Phosphorylation of the D1 Photosystem II Reaction

Center Protein Is Controlled by an Endogenous

Circadian Rhythm1

Isabelle S. Booij-James, W. Mark Swegle, Marvin Edelman, and Autar K. Mattoo*

Vegetable Laboratory, The Henry A. Wallace Beltsville Agricultural Research Center-West, United States
Department of Agriculture-Agricultural Research Service, Beltsville, Maryland 20705–2350 (I.S.B.-J., M.S.,
A.K.M.); and Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel (M.E.)

The light dependence of D1 phosphorylation is unique to higher plants, being constitutive in cyanobacteria and algae. In a
photoautotrophic higher plant, Spirodela oligorrhiza, grown in greenhouse conditions under natural diurnal cycles of solar
irradiation, the ratio of phosphorylated versus total D1 protein (D1-P index: [D1-P]/[D1] ⫹ [D1-P]) of photosystem II is
shown to undergo reproducible diurnal oscillation. These oscillations were clearly out of phase with the period of maximum
in light intensity. The timing of the D1-P index maximum was not affected by changes in temperature, the amount of D1
kinase activity present in the thylakoid membranes, the rate of D1 protein synthesis, or photoinhibition. However, when the
dark period in a normal diurnal cycle was cut short artificially by transferring plants to continuous light conditions, the D1-P
index timing shifted and reached a maximum within 4 to 5 h of light illumination. The resultant diurnal oscillation persisted
for at least two cycles in continuous light, suggesting that the rhythm is endogenous (circadian) and is entrained by an
external signal.

Photosynthetic oxygen evolution involves a su-
pramolecular protein-pigment complex, PSII (Ort
and Yocum, 1996; Mattoo et al., 1999). The PSII reac-
tion center, which includes the D1 and D2 protein
heterodimer, binds most of the nonprotein compo-
nents of the PSII electron transport chain (Nanba and
Satoh, 1987; Michel and Deisenhofer, 1988; Mattoo et
al., 1989; Hankamer et al., 1997). Light is central to
the metabolism of the D1 protein, regulating its syn-
thesis (Mattoo et al., 1984), intramembrane transloca-
tion (Mattoo and Edelman, 1987; Callahan et al.,
1990), posttranslational phosphorylation (Michel et
al., 1988; Elich et al., 1992) and acylation (Mattoo and
Edelman, 1987; Mattoo et al., 1993), and its rate of
degradation (Mattoo et al., 1984; Greenberg et al.,
1987; Aro et al., 1993). The posttranslational phos-
phorylation of D1 occurs at its N-terminal Thr resi-
due, catalyzed by a light-dependent redox-regulated
kinase (Michel et al., 1988; Elich et al., 1992).
Protein phosphorylation is a mechanism used by
eukaryotes to regulate cellular activity (Stone and
Walker, 1995). In plants, protein phosphorylation is a
key response to environmental signals such as
wounding (Usami et al., 1995) and light (Allen, 1992).
The greatest concentration of phosphoproteins in
1
This work was supported in part by the Avron-Wilststter
Minerva Center for Research in Photosynthesis (to M.E.).
* Corresponding author; e-mail mattooa@ba.ars.usda.gov; fax
301–504 –5555.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.013441.
Plant Physiology, December 2002, Vol. 130, pp. 2069–2075, www.plantphysiol.org © 2002 American Society of Plant Biologists
plants is found in the chloroplast membranes (Ben-
nett, 1991). Phosphorylation and dephosphorylation
of the D1 protein are strictly light dependent (Elich et
al., 1993, 1997). Reversible, redox-sensitive phosphor-
ylation of the light-harvesting chlorophyll apopro-
tein is thought to be a mechanism maximizing quan-
tum yield by equalizing electron flow through PSII
and PSI (Allen, 1992); however, the role of phosphor-
ylation of D1 or other PSII proteins is largely un-
known. It has variously been suggested that phos-
phorylation regulates D1 degradation, maintaining it
as a storage form prior to its replacement (Rintamäki
et al., 1995a), or that it regulates dimerization of the
reaction center (Santini et al., 1994).
