added before restacking with Mg
In a first approach, we blocked electron transport by
depleting CO
The aim of this work was to characterize the nature of the
34-kDa (CP34) SDS-PAGE band, which appears in maize thylakoid membranes
following exposure to cold in the presence of light. This cold-induced
polypeptide had been shown to be immunologically related to the
chlorophyll a/b binding protein CP29, one of the minor antenna
complexes of PSII
(7) . Moreover, Mauro et al.
The
Alkaline phosphatase treatment of thylakoids and PSII membranes,
which, respectively, expose stromal and lumenal faces, showed that the
phosphorylation site is located on the stromal side of the membrane.
This post-translational modification is effective in modifying the
protein structure as judged by the limited proteolysis experiment (Fig.
3) and by the change in SDS-PAGE mobility, similar to that described
for other phosphoproteins
(30) . We concluded that the SDS-PAGE
band induced by the light treatment in the cold is the phosphorylation
product of the chlorophyll a/b protein CP29.
Regulation of
function of thylakoid proteins by phosphorylation is a well known
mechanism that drives lateral migration of LHCII antenna complexes in
state I-state II transitions. LHCII kinase has been shown to be
activated by plastoquinol and inactivated by plastoquinone; the role of
plastoquinol has been documented in the phosphorylation of all other
thylakoid proteins, but authors are far from an agreement on the
question of how many kinases exist in thylakoids (for a review, see
Refs. 31 and 32). The redox sensitivity of the LHCII kinase is provided
in vivo (37, 38, 39) , as well as
in vitro (34) , by its association with the cytochrome
b
We tested if the same
kinase was also responsible for the phosphorylation of CP29, another
member of the CAB family. In addition to a wild type line, we
analyzed the maize mutant hcf2, which is known to lack the
activity of the LHCII kinase as a consequence of the absence of the
cytochrome b
On the other hand, PSII core proteins are phosphorylated in isolated
thylakoids while CP29 is not
(41) , thus suggesting that the
complexes are not substrates of the same kinase. Finally, preliminary
results of experiments with DBMIB revealed that this halogenated
quinone analogue clearly enhances phosphorylation of CP29 while it
inhibits both LHCII and PSII polypeptide phosphorylation
(31, 32, 37, 38, 42) . DBMIB
induces the CP29 kinase activity in the concentration range from 1 to
100 µ
M, even in dim light without cold treatment. Taken
altogether, our results bring new evidences for the existence of at
least three thylakoid protein kinases.
Where is the CP29 kinase
located? It has been reported that the 64-kDa LHCII kinase is localized
in grana domains, particularly at the edges of the grana stacks, where
it is associated to the cytochrome b
The redox sensitivity, the inactivation by DCMU, and the activation
by DBMIB indicate that the CP29 kinase must be associated with at least
one quinonic site. On one hand, results obtained with DCMU both in wild
type and hcf2 clearly show that the kinase senses the
electronic state of the Q
Concomitant with the phosphorylation of CP29,
cold treatment induces phosphorylation of the RC subunits CP43, D1, and
D2 and 9 kDa in the monomeric form of PSII. Since this corresponds to a
dissociation of RC dimers into monomers, it can be hypothesized that
this phosphorylation event might be the cause for dissociation of PSII
dimers. Phosphorylation-induced dissociation of neighboring
chlorophyll-proteins has been previously described
(45) ;
similarly, both PSII RC subunits and CP29 phosphorylation can be
related to a regulatory mechanism based on the dissociation of the PSII
supermolecular complex. These events could be part of at least two
regulation phenomena, the first being the dissociation of antenna
complexes from PSII and their migration into the stroma membranes
(1, 45, 46) , with consequent reduction of PSII
antenna size, and the second being PSII cycling whose primary step
could well be the RC dimer dissociation
(47) .
Free lateral
migration of LHCII and PSII RC subpopulations from the grana stacks and
their margins to stroma-exposed thylakoid membrane is an obvious
requirement for such mechanisms. Thylakoid membrane fluidity is
strongly decreased at temperatures lower than 10 °C
(48) ;
we can therefore argue that, under conditions chosen for our
experiments (4 °C), the phosphorylated RC subunits of monomeric
PSII accumulate since subsequent steps in their cycling are impaired.
