557

Absence of the major light-harvesting antenna

proteins alters the redox properties of
photosystem II reaction centres in the chlorina F2
mutant of barley
Marianna Krol, Alexander G. Ivanov, Isabelle Booij-James, Autar K. Mattoo,
P.V. Sane, and Norman P.A. Hüner

Abstract: Although the chlorina F2 mutant of barley specifically exhibits reduced levels of the major light-harvesting pol-
ypeptides associated with photosystem II (PSII), thermoluminescence measurements of photosystem reaction centre photo-
chemistry revealed that S2/S3Q–B charge recombinations were shifted to lower temperatures, while the characteristic peak
of S2Q–A charge recombinations was shifted to higher temperatures compared with wild-type (WT) barley. Thus, we show
that the absence of the major light-harvesting polypeptides affects the redox properties of PSII reaction centres. Radiolab-
eling studies in vivo and in vitro with [32P]orthophosphate or [g-32P]ATP, respectively, demonstrated that the D1 PSII re-
action centre polypeptide is phosphorylated in both the WT and the F2 mutant. In contrast with the radiolabeling results,
phosphorylation of D1 and other PSII proteins, although detected in WT barley, was ambiguous in the F2 mutant when
the phosphothreonine antibody method of detection was used. Thus, caution must be exercised in the use of commercially
available phosphothreonine antibodies to estimate thylakoid polypeptide phosphorylation. Furthermore, in membrano, the
D1 polypeptide of the F2 mutant was less susceptible to trypsin treatment than that of WT barley. The role of the light-
harvesting complex in modulating the structure and function of the D1 polypeptide of PSII reaction centers is discussed.
Key words: chlorina F2, D1, LHCII, phosphorylation, thermoluminescence.
Résumé : Même si les niveaux des polypeptides principaux du complexe collecteur de lumière du photosystème II (PSII)
sont spécifiquement faibles chez le mutant de l’orge chlorina F2, les mesures en thermoluminescence de la photochimie
du centre réactionnel du photosystème ont révélé que les recombinaisons de charges S2/S3Q–B étaient décalées à basse tem-
pérature, alors que le pic caractéristique des recombinaisons de charges de S2Q–A était décalé à haute température, compa-
rativement à l’orge sauvage. Nous montrons ainsi que l’absence des principaux polypeptides collecteurs de lumière affecte
les propriétés d’oxydoréduction des centres réactionnels du PSII. Des études in vivo et in vitro de marquage radioactif
avec le [32P]orthophosphate ou le [g-32P]ATP respectivement, ont démontré que le polypeptide D1 du centre réactionnel
du PSII est phosphorylé tant chez l’orge sauvage que chez le mutant F2. Contrairement aux résultats obtenus avec le mar-
quage radioactif, la détection de la phosphorylation de D1 et des autres protéines du PSII avec un anticorps anti-phospho-
thréonine était ambiguë chez le mutant F2, même si elle était détectée chez l’orge sauvage. Ainsi, les anticorps
commerciaux anti-phosphothréonine devraient être utilisés avec précaution pour estimer la phosphorylation des polypepti-
des du thylacoı̈de. De plus, in membrano, le polypeptide D1 du mutant F2 était moins sensible à un traitement tryptique
que le polypeptide de l’orge sauvage. Le rôle du complexe collecteur de lumière dans la modulation de la structure et de
la fonction du polypeptide D1 des centres réactionnels du PSII est discuté.
Mots-clés : chlorina F2, D1, LHCII, phosphorylation, thermoluminescence.
[Traduit par la Rédaction]

Introduction (Thornber and Highkin 1974). The mutant is deficient in

Lhcb1, Lhcb4, and Lhcb6 light-harvesting polypeptides
The chlorina F2 mutant of barley (Hordeum vulgare L. (LHCII) associated with photosystem II (PSII) and has sig-
‘Dornaria’) (chlorina F22800) is a homozygous single nuclear nificantly reduced amounts of Lhcb2, Lhcb3, and Lhcb4
gene mutant recessive for the accumulation of chlorophyll b polypeptides (Krol et al. 1995, 1999; Bossmann et al.

Received 10 February 2009. Revision received 19 March 2009. Accepted 2 April 2009. Published on the NRC Research Press Web site
at bcb.nrc.ca on 29 May 2009.
M. Krol, A.G. Ivanov, P.V. Sane, and N.P.A. Hüner.1 Department of Biology and the Biotron Experimental Climate Change Research
Centre, University of Western Ontario, 1151 Richmond Street N., London, ON N6A 5B7, Canada.