The effect of light intensity on D1 metabolism has,
without exception, been studied under unnatural
conditions by illuminating plants or thylakoid mem-
branes with radiance of constant intensity. There is
usually a pretreatment of complete darkness or
exposure to a light intensity different from that
ultimately to be used. The intensity of the constant
radiation not only influences the rate of D1 degrada-
tion, but also the ratio of phosphorylated to unphos-
phorylated D1 (Elich et al., 1992; Rintamäki et al.,
1995a). To gain insight into the physiological func-
tion of phosphorylation of D1, we characterized this
process under a natural diurnal cycle, i.e. with natu-
rally fluctuating solar intensity. Antibodies were
raised against synthetic peptides, which selectively
detect each form of D1, phosphorylated and unphos-
phorylated, and allow measurement of the propor-
tion of D1 that is phosphorylated under specific
conditions.
2069
Booij-James et al.
We show here that Spirodela oligorrhiza plants, en-
trained to the natural light/dark cycle in a green-
house, exhibit diurnal oscillation of the ratio of phos-
phorylated to total D1 protein (D1-P index: [D1-P]/
[D1] ⫹ [D1-P]), which is paralleled by de novo D1
phosphorylation in vivo. When plants were shifted
from a light/dark cycle to continuous light condi-
tions, the D1-P index rhythm was maintained for
several cycles. These results show circadian regula-
tion of the phosphorylation of D1, a key PSII reaction
center protein of the chloroplast membranes.
RESULTS
Immunodifferentiation of Phosphorylated and
Non-Phosphorylated D1
In vivo light-dependent phosphorylation of D1
protein is transient and increases with increasing
light intensity (Elich et al., 1992; Rintamaki et al.,
1995a). The light (redox) dependence can be repro-
duced in vitro by incubating isolated thylakoids in
the dark under redox-generating conditions using
ferredoxin and reducing power (Elich et al., 1992,
1993). Thylakoid membranes, extracted from S. oli-
gorrhiza plants that had been held in the dark for 3 d
to allow for protein dephosphorylation, were phos-
phorylated in vitro. Affinity-purified antibodies
(anti-SP1 and anti-SP2) were tested for their abilities
to detect phosphorylated and unphosphorylated D1
(Fig. 1). Dark incubation of thylakoids with ATP,
NADPH, and ferredoxin resulted in the progressive
phosphorylation of D1 as the time of incubation in-
creased. A distinct separation into two D1 forms,
identified as phosphorylated (D1-P) and unphos-
phorylated D1 (Elich et al., 1992), was obtained. Anti-
SP2 recognizes both forms of D1, whereas anti-SP1
recognizes only the unphosphorylated form (Fig. 1B).
Nonrecognition of D1-P by anti-SP1 suggests that the
phosphorylated form of the N-terminal TAILERR. . .
region assumes a more structured, protected confor-
mation than the unphosphorylated form. Such con-
formational changes upon phosphorylation are well
documented (Barford et al., 1991) and have been
evoked for chlorophyll proteins sp29 (Croce et al.,
1996) and light-harvesting chlorophyll apoprotein
(Nilsson et al., 1997).
