Phosphorylation of the D1 Photosystem II Reaction

0 downloads 0 Views 191KB Size Report
Phosphorylation of the D1 Photosystem II Reaction. Center Protein Is Controlled by an Endogenous. Circadian Rhythm. 1. Isabelle S. Booij-James, W. Mark ...

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 supramolecular protein-pigment complex, PSII (Ort and Yocum, 1996; Mattoo et al., 1999). The PSII reaction center, which includes the D1 and D2 protein heterodimer, binds most of the nonprotein components 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 synthesis (Mattoo et al., 1984), intramembrane translocation (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 phosphorylation of D1 occurs at its N-terminal Thr residue, 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 [email protected]; fax 301–504 –5555. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.013441.

plants is found in the chloroplast membranes (Bennett, 1991). Phosphorylation and dephosphorylation of the D1 protein are strictly light dependent (Elich et al., 1993, 1997). Reversible, redox-sensitive phosphorylation of the light-harvesting chlorophyll apoprotein is thought to be a mechanism maximizing quantum yield by equalizing electron flow through PSII and PSI (Allen, 1992); however, the role of phosphorylation of D1 or other PSII proteins is largely unknown. It has variously been suggested that phosphorylation regulates D1 degradation, maintaining it as a storage form prior to its replacement (Rintama¨ki 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 membranes 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 degradation, but also the ratio of phosphorylated to unphosphorylated D1 (Elich et al., 1992; Rintama¨ki et al., 1995a). To gain insight into the physiological function of phosphorylation of D1, we characterized this process under a natural diurnal cycle, i.e. with naturally fluctuating solar intensity. Antibodies were raised against synthetic peptides, which selectively detect each form of D1, phosphorylated and unphosphorylated, and allow measurement of the proportion of D1 that is phosphorylated under specific 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, entrained to the natural light/dark cycle in a greenhouse, exhibit diurnal oscillation of the ratio of phosphorylated 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 conditions, the D1-P index rhythm was maintained for several cycles. These results show circadian regulation 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 reproduced 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. oligorrhiza plants that had been held in the dark for 3 d to allow for protein dephosphorylation, were phosphorylated 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 increased. A distinct separation into two D1 forms, identified as phosphorylated (D1-P) and unphosphorylated D1 (Elich et al., 1992), was obtained. AntiSP2 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 conformation than the unphosphorylated form. Such conformational 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 condition, the ratio of unphosphorylated to phosphorylated 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 inhibitor 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 carried 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 unphosphorylated D1. C, Plants were incubated, for the times indicated, in the light in the absence (lane 0) or presence of 10 ␮M 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU) to inhibit phosphorylation, 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 antiSP1 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, respectively. 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 diurnal 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 oscillations were obtained in the D1-P index, which were clearly out of phase with the period of maximum radiation (Figs. 2A, 3, and 4). Thus, light intensity 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 greenhouse 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

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 sufficient 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 subsequently kept in continuous light (200 ␮mol m⫺2 s⫺1), the free-running period of 24-h oscillation was maintained (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

Figure 2. Rhythmic behavior of the level of phosphorylated D1 in S. oligorrhiza under greenhouse conditions (A). The light intensity (broken 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 temperature 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 continuous light; the cycle was shifted, with the D1-P index peak occurring 4 h after switching the plants to conPlant Physiol. Vol. 130, 2002

The preceding experiments were based on immunological separation of D1 and D1-P to measure the D1-P index. To determine if these steady-state patterns 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 obtained 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 maximum)—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 regulation 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 phosphorylation 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 continuous 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 phosphorylation in vivo. The nonpersistence of rhythmic behavior 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 particular D1 dynamics (Mattoo et al., 1999). 2072

