ABBS 2005,38(02): Transient Decrease of Light-harvesting Complex II Phosphorylation Level by Hypoosmotic Shock in Dark-adapted Dunaliella salina

Abstract        This study investigated
the regulation of major light harvesting chlorophyll a/b protein (LHCII)
phosphorylation by hypoosmotic shock in dark-adapted Dunaliella salina cells.
When the external NaCl concentration decreased in darkness, D. salina
LHCII phosphorylation levels transiently dropped within 20 min and then
restored gradually to basal levels. The transient decrease in LHCII
phosphorylation levels was insensitive to NaF, a phosphatase inhibitor.
Inhibition of intracellular ATP production by addition of an uncoupler or an
ATP synthase inhibitor increased LHCII phosphorylation levels in D. salina
cells exposed to hypoosmotic shock. Taken together, these results indicate that
hypoosmotic shock inhibits the LHCII phosphorylation process. The related
mechanism and physiological significance are discussed.

Key words        ATP content; Dunaliella salina;
hypoosmotic shock; light harvesting chlorophyll a/b protein (LHCII)
phosphorylation

Plants have evolved many
mechanisms for adapting to changes in environmental conditions. One such ­mechanism
is the reversible phosphorylation of the major light ­harvesting chlorophyll
a/b proteins (LHCII), which ­represents a system for balancing the excitation
energy between photosystem I (PSI) and photosystem II (PSII) under fluctuating
light conditions [1–3].
Overexcitation of PSII results in a reduction of the plastoquinone pool and
subsequent plastoquinol occupation of the quinol ­oxidase site in the
cytochrome bIn the unicellular green
alga Chlamydomonas, LHCII kinase remains active in darkness due to
plastoquinone reduction by chlororespiration, which is enhanced under
conditions of ATP depletion [4,5]. In these cells, ­phosphorylation of LHCII in
darkness is accompanied by migration of the Cyt bThe halotolerant green
alga Dunaliella salina is ­distinguished for its adaptation to media
ranging in salinity from Here, we investigated the
regulation of LHCII phosphorylation by hypoosmotic shock, another stress D.
salina occasionally has to deal with in extreme osmotic environmental
conditions. New insights into this system might improve our overall
understanding of the regulation and physiological significance of LHCII
phosphorylation in the halotolerant green alga.

Materials and Methods

Plant materials and
hypoosmotic shock treatments

D. salina cells were
grown in an artificial hypersaline medium containing

ATP content measurement

D. salina cells were
suspended in

Isolation of thylakoid
membranes

Thylakoid membranes were
isolated according to the method of Kim et al. [12]. Briefly,
dark-adapted D. salina cells were suspended in sonication buffer (

Thylakoid membrane
protein analysis and immunoblotting

Thylakoid membranes were
solubilized in

Results

In thylakoid membranes,
phosphorylated proteins mainly belong to PSII core proteins and LHCII.
Different from phosphorylation of PSII core proteins, LHCII phosphorylation is
known to require not only reduction of the plastoquinone pool but also
reduction of the Cyt bPrevious evidence
indicated that hypertonic shock ­induced LHCII phosphorylation in dark-adapted D.
salina [9]. Here, we observed that 20 min of hypoosmotic shock induced the
opposite effects: LHCII phosphorylation ­levels declined as the external NaCl
concentration decreased from 1.5 to 1.0, 0.5 or As shown in Fig. 3,
transfer of D. salina cells to hypoosmotic medium induced a transient
decrease in LHCII phosphorylation levels (20 min), followed by a gradual
restoration to near basal levels within 4 h. This result indicates that the
observed decrease in LHCII phosphorylation levels is a response to transient
decreases in external salt concentration, instead of steady hypoosmotic
conditions.

The phosphorylation
level of LHCII results from the combined effects of LHCII phosphorylation by
LHCII kinase and LHCII dephosphorylation by phosphatase. To investigate the
individual contribution, we tested the effects of NaF, a phosphatase inhibitor,
in vivo and in vitro [15–17] on
hypoosmotic shock-induced decreases in LHCII phosphorylation. In the presence
of In Chlamydomonas,
LHCII phosphorylation is modulated by ATP demand. Depression of respiratory ATP
synthesis in darkness results in the activation of LHCII kinase and LHCII
phosphorylation, while ATP restoration by way of photophosphorylation under
illumination decreases LHCII phosphorylation levels [18]. Thus, we examined the
possible role of ATP content in the regulation of LHCII phosphorylation by
hypoosmotic shock in D. salina cells. It was shown that intracellular
ATP content was increased by 22% when dark-adapted D. salina cells were
subjected to hypoosmotic shock, which may be related to the ­stimulation of
respiration [19]. The ATP content increase in hypoosmotically shocked D.
salina cells was blocked by the addition of 5 mM DCCD, an ATP synthase inhibitor, or 5 mM nigericin, an uncoupler (Fig. 5). Consistent with these observations, addition of DCCD
and nigericin, which block ATP synthesis, also increased LHCII ­phosphorylation
levels in stressed D. salina [Fig.
5(B,C), h and HNig].