Which is the role of CP29 phosphorylation? We approached this
question by using two maize lines phosphorylating CP29 to a different
extent and evaluating their resistance to photoinhibition following
treatment in cold and light. The results showed that the line more
active in CP29 phosphorylation is also more resistant to
photoinhibition. In fact, although CP29 is not the only protein that is
phosphorylated in the cold, PSII core complex subunits and LHCII
polypeptides are phosphorylated both in resistant and sensitive lines.
This is in agreement with previous data obtained by the analysis of two
lines sharing the same genetic background but differing in the level of
CP34 induction.
We therefore propose that CP29 phosphorylation is a
novel photoprotection mechanism, based on the reorganization of
pigments in the chlorophyll-protein complex, in such a way that a lower
amount of excitation energy is funneled to RCII. Further experiments
are in progress to obtain a better characterization of the spectral and
structural changes induced by CP29 phosphorylation.
We are grateful to Dr. David Simpson (Carlsberg
Laboratory, Copenhagen, Denmark) and to Dr. Francis-Andre Wollman
(Institut de Biologie Physico-chimique, Paris, France) for critically
reading the manuscript. We thank Dr. Maria Ruzzene for testing CP29
phosphorylation in vitro by exogenous kinases and Prof.
Arianna Donella Deana for helpful suggestions on the use of phosphatase
enzymes (Dipartimento di Chimica Biologica, Padova, Italy). We also
thank Dr. D. Miles (Dept. of Biology, University of Missouri, Tucker,
Columbia) for the kind gift of the hcf2 maize mutant.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(1) and cytochrome
b
/f (2) complexes between grana-
and stroma-exposed membranes, thus yielding a modulation of cyclic
versus linear photophosphorylation
(2) . A second
mechanism has been described for the thermal dissipation of the
excitation energy excess. This process is dependent on the
deepoxidation of the xanthophyll violaxanthin to zeaxanthin
(3) , which is mainly located in the minor photosystem II (PSII)
subunits CP26 and CP24
(4, 5) .
(
)
In particular, it is not known whether the cold-induced
SDS-PAGE band is the product of a previously silent lhcb gene
or a post-translational modification of an existing chlorophyll protein
such as CP29, with which it shares epitopes
(6, 7) and
pigment composition.
In this study, we have characterized
the cold-induced protein by amino acid sequencing and pulse-chase with
radioactive precursors, showing that it is the result of CP29
phosphorylation. Moreover, we report that this phosphorylation induces
a change in the CP29 conformation and that the kinase activity is
controlled by a quinonic site closely associated to photosystem II RC.
Finally, we show that maize plants unable to perform CP29
phosphorylation are more sensitive to photoinhibition.
Preparation of Thylakoid
Membranes
Zea mays seedlings (cvs. Adon,
DK300, Oh7N, and H93, supplied by Dekalb
Co., IL) were grown for 2 weeks in a growth chamber at 28/21 °C
day/night at a light intensity of 200 µE ms
and 80% humidity. Leaves from 2-week-old
plants were harvested at the end of a 6-h illumination period at 25 or
4 °C, and thylakoids from mesophyll chloroplasts were prepared as
previously described
(8) . PSII membranes (BBY particles) were
obtained according to the method of Berthold et al. (9) with the modification described in Ref. 10. Aliquots were
suspended in 50 m
M Hepes/KOH, pH 7.5, 5 m
M MgCl
, 50% glycerol and frozen at -80 °C
until required. Membrane yield was determined by measuring chlorophyll
content in 80% acetone, using the equations of Porra et al. (11) .