Circadian regulation of a kinase that phosphorylates phosphoenolpyruvate carboxylase in the dark is well documented (Nimmo, 1998) and is exerted at the level of protein abundance linked to transcript accumulation (Hartwell et al., 1999). In our case, oscillations in D1 phosphorylation in vivo were not mirrored 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 synthesis, which continued to rise till late in the afternoon, or photoinhibition. Thus, the oscillations are indicative of an endogenous regulator controlling D1 metabolism and function. Circadian control of D1 phosphorylation adds another dimension to the as yet unsolved role of lightdependent D1 protein turnover or D1 phosphorylation in chloroplast function. D1 phosphorylation has been suggested to be a means of turning off PSII, or rerouting electron transport, to protect the photosystem from damage caused by high light intensity (Rintama¨ ki 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 photosynthesis (Jansen et al., 1996) or initiation of photoinhibition (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 (Mattoo 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 damaged during the light cycle might be substituted at the very beginning of the light period of the following day. If this mechanism were under the control of an endogenous, circadian rhythm, it would enable restoration of thylakoid membrane protein complexes early in the morning to allow optimal photosynthetic reactions during the day. The protein kinase that phosphorylates D1 is localized 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 phosphorylation does not reflect the quantity of the kinase activity measured in thylakoids. It is possible that activation (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 regulation 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

D1 Phosphorylation Is Controlled by a Circadian Rhythm

regulate chloroplast function. This is, perhaps, a means for the chloroplast to anticipate environmental changes. Light intensities supersaturating for photosynthesis 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 chloroplast achieves this. Does it use the D1-P index as a sensor to anticipate the onset of higher light intensities (Mattoo and Edelman, 1985)? Is the phosphorylation state of PSII reaction core proteins a consequence or a determinant of the relative energy distribution between the two photosystems in oxygenic 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 phosphorylation level, as is suggested here for S. oligorrhiza. In cyanobacteria, redox-regulated phosphorylation 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 transcription 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 substrate for circadian clock regulation of PSII core metabolism. The hypothesis can be investigated phylogenetically by viewing atypical species. Prochlorococcus marinus, a ubiquitous, free-living marine cyanobacterium, 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 reported as not being phosphorylated under high irradiance in vivo or under in vitro conditions (Rintamaki 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 extensive, is surprisingly ambiguous concerning phosphorylation of the D1 and D2 proteins. The most recent investigation suggests that neither of these Plant Physiol. Vol. 130, 2002

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 controlled 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 mechanisms in higher plants. More than one kinase is clearly involved with phosphorylation of PSII proteins (Elich et al., 1997). We have purified and cloned a S. oligorrhiza kinase that phosphorylates a synthetic peptide mimicking the D1 protein in a calciumdependent manner (A. Raskind, M. Swegle, I. BooijJames, V. Kumar, M. Edelman, and A. Mattoo, unpublished 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) containing 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 frozen on dry ice, and then stored at ⫺80°C until thylakoids were isolated. Three sets of samples were used for analysis of phosphorylated and unphosphorylated 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 experiments, 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; Zurawski 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 conjugates 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; accepted September 19, 2002.

LITERATURE CITED Allen JF (1992) Protein phosphorylation in regulation of photosynthesis. Biochim Biophys Acta 1098: 275–335 Anderson SL, Kay SA (1996) Illuminating the mechanism of the circadian clock in plants. Trends Plant Sci 1: 51–57 Andronis C, Kruse O, Deak Z, Vass I, Diner BA, Nixon PJ (1998) Mutation of residue threonione-2 of the D2 polypeptide and its effect on PSII function in Chlamydomonas reinhardtii. Plant Physiol 117: 515–524 Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 Aro E-M, Virgin I, Andersson B (1993) Photoinhibition of photosystem II inactivation, protein damage and turnover. Biochim Biophys Acta 1143: 113–134 Barford D, Hu S-H, Johnson LN (1991) Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP. J Mol Biol 218: 233–260 Bennett J (1991) Protein phosphorylation in green plant chloroplasts. Annu Rev Plant Physiol Plant Mol Biol 42: 281–311 Bustos SA, Schaefer MR, Golden SS (1990) Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity. J Bacteriol 172: 1998–2004

