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. 2002 Dec;130(4):2069-75.
doi: 10.1104/pp.013441.

Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm

Isabelle S Booij-James  1 , W Mark Swegle, Marvin Edelman, Autar K Mattoo
Affiliations

Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm

Isabelle S Booij-James et al. Plant Physiol. 2002 Dec.

Abstract

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.

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Figures

Figure 1
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 μm 3-(3,4-dichlorophenyl)-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 anti-SP1 antibody as indicated.
Figure 2
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 (▪). At the end of the light period and 2 h into darkness, a set of plants was exposed for 5 min to 300 μmol m−2 s−1 fluorescent light (arrow) and thereafter returned to darkness (□). 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 μmol m−2 s−1 until the end of the experiment. Error bars indicate ses based on a sample size of three.
Figure 3
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.
Figure 4
Figure 4
D1-P index (▪) parallels in vivo labeling of D1 with [32P]orthophosphate (●), but is independent of de novo D1 synthesis (○)—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.
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