PAGE and Immunoblotting
For the
purification of CP29 and CP34 apoproteins, two consecutive preparative
SDS-PAGE were used to avoid minor contaminations. The first
elecrophoresis was run on a 12-18% polyacrylamide gradient gel
containing 6
M urea with the Tris/sulfate buffer system, as
previously described
(8) . Bands were excised and rerun on a
second gel of uniform polyacrylamide concentration (10%) without urea
in the Tris/tricine buffer
(12) . Two-dimensional
electrophoresis was performed using the non-denaturing Deriphat-PAGE
(on a 5-12% polyacrylamide gradient gel)
(13) for the
first dimension and the SDS-urea-PAGE (9-16% gradient gel)
according to Laemmli
(14) , with double buffer concentration for
the second dimension. Individual spots in the two-dimensional gels were
identified by immunoblotting with specific antibodies as previously
described
(13) . One-dimensional electrophoresis was always
performed by the Tris/sulfate, 6
M urea system mentioned
above. For immunoblot assays, samples were separated by PAGE and
electro-transferred to nitrocellulose filters (Hoefer). Filters were
then probed with -CP29 antibodies, and antibody binding was
detected by alkaline phosphatase-conjugated
-rabbit IgG
(Boehringer Mannheim). Antibodies were raised in rabbits, using
poly(A)
poly(U) as adjuvant, and characterized as previously
described
(15) .
Purification of Proteolytic Peptides and Amino Acid
Sequencing
The samples were purified by SDS-PAGE as
described above and electrotransferred to Immobilon-P membranes
(Millipore)
(17) . The proteins were then visualized by staining
with 0.2% Ponceau S in 2% acetic acid. The bands of interest were
excised and digested 24 h with trypsin in 0.1
M ammonium
bicarbonate containing 5 m
M calcium chloride and 8%
acetonitrile. The resulting tryptic fragments were first purified by
utilizing an ABI 130 HPLC and an aquapore RP-300 column equilibrated
with 0.1
M potassium phosphate, pH 7.0, followed by a 17-min
linear gradient from 0 to 40% acetonitrile. Individual peaks were then
rechromatographed using the same column equilibrated with 0.1% (v/v)
trifluoroacetic acid and a linear gradient from 0 to 40% acetonitrile
in 0.07% (v/v) trifluoroacetic acid. Samples were sequenced with an
Applied Biosystems model 475A sequencer equipped with an ``on
line'' model 120A PTH amino acid analyzer.
In Vivo Labeling
Maize seedlings of
2-week-old plants were cut under water at the base of their epicotyl
and quickly immersed into 0.5 ml of 40 m
M Hepes/KOH (pH 7.5),
in small glass vials (height, 40 mm; diameter, 5 mm). 300 µCi of
[S]methionine or 200 µCi of
[
P]orthophosphoric acid were added to each vial,
and seedlings were allowed to incorporate radioactivity for 3 h at 25
°C in the light, with the evaporative demand increased by cool air
from a hair drier. When solutions became low during this period, 40
m
M Hepes/KOH was added to each vial. Following incorporation,
seedlings were kept in the light for 6 h at either 4 or 25 °C.
Leaves, apart from the first one, were then cut, and thylakoids were
immediately prepared as described
(8) . Densitometry of the
relative Coomassie-stained gels (treated with
EN
HANCE
autoradiography enhancer, DuPont NEN)
and autoradiographs (Hyperfilm-
Amersham films) were
performed with a Bio-Rad GS670 imaging densitometer according to the
company's protocol.
Limited Proteolysis of Thylakoid
Membranes
Thylakoid membranes (25 µg of chlorophyll)
were washed once in 5 m
M EDTA, 10 m
M Hepes/KOH, pH
7.5, and resuspended in 0.1% DM, 10 m
M Hepes/KOH, pH 7.5, at a
chlorophyll concentration of 0.5 mg/ml. Proteolytic digestion was
carried out at 37 or 25 °C for 30 min by addition of chymotrypsin
to a final concentration of 100 µg/ml. The reaction was stopped by
adding 1 volume of solubilization buffer (containing 6
M urea,
5% SDS, 1% 2-mercaptoethanol, and 20% glycerol), and the fragments were
loaded on a 12% acrylamide gel using the Tris/tricine system.
In Vitro Dephosphorylation
Membranes, in
aliquots of 5 µg of chlorophyll, were suspended in 50 m
M Tris/HCl (pH 7.0 or 8.5) and incubated with 0.5 units of alkaline
phosphatase (type VII-SA, Sigma) at 30 °C. Where indicated,
membranes were previously washed twice in 50 m
M Tricine, pH
7.8, 5 m
M EDTA, and/or MgClwas added to the
sample to a final concentration of 5 m
M. After incubation, the
reactions were stopped by addition of the solubilization buffer;
samples were boiled for 2 min and loaded onto the gel.
