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Keywords:

  • 1 – qP;
  • CP29;
  • LHCII;
  • phosphorylation;
  • photosystem II;
  • redox regulation

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Differential redox regulation of thylakoid phosphoproteins was studied in winter rye plants in vivo. The redox state of chloroplasts was modulated by growing plants under different light/temperature conditions and by transient shifts to different light/temperature regimes. Phosphorylation of PSII reaction centre proteins D1 and D2, the chlorophyll a binding protein CP43, the major chlorophyll a/b binding proteins Lhcb1 and Lhcb2 (LHCII) and the minor light-harvesting antenna protein CP29 seem to belong to four distinct regulatory groups. Phosphorylation of D1 and D2 was directly dependent on the reduction state of the plastoquinone pool. CP43 protein phosphorylation generally followed the same pattern, but often remained phosphorylated even in darkness. Phosphorylation of CP29 occurred upon strong reduction of the plastoquinone pool, and was further enhanced by low temperatures. In vitro studies further demonstrated that CP29 phosphorylation is independent of the redox state of both the cytochrome b6/f complex and the thiol compounds. Complete phosphorylation of Lhcb1 and 2 proteins, on the contrary, required only modest reduction of the plastoquinone pool, and was subject to inhibition upon increase in the thiol redox state of the stroma. Furthermore, the reversible phosphorylation of Lhcb1 and 2 proteins appeared to be an extremely dynamic process, being rapidly modulated by short-term fluctuations in chloroplast redox conditions.


Abbreviations
DBMIB

2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone

DCMU

3-(3,4-dichlorophenyl)-1,1-dimethylurea

Fv/Fmax

variable/maximal fluorescence ratio

LHCII

light-harvesting chlorophyll a/b complex of PSII

PSII

photosystem II

PSI

photosystem I

PFD

photon flux density

P-LHCII

P-CP29, P-CP43, P-D2 and P-D1, phosphorylated forms of LHCII, CP29, CP43, D2 and D1, respectively

P-Thr

phosphothreonine

qP

photochemical quenching of chlorophyll fluorescence.

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Photosynthetic organisms have evolved various regulatory mechanisms that, on one hand ensure an efficient capture of light energy under limiting irradiance levels, and on the other hand provide protection against the potentially damaging effects of excess irradiance. One such mechanism is a reversible thylakoid protein phosphorylation. The main phosphoproteins in the thylakoid membrane include the photosystem II (PSII) core proteins D1, D2, CP43 and the psbH gene product, as well as the two major chlorophyll a/b binding proteins of the light-harvesting antenna (LHCII). Additionally, a chill-induced and light-dependent phosphorylation of a minor chlorophyll a/b binding protein CP29 has been shown to occur in maize, barley and rye leaves (Bergantino et al. 1995, 1998; Pursiheimo et al. 2001). Recently, a 12 kDa thylakoid phosphoprotein was demonstrated to partially release from the membrane upon phosphorylation (Carlberg et al. 2003). The exact location of this protein, however, still remains unknown.

Reversible phosphorylation of the PSII core proteins D1, D2 and CP43 is likely to be involved in the regulation of the PSII damage and repair cycle (Rintamäki, Kettunen & Aro 1996; Baena-Gonzalez, Barbato & Aro 1999), whereas reversible LHCII protein phosphorylation has been proposed to potentiate state-transitions, either by balancing the distribution of excitation energy between the two photosystems (Allen 1992) or by enhancing the production of ATP via cyclic electron flow (Finazzi et al. 1999).

The identity and number of kinases responsible for thylakoid protein phosphorylation is not yet fully resolved. Numerous studies have, however, demonstrated the importance of plastoquinol redox poise in regulation of all thylakoid protein kinase activities (Bennett 1991; Allen 1992; Gal, Zer & Ohad 1997). Phosphorylation of the PSII core proteins D1, D2 and CP43 has been suggested to be controlled by the reduction of the plastoquinone pool (Bennett, Shaw & Michel 1988). Phosphorylation of CP29 has been observed only under rather severe environmental conditions in vivo (Bergantino et al. 1995, 1998; Pursiheimo et al. 2001). Nevertheless, the studies on regulation mechanisms of CP29 have been complicated by a lack of methods to phosphorylate CP29 in vitro.

