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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- plants is found in the chloroplast membranes (Ben-
pramolecular protein-pigment complex, PSII (Ort nett, 1991). Phosphorylation and dephosphorylation
and Yocum, 1996; Mattoo et al., 1999). The PSII reac- of the D1 protein are strictly light dependent (Elich et
tion center, which includes the D1 and D2 protein al., 1993, 1997). Reversible, redox-sensitive phosphor-
heterodimer, binds most of the nonprotein compo- ylation of the light-harvesting chlorophyll apopro-
nents of the PSII electron transport chain (Nanba and tein is thought to be a mechanism maximizing quan-
Satoh, 1987; Michel and Deisenhofer, 1988; Mattoo et tum yield by equalizing electron flow through PSII
al., 1989; Hankamer et al., 1997). Light is central to and PSI (Allen, 1992); however, the role of phosphor-
the metabolism of the D1 protein, regulating its syn- ylation of D1 or other PSII proteins is largely un-
thesis (Mattoo et al., 1984), intramembrane transloca- known. It has variously been suggested that phos-
tion (Mattoo and Edelman, 1987; Callahan et al., phorylation regulates D1 degradation, maintaining it
1990), posttranslational phosphorylation (Michel et as a storage form prior to its replacement (Rintamäki
al., 1988; Elich et al., 1992) and acylation (Mattoo and et al., 1995a), or that it regulates dimerization of the
Edelman, 1987; Mattoo et al., 1993), and its rate of reaction center (Santini et al., 1994).
degradation (Mattoo et al., 1984; Greenberg et al., The effect of light intensity on D1 metabolism has,
1987; Aro et al., 1993). The posttranslational phos- without exception, been studied under unnatural
phorylation of D1 occurs at its N-terminal Thr resi- conditions by illuminating plants or thylakoid mem-
branes with radiance of constant intensity. There is
due, catalyzed by a light-dependent redox-regulated
usually a pretreatment of complete darkness or
kinase (Michel et al., 1988; Elich et al., 1992).
exposure to a light intensity different from that
Protein phosphorylation is a mechanism used by
ultimately to be used. The intensity of the constant
eukaryotes to regulate cellular activity (Stone and radiation not only influences the rate of D1 degrada-
Walker, 1995). In plants, protein phosphorylation is a tion, but also the ratio of phosphorylated to unphos-
key response to environmental signals such as phorylated D1 (Elich et al., 1992; Rintamäki et al.,
wounding (Usami et al., 1995) and light (Allen, 1992). 1995a). To gain insight into the physiological func-
The greatest concentration of phosphoproteins in tion of phosphorylation of D1, we characterized this
process under a natural diurnal cycle, i.e. with natu-
1
rally fluctuating solar intensity. Antibodies were
This work was supported in part by the Avron-Wilststter
Minerva Center for Research in Photosynthesis (to M.E.).
raised against synthetic peptides, which selectively
* Corresponding author; e-mail mattooa@ba.ars.usda.gov; fax detect each form of D1, phosphorylated and unphos-
301–504 –5555. phorylated, and allow measurement of the propor-
Article, publication date, and citation information can be found tion of D1 that is phosphorylated under specific
at www.plantphysiol.org/cgi/doi/10.1104/pp.013441. conditions.
Plant Physiology, December 2002, Vol. 130, pp. 2069–2075, www.plantphysiol.org © 2002 American Society of Plant Biologists 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.