Plants rapidly dephosphorylate D1-P in the light in
the presence of DCMU, which inhibits D1 kinase
activity (Elich et al., 1993, 1997). Under this condi-
tion, the ratio of unphosphorylated to phosphory-
lated D1 should increase. Such was the case when
immunoblots of the DCMU-treated samples were
probed with anti-SP2 and anti-SP1 (Fig. 1C,
⫹DCMU). In a converse manner, under conditions
where phosphorylation is inhibited by DCMU and
D1-P dephosphorylation is inhibited by NaF (an in-
hibitor of phosphatase), D1 and D1-P levels should
remain unchanged. This was the case, as shown in a
separate experiment with anti-SP2 (Fig. 1C,
2070
Figure 1. A, Amino acid sequences and location along the protein
chain of the synthetic peptides used to produce D1 antibodies. Cys
(C) residue at the C terminus of SP1 and N terminus of SP2 is not
present in the native sequence. B, In vitro phosphorylation of D1 in
thylakoids isolated from S. oligorrhiza plants that had been held in
the dark for 3 d. After SDS-PAGE of duplicate samples on a single gel,
proteins were electrotransferred to a nitrocellulose membrane, which
was later cut in half and developed with anti-SP2 and anti-SP1
antibodies. Thylakoid protein phosphorylation in the dark was car-
ried out in reaction mixtures primed to generate redox conditions
using ferredoxin, ATP, and NADPH as detailed by Elich et al. (1992,
1993) and described in the text. Time of phosphorylation in minutes
is indicated. The aligned blots show that the bottom band is unphos-
phorylated D1. C, Plants were incubated, for the times indicated, in
the light in the absence (lane 0) or presence of 10 ␮M 3-(3,4-
dichlorophenyl)-1,1-dimethylurea (DCMU) to inhibit phosphoryla-
tion, and, in a separate experiment, in 10 mM NaF, which inhibits D1
dephosphorylation (Elich et al., 1993). Thylakoids were isolated and
analyzed by SDS-PAGE and immunodecorated with anti-SP2 or anti-
SP1 antibody as indicated.
⫹DCMU⫹NaF). These observations confirm the
specificity of the antibodies and the identification of
the upper and lower immunoreactive bands in Figure
1, B and C, as D1-P and unphosphorylated D1, re-
spectively. These results are consistent with previous
conclusions (Elich et al., 1992, 1993).
Diurnal Oscillations of the D1-P Index
Thylakoid samples isolated from S. oligorrhiza
plants grown in the greenhouse under natural diur-
nal cycles of solar irradiation were immunoblotted
and analyzed for D1 and D1-P. The data in Figure 2
are plotted as the D1-P index, which is the apparent
percentage of D1 in the phosphorylated form, versus
time over three light/dark cycles. Reproducible os-
cillations were obtained in the D1-P index, which
were clearly out of phase with the period of maxi-
mum radiation (Figs. 2A, 3, and 4). Thus, light inten-
sity per se does not directly correlate with the ratio of
phosphorylated versus total D1.
The D1-P index maximum consistently preceded
the peak of maximum light intensity (Fig. 2A). This
24-h oscillation was observed on three consecutive
days and on all the additional occasions when the
experiment was repeated (Figs. 3 and 4). During
these experiments, the temperature in the green-
house was kept constant (at 13°C, 20°C, or 26°C; Fig.
3B presents data with plants maintained at 26°C) or
Plant Physiol. Vol. 130, 2002

Figure 2. Rhythmic behavior of the level of phosphorylated D1 in S.
oligorrhiza under greenhouse conditions (A). The light intensity (bro-
ken lines) and the D1-P index are shown at indicated times over three
day/night cycles (f). At the end of the light period and 2 h into
darkness, a set of plants was exposed for 5 min to 300 ␮mol m⫺2 s⫺1
fluorescent light (arrow) and thereafter returned to darkness (䡺). Error
bars indicate SEs based on a sample size of four. B, D1-P index
maintains oscillations in free-running conditions in continuous light.
Plants were grown in the greenhouse under natural light/dark cycles
for a week in medium lacking Suc. Then, at the end of the light cycle
and 2 h into darkness, a set of plants was left in the greenhouse until
the end of the experiment (arrow), whereas another was brought into
the laboratory and incubated in continuous light at 200 ␮mol m⫺2
s⫺1 until the end of the experiment. Error bars indicate SEs based on
a sample size of three.
was allowed to rise and fall with changing sunlight
intensity (Fig. 3A). The oscillations and D1-P index
pattern seen in Figure 2A were indifferent to temper-
ature within the range tested (Fig. 3, A versus B).
These data show that the phase of the rhythm as
entrained to the light/dark cycle is not much affected
by different temperatures.
At the end of the light period and 2 h in the dark
(early night), some plants were moved to continuous
light conditions (artificial fluorescent light, 200 ␮mol
m⫺2 s⫺1; Fig. 2B) or were given a 5-min light pulse
(indicated by an arrow in the black bar in the middle
of Fig. 2A). The clock was rapidly reset in plants
where the dark period was interrupted with contin-
uous light; the cycle was shifted, with the D1-P index
peak occurring 4 h after switching the plants to con-
Plant Physiol. Vol. 130, 2002
D1 Phosphorylation Is Controlled by a Circadian Rhythm
tinuous light (Fig. 2B). However, a 5-min light pulse
(300 ␮mol m⫺2 s⫺1) in the dark period was not suf-
ficient to entrain the clock (Fig. 2A, open rectangles).