Fluorescence Analysis
The cultivars
Oh7N and H93 were grown exactly in the same
conditions described for the preparation of thylakoid membranes, in a
Haereus growth cabinet at a light intensity of 300 µE
ms
. Changes in chlorophyll
fluorescence yield were measured using a commercial pulsed fluorimeter
(PAM 2000, Walz, Effeltrich, Germany) with the light guide that
delivers the measuring and saturating light held at 5 mm from the upper
side of the third maize leaf. The leaf was fixed on a copper support
while still in the pot in a cold room. For the photoinhibitory
treatment at 4 °C, strong white light (1000 µE m
s
at the surface of the leaf) was delivered by
a cold light illuminator (KL1500 electronic, Schott, France). The
temperature of the leaf (measured by a thermocouple) could be rapidly
changed from 4 to 25 °C. The minimum
( F
) and maximum
( F
) fluorescence yield in the dark, and
the ratio ( F
-
F
)/ F
=
F
/ F
, which
reflects the potential yield of the photochemical reaction of
photosystem II
(18) was recorded at 25 °C before each
chilling period. The standard protocol consisted of four periods of 2 h
of light at 4 °C, separated by 15 min of dark at 25 °C. During
this dark period, that part of non-photochemical quenching not related
to photoinhibition could relax
(19) . After the last
photoinhibitory treatment, the plants were allowed to recover at 25
°C for 15 h, and the fluorescence parameters were measured again
(24 h after the first measurements).
Characterization of the CP34
Protein
When maize leaves are chilled in the light, a new
SDS-PAGE band appears at a position corresponding to 34 kDa. The
process is completely reversible upon recovery at room temperature for
1 h (Fig. 1). This protein is related to the chlorophyll a/b binding protein CP29, as recognized by immunological
cross-reaction
(6, 7) and spectroscopic analysis of the
isolated native protein. This band was called CP34 on the basis of its
apparent molecular weight.The relationship between the
CP29 and CP34 proteins is unclear. To assess the origin of this
cold-induced thylakoid protein, we first determined the primary
sequence of both CP29 and CP34.
Figure 2:
N-terminal sequences of tryptic fragments
from digestions of CP29 and CP34 apoproteins. The alignment was
obtained with the FASTA program. Vertical bars stress
amino acid identities. Amino acid positions on the primary sequence of
the barley protein (20) are indicated. X, undetermined amino
acid.
These results show that the two
SDS-PAGE bands recognized by the -CP29 antibodies in cold-treated
maize thylakoids are the products of genes very similar if not
identical to lhcb4. As far as the sequence analysis, CP29 and
CP34 are identical. In Vivo Labeling of Thylakoid Proteins with
[
S]Methionine-In the light of the
above results, we could then address questions on the biogenesis of
CP34, i.e. whether it was ( a) the product of an
alternative splicing of the lhcb4 gene transcript,
( b) the unprocessed precursor of CP29, or ( c) a
post-translationally modified CP29. The first two hypotheses imply that
the cold-induced protein is synthesized de novo following cold
stress, while a post-translational modification would act on a protein
already existing in the thylakoids.
S]methionine to seedlings.
Maize seedlings, loaded with [
S]methionine for 3
h at 25 °C, were then incubated for 6 h at either 25 or 4 °C
under 200 µE m
s
illumination. Following this treatment, thylakoids were isolated
from mesophyll chloroplasts, and their polypeptide content was analyzed
by two-dimensional gel electrophoresis and autoradiography. Membranes
were first fractionated on non-denaturing Deriphat-PAGE
(13, 21) and then by SDS-urea-PAGE in the second dimension. This
combined electrophoretic system allowed a clear-cut separation of CP29
from the D1 apoprotein, a PSII core component comigrating with CP29,
which is known to have a very fast turnover, and was therefore expected
to be heavily labeled
(22, 23) . As for their amino acid
content, CP29 and D1 proteins, respectively, contain 3 and 12
methionines in their primary sequences in barley
(20, 24, 25) .
S]methionine incorporation in CP34
following induction by cold shows that this polypeptide is neither the
precursor of CP29 nor the product of an alternative splicing of the
lhcb4 maize messenger, since both cases imply de novo synthesis and consequent incorporation of radioactivity.