Plastoquinone reduction is likewise required for phosphorylation of LHCII proteins even though occurring via a distinct mechanism. Although the identity of thylakoid kinases has in general remained elusive, the recent progress with LHCII kinases has been very promising. A novel thylakoid kinase active in LHCII protein phosphorylation was reported from Chlamydomonas (Depège, Bellafiore & Rochaix 2003) which, however, did not show any homology with the TAK kinases reported earlier to phosphorylate the LHCII proteins in plants (Snyders & Kohorn 1999). Much more is known about the regulation of the LHCII kinase, which for activation requires an interaction with the cytochrome b6/f (cyt b6/f) complex and binding of reduced plastoquinol to the Qo site (Vener et al. 1995, 1997; Gal et al. 1997; Zito et al. 1999). More recently, it has been shown that dynamic oxidoreduction of plastoquinol of the Qo site is essential in the activation process (Finazzi et al. 2001; Hou, Rintamäki & Aro 2003). When plants are shifted to high light or low temperature, LHCII protein phosphorylation becomes down-regulated by accumulation of thiol reductants in chloroplasts (Rintamäki et al. 1997, 2000; Pursiheimo et al. 2001; Hou et al. 2002). Long-term growth of winter rye plants under high irradiance, and particularly under low temperature in turn restored the capacity for LHCII protein phosphorylation as a result of metabolic acclimation (Pursiheimo et al. 2001).

In this study, we have explored the dynamics of PSII and LHCII protein phosphorylation in differentially light/temperature-acclimated plants. Various acclimation states of winter rye plants were first induced by long-term growth under contrasting light/temperature regimes, and the dynamics of phosphorylation of various PSII proteins was subsequently monitored upon a shift in environmental conditions. Phosphorylation strategies of various PSII phosphoproteins were dissected into four distinct groups with the LHCII kinase being the most delicate sensor of environmental and metabolic cues. Regulation mechanisms were also mimicked by studies with isolated thylakoids in vitro.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Material and growth conditions

For long-term acclimation to different light conditions, the winter rye (Secale cereale L. cv. Voima) plants were grown for 3 weeks under photon flux densities (PFDs) of 300 (control) and 600 (high light) µmol photons m−2 s−1 (20 °C day/13 °C night). To achieve a similar physiological state, the growth of rye plants under low temperature conditions (5 °C day/5 °C night) took 8 weeks at a PFD of 300 µmol photons m−2 s−1. A light/dark rhythm of 16/8 h was maintained during growth and also during the short-term light and temperature shift experiments when lasting longer than 16 h. For all experiments, samples were taken from the third and fourth mature leaves, and a 5 cm segment was discarded from both ends of the leaf.

Light and temperature shift treatments of the plants

Control rye plants grown at 300 µmol photons m−2 s−1/20 °C were shifted to 600 µmol photons m−2 s−1/20 °C (high light) or to 300 µmol photons m−2 s−1/5 °C (low temperature) for 24 h. High-light-plants, grown at 600 µmol photons m−2 s−1/20 °C, were shifted to 300 µmol photons m−2 s−1/5 °C. The effect of high light on low-temperature-acclimated plants was studied by increasing the light intensity from 300 to 400 or 700 µmol photons m−2 s−1 at 5 °C. When PSII light (650 ± 10 nm, 30 µmol photons m−2 s−1) was used, the plants were illuminated through an S25-650 filter (Corion, Franklin, MA, USA) with a slide projector (250 W lamp) as a light source.

Fluorescence measurements

The redox state of PSII (1 − qP) was monitored by measuring the photochemical quenching of chlorophyll fluorescence (qP) with a PAM 101 fluorometer (Heinz Walz, Effeltrich, Germany) (van Kooten & Snel 1990; Öquist & Huner 1993). After transferring the leaf to the PAM fluorometer, steady-state fluorescence (Fs) was attained within 30 min at the same light intensity and temperature that prevailed during the experimental treatment of plants, and 1 − qP was measured as (Fs − F0′)/(Fm′ − Fo′). The photochemical efficiency of PSII was monitored as a ratio of variable to maximal fluorescence, Fv/Fmax (Fv is a difference between maximal, Fmax, and initial fluorescence, Fo), measured from intact leaves with a Hansatech (Kings Lynn, UK) PEA fluorometer, after a 30-min dark incubation.

Isolation of thylakoid membranes

Directly after the in vivo treatment, the leaves were frozen and crushed in liquid nitrogen. The freezing step was, however, omitted when isolated thylakoids were used for subsequent in vitro assays. Grinding of the sample with an Ultra Turrax (Janko and Kungel IKA, Germany) was performed rapidly in a small volume of ice-cold isolation buffer (330 mm sucrose, 25 mm Tris-HCl, pH 8.5, 10 mm MgCl2, 10 mm NaF). After filtration through Miracloth (Calbiochem, San Diego, CA, USA), the thylakoids were collected by centrifugation at 6000 × g, for 2 min at 4 °C, washed in 25 mm Tris-HCl, pH 8.5, 10 mm MgCl2, 10 mm NaF, and finally suspended in the isolation buffer and stored at −80 °C. The chlorophyll content was determined according to Porra, Thompson & Kriedemann (1989).