Discussion

Previous studies with isolated
thylakoid membranes have demonstrated that LHCII kinase is activated when the ­intersystem
electron carriers are reduced. Thus, LHCII phosphorylation is traditionally
induced by exposing the thylakoids to white light in the presence of ATP ­[14,20–23], while the inactivation of LHCII kinase is
usually ­obtained by oxidizing plastoquinone with a light-dark ­transition
[24]. However, dark-adapted green algae (Chlamydomonas and Dunaliella)
still exhibit LHCII phosphorylation activity, probably due to chlororespiration-dependent
reduction of the plastoquinone pool [8,25,26]. Nevertheless, the inactivation process in
dark-adapted green algae is not well understood. Here, our results reveal that
a decrease in external osmotic pressure leads to a transient decrease in LHCII
phosphorylation levels in dark-adapted D. salina, providing a useful
system for future in vivo studies of “dark” down-regulation of
LHCII phosphorylation in green alga.

Rokka et al. [24]
reported that abrupt transfer of ­isolated spinach thylakoids to heat-shock
induced a rapid decrease in the phosphorylation levels of the LHCII and PSII
core proteins, and attributed this effect to activation of phosphatase. Here,
our results show that hypoosmotic shock-induced decrease of phosphorylated LHCII
is insensitive to phosphatase inhibitor in dark-adapted D. salina cells.
Thus, the decrease in LHCII phosphorylation levels induced by hypoosmotic shock
in dark-adapted D. salina cells is probably due to inhibition of LHCII
phosphorylation, but not stimulation of LHCII dephosphorylation.

Bult et al. [18]
have investigated ATP control on LHCII phosphorylation in dark-adapted Chlamydomonas
and found that a decrease in ATP content activates LHCII kinase while the
inactivation process needs ATP synthesis. Here we observed that the changes in
ATP content were correlated with LHCII phosphorylation levels in
hypoosmotically shocked D. salina cells in the presence or absence of
DCCD or nigericin, suggesting that increases in ATP ­content might be related
to the inactivation of LHCII ­kinase in D. salina cells following
hypoosmotic shock. Conversely, we have reported salt-induced activation of
LHCII kinase in D. salina [9,27], and the present results reveal that
hypoosmotical treatment of D. salina cells with non-ionic medium
decreases LHCII phosphorylation ­levels more effectively than treatment with
ionic medium (Fig. 2).
Thus, transient decreases in intracellular and ­extracellular ion concentration
upon hypoosmotic shock may also contribute to the inactivation of LHCII kinase.

Notably, treating
hypoosmotically shocked cells with DCCD led to a large decrease in ATP content
without a further increase in LHCII phosphorylation levels compared with that
in un-shocked cells. This observation was ­different from the results obtained
with Chlamydomonas [18] and un-shocked D. salina cells [27] where
a decrease in ATP content caused LHCII phosphorylation. These ­results suggest
that other factors might also be involved in hypoosmotic shock-induced
decreases in LHCII phosphorylation. Zer et al. [28,29] reported that
LHCII ­phosphorylation was regulated not only by kinase activity but also by
the exposure and access of the LHCII ­phosphorylation site to protein kinase.
Maeda and ­Thompson [30] reported that chloroplast envelope ­expanded in Dunaliella
in the case of hypoosmotic shock. Taken together, the conformational changes in
chloroplast structure may be unfavorable to the access of the LHCII
phosphorylation site to LHCII kinase. However, further experiments will be required
to confirm these points.

We have reported that
salt shock decreases intracellular ATP content and induces state II transition
associated with LHCII phosphorylation [9]. On the contrary, the present results
show that hypoosmotic shock increases ­intracellular ATP content and induces a
transient decrease in LHCII phosphorylation. The dephosphorylated LHCII is
suggested to interact with PSII. Such reorganization of photosynthetic
apparatus will optimize relative efficiency of PSII activity and favor linear
electron flow upon ­re-illumination [11], and thus decrease ATP synthesis to ­balance
the ATP supply in the case of hypoosmotic shock. In this way, state transitions
enable D. salina to adjust the NADPH/ATP ratio according to
physiological conditions when it is subjected to osmotic shocks.

é

Acknowledgements

We gratefully
acknowledge Prof. Harold MEIJER for his kind help in calculating the
osmolalities of various components, Prof. Ji-Rong HUANG for his critical ­comments
on the manuscript, and Prof. Jia-Mian WEI for his kind help during the process
of experiments.