I. Booij-James and A.K. Mattoo. Henry A. Wallace Beltsville Agricultural Research Center, USDA/ARS, 10300 Baltimore Avenue,
Beltsville, MD 20705, USA.
1Corresponding author (e-mail: nhuner@uwo.ca).

Biochem. Cell Biol. 87: 557–566 (2009) doi:10.1139/O09-013 Published by NRC Research Press
558 Biochem. Cell Biol. Vol. 87, 2009

Fig. 1. Time course of D1 protein degradation in (A and B) WT and (C and D) chlorina F2 mutant barley leaves under photoinhibitory
conditions of 1200 mmol photons
m–2
s–1 at 20 8C in the (A and C) absence or (B and D) presence of chloramphenicol (CAP). Leaves of
WT and the F2 mutant were subjected to 30 mg/mL CAP for 2 h at 20 8C prior to photoinhibitory treatment. (E) Relative changes of PSII
photochemical efficiency measured as Fv/Fm in WT and the F2 mutant during exposure to high-light treatment and recovery from photoin-
hibition. Recovery of WT and F2 leaves from photoinhibition was performed at room temperature under a low light intensity of 30 mmol
photons
m–2
s–1. All data were normalized to the Fv/Fm values of control dark-adapted leaves. Means ± SE were calculated from six to eight
independent measurements.

1997). The reduced level of Lhcb polypeptides in the barley
F2 mutant results in 20% and 80% reduction of the effective
antenna sizes of PSI and PSII, respectively (Harrison et al.
1993). In addition, the lower PSII antenna size in the chlor-
ina F2 mutant is counterbalanced by an increase in the num-
ber of PSII reaction centres compared with wild-type (WT)
barley on a chlorophyll basis (Ghirardi et al. 1986). This is
consistent with freeze-fracture electron microscopy, which
demonstrated a decreased PSII unit size and increased den-
sity of PSII units in the chlorina F2 mutant compared with
WT (Simpson 1979). Thus, comparisons of the chlorina F2
mutant of barley with its WT counterpart have been impor-
tant in our initial understanding of the structure and function
of the major light-harvesting system (LHCII) of terrestrial
plants. Recent reports of the crystal structures of LHCII
from pea (Standfuss et al. 2005) and spinach (Liu et al.
2004) at 2.5 and 2.72 Å resolution, respectively, as well as
cyanobacterial PSII at 3.0 (Loll et al. 2005) and 3.5 Å (Fer-
reira et al. 2004) have provided considerable insight into the
molecular structure and organization of the PSII reaction
centre, its core antenna complexes, and its associated LHCII
as well as the regulation of light absorption, energy transfer,
and photoprotection through antenna quenching (Horton et
al. 2008).
Since the first report of thylakoid polypeptide phosphory-
lation (Bennett 1977), a major focus of photosynthesis re-
search has been not only to identify and characterize the
family of phosphopeptides present in thylakoid membranes
(Rintamäki and Aro 2001; Vener 2006, 2007) but also to
elucidate the mechanism(s) controlling their phosphorylation

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Krol et al.
Fig. 2. Thermoluminescence glow curves of (A) WT and (B)
chlorina F2 mutant barley leaves after illumination with two single
saturating turnover flashes of white light at 2 8C. Solid lines, S2/
– –
3QB charge recombinations; broken lines, S2/3QA charge recombi-
nations measured in DCMU-treated leaves (10 mmol/L). The traces
represent averages of four to six independent measurements.
(Horton and Black 1980; Allen 1992; Ohad et al. 2001;
Snyders and Kohorn 2001; Wollman 2001; Depège et al.
2003; Rochaix 2004) as well as the function of these phos-
phorylation events (Horton and Black 1980; Allen 1992;
Ohad et al. 2001; Kargul et al. 2005; Takahashi et al. 2006;
Edelman and Mattoo 2008; Kargul and Barber 2008).
Thylakoid polypeptide phosphorylation has been reported
to be an important response to imbalances in intersystem
photosynthetic electron flow between PSII and PSI. State
transitions are considered a mechanism to regulate the distri-
bution of excitation energy between PSII and PSI to ensure
equal excitation of PSII and PSI and maximum efficiency of
linear electron transport (Allen 1992; Ohad et al. 2001;
Wollman 2001; Rochaix 2004; Kargul and Barber 2008). It
appears that the interaction of the redox state of the intersys-
tem PQ pool and the cyt b6/f complex acts as the sensor for
state transitions. The binding of PQH2 to the Qo site of
cyt b6 induces the rotation of the Rieske Fe-S protein as
well as dimerization of the cyt b6/f complex (Zito et al.