Figure 1. A, Amino acid sequences and location along the protein
chain of the synthetic peptides used to produce D1 antibodies. Cys
RESULTS (C) residue at the C terminus of SP1 and N terminus of SP2 is not
Immunodifferentiation of Phosphorylated and present in the native sequence. B, In vitro phosphorylation of D1 in
thylakoids isolated from S. oligorrhiza plants that had been held in
Non-Phosphorylated D1
the dark for 3 d. After SDS-PAGE of duplicate samples on a single gel,
In vivo light-dependent phosphorylation of D1 proteins were electrotransferred to a nitrocellulose membrane, which
protein is transient and increases with increasing was later cut in half and developed with anti-SP2 and anti-SP1
antibodies. Thylakoid protein phosphorylation in the dark was car-
light intensity (Elich et al., 1992; Rintamaki et al.,
ried out in reaction mixtures primed to generate redox conditions
1995a). The light (redox) dependence can be repro- using ferredoxin, ATP, and NADPH as detailed by Elich et al. (1992,
duced in vitro by incubating isolated thylakoids in 1993) and described in the text. Time of phosphorylation in minutes
the dark under redox-generating conditions using is indicated. The aligned blots show that the bottom band is unphos-
ferredoxin and reducing power (Elich et al., 1992, phorylated D1. C, Plants were incubated, for the times indicated, in
1993). Thylakoid membranes, extracted from S. oli- the light in the absence (lane 0) or presence of 10 ␮M 3-(3,4-
gorrhiza plants that had been held in the dark for 3 d dichlorophenyl)-1,1-dimethylurea (DCMU) to inhibit phosphoryla-
to allow for protein dephosphorylation, were phos- tion, and, in a separate experiment, in 10 mM NaF, which inhibits D1
phorylated in vitro. Affinity-purified antibodies dephosphorylation (Elich et al., 1993). Thylakoids were isolated and
(anti-SP1 and anti-SP2) were tested for their abilities analyzed by SDS-PAGE and immunodecorated with anti-SP2 or anti-
SP1 antibody as indicated.
to detect phosphorylated and unphosphorylated D1
(Fig. 1). Dark incubation of thylakoids with ATP,
NADPH, and ferredoxin resulted in the progressive ⫹DCMU⫹NaF). These observations confirm the
phosphorylation of D1 as the time of incubation in- specificity of the antibodies and the identification of
creased. A distinct separation into two D1 forms, the upper and lower immunoreactive bands in Figure
identified as phosphorylated (D1-P) and unphos- 1, B and C, as D1-P and unphosphorylated D1, re-
phorylated D1 (Elich et al., 1992), was obtained. Anti- spectively. These results are consistent with previous
SP2 recognizes both forms of D1, whereas anti-SP1 conclusions (Elich et al., 1992, 1993).
recognizes only the unphosphorylated form (Fig. 1B).
Nonrecognition of D1-P by anti-SP1 suggests that the Diurnal Oscillations of the D1-P Index
phosphorylated form of the N-terminal TAILERR. . .
region assumes a more structured, protected confor- Thylakoid samples isolated from S. oligorrhiza
mation than the unphosphorylated form. Such con- plants grown in the greenhouse under natural diur-
formational changes upon phosphorylation are well nal cycles of solar irradiation were immunoblotted
documented (Barford et al., 1991) and have been and analyzed for D1 and D1-P. The data in Figure 2
evoked for chlorophyll proteins sp29 (Croce et al., are plotted as the D1-P index, which is the apparent
1996) and light-harvesting chlorophyll apoprotein percentage of D1 in the phosphorylated form, versus
(Nilsson et al., 1997). time over three light/dark cycles. Reproducible os-
Plants rapidly dephosphorylate D1-P in the light in cillations were obtained in the D1-P index, which
the presence of DCMU, which inhibits D1 kinase were clearly out of phase with the period of maxi-
activity (Elich et al., 1993, 1997). Under this condi- mum radiation (Figs. 2A, 3, and 4). Thus, light inten-
tion, the ratio of unphosphorylated to phosphory- sity per se does not directly correlate with the ratio of
lated D1 should increase. Such was the case when phosphorylated versus total D1.
immunoblots of the DCMU-treated samples were The D1-P index maximum consistently preceded
probed with anti-SP2 and anti-SP1 (Fig. 1C, the peak of maximum light intensity (Fig. 2A). This
⫹DCMU). In a converse manner, under conditions 24-h oscillation was observed on three consecutive
where phosphorylation is inhibited by DCMU and days and on all the additional occasions when the
D1-P dephosphorylation is inhibited by NaF (an in- experiment was repeated (Figs. 3 and 4). During
hibitor of phosphatase), D1 and D1-P levels should these experiments, the temperature in the green-
remain unchanged. This was the case, as shown in a house was kept constant (at 13°C, 20°C, or 26°C; Fig.