The results were independent of the light intensity
used for the pulse (data not shown). In plants subse-
quently kept in continuous light (200 ␮mol m⫺2 s⫺1),
the free-running period of 24-h oscillation was main-
tained (Fig. 2B, days 2 and 3). The shifted peaks were
lower in amplitude than that observed in a natural
light/dark cycle. Such damping has been seen upon
imposition of similar conditions in other biological
systems (Hennessey et al., 1993).
In Vivo D1 Phosphorylation Mirrors D1-P
Index Oscillations
The preceding experiments were based on immu-
nological separation of D1 and D1-P to measure the
D1-P index. To determine if these steady-state pat-
terns reflected de novo phosphorylation, the in vivo
patterns of D1 synthesis and phosphorylation were
determined through the diurnal cycle using [35S]Met
and [32P]orthophosphate, respectively. The pattern
for D1 phosphorylation mirrored that for the D1-P
index, both peaking 4 h into the light phase (at 10
am). In contrast to its phosphorylation, D1 continued
to be synthesized as the day progressed, peaking
after about 10 h of the light phase (at about 4 pm), and
well beyond the maximum in light intensity (Fig. 4,
high light). The steady-state level of D1 as quantified
by immunoblot analysis of these samples did not
show any major alterations.
Similar patterns and identical peak times were ob-
tained on a cloudy day, with maximum greenhouse
illumination at 200 ␮mol m⫺2 s⫺1 and peaks for D1
phosphorylation, D1-P index and D1 synthesis all
Figure 3. Temperature independence of D1-P index in S. oligorrhiza
plants analyzed over a day/night cycle. A, Data from experiment
where the temperature was not controlled. B, Data from plants
maintained at a constant temperature of 26°C. Light intensity at the
indicated times is shown in micromoles per meter per second. Error
bars indicate SEs based on a sample size of four.
2071
Booij-James et al.
Figure 4. D1-P index (f) parallels in vivo labeling of D1 with
[32P]orthophosphate (F), but is independent of de novo D1 synthesis
(E)—quantified by isotope incorporation into D1 (percentage of max-
imum)—and the daylight maximum (broken lines). Light intensity at
the indicated times is shown in micromoles per meter per second.
Data points were averaged from four independent experiments. High
light (on the left) indicates experiments done on a sunny day. Low light
(on the right) indicates experiments done on a cloudy day.
occurring at 100 ⫾ 25 ␮mol m⫺2 s⫺1 (Fig. 4, low
light). These intensities are well below the initiation
point for photoinhibition in S. oligorrhiza (⬎500 ␮mol
m⫺2 s⫺1; Jansen et al., 1996).
DISCUSSION
We demonstrate that beyond the known redox reg-
ulation of phosphorylation of the D1 photosystem II
reaction center protein (Elich et al., 1992; Silverstein
et al., 1993), the overriding control is exerted by an
internal, circadian clock. Diurnal rhythm of D1 phos-
phorylation follows parameters that are fundamental
to circadian rhythms (Golden et al., 1997; Dunlap,
1998; McClung, 2001), i.e. it has a 24-h periodicity
and can be entrained. In most circadian systems, light
is a primary signal for entraining rhythmic activity to
the daily light/dark cycle by resetting the phase of
the clock without altering the cycle length (Feldman,
1982; Wilkins, 1992; Anderson and Kay, 1996). This
appears true for the D1-P index as well. The clock
was reset by interrupting the dark period with con-
tinuous light of ⱖ200 ␮mol m⫺2 s⫺1. Therefore, it
appears that light resets the phase and then acts in
concert with the clock to regulate D1 phosphoryla-
tion in vivo. The nonpersistence of rhythmic behav-
ior in the D1-P index in total darkness is likely due to
the fact that light is an absolute requirement for most
chloroplast activities (Ort and Yocum, 1996), in par-
ticular D1 dynamics (Mattoo et al., 1999).