1999; Wollman 2001; Finazzi 2005). The dimerization of
the cyt b6/f complex activates a thylakoid protein kinase
that regulates the reversible phosphorylation of polypeptides
associated with the mobile, peripheral LHCII of PSII, which,
559
in turn, results in the dissociation of LHCII from PSII with
its concomitant reassociation with PSI (Allen 1992; Woll-
man 2001; Takahashi et al. 2006). Two orthologous thyla-
koid serine/threonine protein kinases have been identified in
Chlamydomonas reinhardtii (STT7) and Arabidopsis thali-
ana (STN7) that are required for the regulation of state tran-
sitions (Depège et al. 2003; Bellafiore et al. 2005). In
addition, A. thaliana deletion mutants of the PSI polypepti-
des PsaE, PsaH, and PsaL exhibit an impaired ability to
undergo state transitions (Lunde et al. 2000; Haldrup et al.
2001). Although it is clear that thylakoid protein phosphory-
lation is required for the regulation of state transitions (De-
pège et al. 2003; Bellafiore et al. 2005), the direct role of
LHCII phosphorylation in the regulation of state transitions
remains equivocal (Elich et al. 1997; Rintamäki and Aro
2001; Kargul et al. 2005; Takahashi et al. 2006; Vener
2006, 2007; Kargul and Barber 2008). Recent data indicate
that the phosphorylation status of monomeric CP29 (Lhcb4)
that links trimeric LHCII to the PSII core complex actually
regulates the association/disassociation of LHCII from PSII
and its subsequent binding to PSI rather than the phosphory-
lation status of LHCII itself (Kargul et al. 2005; Takahashi
et al. 2006; Vener 2007; Kargul and Barber 2008).
The D1 protein of PSII reaction centres is encoded by
psbA, which is highly conserved in all plants (Mattoo et al.
1989; Ferreira et al. 2004). D1, and D2 encoded by psbD,
form the PSII heterodimer that binds the redox cofactors re-
quired for primary charge separation and PQ reduction and
catalyzes the photooxidation of water through the PSII-asso-
ciated oxygen-evolving complex (Mattoo et al. 1989; Li and
Burnap 2002; Ferreira et al. 2004; Merchant and Sawaya
2005; Edelman and Mattoo 2008). The D1/D2 heterodimer
is characterized by a rapid, light-dependent turnover of the
D1 reaction centre polypeptide (Edelman and Mattoo 2006).
The rapid turnover of D1 appears to have evolved to com-
pensate for the inevitable photoinactivation of PSII after a
leaf has been exposed to 106 to 107 photons (Anderson et
al. 1998). Since the photoinactivation of PSII is dependent
on the number of photons absorbed rather than the rate of
photon absorption, PSII photoinactivation can occur at low
as well as high irradiance. Two families of thylakoid pro-
teases appear to regulate the degradation of D1: the Deg
family of ATP-independent serine endoproteases (Huesgen
et al. 2005) and the FtsH family of ATP-dependent zinc
metalloproteases (Adam et al. 2006). Arabidopsis thaliana
exhibits 16 Deg genes, and four of the Deg proteases
(Deg1, Deg2, Deg5, and Deg8) are localized to the chloro-
plast. Arabidopsis thaliana also contains 12 FtsH genes,
and four of these proteases (FstH1, FstH2, FstH5, and
FstH8) are also chloroplastic. Although D1 is a thylakoid
phosphoprotein in higher plants and perhaps also in the
green alga C. reinhardtii (Turkina et al. 2006), it is not
phosphorylated in cyanobacteria (Allen 1992). The role of
reversible phosphorylation of D1 in higher plants remains
controversial. It has been suggested that the light-dependent
phosphorylation of D1 is critical in the regulation of repair
cycle of photoinhibited PSII in leaves of terrestrial plants
exposed to high irradiance (Rintamäki et al. 1997; Aro et
al. 2005) and facilitates the repair of photodamaged PSII
centres at high light (Tikkanen et al. 2008). However,
Booij-James et al. (2002) reported that in Spirodela, D1
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560
Fig. 3. (A) Immunoblot analysis of D1 protein abundance in WT barley and the chlorina F2 mutant. (B) The PSII phosphoproteins were
analyzed with phosphothreonine (PhThr) specific antibody. (C and D) Effects of Mg2+on thylakoid protein phosphorylation detected by
PhThr antibody in the presence (L) or absence (D) of light in (C) WT and (D) the F2 mutant.
phosphorylation is regulated by a circadian rhythm at both
high and low irradiance. In A. thaliana, the thylakoid protein
kinase STN8 appears to be specific for the phosphorylation
of D1, D2, and CP43 (Bonardi et al. 2005; Vainonen et al.