separate experiment with anti-SP2 (Fig. 1C, 3B presents data with plants maintained at 26°C) or
2070 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
Figure 2. Rhythmic behavior of the level of phosphorylated D1 in S. well beyond the maximum in light intensity (Fig. 4,
oligorrhiza under greenhouse conditions (A). The light intensity (bro- high light). The steady-state level of D1 as quantified
ken lines) and the D1-P index are shown at indicated times over three by immunoblot analysis of these samples did not
day/night cycles (f). At the end of the light period and 2 h into show any major alterations.
darkness, a set of plants was exposed for 5 min to 300 ␮mol m⫺2 s⫺1 Similar patterns and identical peak times were ob-
fluorescent light (arrow) and thereafter returned to darkness (䡺). Error tained on a cloudy day, with maximum greenhouse
bars indicate SEs based on a sample size of four. B, D1-P index illumination at 200 ␮mol m⫺2 s⫺1 and peaks for D1
maintains oscillations in free-running conditions in continuous light.
phosphorylation, D1-P index and D1 synthesis all
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
Figure 3. Temperature independence of D1-P index in S. oligorrhiza
(indicated by an arrow in the black bar in the middle plants analyzed over a day/night cycle. A, Data from experiment
of Fig. 2A). The clock was rapidly reset in plants where the temperature was not controlled. B, Data from plants
where the dark period was interrupted with contin- maintained at a constant temperature of 26°C. Light intensity at the
uous light; the cycle was shifted, with the D1-P index indicated times is shown in micromoles per meter per second. Error
peak occurring 4 h after switching the plants to con- bars indicate SEs based on a sample size of four.

Plant Physiol. Vol. 130, 2002 2071


Booij-James et al.

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
Figure 4. D1-P index (f) parallels in vivo labeling of D1 with
[32P]orthophosphate (F), but is independent of de novo D1 synthesis
(Rintamäki et al., 1995a). However, we show here
(E)—quantified by isotope incorporation into D1 (percentage of max- that the greatest amount of phosphorylation occurs
imum)—and the daylight maximum (broken lines). Light intensity at hours before maximal light intensity, and at light
the indicated times is shown in micromoles per meter per second. intensities well below those saturating for photosyn-
Data points were averaged from four independent experiments. High thesis (Jansen et al., 1996) or initiation of photoinhi-
light (on the left) indicates experiments done on a sunny day. Low light bition (Jansen et al., 1999). We note here that the rate
(on the right) indicates experiments done on a cloudy day. 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
occurring at 100 ⫾ 25 ␮mol m⫺2 s⫺1 (Fig. 4, low circadian oscillations in D1 phosphorylation are part
light). These intensities are well below the initiation of a regulatory system controlling the operation of
point for photoinhibition in S. oligorrhiza (⬎500 ␮mol the photosynthetic apparatus, as well as a signal to
m⫺2 s⫺1; Jansen et al., 1996). alter the metabolism of the D1 protein. Riesselmann
and Piechulla (1992) suggested that proteins dam-
aged during the light cycle might be substituted at
DISCUSSION the very beginning of the light period of the follow-
We demonstrate that beyond the known redox reg- ing day. If this mechanism were under the control of
ulation of phosphorylation of the D1 photosystem II an endogenous, circadian rhythm, it would enable
reaction center protein (Elich et al., 1992; Silverstein restoration of thylakoid membrane protein com-
et al., 1993), the overriding control is exerted by an plexes early in the morning to allow optimal photo-
internal, circadian clock. Diurnal rhythm of D1 phos- synthetic reactions during the day.