2072
Circadian regulation of a kinase that phosphory-
lates phosphoenolpyruvate carboxylase in the dark is
well documented (Nimmo, 1998) and is exerted at the
level of protein abundance linked to transcript accu-
mulation (Hartwell et al., 1999). In our case, oscilla-
tions in D1 phosphorylation in vivo were not mir-
rored by in vitro D1-peptide kinase activity, which
remained nearly constant during the course of the
day (data not shown), the rate of D1 protein synthe-
sis, which continued to rise till late in the afternoon,
or photoinhibition. Thus, the oscillations are indica-
tive of an endogenous regulator controlling D1 me-
tabolism and function.
Circadian control of D1 phosphorylation adds an-
other dimension to the as yet unsolved role of light-
dependent D1 protein turnover or D1 phosphoryla-
tion in chloroplast function. D1 phosphorylation has
been suggested to be a means of turning off PSII, or
rerouting electron transport, to protect the photosys-
tem from damage caused by high light intensity
(Rintamäki et al., 1995a). However, we show here
that the greatest amount of phosphorylation occurs
hours before maximal light intensity, and at light
intensities well below those saturating for photosyn-
thesis (Jansen et al., 1996) or initiation of photoinhi-
bition (Jansen et al., 1999). We note here that the rate
of D1 degradation is least in the dark but increases
during the day with increase in light intensity (Mat-
too et al., 1984; Jansen et al., 1999). It is possible that
circadian oscillations in D1 phosphorylation are part
of a regulatory system controlling the operation of
the photosynthetic apparatus, as well as a signal to
alter the metabolism of the D1 protein. Riesselmann
and Piechulla (1992) suggested that proteins dam-
aged during the light cycle might be substituted at
the very beginning of the light period of the follow-
ing day. If this mechanism were under the control of
an endogenous, circadian rhythm, it would enable
restoration of thylakoid membrane protein com-
plexes early in the morning to allow optimal photo-
synthetic reactions during the day.
The protein kinase that phosphorylates D1 is local-
ized in the chloroplast membranes (Elich et al., 1992),
but is likely encoded by a nuclear gene because no
open reading frame for a kinase-like gene has been
identified in the chloroplast genome (Sugiura, 1992).
The endogenous rhythm in D1 protein phosphoryla-
tion does not reflect the quantity of the kinase activ-
ity measured in thylakoids. It is possible that activa-
tion (plastoquinone redox) state of the D1 kinase
(Elich et al., 1992) or the phosphatase (Elich et al.,
1993; Vener et al., 1999) may be involved. However,
whether it is a consequence of redox versus direct
regulation of the kinase, the outcome still is clock
regulation of D1 phosphorylation. The circadian reg-
ulation of the expression of a possibly nuclear D1
kinase gene, or its gene product, could provide a
crosstalk mechanism by which a biological clock in
the nucleus uses a nuclear-encoded protein kinase to
Plant Physiol. Vol. 130, 2002

regulate chloroplast function. This is, perhaps, a
means for the chloroplast to anticipate environmental
changes. Light intensities supersaturating for photo-
synthesis cause deleterious effects in the chloroplast;
therefore, there is a need for a sensing mechanism to
down-regulate PSII. It is not known how the chloro-
plast achieves this. Does it use the D1-P index as a
sensor to anticipate the onset of higher light intensi-
ties (Mattoo and Edelman, 1985)? Is the phosphory-
lation state of PSII reaction core proteins a conse-
quence or a determinant of the relative energy
distribution between the two photosystems in oxy-
genic photosynthesis? Because radiance-dependent
D1 turnover is a fact of life for PSII and because
phototrophs are normally subjected to a daily light/
dark cycle, circadian regulation of D1 metabolism is
not unexpected. However, how regulation is achieved
may differ for different photosynthetic forms. In
higher plants, where D1 is reversibly phosphorylated,
circadian regulation of metabolism can be at the phos-
phorylation level, as is suggested here for S. oligor-
rhiza. In cyanobacteria, redox-regulated phosphoryla-
tion of D1 does not appear to occur, but these oxygenic
bacterial phototrophs often possess multiple copies of
the psbA gene coding for D1 (Golden, 1995; Chen et
al., 1999), with different D1 isoforms adapted in vivo
to varying photon irradiation, one dominant at lower
and another at higher light intensities (Bustos et al.,
1990; Clarke et al., 1993; Kulkarni and Golden, 1994).