In vitro phosphorylation of thylakoid proteins

Thylakoids from dark-adapted plants were suspended in an assay buffer (50 mm Hepes-KOH, pH 8, 330 mm sorbitol, 5 mm NaCl, 10 mm MgCl2) at a final chlorophyll concentration of 0.4 mg Chl mL−1. The suspension was pre-incubated for 10 min with or without 10 mm NaF in the presence of the following chemicals: 10 mm Na-EDTA, 5 mm CaCl2, 5 mm KCl, 10 µm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) + 5 mm l-ascorbic acid, 20 µm 3-(3,4-dichlorophenyl)-1,1-dimethylurea DCMU, 2 mm reduced DTT (DTTred), 2 mm oxidized DTT (DTTox) or 5 µm nigericin. Phosphorylation was initiated by addition of 0.4 mm ATP, and samples were illuminated under 50 µmol photons m−2 s−1 of white light or under 30 µmol photons m−2 s−1 of PSII light at 20 °C for 15 min.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and detection of thylakoid phosphoproteins

Thylakoid polypeptides were solubilized and separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) essentially according to Laemmli (1970), using 15% acrylamide and 6 m urea in the separation gel. The proteins were electroblotted onto a PVDF membrane (Immobilon P; Millipore, Bedford, MA, USA), which was blocked with 1% bovine serum albumin (fatty acid free; SigmaAldrich, Finland). The phosphoproteins were immunodetected with a commercial polyclonal phospho-threonine (P-Thr) antibody either from Zymed Laboratories Inc. (San Francisco, CA, USA) or from New England Biolabs (Beverly, MA, USA) using an Immuno-Lite Assay Kit (Bio-Rad, Hercules, CA, USA) (Rintamäki et al. 1997). Phosphoproteins detected with the phosphothreonine antibodies were previously isolated and identified as the PSII core proteins P-CP43, P-D2, P-D1, and the light-harvesting antenna proteins P-Lhcb1 and P-Lhcb2 (P-LHCII) as well as P-CP29 (Bergo et al. 2002). The level of protein phosphorylation was quantified by using an image program (Imaging Research Inc., St. Catherine’s, Ontario, Canada). D1 protein phosphorylation was also studied by immunodetection after electrophoretic separation of the non-phosphorylated (D1) and phosphorylated (P-D1) forms (Koivuniemi, Aro & Andersson 1995; Rintamäki et al. 1996).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Steady-state level of PSII protein phosphorylation during the light phase in winter rye plants grown under different light/temperature regimes

The steady-state level of PSII protein phosphorylation was first explored in winter rye plants grown under different light regimes. Despite differences in the redox state of PSII (1 − qP) in plants grown under 300 and 600 µmol photons m−2 s−1/20 °C being 0.08 and 0.18, respectively (Fig. 1), quite similar phosphorylation levels of D1, D2, CP43 and LHCII proteins were detected in these plants, measured after 4 h of illumination under growth conditions. Low-temperature-acclimated (300/5) plants were characterized by even higher 1 − qP of 0.23 and slightly reduced Fv/Fmax of 0.79. These plants exhibited slightly increased steady-state phosphorylation levels of the PSII core proteins and LHCII (Fig. 1). Phosphorylated CP29 was detected exclusively in the thylakoids of low-temperature-acclimated plants (Fig. 1).

image

Figure 1. PSII protein phosphorylation, 1 − qP and Fv/Fmax in winter rye plants after long-term growth under different light and temperature regimes (300 µmol photons m−2 s−1/20 °C, 300/20; 600 µmol photons m−2 s−1/20 °C, 600/20; 300 µmol photons m−2 s−1/5 °C, 300/5). Leaf samples were collected 4 h after the lights were turned on. Phosphothreonine antibody (Zymed) was used to detect P-CP43, P-D2, P-D1, P-CP29 and P-LHCII, the phosphorylated forms of CP43, D2, D1, CP29 and LHCII, respectively. The relative proportion of phosphorylated form of D1 protein (% P-D1), was measured with a D1 protein-specific antibody and presented as a percentage of total D1 protein. The results are means ± SE of at least three independent experiments. For 1 − qP and Fv/Fmax, the SE values were less than 5%.