2005). However, Bonardi et al. (2005) reported that D1 turn-
over in stn8 mutants exposed to high light was indistinguish-
able from that of WT, indicating that D1 phosphorylation
may not be required for D1 repair. Also, it has now been
demonstrated that D1 phosphorylation is not linked to D1
degradation (Booij-James et al. 2009).
It has been assumed that the mutation associated with
chlorina F2 specifically affects the accumulation of LHCII
and core antenna polypeptides (Lhcb1, Lhcb2, Lhcb3,
Lhcb4, and Lhcb6) but does not affect the structure and
function of the PSII reaction centre. By combining the de-
tection of thylakoid phosphoproteins by in vivo and in vitro
32P-labeling with measurements of PSII photochemistry us-
ing thermoluminescence, we show that the F2 mutation has
a significant pleiotropic affect on the structure and function
of PSII reaction centres of barley. In addition, we show that
the use of phosphothreonine (PhThr) antibodies to assess
thylakoid polypeptide phosphorylation can be misleading
and should be confirmed by 32P-labeling. A possible role
for the phosphorylation of D1 is discussed.
Materials and methods
Plant material
Seeds of WT barley (H. vulgare ‘Dornaria.’) and the
chlorina F2 mutant chlorina F22800 were obtained from the
Carlsberg Research Center laboratories (Copenhagen, Den-
mark). Plants were grown for 5 weeks in controlled environ-
ment growth chambers under a 16 h photoperiod at an
Biochem. Cell Biol. Vol. 87, 2009
irradiance of 250 mmol photons
m–2
s–1 and a day/night tem-
perature of 22 8C/16 8C. All experiments were carried out
using fully expanded third and fourth leaves.
High-light treatment of fully expanded WT and chlorina
F2 leaves was performed at 20 8C and photosynthetic pho-
ton flux density of 1200 mmol photons
m–2
s–1. D-Chloram-
phenicol (1 mmol/L) was applied to WT and F2 leaves for
2 h before their exposure to high light. Photoinhibited leaves
were allowed to recover at room temperature and low irradi-
ance (50 mmol photons
m–2
s–1).
Thylakoid membrane isolation and SDS–PAGE
Thylakoid membranes from the middle portion of leaves
were isolated according to Harrison et al. (1993) in the pres-
ence of 10 mmol/L NaF. The protease inhibitors benzami-
dine (2 mmol/L) and 3-aminocapronic acid (2 mmol/L)
were also added to the isolation buffer consisting of
0.1 mol/L tricine, 0.4 mol/L sorbitol, 5 mmol/L NaCl, and
25 mmol/L MgCl2 (pH 7.5). Resuspension buffer contained
0.05 mol/L sorbitol and 5 or 50 mmol/L EDTA (pH 7.8) in
WT or F2 respectively.
SDS–PAGE was performed as described previously (Krol
et al. 1999) using12% (w/v) polyacrylamide and 6 mol/L
urea in the separating gel. The solubilization of thylakoid
proteins was carried out at room temperature for 30 min at
a SDS to chlorophyll ratio of 20:1 or SDS to protein ratio
of 4:1. Gel slots were loaded with equal amounts of protein
(20 mg).
Immunoblotting
Immunoblotting was performed by electrotransfer of the
proteins from SDS–PAGE to nitrocellulose membranes
(0.2 mm pore size) (Bio-Rad) at 5 8C for 1 h at a constant
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Krol et al. 561

Fig. 4. In vivo radiolabeling of thylakoid proteins in WT and chlorina F2 mutant barley leaves. Leaves were incubated with (A)
[35S]methionine and (B) [32P]orthophosphate for 3 and 16 h. (C) In vitro phosphorylation of isolated thylakoids from WT and the F2 mutant
was stimulated (+) by exposure to white actinic light (100 mmol photons
m–2
s–1) for 6, 10, and 60 min. D1-P, phosphorylated D1; D2-P,
phosphorylated D2; LHCII-P, phosphorylated LHCII.

Fig. 5. Effects of trypsin treatment on thylakoid protein degradation
in thylakoid membranes isolated from WT and chlorina F2 mutant
barley leaves. The cleavage of D1 protein was detected by D1 anti-
body in the control (lane 0) and trypsin-treated membranes (lanes 1,
3, and 5).
current of 100 A using a transfer medium containing Tris–
glycine buffer (0.25 mol/L Tris base and 1.92 mol/L gly-
cine) and 20% (v/v) methanol. The membranes were probed
with antibodies raised against D1 and Lhcb5 (Agrisera Swe-
den). Dilutions used were 1:2500 for D1 and 1:2000 for
Lhcb5. Polyclonal anti-PhThr antibody was purchased from
Zymed Labs, Inc., USA. Western blotting detection reagent
and ECL (Amersham) were used to immunodetect thylakoid
phosphoproteins according to the manufacture’s specifica-
tions.