2074

Callahan FE, Ghirardi ML, Sopory SK, Mehta AM, Edelman M, Mattoo AK (1990) A novel metabolic form of the 32-kDa-D1 protein in the grana-localized reaction center of photosystem II. J Biol Chem 265: 15357–15360 Chen Y-B, Dominic B, Zani S, Mellon MT, Zehr JP (1999) Expression of photosynthesis genes in relation to nitrogen fixation in the diazotrophic filamentous nonheterocystous cyanobacterium Trichdesmium sp. Plant Mol Biol 41: 89–104 Clarke AK, Soitamo A, Gustafsson P, Oquist G (1993) Rapid interchange between two distinct forms of cyanobacterial photosystem II reaction center protein D1 in response to photoinhibition. Proc Natl Acad Sci USA 90: 9973–9977 Croce R, Breton J, Bassi R (1996) Conformational changes induced by phosphorylation in the CP29 subunit of photosystem II. Biochemistry 35: 11142–11148 Dunlap JC (1998) Common threads in eukaryotic circadian systems. Curr Opin Genet Dev 8: 400–406 Elich TD, Edelman M, Mattoo AK (1992) Identification, characterization and resolution of the in vivo phosphorylated form of the D1 photosystem II reaction center protein. J Biol Chem 267: 3523–3529 Elich TD, Edelman M, Mattoo AK (1993) Dephosphorylation of photosystem II core proteins is light-regulated in vivo. EMBO J 12: 4857–4862 Elich TD, Edelman M, Mattoo AK (1997) Evidence for light-dependent and light-independent protein dephosphorylation in chloroplasts. FEBS Lett 411: 236–238 Feldman JF (1982) Genetic approaches to circadian clocks. Annu Rev Plant Physiol 33: 583–608 Garcia-Fernandez JM, Hess WR, Houmard J, Partensky F (1998) Expression of the psbA gene in the marine oxyphotobacteria Prochlorococcus spp. Arch Biochem Biophys 359: 17–23 Golden SS (1995) Light responsive gene expression in cyanobacteria. J Bacteriol 177: 1651–1654 Golden SS, Ishiura M, Johnson CH, Kondo T (1997) Cyanobacterial circadian rhythms. Annu Rev Plant Physiol Plant Mol Biol 48: 327–354 Greenberg BM, Gaba V, Mattoo AK, Edelman M (1987) Identification of a primary in vivo degradation product of the rapidly turning-over 32-kDa protein of photosystem II. EMBO J 6: 2865–2869 Hankamer B, Barber J, Boekema EJ (1997) Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo. Annu Rev Plant Physiol Plant Mol Biol 48: 641–671 Hartwell J, Gill A, Nimmo GA, Wilkins MB, Jenkins GI, Nimmo HG (1999) Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression. Plant J 20: 333–342 Hennessey TL, Freeden A, Field CB (1993) Environmental effects on circadian rhythms in photosynthesis and stomatal opening. Planta 189: 369–376 Hwang S, Kawazoe R, Herrin DL (1996) Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas. Proc Natl Acad Sci USA 93: 996–1000 Jansen MAK, Gaba V, Greenberg BM, Mattoo AK, Edelman M (1996) Low threshold levels of ultraviolet-B in a background of photosynthetically active radiation trigger rapid degradation of the D2 protein of photosystem II. Plant J 9: 693–699 Jansen MAK, Mattoo AK, Edelman M (1999) D1–D2 protein degradation in the chloroplast: complex light saturation kinetics. Eur J Biochem 260: 527–532 Kawazoe R, Hwang S, Herrin DL (2000) Requirements for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplastencoded genes in Chlamydomonas. Plant Mol Biol 44: 699–709 Kulkarni RD, Golden SS (1994) Adaptation to high light intensity in Synechococcus sp. strain PCC 7942: regulation of three psbA genes and two forms of D1 protein. J Bacteriol 176: 959–965 Liu Y, Golden SS, Kondo T, Ishiura M, Johnson CH (1995) Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria. J Bacteriol 177: 2080–2086 Mattoo AK, Edelman M (1985) Photoregulation and metabolism of a thylakoidal herbicide-receptor protein. In JB St. John, E Berlin, PC Jackson, eds, Frontiers of Membrane Research in Agriculture. Rowman and Allanheld, Totowa, NJ, pp 23–34 Mattoo AK, Edelman M (1987) Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein. Proc Natl Acad Sci USA 84: 1497–1501