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Modulations in PSII protein phosphorylation upon sudden environmental shifts

The steady-state phosphorylation levels of the PSII core and LHCII proteins were similar after long-term acclimation of plants to different light and temperature conditions, despite small differences in the redox states of PSII (Fig. 1). Next we studied whether a sudden increase in the 1 − qP value is reflected in PSII protein phosphorylation. Winter rye plants were shifted from normal growth conditions (300/20) to low temperature (5 °C, without any change in light intensity) or to a two-fold higher light intensity (600 µmol photons m−2 s−1, constant temperature). These shifts in environmental conditions induced an increase in 1 − qP, from 0.08 to 0.22 and 0.25, respectively, and an increase in the phosphorylation level of the PSII core proteins D1, D2 and CP43, measured 2 h after the environmental shifts (Fig. 2). Such increased levels of both 1 − qP and the PSII core protein phosphorylation were acquired rapidly, already during the first hour after the environmental shifts, and remained quite invariable throughout the 4 h illumination period (data not shown). The photochemical efficiency of PSII, expressed as Fv/Fmax showed no major changes (Fig. 2). Shifts to low temperature or to high light were already previously shown to induce down-regulation of LHCII protein phosphorylation, which at low temperature was also due to a change in the chloroplast redox state rather than to a low-temperature-induced change in thylakoid membrane fluidity (Fig. 2, Pursiheimo et al. 2001).

image

Figure 2. Changes in PSII protein phosphorylation, redox state of PSII (1 − qP) and Fv/Fmax upon short-term (2 h) shifts of winter rye plants to contrasting environmental conditions. After 4 h illumination at normal growth conditions (300 µmol photons m−2 s−1/20 °C, 300/20, control plants indicated in bold below the figure), the plants were shifted to 50 µmol photons m−2 s−1/20 °C (50/20), to 300 µmol photons m−2 s−1/5 °C (300/5), to 600 µmol photons m−2 s−1/20 °C (600/20) or to 2000 µmol photons m−2 s−1/20 °C (2000/20) for 2 h. A dark sample was collected from control plants at the end of the 8 h dark period. P-Thr antibody (Zymed) was used to detect P-CP43, P-D2, P-D1 and P-LHCII, the phosphorylated forms of CP43, D2, D1 and LHCII, respectively. Phosphorylation levels are indicated relative to control plants designated as 100%. The results are means ± SE of at least three independent experiments.

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Quite opposite effects on PSII protein phosphorylation were acquired by a shift of plants to low light conditions (50 µmol photons m−2 s−1). This shift induced a decline in 1 − qP from 0.08 to 0.03, a lower level of PSII core protein phosphorylation, and strong phosphorylation of LHCII (Fig. 2). Only low levels of P-D1, P-D2 and P-LHCII protein were detected in darkness at the end of the diurnal 8 h dark period. CP43, on the other hand, showed a surprisingly high level of phosphorylation even at the end of the dark period (Fig. 2).

Modulations of PSII protein phosphorylation under photo-inhibitory conditions were studied by illuminating rye plants at a PFD of 2000 µmol m−2 s−1 for 2 h (Fig. 2). This treatment caused a 26% decline in Fv/Fmax and a dramatic increase in 1 − qP, from 0.08 to 0.7. Such high light treatment also induced an approximately two-fold increase in the phosphorylation level of D1, and up to 20- to 30-fold increase in D2 and CP43 protein phosphorylation, whereas the LHCII proteins turned completely dephosphorylated (Fig. 2).

Oscillations in the redox state of PSII induce opposite oscillations in the level of LHCII protein phosphorylation

We next investigated how winter rye plants that were already acclimated to elevated PSII redox state during growth at 600 µmol photons m−2 s−1/20 °C respond to a shift to low temperature. A shift of such high-light-acclimated winter rye plants to low temperature together with a 50% reduction in the light intensity (300 µmol photons m−2 s−1/5 °C), induced oscillations in 1 − qP during the first hours after the shift (Fig. 3). One hour after the shift a two-fold increase in 1 − qP was recorded. During the next hour, 1 − qP almost returned to the level before the shift, and then increased again during the third hour after the shift. The shift also induced an increase in the phosphorylation level of D1 (Fig. 3) as well as that of the D2 and CP43 proteins (data not shown) within the first hour after the shift but no distinct oscillations were recorded thereafter, as was observed for 1 − qP.

image

Figure 3. Oscillations in the redox state of PSII (1 − qP) and in the level of LHCII protein phosphorylation in vivo. After 4 h illumination at high light growth conditions (600 µmol photons m−2 s−1/20 °C; time 4 h in the figure), the plants were shifted to 300 µmol photons m−2 s−1/5 °C. Leaf samples were collected at indicated time points and the level of LHCII and D1 protein phosphorylation was explored by using a P-Thr antibody (Zymed) and a D1 protein specific antibody, respectively. 1 − qP was measured with a PAM fluorometer. Results are the means ± SD of three independent experiments.