Radiolabelling of thylakoid membrane proteins
Fully developed leaves from WT and the chlorina F2 mu-
tant plants were cut into 1 cm pieces and incubated with
5 mL of Huntner’s medium (Posner 1967) containing
300 mCi/mL [32P]orthophosphate or 100 mCi/mL
[35S]methionine (1 mCi = 37 kBq) for 3, 12, and 24 h under
low light intensity (50 mmol photons
m–2
s–1). Leaf samples
were washed three times with cold distilled water and isola-
tion of thylakoids was performed as described above. Thyla-
koid polypeptide phosphorylation was analyzed by SDS–
PAGE and autoradiography according to Elich et al. (1992).
In vitro phosphorylation was performed on isolated thyla-
koids at a chlorophyll concentration of 0.2 mg/mL. Isolation
buffer was supplemented with 10 mmol/L NaF. An ATP
stock solution containing [g-32 P]ATP was added to a final
concentration of 0.2 mmol/L and 50 mCi/mL. Thylakoids
were incubated in the light (50 mmol photons
m–2
s–1) at
room temperature for various times and analyzed by SDS–
PAGE and autoradiography according to Elich et al. (1992).

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562
Trypsin treatment
Thylakoids were isolated from leaves of WT and the
chlorina F2 mutant as described above. The equivalent of
25 mg of chlorophyll was suspended in 100 mL of isolation
buffer containing 50 mmol/L Tris–HCl (pH 7.5), 5 mmol/L
MgCl2, and 10 mmol/L NaCl. Trypsin (type XIII, bovine
pancreas) (Sigma) was added to the samples to a final con-
centration of 0.2 mg/mL and subsequently the samples were
incubated for 1, 3, or 5 min in the light (50 mmol photo-
ns
m–2
s–1). Proteolysis was terminated by addition of soy-
bean trypsin inhibitor (Sigma) at a ratio of 4:1 inhibitor–
protein (w/w) (Zer et al. 1999). Proteolytic experiments
were performed three times.
Chlorophyll fluorescence
Modulated chlorophyll a fluorescence of dark-adapted
leaves was measured under ambient O2 and CO2 concentra-
tions with a PAM 101 chlorophyll fluorescence measuring
system (Heinz Walz GmbH, Effeltrich, Germany) as de-
scribed previously (Ivanov et al. 2001). Chlorophyll fluores-
cence at open PSII centres (Fo) was excited by a modulated
measuring beam (650 nm, 0.12 mmol photons
m–2
s–1) at
1.6 kHz in the dark and 100 kHz in the light. Maximum flu-
orescence at closed PSII centres (Fm) in a dark-adapted state
was induced by saturating white light pulses (800 ms,
7500 mmol photons
m–2
s–1) provided by a Schott lamp
(KL 1500) (Schott Glaswerke, Mainz, Germany). Maximum
photochemical efficiency (Fv/Fm) was calculated as (Fm –
Fo)/Fm.
Thermoluminescence
Thermoluminescence (TL) measurements of intact leaves
from WT and chlorina F2 mutant barley plants were per-
formed using a custom-designed, personal-computer-based
TL data acquisition and analysis system as described previ-
ously (Ivanov et al. 2001). A xenon-discharge flash lamp
(XST103) (Heinz Walz GmbH, Effeltrich, Germany) was
used to expose the samples to a single turnover flash
(1.5 ms peak width at 50% of maximum). Dark-adapted
leaves were cooled to 2 8C prior to exposing the samples to
the flashes. To measure S2/S3Q–A recombinations, leaves
were vacuum infiltrated with 3-(3,4-dichlorophenyl)-1,1-di-
methylurea (DCMU) (20 mmol/L) in darkness before the
flash illumination. TL glow curves were collected at a heat-
ing rate of 0.6 8C/s.
Results
Sensitivity of PSII to photoinhibition in vivo
D1 protein abundance in leaves of WT and the F2 mutant
during exposure to high light (1200 mmol photons
m–2
s–1)
at 20 8C was assessed by immunoblotting using antibodies
against D1 protein (Figs. 1A–1D). As expected, the addition
of chloramphenicol to prevent protein synthesis during the
photoinhibitory treatments resulted in a gradual decrease of
D1 content in both WT and the F2 mutant (Figs. 1B and
1D) relative to controls (Figs. 1A and 1C). However, the rel-
ative abundance of D1 decreased to a greater extent in
leaves of the F2 mutant (Fig. 1D) than in WT leaves
(Fig. 1B).