In Vivo Labeling of Thylakoid Proteins with
H333PO
We then checked the remaining hypothesis,
aiming at identifying the specific post-translational modification of
CP29. It has been already reported that the cold-induced polypeptide is
not glycosylated
(7) . Moreover, the putative modification
appeared to be rapidly inducible and reversible. This was shown by the
rate of appearance of the 34-kDa band on exposing leaves to chilling
and high light,as well as the rate of degradation of the
polypeptide on returning leaves to 25 °C
(7) (Fig. 1). These kinetics are consistent with a
phosphorylation-dephosphorylation mechanism that could also explain the
electrophoretic upshift, a behavior common to other phosphoproteins
(30) . In vitro phosphorylation of the purified,
non-denatured form of the CP29 protein by various kinases was not
successful (not shown). We then proceeded to in vivo labeling
of maize leaves with H333PO
. The radioactive phosphate was
fed to excised seedlings as described for
[
S]methionine. Seedlings were then exposed to
chilling treatment in the light, and thylakoid membranes were analyzed
by two-dimensional PAGE and autoradiography. The results are shown in
Fig. 3 c; CP34, but not CP29, was heavily labeled. The
two-dimensional analysis allowed a sharp distinction between CP34 and
the phosphorylated form of the D2 apoprotein, which comigrate in
one-dimensional PAGE.
Figure 1:
Polypeptide profile and immunoblot of
thylakoid membranes from control ( C), cold-treated
( T), and recovered at room temperature for 1 h ( RT)
maize leaves. Coomassie-stained SDS-PAGE ( A) and immunoblot
assayed with -CP29 polyclonal antibodies ( B) are shown;
the chlorophyll a/b binding protein CP29 and the cold-induced
34-kDa band are indicated, together with some other major
bands.
In the autoradiograph, all of the known major
thylakoid phosphoproteins
(31, 32) could be identified:
the light-harvesting chlorophyll a/b proteins (three between
28 and 30 kDa)
(8) and the four proteins of the PSII core CP43
(43 kDa), D2 (34 kDa), D1 (32 kDa), and the 9-kDa product of the
psbH gene (not visible in the figure but evident at the lower
edge of the gel in the original film). Apart from CP34, the
autoradiographic pattern of control and cold-treated samples also
differed with respect to the relative intensity of the labeling of the
other subunits; LHCII apoproteins were more heavily phosphorylated in
the cold-treated samples by a factor of 2.5, as determined by
densitometry of the film. Moreover, labeling of the PSII core subunits
CP43, D1, D2, and 9 kDa was barely detectable in the control sample,
while it was very strong in the cold-treated one. It is worth noting
that only the PSII core subunits deriving from the monomeric form of
the complex are phosphorylated, while those associated to the dimeric
form are not.
Limited Proteolysis of CP29 and CP34 in Destacked
Thylakoids
Which is the effect of the phosphorylation on
CP29? Protein phosphorylation has been shown to be one of the most
general mechanisms in the regulation of protein function through
changes in their conformational state
(30) . To verify if this
is also the case for CP29, we analyzed the two proteins by limited
proteolysis at 37 and 25 °C. Control and cold-treated membranes
were partially solubilized with 0.1% DM and digested with chymotrypsin.
The results of the proteolytic treatment (identical at the two
temperatures) were analyzed by SDS-PAGE and immunoblotting (Fig. 4). It
was shown that the digestion produced different patterns in the two
samples; the smaller fragment detected (about 16 kDa) was more abundant
in the control membranes, while an additional 23-kDa band appeared in
the cold-treated sample containing CP34. This may indicate that at
least one proteolytic site was protected in the phosphorylated form of
the protein due to a conformational change induced by the
phosphorylation. Similar results, in fact, were obtained with other
endoproteinases cutting at different sites (not shown), ruling out the
possibility that the added phosphate group could sterically constrain
proteolysis at a nearby target site.
In Vitro Dephosphorylation of CP34
As
shown above, the phosphorylation of CP29 is accompanied by a change of
its mobility in SDS-urea-PAGE possibly due to changes in the secondary
structure of the polypeptide in the gel, owing to the addition of the
highly charged phosphate group. It can be asked whether the change of
electrophoretic mobility is exclusively due to phosphorylation or if
other modifications such as aggregation with surrounding small peptides
are involved, similar to the case of D1 and the cytochrome
b-subunits
(33) .