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LHCII protein phosphorylation responded quite differently upon a similar shift in environmental conditions, and showed strong oscillations with a pattern opposite to that of 1 − qP (Fig. 3). The abrupt increase in 1 − qP, which occurred during the first hour after the shift, was accompanied with a prominent decrease in LHCII protein phosphorylation. However, during the next hour, the level of phosphorylation recovered almost to the initial control level, and then declined again during the third hour after the shift. High values of 1 − qP and low LHCII protein phosphorylation were recorded also the next day, after an 8 h dark period (measured 4 h after the onset of illumination, 24 h time point in Fig. 3). Phosphorylation of CP29 was induced upon a shift of plants to low temperature, but no marked oscillations in the level of P-CP29 could be detected (data not shown).

Phosphorylation of CP29 in vivo

Light-induced phosphorylation of CP29 (identification demonstrated in Bergo et al. 2002), occurred in winter rye plants grown under low temperature (Fig. 1) as well as in plants shifted from normal growth conditions (300 µmol photons m−2 s−1/20 °C) to low temperature (300 µmol photons m−2 s−1/5 °C) (Fig. 4b). We also studied how the phosphorylation of CP29 is modulated by environmental shifts in plants grown under low temperature. Increasing the light intensity from 300 to only 400 µmol photons m−2 s−1 at low temperature was sufficient to increase the level of CP29 phosphorylation and that of the PSII core protein phosphorylation, and to decrease LHCII protein phosphorylation (Fig. 4a). The opposite effect of an environmental shift on the phosphorylation level of CP29 and that of LHCII was particularly pronounced when the light intensity was increased to 700 µmol photons m−2 s−1 at low temperature (Fig. 4a).

image

Figure 4. Modulation of CP29 phosphorylation in vivo by changing the temperature, light intensity and light quality conditions. (a) An immunoblot demonstrating thylakoid protein phosphorylation upon increasing light intensity under low temperature. Four hours after the lights were turned on, the low-temperature-grown plants (300 µmol photons m−2 s−1/5 °C, 300/5) were shifted for 2 h to 400 and 700 µmol photons m−2 s−1 at 7 and 11 °C, respectively. (b) An immunoblot demonstrating the induction of CP29 phosphorylation upon change in environmental conditions of plants grown at 300 µmol photons m−2 s−1/20 °C (300/20). Leaf samples were collected 2 h after a shift of winter rye plants from normal growth conditions (300/20) to 50 µmol photons m−2 s−1/20 °C (50/20), to 2000 µmol photons m−2 s−1/20 °C (2000/20), to 300 µmol photons m−2 s−1/5 °C (300/5) or to light-favouring PSII excitation at 20 °C. P-Thr antibodies from Zymed (a) and from New England Biolabs (b) were used to immunodetect P-CP43, P-D2, P-D1, P-CP29 and P-LHCII. Note that the P-Thr antibody from New England Biolabs exhibits a particularly strong immunoreaction with P-CP29.

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To investigate in more detail the conditions that induce CP29 phosphorylation in vivo, we took advantage of a new P-Thr antibody from New England Biolabs, which was found to exhibit a particularly strong immunoreaction with the phosphorylated form of CP29. With this P-Thr antibody, minor amounts of P-CP29 could be detected even at moderate light intensities, and the level of phosphorylation clearly increased when increasing reduction of the plastoquinone pool was induced by illumination of leaves under high light intensity or at low temperature (Fig. 4b). Illumination of winter rye leaves under PSII light for 2 h induced pronounced phosphorylation of CP29, as well as the phosphorylation of D1, D2, CP43 and LHCII proteins (Fig. 4b).