Several studies have shown that light-induced tran-
scription of cyanobacterial psbA occurs within the
larger framework of circadian control (Liu et al., 1995;
Chen et al., 1999). Thus, a generalized hypothesis can
be forwarded that reversible phosphorylation of D1
(and maybe, other phosphorylated PSII proteins) in
higher plants evolutionarily replaced multiple DNA
copies in cyanobacteria as a more energy-efficient sub-
strate for circadian clock regulation of PSII core
metabolism.
The hypothesis can be investigated phylogeneti-
cally by viewing atypical species. Prochlorococcus ma-
rinus, a ubiquitous, free-living marine cyanobacte-
rium, is unusual in that it contains chlorophyll a, b,
and c, but no phycobilisomes. In addition, it has only
a single psbA gene and one type of D1 protein
(Garcia-Fernandez et al., 1998). It will be interesting
to determine if D1 can be phosphorylated in this
organism. The state of D1 phosphorylation in lower
plants and algae also bears further investigation. The
D1 protein of the moss Ceratodon purpureus is re-
ported as not being phosphorylated under high irra-
diance in vivo or under in vitro conditions (Rinta-
maki et al., 1995b). However, information on gene
copy number and regulation of D1 metabolism in this
model lower plant organism is currently lacking. The
literature for Chlamydomonas reinhardtii, although ex-
tensive, is surprisingly ambiguous concerning phos-
phorylation of the D1 and D2 proteins. The most
recent investigation suggests that neither of these
Plant Physiol. Vol. 130, 2002
D1 Phosphorylation Is Controlled by a Circadian Rhythm
PSII core proteins is phosphorylated in this green
alga (Andronis et al., 1998). The psbA gene of C.
reinhardtii maps to the edge of the inverted repeat
region; thus, there are two identical copies of the
gene and a single type of D1 protein. Transcription of
chloroplast-encoded genes, including psbA, is con-
trolled by a nuclear-regulated circadian clock in C.
reinhardtii (Hwang et al., 1996; Kawazoe et al., 2000),
which would be predicted by our hypothesis above.
The isolation of a D1 kinase gene and elucidation of
its role could be a step toward understanding the role
of D1 phosphorylation and its regulative mecha-
nisms in higher plants. More than one kinase is
clearly involved with phosphorylation of PSII pro-
teins (Elich et al., 1997). We have purified and cloned
a S. oligorrhiza kinase that phosphorylates a synthetic
peptide mimicking the D1 protein in a calcium-
dependent manner (A. Raskind, M. Swegle, I. Booij-
James, V. Kumar, M. Edelman, and A. Mattoo, un-
published data). However, it is yet to be ascertained
if this is the protein kinase that phosphorylates D1 in
vivo. One approach being followed is the antisense
RNA technology to silence this protein and check if
the knockout transformants phosphorylate D1.
MATERIALS AND METHODS
Plant Material and Thylakoid Membrane Preparation
Spirodela oligorrhiza plants were maintained as described (Elich et al.,
1992) at 25°C in one-half strength Huntner’s medium (Posner, 1967) con-
taining 0.5% (w/v) Suc under 25 ␮mol m⫺2 s⫺1 cool-white fluorescent light.
Plants were transferred to medium lacking Suc for at least 48 h before each
experiment. Thylakoids were prepared according to published methods
(Elich et al., 1992). The final thylakoid pellet was suspended in a small
volume of buffer A (10 mm Tricine-NaOH, pH 7.8, 100 mm sorbitol, 10 mm
MgCl2, and 10 mm NaCl) such that the chlorophyll concentration was
greater than 250 ␮g mL⫺1. Chlorophyll concentrations were determined in
80% (w/v) acetone (Arnon, 1949).