Functionally, the sensitivity to photoinhibition was as-
Biochem. Cell Biol. Vol. 87, 2009
sessed concomitantly by monitoring the decrease in the
maximum photochemical efficiency of PSII measured as Fv/
Fm. The greater decrease of D1 content during photoinhibi-
tion in the F2 mutant correlated with much lower photo-
chemical efficiency of PSII, measured as Fv/Fm, in the F2
mutant (Fig. 1E) compared with WT (Fig. 1E). After 4 h of
high-light treatment, Fv/Fm values were reduced by about
95% in the F2 mutant and only 55% in the WT. The recov-
ery of Fv/Fm from photoinhibition in WT after exposure to
low light (20 mmol photons
m–2
s–1) at 20 8C was rapid and
almost complete after 1 h (Fig. 1E). In contrast, the recovery
in the F2 mutant was not complete even after 4 h at low ir-
radiance (Fig. 1E), indicating that the PSII repair cycle in
the F2 mutant was more sensitive to high light than WT bar-
ley. Thus, in contrast with our initial expectations, the re-
sults illustrated in Fig. 1 indicate that, as a result of the
absence of the major Lhcb light-harvesting polypeptides in
the F2 mutant, the PSII reaction centres in leaves of this
barley mutant are more sensitive to high light than those in
WT barley, which exhibit a complete complement of Lhcb
light-harvesting polypeptides.
In vivo thermoluminescence
PSII TL represents a very sensitive method to assess PSII
photochemistry (Inoue 1996; Vass and Govindjee 1996; Du-
cruet 2003; Sane 2004). This spectroscopic technique pro-
vides information on the activation energies associated with
the charge recombination processes between the electron ac-
ceptors QA and QB on the reducing side of PSII reaction
centres with the electron donors, the S2 and S3 states of the
oxygen evolving complex of PSII reaction centres (Inoue
1996; Ducruet 2003; Sane 2004). Flash excitation in the ab-
sence of DCMU provides information on the S2Q–B and S3Q–B
charge recombinations whereas flash excitation in the pres-
ence of DCMU provides information on the S2Q–A and S3Q–A
charge recombinations (Inoue 1996; Vass and Govindjee
1996; Ducruet 2003; Sane 2004). The temperature maxima
(TM) of the TL peaks related to the recombination of these
charge pairs reflect the activation energies and hence are a
measure of the redox potentials of the participating oxidized
and reduced donors (DeVault and Govindjee 1990).
Typical glow curves obtained following excitation with
two successive single turnover flashes in the absence and
presence of DCMU are shown in Fig. 2. The TL glow curve
of WT barley leaves in the absence of DCMU exhibited a
peak with a characteristic TM of 25.2 8C (Fig. 2A, solid
line). Since the TL peak at 25.2 8C disappeared in DCMU-
treated WT leaves (Fig. 2A, broken line), this emission band
was assigned to S2/S3Q–B charge recombinations for WT bar-
ley leaves (Vass and Govindjee 1996; Sane 2004). As ex-
pected, WT barley leaves incubated in the presence of
DCMU yielded a new TL peak with a TM of 9.3 8C
(Fig. 2A, broken line) with a lower total area and amplitude
than that observed for S2/S3Q–B charge recombinations
(Fig. 2A, solid line). Consequently, the emission band at
9.3 8C was assigned to S2/S3Q–A charge recombinations for
WT barley leaves (Vass and Govindjee 1996; Sane 2004).
Thus, the DTM between S2/S3Q–B and S2/S3Q–A charge recom-
binations was 15.9 8C in WT barley leaves, where DTM =
(TMS2/S3Q–B) – (TMS2/S3Q–A).
In contrast with WT, the total TL emission for S2/S3Q–B
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Krol et al.
charge recombinations detected in the absence of DCMU in
the F2 mutant (Fig. 2B, solid line) was about fourfold lower
than that of WT (Fig. 2A, solid line) and exhibited a TM of
15.3 8C (Fig. 2B, solid line). Furthermore, the S2/S3Q–A re-
combinations detected in the presence of DCMU in leaves
of the F2 mutant exhibited a TM of 12.2 8C (Fig. 2B, broken
line). The temperature gap (DTM = 3.1 8C) for leaves of the
F2 mutant decreased by 12.8 8C relative to that of WT bar-
ley leaves (DTM = 15.9 8C). Thus, the temperature gap be-
tween the two peaks representing S2/S3Q–B and S2/S3Q–A
charge recombinations measured as the DTM was much nar-
rower in the PSII reaction centre of the F2 mutant (3.1 8C)
compared with that of WT barley leaves (15.9 8C). The acti-
vation energy for charge recombination appears to be signif-
icantly lower in PSII reaction centres of the F2 mutant
compared with the WT barley leaves.