In vitro phosphorylation of CP29

In vitro phosphorylation of CP29 has not been reported earlier. Using the P-Thr antibody from New England Biolabs we were able for first time to study the induction of CP29 phosphorylation in vitro in isolated thylakoid membranes (Fig. 5). Consequently, the phosphorylation/dephosphorylation characteristics of CP29 were tested using several chemicals known to regulate the reversible phosphorylation of other PSII phosphoproteins (Carlberg et al. 1999; Rintamäki et al. 2000). For these experiments the thylakoids were isolated from dark-adapted plants, and were first pre-incubated in darkness with or without the addition of various chemicals, and only thereafter ATP was added and phosphorylation induced by turning white light on.

image

Figure 5. Phosphorylation of CP29 in isolated thylakoids in vitro. (a) Thylakoids were isolated from dark-acclimated plants grown at 300 µmol photons m−2 s−1/20 °C (dark thyl) and then pre-incubated in darkness for 10 min with (+) or without (–) 10 mm NaF in the absence (ctl) or presence of 5 mm CaCl2, 5 mm KCl, 2 mm reduced DTT (DTTred) or 2 mm oxidized DTT (DTTox). Thereafter phosphorylation was initiated by addition of 0.4 mm ATP, and illuminating the thylakoids under 50 µmol photons m−2 s−1 for 15 min. (b) An immunoblot demonstrating the in vitro phosphorylation of CP29 upon modulation of the photosynthetic electron transfer chain. Thylakoids were isolated from dark-acclimated plants grown at 300 µmol photons m−2 s−1/20 °C (dark thyl) and then pre-incubated for 10 min in darkness with 10 mm NaF in the absence (white light ctl) or presence of 10 µm DBMIB + 5 mm ascorbate (DBMIB), 5 mm ascorbate, 20 µm DCMU or 5 µm nigericin. Thereafter phosphorylation was initiated by addition of 0.4 mm ATP, and by illumination under 50 µmol photons m−2 s−1 white light or under 30 µmol photons m−2 s−1 PSII light at 20 °C. A P-Thr antibody (New England Biolabs) exhibiting a particularly strong immunoreaction with the phosphorylated form of CP29 was used to detect P-CP43, P-D2, P-D1, P-CP29 and P-LHCII, the phosphorylated forms of CP43, D2, D1, CP29 and LHCII, respectively.

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Phosphorylation of CP29 could be induced both in isolated thylakoids (Fig. 5) and in intact chloroplasts (data not shown) under low-intensity white light. Addition of NaF in the reaction media slightly increased the level of phosphorylation. Interestingly, a decreased level of CP29 phosphorylation was observed when the phosphorylation reaction was performed in the presence of 5 mm CaCl2 but omitting NaF (Fig. 5a). On the contrary, CaCl2 exhibited no inhibitory effects over CP29 phosphorylation if protein phosphatase activity was inhibited by including NaF in the reaction mixture. Furthermore, 5 mm KCl had no effect on the in vitro phosphorylation pattern of any of the PSII phosphoproteins detected with the antibody. Neither was the phosphorylation of CP29 affected by reduced or oxidized forms of DTT, thus showing again a distinct difference to the regulation of LHCII proteins (Fig. 5a, Rintamäki et al. 2000).

Illumination of isolated thylakoids under PSII light in the presence of ATP induced strong phosphorylation of CP29 (Fig. 5b). The presence of DCMU in the reaction mixture completely abolished the phosphorylation of CP29, whereas DBMIB showed no effect in illuminated thylakoids (Fig. 5b), again in variance with the phosphorylation of LHCII proteins (Rintamäki et al. 2000). Furthermore, CP29 phosphorylation was not affected by the presence of ascorbate or nigericin (Fig. 5b).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Reversible phosphorylation of thylakoid proteins has been under intense research in attempts to reveal the physiological roles of such intriguing phenomenon (For recent reviews, see Gal et al. 1997; Rintamäki & Aro 2001; Wollman 2001; Aro & Ohad 2003). In intact plants in vivo, the steady-state phosphorylation levels of thylakoid proteins are determined by simultaneous activities of both the protein kinases and phosphatases, both of which are likely to be subject to distinct regulation mechanisms (Ebbert & Godde 1994; Rokka et al. 2000; Rintamäki & Aro 2001). A wealth of biochemical and genetic data suggests that the PSII core proteins and the LHCII proteins are phosphorylated by two distinct protein kinases (Gal et al. 1987; Bennett et al. 1988; Rintamäki et al. 1997), and a yet different kinase has been postulated for the phosphorylation of CP29 (Bergantino et al. 1998). A promising candidate for LHCII kinase, TAK-1, co-purifies with the cyt b6/f complex and was shown to phosphorylate LHCII proteins but not the PSII core proteins in vitro (Snyders & Kohorn 1999). TAK-1 antisence plants, however, exhibited reduced levels of phosphorylation of all PSII phosphoproteins (Snyders & Kohorn 2001), thereby challenging the target specificity of TAK-1 towards LHCII. Very recently, a different protein kinase required for LHCII protein phosphorylation, Stt7, was identified by analysing Chlamydomonas mutants that are unable to perform state transitions (Depège et al. 2003). Similar protein kinase(s) may also have a role in the phosphorylation of LHCII in higher plants: Stt7 is related to two unknown Arabidopsis serine-threonine protein kinases, both of which possess a predicted chloroplast target signal (Depège et al. 2003). As to the dephosphorylation of PSII phosphoproteins, both soluble and membrane-bound phosphatases have been found in chloroplasts (reviewed in Rintamäki & Aro 2001). However, most of these enzymes remain only poorly characterized.