phorylation follows parameters that are fundamental The protein kinase that phosphorylates D1 is local-
to circadian rhythms (Golden et al., 1997; Dunlap, ized in the chloroplast membranes (Elich et al., 1992),
1998; McClung, 2001), i.e. it has a 24-h periodicity but is likely encoded by a nuclear gene because no
and can be entrained. In most circadian systems, light open reading frame for a kinase-like gene has been
is a primary signal for entraining rhythmic activity to identified in the chloroplast genome (Sugiura, 1992).
the daily light/dark cycle by resetting the phase of The endogenous rhythm in D1 protein phosphoryla-
the clock without altering the cycle length (Feldman, tion does not reflect the quantity of the kinase activ-
1982; Wilkins, 1992; Anderson and Kay, 1996). This ity measured in thylakoids. It is possible that activa-
appears true for the D1-P index as well. The clock tion (plastoquinone redox) state of the D1 kinase
was reset by interrupting the dark period with con- (Elich et al., 1992) or the phosphatase (Elich et al.,
tinuous light of ⱖ200 ␮mol m⫺2 s⫺1. Therefore, it 1993; Vener et al., 1999) may be involved. However,
appears that light resets the phase and then acts in whether it is a consequence of redox versus direct
concert with the clock to regulate D1 phosphoryla- regulation of the kinase, the outcome still is clock
tion in vivo. The nonpersistence of rhythmic behav- regulation of D1 phosphorylation. The circadian reg-
ior in the D1-P index in total darkness is likely due to ulation of the expression of a possibly nuclear D1
the fact that light is an absolute requirement for most kinase gene, or its gene product, could provide a
chloroplast activities (Ort and Yocum, 1996), in par- crosstalk mechanism by which a biological clock in
ticular D1 dynamics (Mattoo et al., 1999). the nucleus uses a nuclear-encoded protein kinase to
2072 Plant Physiol. Vol. 130, 2002
D1 Phosphorylation Is Controlled by a Circadian Rhythm

regulate chloroplast function. This is, perhaps, a PSII core proteins is phosphorylated in this green
means for the chloroplast to anticipate environmental alga (Andronis et al., 1998). The psbA gene of C.
changes. Light intensities supersaturating for photo- reinhardtii maps to the edge of the inverted repeat
synthesis cause deleterious effects in the chloroplast; region; thus, there are two identical copies of the
therefore, there is a need for a sensing mechanism to gene and a single type of D1 protein. Transcription of
down-regulate PSII. It is not known how the chloro- chloroplast-encoded genes, including psbA, is con-
plast achieves this. Does it use the D1-P index as a trolled by a nuclear-regulated circadian clock in C.
sensor to anticipate the onset of higher light intensi- reinhardtii (Hwang et al., 1996; Kawazoe et al., 2000),
ties (Mattoo and Edelman, 1985)? Is the phosphory- which would be predicted by our hypothesis above.
lation state of PSII reaction core proteins a conse- The isolation of a D1 kinase gene and elucidation of
quence or a determinant of the relative energy its role could be a step toward understanding the role
distribution between the two photosystems in oxy- of D1 phosphorylation and its regulative mecha-
genic photosynthesis? Because radiance-dependent nisms in higher plants. More than one kinase is
D1 turnover is a fact of life for PSII and because clearly involved with phosphorylation of PSII pro-
phototrophs are normally subjected to a daily light/ teins (Elich et al., 1997). We have purified and cloned
dark cycle, circadian regulation of D1 metabolism is a S. oligorrhiza kinase that phosphorylates a synthetic
not unexpected. However, how regulation is achieved peptide mimicking the D1 protein in a calcium-
may differ for different photosynthetic forms. In dependent manner (A. Raskind, M. Swegle, I. Booij-
higher plants, where D1 is reversibly phosphorylated, James, V. Kumar, M. Edelman, and A. Mattoo, un-
circadian regulation of metabolism can be at the phos- published data). However, it is yet to be ascertained
phorylation level, as is suggested here for S. oligor- if this is the protein kinase that phosphorylates D1 in
rhiza. In cyanobacteria, redox-regulated phosphoryla- vivo. One approach being followed is the antisense
tion of D1 does not appear to occur, but these oxygenic RNA technology to silence this protein and check if
bacterial phototrophs often possess multiple copies of the knockout transformants phosphorylate D1.