In Vitro Phosphorylation
In vitro phosphorylation of S. oligorrhiza thylakoids was carried out in the
dark essentially as described (Elich et al., 1992). Thylakoids were diluted to
a chlorophyll concentration of 200 ␮g mL⫺1 in buffer A containing 10 mm
NaF, 0.5 mm NADPH, 3.5 ␮m ferredoxin, and 0.2 mm ATP. Reactions were
stopped at the indicated times by addition of 0.5 volumes of 3⫻ SDS sample
buffer (Mattoo et al., 1981).
Diurnal Oscillations Experiments
S. oligorrhiza plants were maintained on medium lacking Suc under
fluorescent lighting as above and were then shifted to the greenhouse at
Beltsville, MD for at least 3 d on the same medium before experiments were
started. Temperature and light intensity were recorded at 1- or 2-h intervals.
Samples (10–20 plants) were harvested in quadruplicate, immediately fro-
zen on dry ice, and then stored at ⫺80°C until thylakoids were isolated.
Three sets of samples were used for analysis of phosphorylated and un-
phosphorylated D1 by SDS-PAGE/immunoblotting, and one set for in vitro
kinase assays. For constant temperature experiments, plants were held in
temperature-controlled water baths at 15°C or 26°C. For cycle shift experi-
ments, 1-week-old greenhouse-grown plants were divided into five sets
and, at nightfall, were transferred in darkness to an indoor controlled
growth chamber where they were maintained in darkness for 1.5 h. One set
of plants was left in darkness for the duration of the experiment, and the
other four were exposed to 200 or 300 ␮mol m⫺2 s⫺1 fluorescent light in
2073
Booij-James et al.
controlled growth chambers. Two of the four sets were returned to darkness
following a 5-min light pulse, whereas the remaining two were left in
continuous light for the duration of the experiment.
In Vivo Pulse-Labeling Experiments
S. oligorrhiza plants were transferred for at least 2 d to medium lacking
Suc and phosphate and were maintained under fluorescent lighting as
above. Plants were then shifted to the greenhouse and were maintained at
constant temperature on the same medium for 2 d prior to the experiment.
Plants were pulse-labeled for 1 h with 0.5 mCi mL⫺1 of [32P]orthophosphate
(Elich et al., 1992) or 0.1 mCi mL⫺1 of [35S]Met (Mattoo et al., 1981). The
plants were subsequently washed three times with ice-cold water, collected
on dry ice, and stored at ⫺80°C until thylakoids were isolated.
Preparation of Antisera
Based on the archetype D1 sequence of spinach (Spinacia oleracea; Zuraw-
ski et al., 1982), two peptides corresponding to amino acids 1 through 17 and
57 through 85 of the mature protein were synthesized and designated SP1
and SP2, respectively (Fig. 1A). The SP2 peptide contains at its N terminus
a Cys added for conjugation purposes. These two synthetic peptides were
conjugated to bovine serum albumin through Cys residues, and the conju-
gates were used to immunize rabbits. The resulting antisera were purified
by immunoaffinity using Sulfolink columns (Pierce, Rockford, IL) to which
the individual peptides had been conjugated.
Electrophoresis and Immunoblots
Thylakoid proteins were solubilized in 1⫻ SDS sample buffer for 1 h at
room temperature and were separated by SDS-PAGE on 10% to 20% (w/v)
acrylamide gradient gels (Elich et al., 1992). The samples were loaded on an
equal chlorophyll basis (1 ␮g of chlorophyll⫺1 lane). The gels were stained
with Coomassie Blue R-250 or were electrotransferred to nitrocellulose
membranes for at least 5 h at 0.2 mA. The blots were immunodecorated with
immunoaffinity-purified anti-SP1 or anti-SP2 as primary antibodies, and an
alkaline phosphatase-conjugate as secondary antibody (Pierce).
ACKNOWLEDGMENTS
We thank Dr. Tedd Elich for preparing and testing the antibodies, his
input into Figure 1, and many discussions. We also thank Prof. Susan
Golden for constructive comments.
Received August 21, 2002; returned for revision September 13, 2002; ac-
cepted September 19, 2002.
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