Determination of the phosphorylated pool of PSII proteins is likely to give valuable information on the physiological significance of thylakoid protein phosphorylation under various environmental conditions. It is not, however, a trivial measurement (see, e.g. Vener et al. 2001), and has thus been performed only in a few studies. Maximal phosphorylation levels in illuminated leaves reported so far are 80% for the D1 protein (Rintamäki et al. 1997), 40% for CP43 (Vener et al. 2001) and only 20% for LHCII proteins (Islam 1987). We used here the immunodetection with phosphothreonine antibodies to determine changes in the steady-state phosphorylation levels of PSII proteins. Although this method does not reveal the absolute amounts of phosphorylated proteins, it has been shown to be excellent in monitoring the changes in thylakoid protein phosphorylation upon changes in environmental conditions. In the present study we investigated modulations in PSII protein phosphorylation in a range of experimental conditions, and provide evidence that the D1 and D2 proteins, CP43, CP29 and LHCII proteins belong in four distinct groups with respect to the regulatory patterns of phosphorylation/dephosphorylation.

Phosphorylation of CP29 is enhanced under harsh environmental conditions

Phosphorylation of the minor light-harvesting antenna protein CP29 was first discovered in a low-temperature-tolerant cultivar of maize, and has recently been reported to occur also in barley and in winter rye (Bergantino et al. 1995, 1998; Mauro et al. 1997, Bergo et al. 2002). In all these species, strong phosphorylation of CP29 is induced when leaves are illuminated under low temperature (Fig. 4, Bergantino et al. 1998). The kinase responsible for CP29 protein phosphorylation has been postulated to be under a redox control of the plastoquinone pool (Bergantino et al. 1995). Our experiments with winter rye suggest that the phosphorylation of CP29 is activated by reduction of the plastoquinone pool, but the activity is further enhanced via some yet unknown mechanism under low temperature in vivo. At ambient temperature, the PSII light was most efficient in inducing the phosphorylation of CP29 in vivo (Fig. 4, Pursiheimo et al. 2001). Moreover, in vitro studies by immunoblotting with a P-Thr antibody demonstrated that the CP29 phosphorylation is completely abolished by addition of DCMU, whereas DBMIB is of no effect on CP29 phosphorylation (Fig. 5b). Of particular interest was that reduced DTT, which completely down-regulates Lhcb1 and 2 protein phosphorylation, has no role in the regulation of CP29 phosphorylation (Fig. 5a). Moreover, under low temperatures in vivo, a strong phosphorylation of CP29 is observed already at relatively low reduction state of the plastoquinone pool (Figs 2 & 4b). This may be due to an inhibition of CP29 phosphatase activity or, alternatively, to an enhancement of the CP29 kinase activity, possibly via a conformational change upon low-temperature-induced change in the fluidity of the thylakoid membrane.

CP29 is a calcium-binding protein with the binding domain located at the lumenal side of the thylakoid membrane (Jegerschöld et al. 2000). The phosphorylatable threonine residue of CP29 resides within a CK2 phosphorylation motif, which is generally recognized as a target for calcium-dependent protein kinases (Testi et al. 1996), thus indirectly implicating a role for calcium in the regulation of CP29 phosphorylation. Our results from the in vitro experiments suggest that calcium may also be involved in the regulation of CP29 phosphatase activity. This interpretation was deduced from lower levels of CP29 protein phosphorylation in the presence of CaCl2 only when protein phosphatase activity was allowed during the phosphorylation reaction (Fig. 5a).

CP29 phosphorylation has been implicated in dissipation of excess excitation energy and thus providing protection against photo-inhibition under low temperature (Bergantino et al. 1995). Phosphorylation of purified CP29, however, did not induce any marked changes in fluorescence characteristics of CP29 in vitro, suggesting that CP29 phosphorylation may not be crucial for the energy dissipation process (Crimi et al. 2001). Moreover, nigericin was observed not to exert any effect over in vitro phosphorylation of CP29, indicating that the trans-thylakoid proton gradient, which is required for xantophyll-dependent dissipation of excitation energy (Demming-Adams & Adams 1996), is not a prerequisite for phosphorylation of CP29 (Fig. 5b).