References

 1   Allen JF. Protein phosphorylation in
regulation of photosynthesis. Biochim Biophys Acta 1992, 1098: 275–335

 2   Bennett J. Protein phosphorylation in green plant
chloroplast. Annu Rev Plant Physiol Plant Mol Biol 1991, 42: 281–311

 3   Aro EM, Ohad I. Redox regulation of thylakoid
protein phosphorylation. Antioxid Redox Signal 2003, 5: 55–67

 4   Wollman FA. State transitions reveal the dynamics
and flexibility of the photosynthetic apparatus. EMBO J 2001, 20: 3623–3630

 5   Rochaix JD. Chlamydomonas, a model
system for studying the assembly and dynamics of photosynthetic complexes. FEBS
Lett 2002, 529: 34–38

 6   Ben-Amotz A, Avron M. The role of gylcerol in
the osmotic regulation of the halophilic alga Dunaliella parva. Plant
Physiol 1973, 51: 875–878

 7   Gilmour DJ, Hipkins MF, Webber AN, Baker NR,
Boney AD. The effect of ionic stress on photosynthesis in Dunaliella
tertiolecta. Planta 1985, 163: 250–256

 8    9   Liu XD, Shen YG. NaCl-induced phosphorylation
of light harvesting chlorophyll a/b proteins in thylakoid membranes from the
halotolerant green alga, Dunaliella salina. FEBS Lett 2004, 569:
337–340

10  Arnon DI. Copper enzymes in isolated
chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 1949,
24: 1–15

11  Liu XD, Shen YG. Hypoosmotic shock induces a
state I transition of photosynthetic apparatus in Dunaliella salina.
Chin Sci Bull 2004b, 49: 672–675

12  Kim JH, Nemson JA, Melis A. Photosystem II
reaction center damage and repair in Dunaliella salina (Green alga).
Analysis under physiological and irradiance-stress conditions. Plant Physiol
1993, 103: 181–189

13  Liu XD, ShenYG. Salt-induced redox-independent
phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella
salina thylakoid membranes. Biochim Biophys Acta 2005, 1706: 215–219

14  Rintam鋕i E, Salonen M, Suoranta UM,
Carlberg I, Andersson B, Aro EM. Phosphorylation of light-harvesting complex II
and photosystem II core proteins shows different irradiance-dependent
regulation in vivo. Application of phosphothreonine antibodies to
analysis of thylakoid phosphoproteins. J Biol Chem 1997, 272: 30476–30482

15  Elich TD, Edelman M, Mattoo AK.
Identification, characterization, and resolution of the in vivo
phosphorylated form of the D1 photosystem II reaction center protein. J Biol
Chem 1992, 267: 3523–3529

16  Elich TD, Edelman M, Mattoo AK.
Dephosphorylation of photosystem II core proteins is light-regulated in vivo.
EMBO J 1993, 12: 4857–4862

17  Ebbert V, Godde D. Regulation of thylakoid
protein phosphorylation in intact chloroplasts by the activity of kinases and
phosphatases. Biochim Biophys Acta 1994, 1187: 335–346

18  Bulté L,
Gans P, RebéilléF, Wollman FA. ATP control on state
transitions in vivo in Chlamydomonas reinhardtii. Biochim Biophys
Acta 1990, 1020: 72–80

19  Gilmour DJ, Hipkins MF, Boney AD. The effect
of decreasing the external salinity on the primary processes of photosynthesis
in Dunaliella tertiolecta. J Exp Bot 1984, 35: 28–35

20  Bennett J. Chloroplast phosphoproteins. The
protein kinase of thylakoid membranes is light-dependent. FEBS Lett 1979, 103:
342–344

21  Allen JF, Bennett J, Steinback KE, Arntzen CJ.
Chloroplast protein phosphorylation couples plastoquinone redox state to
distribution of excitation energy between photosystems. Nature 1981, 291: 25–29

22  Michel H, Bennett J. Use of synthetic peptides
to study the substrate specificity of a thylakoid protein kinase. FEBS Lett 1989,
254: 165–170

23  Rintamäki E, Martinsuo P, Pursiheimo S, Aro EM. Cooperative
regulation of light-harvesting complex II phosphorylation via the plastoquinol
and ferredoxin-thioredoxin system in chloroplast. Proc Natl Acad Sci USA 2000,
97: 11644–11649

24  Rokka A, Aro EM, Herrmann RG, Andersson B,
Vener AV. Dephosphorylation of photosystem II reaction center proteins in plant
photosynthetic membranes as an immediate response to abrupt elevation of
temperature. Plant Physiol 2000, 123: 1525–1536

25  Wollman FA, Delepelaire P. Correlation between
changes in light energy distribution and changes in thylakoid membrane
polypeptide phosphorylation in Chlamydomonas reinhardti. J Cell Biol
1984, 98: 1–7

26  Morgan-Kiss R, Ivanov AG, Huner NP. The
Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state
I-state II transitions. Planta 2002, 214: 435–445

27  Liu XD, ShenYG. Salt shock induces state II
transition of the photosynthetic apparatus in dark-adapted Dunaliella salina
cells. Environ Exp Bot 2006, in press

28  Zer H, Vink M, Keren N, Dilly-Hartwig HG, Paulsen
H, Herrmann RG, Andersson B et al. Regulation of thylakoid protein
phosphorylation at the substrate level: Reversible light-induced conformational
changes expose the phosphorylation site of the light-harvesting complex II.
Proc Natl Acad Sci 29  Zer H, Vink M, Shochat S, Herrmann RG,
Andersson B, Ohad I. Light affects the accessibility of the thylakoid light
harvesting complex II (LHCII) phosphorylation site to the membrane protein
kinase(s). Biochemistry 2003, 42: 728–738

30  Maeda M,