. Accordingly, the
treatment of BBY particles was ineffective. These results, shown in
Fig. 5B, indicate that the phosphorylation site is most
probably exposed on the stromal side of the membrane in the stacked
domain.
Figure 5:In vitro dephosphorylation of
CP34. Immunoblot analysis with -CP29 polyclonals of thylakoid
membrane samples from cold-treated leaves, after in vitro dephosphorylation treatment is shown. A, membranes were
suspended in Tris buffer at the indicated pH values and incubated with
alkaline phosphatase at 30 °C for different times, in the absence
or presence ( +DM) of 0.1% dodecyl maltoside. B,
EDTA-washed thylakoids or PSII membranes were incubated for 20 min with
Mg
, alkaline phosphatase ( AP), or both (in
these cases, the first component was added, the sample was kept for 5
min on ice, and the incubation was started as the second component was
mixed). Control membranes, not treated and kept for 20 min at 30
°C, are indicated by C.
Identification of the Site Controlling CP29
Phosphorylation
Previous work on thylakoid kinases showed
that the activity of the LHCII-specific enzyme is controlled by the
redox state of the plastoquinone pool, thus acting in the regulation of
the electron transport between PSII and PSI
(1) . Activation of
the kinase appears to be dependent on the formation of a complex with
the cytochrome b /f (34, 35) . We tested the effect of blocking the
photosynthetic electron transport chain at different sites. This was
performed by using a mutated line and treatments with chemical
inhibitors.
, the terminal electron acceptor of
photosynthesis; seedlings were illuminated in N
atmosphere
for 10 min at room temperature. Thylakoids were then isolated and
analyzed by SDS-PAGE and immunoblotted with
-CP29 polyclonals,
showing that the phosphorylated form of CP29 was induced in those
conditions even without cold treatment (Fig. 6, panel A, lane c). This suggests that
phosphorylation is activated by the reduction of a component of the
electron transport chain. Further insights were obtained by comparing
the wild type with hcf2 mutant seedlings lacking cytochrome
b
/f (36) . In this mutant, CP34
was present following illumination, even at room temperature ( lanes f and g). 20 µ
M DCMU was used to
block electron transport between PSII reaction center and
plastoquinone. This treatment completely prevented CP29 phosphorylation
in both wild type and hcf2 mutant whether at 4 °C or room
temperature ( lanes d, h, and i).
These results suggest that the CP29 phosphorylation is controlled by
the redox state of plastoquinone.
Fluorescence Analysis
Which is the role
of cold-induced CP29 phosphorylation? A possible function would be
protection against photoinhibition. We compared the sensitivity to
photoinhibition of maize lines differing for their ability to
phosphorylate CP29 following cold stress in the light. Several
available lines were screened by exposing seedlings to the
photoinhibitory treatment, followed by SDS-PAGE and immunoblot analysis
(not shown) or fluorescence analysis. For example, the two lines
H93 and Oh7N differed substantially in the level of
CP34 after the cold treatment. In H93, CP34 represented 9% of
the sum (CP29 + CP34), while in Oh7N CP34 represented
33%. As shown in Fig. 7, under high light (1000 µE s
) at 4 °C, the
F
/ F
fluorescence ratio decreased rapidly in the line H93 (from 0.75 to 0.48 in 6 h), mainly due to a rapid decrease of
F
. The decrease in
F
/ F
was
much smaller in Oh7N, from 0.79 to only 0.68, even after 8 h
of treatment. This shows that the photochemistry of PSII in the line
Oh7N was much better protected under stress conditions than in
the line H93. The recovery of the
F
/ F
ratio
after 15 h at room temperature confirms that the PSII reaction centers
were more irreversibly photoinhibited at 4 °C in H93 than
Oh7N. Moreover, the resistant line Oh7N has a
capacity to recover from the stress, which is almost absent in
H93. This overall sensitivity of the photochemistry to cold
stress was observed in several lines and was correlated with their
ability to phosphorylate CP29, suggesting that this modification of the
protein could be involved in a photoprotective mechanism. Although
fluorescence results must be cautiously compared between different
leaves, we are confident that the differences in fluorescence induction
values of the two maize lines are significant. In fact, the same figure
was obtained for 14-, 18-, and 21-day-old plants while, for each age,
chlorophyll content and deepoxidation state of xanthophyll at the end
of the treatment showed very small differences, if any (within 3 and
2%, respectively).
isolated the two com-plexes in their native forms and showed that
the spectral properties and the pigment composition were very similar.