D1 phosphorylation
The immunoblot for the D1 polypetide using D1-specific
antibody illustrates that the abundance of this polypeptide is
similar in WT and the F2 mutant (Fig. 3A). Furthermore,
the polyclonal antibody (PhThr), used to detect threonine
phosphorylation of proteins (Rintamäki et al. 1997), sug-
gested that the D1 polypeptide, in addition to the core com-
plexes CP43 and CP47 as well as the major light-harvesting
polypeptides (Lhcb), was phosphorylated in WT barley
(Fig. 3B) but not in the F2 mutant. Since the F2 mutant is
severely impaired in the accumulation of LHCII and specific
core antenna proteins of the PSII (Krol et al. 1995, 1999),
the inability to detect phosphorylation of CP43, CP47, and
Lhcb proteins using PhThr antibodies was not surprising
(Fig. 3B). However, phosphorylation of D1 or other PSII re-
action centre proteins was undetectable in the F2 mutant us-
ing the PhThr antibody detection method.
Light and Mg2+ are important regulators of thylakoid pro-
tein phosphorylation (Bennett 1977; Rintamäki et al. 1995).
The effects of MgCl2 on in vitro phosphorylation of D1 and
Lhcb proteins under light or dark conditions were tested and
are presented in Figs. 3C and 3D. Figure 3C, lanes 1 and 3,
shows that minimal phosphorylation of both D1 and Lhcb
was detected in WT thylakoids incubated in the dark regard-
less of the presence or absence of Mg2+ in WT barley. The
results illustrated in Fig. 3C, lane 2, indicate that light and
Mg2+ stimulate phosphorylation of D1 and Lhcb in WT bar-
ley. Regardless of the experimental conditions employed,
phosphorylation of PSII-related proteins was not detected in
the F2 mutant using the PhThr antibody technique (Fig. 3D).
Thus, the absence of detectable phosphorylation of D1 in the
F2 mutant using immunoblotting with PhThr antibodies ap-
pears to be independent of the experimental conditions
under which protein phosphorylation is assayed.
We pursued this line of investigation further through ra-
diolabeling experiments. Figure 4A illustrates that both WT
and the F2 mutant are capable of synthesizing D1 as esti-
mated by in vivo pulse labeling with [35S]methionine, data
consistent with the D1 antibody based data (Fig. 1). To ana-
lyze the apparent lack of D1 phosphorylation using PhThr
antibodies, we used an in vivo [32P]orthophosphate labeling
approach (Elich et al. 1992; Booij-James et al. 2002). In
vivo pulse labeling with 32P demonstrated that, indeed, D1
is phosphorylated in both WT and the F2 mutant (Fig. 4,
563
lanes 2 and 4). While D2 is already phosphorylated after
3 h of illumination, D1 phosphorylation is prominent only
by 16 h of illumination. In contrast, Lhcb (LHCII) protein
phosphorylation decreased after 16 h of illumination in WT
plants (Fig. 4B, lanes 1 and 2) and was not detected in the
F2 mutant (Fig. 4B, lanes 3 and 4), as expected. In vitro
phosphorylation performed by incubation of thylakoid mem-
branes isolated from WT and F2 with [g-32P]ATP resulted
in rapid phosphorylation of D1 protein in both WT and the
F2 mutant (Fig. 4C), confirming that D1 is phosphorylated
in the F2 mutant. Cleary, these in vivo and in vitro radiolab-
eling data with [32P]orthophosphate and [32P]ATP, respec-
tively, are inconsistent with the inability to detect
phosphorylation of D1 and other chloroplast proteins in the
F2 mutant using the PhThr antibody method (compare
Fig. 3B with Figs. 4B and 4C).
Configuration of thylakoid proteins
How can these apparently conflicting data be reconciled?
We hypothesized that the discrepancy between the data for
D1 phosphorylation detected by the use of the PhThr anti-
body versus 32P-labeling may be accounted for by a differ-
ential accessibility of the N-terminal phosphorylation site
on D1 to the PhThr antibody in thylakoids of WT versus
the F2 mutant. To test if PSII proteins in F2 mutant thyla-
koids maintain a different configuration in the absence of
the LHCII protein, thylakoids from the barley WT and F2
mutant were incubated in the presence of trypsin and then
proteins were solubilized, fractionated by SDS–PAGE, and
immunodetection carried out with D1-specific antibody. The
immunoblots indicated that the D1 polypeptide of WT bar-
ley was more accessible and therefore relatively more sus-
ceptible to trypsin digestion than that of the F2 mutant
(Fig. 5).