Plant Physiol. Vol. 130, 2002

D1 Phosphorylation Is Controlled by a Circadian Rhythm

Mattoo AK, Elich TD, Ghirardi ML, Callahan FE, Edelman M (1993) Post-translational modification of chloroplast proteins and the regulation of protein turnover. In NH Battey, HG Dickinson, AM Hetherington, eds, Posttranslational Modifications in Plants. Cambridge University Press, Cambridge, UK, pp 65–78 Mattoo AK, Giardi M-T, Raskind A, Edelman M (1999) Dynamic metabolism of photosystem II reaction center proteins and pigments. Physiol Plant 107: 454–461 Mattoo AK, Hoffman-Falk H, Marder JB, Edelman M (1984) Regulation of protein metabolism: coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kDa protein of the chloroplast membranes. Proc Natl Acad Sci USA 81: 1380–1384 Mattoo AK, Marder JB, Edelman M (1989) Dynamics of the photosystem II reaction center. Cell 56: 241–246 Mattoo AK, Pick U, Hoffman-Falk H, Edelman M (1981) The rapidly metabolized 32,000-dalton polypeptide of the chloroplast is the “proteinaceous shield” regulating photosystem II electron transport and mediating diuron herbicide sensitivity. Proc Natl Acad Sci USA 78: 1572–1576 McClung CR (2001) Circadian rhythms in plants. Annu Rev Plant Physiol Plant Mol Biol 52: 139–162 Michel H, Deisenhofer J (1988) Relevance of the photosynthetic reaction center from purple bacteria to the structure of photosystem II. Biochemistry 27: 1–7 Michel H, Hunt DF, Shabanowitz J, Bennett J (1988) Tandem mass spectrometry reveals that three photosystem II proteins of spinach chloroplast contain N-acetyl-O-phosphothreonine at their N-termini. J Biol Chem 263: 1123–1130 Nanba O, Satoh K (1987) Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559. Proc Natl Acad Sci USA 84: 109–112 Nilsson A, Stys D, Drakenberg T, Spangfort MD, Forsen S, Allen JF (1997) Phosphorylation controls the three-dimensional structure of plant light harvesting complex II. J Biol Chem 272: 18350–18357 Nimmo HG (1998) Circadian regulation of a plant protein kinase. Chronobiol Int 15: 109–118

Plant Physiol. Vol. 130, 2002

Ort DR, Yocum CF (1996) Advances in Photosynthesis, Vol 4. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Boston, MA Posner HB (1967) Aquatic vascular plants. In FA Witt, NK Wessels, eds, Methods in Developmental Biology. Crowell, NY, pp 301–317 Riesselmann S, Piechulla B (1992) Diurnal and circadian light-harvesting complex and quinone B-binding protein synthesis in leaves of tomato (Lycopersicon esculentum). Plant Physiol 100: 1840–1845 Rintama¨ki E, Kettunen R, Tyystja¨rvi E, Aro E-M (1995a) Light-dependent phosphorylation of D1 reaction centre protein of photosystem II: hypothesis for the functional role in vivo. Physiol Plant 93: 191–195 Rintama¨ki E, Salo R, Lehtonen E, Aro E-M (1995b) Regulation of D1 protein degradation during photoinhibition of photosystem II in vivo: phosphorylation of the D1 protein in various plant groups. Planta 195: 379–386 Santini C, Tidu V, Tognon G, Ghiretti Magaldi A, Bassi R (1994) Threedimensional structure of the higher-plant photosystem II reaction centre and evidence for its dimeric organization in vivo. Eur J Biochem 221: 307–315 Silverstein T, Cheng L, Allen JF (1993) Redox titration of multiple protein phosphorylations in pea chloroplast thylakoids. Biochim Biophys Acta 1183: 215–220 Stone JM, Walker JC (1995) Plant protein kinase families and signal transduction. Plant Physiol 108: 451–457 Sugiura M (1992) The chloroplast genome. Plant Mol Biol 19: 149–168 Usami S, Hiroharu B, Ito Y, Nishihama R, Machida Y (1995) Cutting activates a 46-kilodalton kinase in plants. Proc Natl Acad Sci USA 92: 8660–8664 Vener AV, Rokka A, Fulgosi H, Andersson B, Herrmann RG (1999) A cyclophilin-regulated PP2A-like protein phosphatase in thylakoid membranes of plant chloroplasts. Biochemistry 38: 14955–14965 Wilkins MB (1992) Circadian rhythms: their origin and control. New Phytol 121: 347–375 Zurawski G, Bohnert HJ, Whitfeld PR, Bottomley W (1982) Nucleotide sequence of the gene for the Mr 32000 thylakoid protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of Mr 38950. Proc Natl Acad Sci USA 79: 7699–7703

2075

Suggest Documents