the psbA gene coding for D1 (Golden, 1995; Chen et
al., 1999), with different D1 isoforms adapted in vivo MATERIALS AND METHODS
to varying photon irradiation, one dominant at lower
Plant Material and Thylakoid Membrane Preparation
and another at higher light intensities (Bustos et al.,
1990; Clarke et al., 1993; Kulkarni and Golden, 1994). Spirodela oligorrhiza plants were maintained as described (Elich et al.,
Several studies have shown that light-induced tran- 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.
scription of cyanobacterial psbA occurs within the Plants were transferred to medium lacking Suc for at least 48 h before each
larger framework of circadian control (Liu et al., 1995; experiment. Thylakoids were prepared according to published methods
Chen et al., 1999). Thus, a generalized hypothesis can (Elich et al., 1992). The final thylakoid pellet was suspended in a small
be forwarded that reversible phosphorylation of D1 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
(and maybe, other phosphorylated PSII proteins) in greater than 250 ␮g mL⫺1. Chlorophyll concentrations were determined in
higher plants evolutionarily replaced multiple DNA 80% (w/v) acetone (Arnon, 1949).
copies in cyanobacteria as a more energy-efficient sub-
strate for circadian clock regulation of PSII core
In Vitro Phosphorylation
metabolism.
The hypothesis can be investigated phylogeneti- In vitro phosphorylation of S. oligorrhiza thylakoids was carried out in the
cally by viewing atypical species. Prochlorococcus ma- 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
rinus, a ubiquitous, free-living marine cyanobacte- NaF, 0.5 mm NADPH, 3.5 ␮m ferredoxin, and 0.2 mm ATP. Reactions were
rium, is unusual in that it contains chlorophyll a, b, stopped at the indicated times by addition of 0.5 volumes of 3⫻ SDS sample
and c, but no phycobilisomes. In addition, it has only buffer (Mattoo et al., 1981).
a single psbA gene and one type of D1 protein
(Garcia-Fernandez et al., 1998). It will be interesting Diurnal Oscillations Experiments
to determine if D1 can be phosphorylated in this
organism. The state of D1 phosphorylation in lower S. oligorrhiza plants were maintained on medium lacking Suc under
fluorescent lighting as above and were then shifted to the greenhouse at
plants and algae also bears further investigation. The Beltsville, MD for at least 3 d on the same medium before experiments were
D1 protein of the moss Ceratodon purpureus is re- started. Temperature and light intensity were recorded at 1- or 2-h intervals.
ported as not being phosphorylated under high irra- Samples (10–20 plants) were harvested in quadruplicate, immediately fro-
diance in vivo or under in vitro conditions (Rinta- 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-
maki et al., 1995b). However, information on gene phosphorylated D1 by SDS-PAGE/immunoblotting, and one set for in vitro
copy number and regulation of D1 metabolism in this kinase assays. For constant temperature experiments, plants were held in
model lower plant organism is currently lacking. The temperature-controlled water baths at 15°C or 26°C. For cycle shift experi-
literature for Chlamydomonas reinhardtii, although ex- ments, 1-week-old greenhouse-grown plants were divided into five sets
and, at nightfall, were transferred in darkness to an indoor controlled
tensive, is surprisingly ambiguous concerning phos- growth chamber where they were maintained in darkness for 1.5 h. One set
phorylation of the D1 and D2 proteins. The most of plants was left in darkness for the duration of the experiment, and the
recent investigation suggests that neither of these other four were exposed to 200 or 300 ␮mol m⫺2 s⫺1 fluorescent light in

Plant Physiol. Vol. 130, 2002 2073


Booij-James et al.

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