LHCII protein phosphorylation is delicately modulated

Just opposite to CP29 phosphorylation, the LHCII protein phosphorylation appeared to be under a delicate control of the chloroplast redox environment, and fluctuated distinctly with changes in the redox state of PSII (Fig. 3). Increasing reduction state, induced either by a shift to high light or by a shift to low temperature, decreased the phosphorylation level of LHCII (Figs 2 & 3, see also Pursiheimo et al. 2001). The regulatory mechanism of LHCII protein phosphorylation fundamentally differs from those of CP29 and the PSII core protein phosphorylation. Unique for the LHCII kinase are the cyt b6/f-dependent activation (Vener et al. 1995, 1997) and the thioredoxin-mediated down-regulation mechanisms (Rintamäki et al. 1997). Regarding the regulation of LHCII protein phosphorylation in vivo, illumination of leaves under PSII light does not correspond to illumination under high light (Fig. 4b). It is important to note that illumination under PSII light does not allow accumulation of inhibitory thiol compounds that are crucial modulators of the activation state of the LHCII kinase under in vivo conditions (Hou et al. 2002).

Oscillations in LHCII protein phosphorylation, occurring when high-light-acclimated plants were shifted to low temperature (Fig. 5), clearly demonstrate that LHCII protein phosphorylation is a dynamic process rapidly sensing the fluctuations in the redox state of chloroplasts. Such oscillations might be related to sucrose metabolism, which slows down upon a sudden low-temperature-shift of plants (Stitt & Grosse 1988; Labate & Leegood 1989; Strand et al. 1999). This in turn results in accumulation of sugar-phosphate intermediates in the cytoplasm with distinct consequences on the metabolic state of chloroplast stroma. Such metabolic changes in chloroplasts also modify the redox environment, which is subsequently reflected in the phosphorylation status of LHCII proteins (Hou et al. 2002).

PSII core protein phosphorylation

Winter rye plants exhibit a relatively low proportion of D1 protein phosphorylation under normal growth conditions (Fig. 1), as compared, for example, with pumpkin with maximal level of D1 protein phosphorylation already at growth light intensity (Rintamäki et al. 1996). Although photo-acclimation of plants attempts to maintain homeostasis, the relationship between 1 − qP and the steady-state phosphorylation level of D1, D2 and CP43 is, however, not always so straightforward. This was evident when comparing these two parameters in winter rye plants grown at high light or at normal conditions (Fig. 1). Despite slightly elevated 1 − qP in high-light-grown plants, the phosphorylation status of PSII proteins remained at similar levels as in plants grown under normal conditions. At the moment it is not clear whether this observation is merely related to the anatomy of the differentially light-acclimated leaves, the measured 1 − qP describing only the uppermost cell layers in high- and low-light-acclimated thick and thin leaves, respectively. Another alternative is that acclimation of leaves to high light intensity involves adjustments in PSII protein phosphorylation via an additional, yet unrevealed mechanism.

It is intriguing that the CP43 protein phosphorylation is observed also in darkness (Figs 2 & 5a), quite differently from that of the D1 and D2 proteins. Such a regulatory pattern of CP43 phosphorylation may be connected to its location adjacent to the D1 protein, in the peripheral part of the PSII core complex (Zouni et al. 2001). CP43 is readily dissociated from the PSII complex during light-induced PSII photoinhibition–repair cycle (Barbato et al. 1992; Baena-Gonzalez et al. 1999). Phosphorylation of CP43 has been implicated in preventing premature dissociation of CP43 from the PSII complex in granal thylakoids (Baena-Gonzalez et al. 1999), and may thus be involved also in maintaining the integrity of PSII during the dark period. Mechanistically, the maintenance of CP43 protein phosphorylation in darkness may depend on regulation at the phosphatase level.

In conclusion, the phosphorylation pattern of thylakoid proteins under varying redox conditions in chloroplasts implies a presence of different regulatory pathways for phosphorylation and possibly also for de-phosphorylation. PSII core protein phosphorylation is robust and remains at a high level under conditions that induce reduction of the plastoquinone pool. Phosphorylation of CP29 requires even more reducing, if not stressful, conditions for induction. On the contrary, LHCII protein phosphorylation, being maximal under the low reduction state of the plastoquinone pool, has two unique features by requiring reduction of cyt b6/f for activation and being susceptible to inhibition by chloroplast thiol reductants.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This work was financially supported by Academy of Finland, Finnish Ministry of Agriculture and Forestry, and Nordisk Kontaktorgan för Jordbruksforskning.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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