We further characterized CP29 and CP34 by amino acid sequencing,
showing that the two polypeptides, homologous to the barley CP29, were
identical over the 32- and 20-amino acid-long stretches determined.
This strongly suggested that CP34 was not the product of a different
gene but rather of an alternative splicing of the lhcb4 messenger, the pre-cursor of CP29, or a post-translational
modification of it.
S labeling experiment
discriminated between these hypotheses since the synthesis of CP34 was
not enhanced, as would be the case if it was either the product of
alternative splicing or the precursor complex. We thus concluded that
CP34 is a post-translationally modified form of CP29 and proceeded to
the identification of the nature of the modification. This was made
difficult by the fact that only in vivo treatment is effective
in inducing CP34 accumulation while treatment of isolated thylakoids is
not. As suggested by the rapid formation and degradation of the
protein, we succeeded in labeling CP34 by feeding H333PO
to
the plant prior to treatment in the light. Not only was CP34, in
contrast to CP29, heavily labeled, but the treatment of the membranes
with alkaline phosphatase restored CP29 mobility, thus confirming that
the post-translational modification of CP29 was a phosphorylation.
/f complex.
/f complex, while the
phosphorylation of the four PSII core proteins is largely unaffected
(37, 39) . We noticed that the phospho-CP29 (CP34) was
constitutively present in the thylakoid membranes of this mutant. A
pretreatment with DCMU, which prevents all known thylakoid protein
phosphorylation by blocking the reduction of plastoquinone
(40) , totally abolished phosphorylation of CP29 in both wild
type and hcf2 lines even when chilling treatment was applied.
The sensitivity of the CP29 kinase to inhibition by DCMU points to a
redox control at the level of plastoquinone, with plastoquinol being
the activator of the enzyme. However, in contrast to the LHCII kinase,
referred to as the 64-kDa kinase, and likewise the putative RC kinase
of PSII core proteins, the activation of the CP29 kinase does not
require an active cytochrome b
/f complex.
/f complex, which modulates its activity
(34) . In the mutant
lacking this complex, we noticed that the CP29 kinase is constitutively
activated, but it does retain its redox sensitivity since DCMU,
i.e. oxidation of plastoquinol to plastoquinone, turns it off.
site of PSII.
On the other hand, the activation by DBMIB suggests a negative control
played by the Q
site of the cytochrome
b
/f, to which DBMIB binds in wild type
(43) , displacing the quinone on the cytochrome complex and thus
mimicking the hcf2 condition. We propose as a working
hypothesis that the CP29 protein kinase is located in grana partitions,
in between PSII (where it is in contact with the
Q
site) and the cytochrome
b
/f complex (in contact with the
Q
site). This would also mean an
intermediate position between the RC kinase and the LHCII kinase,
respectively. If we consider the models so far proposed for the
topological organization of photosystem II
(21, 44) ,
our hypothesis is consistent with the position of CP29 between the RC
and LHCII, which are substrates of phosphorylation by the different
kinase (Fig. 8). An integrated scheme for the redox control of
thylakoid protein phosphorylation cannot be drawn as yet and is beyond
the aim of this work.
In that study, it was also shown that the
protective effect was located in the antenna system rather than in the
RC complex and consisted in a decreased efficiency in the energy
transfer from the antennas to the RC. CP29 phosphorylation isolates an
important portion (30%) of PSII antennas, which are connected to the RC
through CP29
(32, 45) , and may influence chlorophyll
and/or carotenoid organization within the complex, thus leading to a
decreased energy transfer to PSII RC. While the disconnection of PSII
chlorophyll proteins seems to be a general consequence of
photoinibitory conditions
(47) irrespective of CP29
phosphorylation, spectral differences were actually detected between
CP29 and CP34,
with a decrease in the absorption of the
Chla spectral forms, which are more effective in the energy transfer to
PSII RC. This hypothesis is consistent with the observed increase in
F
during the treatment in the cold
(18) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.