Discussion
Reversible D1 phosphorylation appears to occur in all
higher plants (Edelman and Mattoo 2008). However, D1
phosphorylation does not appear to be universal, since it is
absent in cyanobacteria (Allen 1992). In addition, Rintamäki
et al. (1995) reported that D1 phosphorylation does not oc-
cur in lower plants such as the moss Ceratodon purpureus.
However, we question the validity of the conclusions regard-
ing the lack of D1 phosphorylation in lower plants (Rinta-
mäki et al. 1995; Pursiheimo et al. 1998). In the latter case,
PhThr antibodies were used to detect D1 phosphorylation.
Our data for the F2 mutant and WT barley clearly indicate
that the absence of phosphorylation as detected by the use
of PhThr antibodies does not necessarily indicate the ab-
sence of D1 phosphorylation. Thus, caution must be exer-
cised in the use of commercially available PhThr antibodies
to estimate thylakoid polypeptide phosphorylation.
How can the F2 mutation associated with chlorophyll b
biosynthesis and the accumulation of LHCII and PSII core
polypeptides affect a change in structure of the PSII reaction
centre polypeptide, D1? The localization of individual chlor-
ophyll–protein complexes within the LHCII–PSII supercom-
plex revealed that the monomeric minor light-harvesting
proteins CP29 (Lhcb4) and CP26 (Lhcb5) are localized at
the interface between the PSII core (D1/D2/CP43/CP47)
Published by NRC Research Press
564
and the peripheral major LHCII complex (Yakushevska et
al. 2003). It has also been demonstrated that CP29 is critical
for the stabilization of the oligomeric structure of PSII and
formation of the LHCII–PSII supercomplex (Yakushevska
et al. 2003). Reversible phosphorylation of the hydrophilic,
stroma-exposed N-terminal domain of CP29 causes confor-
mational changes of CP29 (Croce et al. 1996). More re-
cently, multiple phosphorylation sites at seven distinct
residues were reported for CP29 depending on the environ-
mental conditions, two of them (Thr7 and Thr33) being con-
stitutively phosphorylated (Turkina et al. 2006; Vener 2007).
The positioning of major PSII proteins within the LHCII–
PSII supercomplex model situates CP29 closest to the D1
protein, especially to its phosphorylation site (Yakushevska
et al. 2003; Kargul and Barber 2008). Apparently, this prox-
imity creates massive clustering of negatively charged phos-
phorylated sites at the interface between the D1 and CP29
proteins (Turkina et al. 2006), which may be essential for
keeping the optimal conformation of the PSII oligomeric
structure (Yakushevska et al. 2003). In light of these find-
ings, the lack of CP29 (Lhcb4) (Bossmann et al. 1997) bear-
ing at least two constitutively phosphorylated and negatively
charged residues as well as the absence of Lhcb1 and Lhcb2
within the PSII complex of the F2 mutant will affect the lo-
cal electrostatic environment of the closest neighbours in-
cluding the D1 protein. We suggest that, as a consequence
of the modified electrostatic environment, a pleiotropic ef-
fect in the tertiary structure and (or) conformation of the D1
protein may be induced by the F2 mutation.
That the tertiary structure and (or) conformation of the D1
protein of the F2 reaction centre may be altered by the F2
mutation is consistent with the differential susceptibility of
the D1 polypeptide of the F2 reaction centre to proteolysis
by trypsin (Fig. 5). Functionally, it is correlated with signifi-
cant alterations in PSII photochemistry as detected by TL
(Fig. 2). The DTM for S2Q–B and S2Q–A recombinations were
reduced from about 16 8C for WT to 3 8C for the PSII reac-
tion centre of the F2 mutant. This may be interpreted to in-
dicate that the activation energy for these charge
recombination events has been significantly lowered in the
F2 reaction centre compared with WT as a consequence of
alternations in the redox potentials of QA and QB (Ivanov et
al. 2008). These TL results are consistent with enhanced re-
action centre quenching (Ivanov et al. 2006, 2008). Thus, we
suggest that D1 phosphorylation may be associated with an
increased potential for reaction centre quenching. Thus,
although the F2 mutation clearly affects the accumulation
of the major LCHII and PSII core antenna polypeptides,
this mutation appears to alter the redox properties of the
PSII reaction centre, possibly via an effect on the conforma-
tion of the supramolecular complex of PSII.
Acknowledgements
This work was financially supported by a grant from the
Natural Sciences and Engineering Research Council of Can-
ada to N.P.A.H. Mention of trade names or commercial
products in this article is solely for the purpose of providing
specific information and does not imply recommendation or
endorsement by the US Department of Agriculture.
Biochem. Cell Biol. Vol. 87, 2009
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