ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 943-955 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 1028-1042.
943
The Effect of Hydrogen Peroxide on the Redistribution
of Antenna Complexes Between Photosystems
in Higher Plants
Nikolai V. Balashov
1
, Maria M. Borisova-Mubarakshina
1
,
and Daria V. Vetoshkina
1,a
*
1
Institute of Basic Biological Problems, Pushchino Scientific Center for Biological Research,
Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
a
e-mail: vetoshkina_d@mail.ru
Received May 5, 2025
Revised July 1, 2025
Accepted July 1, 2025
Abstract— One of the adaptive mechanisms used by photosynthetic organisms in response to changing light
conditions is redistribution of antenna complexes between the photosystems, a process known as state tran-
sitions (ST). This mechanism allows to regulate the amount of light energy absorbed by the photosystems.
Numerous studies have reported inhibition of ST at the elevated light intensity; however, the mechanism
underlying this process is still debated. We evaluated the effect of H
2
O
2
at various concentrations on the ST
process in functionally active thylakoids isolated from Arabidopsis thaliana leaves and investigated which
stage of this process is affected by H
2
O
2
. To assess the extent of ST, we measured low-temperature chloro-
phylla fluorescence spectra (650-780nm) and calculated the F745/F685 ratio, whose changes can serve as an
indicator of ST progression. H
2
O
2
inhibited ST under the low-intensity light conditions and, furthermore, led
to a decrease in the accumulation of phosphorylated Lhcb1 and Lhcb2 proteins involved in ST. This suggests
that the observed ST inhibition resulted from the suppression of STN7 kinase activity. Importantly, H
2
O
2
in the
tested concentrations did not affect the electron transport rate, indicating that the inhibition of STN7 kinase
activity was not associated with suppression of the photosynthetic electron transport chain (PETC) activity.
The treatment with H
2
O
2
did not reduce the level of phosphorylated D1 protein (a product of phosphorylation
by the thylakoid STN8 kinase). Taken together, these results demonstrate for the first time the mechanism by
which H
2
O
2
inhibits STN7 kinase activity and, consequently, the process of ST.
DOI: 10.1134/S0006297925601443
Keywords: photosynthesis, phosphorylation, state transitions, light-harvesting antenna, hydrogen peroxide
* To whom correspondence should be addressed.
INTRODUCTION
Environmental changes trigger adaptive mecha-
nisms, thus enabling plants to regulate the amount of
light quanta absorbed by the photosynthetic apparatus
(PA). The efficiency of light energy absorption is de-
termined by the functional size of the light-harvesting
antenna of each photosystem, which can dynamically
adjust in response to varying environmental condi-
tions. The light-harvesting antenna complexes (LHC)
of the photosystems are pigment–protein complexes.
In higher plants, they consist of Lhcb1-6 proteins in
photosystem  II (PSII) and Lhca1-4 proteins in photo-
systemI (PSI) capable of non-covalent binding of chlo-
rophylls (Chls) and carotenoids [1-3].
One of the key adaptive responses to changes in
the light conditions which allows simultaneous regu-
lation of the functional antenna size in both PSI and
PSII, is a process known as state transitions (ST). This
process is considered to be a mechanism by which
plants adjust to changes in the spectral composition of
light. Under illumination predominantly exciting PSII,
the thylakoid membrane-associated enzyme STN7 ki-
nase becomes activated and phosphorylates the outer
light-harvesting complex II (LHCII) proteins, resulting
in the electrostatic repulsion of the antenna complexes
BALASHOV et al.944
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
from PSII, followed by their association with PSI, and
formation of state  2. Subsequent shading or illumina-
tion with light that predominantly excites PSI leads to
the dephosphorylation of these proteins and return of
LHCII to PSII, and formation of state  1. Dephosphor-
ylation is catalyzed by the PPH1/TAP38 phosphatase,
also associated with the thylakoid membrane [4, 5].
Later studies confirmed that ST can proceed under
low-intensity white light also (LL).
Substantial progress has been made in under-
standing the molecular mechanism of ST, including
identification of key components involved in this
process. Loosely bound LHCII trimeric complexes
(L-trimers) migrate between the photosystems, while
strongly (S-) and moderately (M-) bound trimers re-
main associated with PSII [6, 7]. The L-trimers consist
of Lhcb1 and Lhcb2 proteins, both of which undergo
phosphorylation. However, the roles of these proteins
differ: phosphorylated Lhcb2 (Lhcb2-P) is crucial for
the LHCII association with PSI [8, 9], whereas phos-
phorylated Lhcb1 (Lhcb1-P) is involved in the disas-
sembly of grana stacking [10, 11].
Exposure to high-intensity light (HL) leads to
a reduced phosphorylation of LHCII proteins, and,
consequently, suppression of LHCII migration to PSI,
meaning that the ST is inhibited under these condi-
tions [12-14]. In our previous work, we demonstrated
that the light intensity at which ST become inhibit-
ed varies between plant species [15]. For example,
in the model plant Arabidopsis thaliana, inhibition
of ST occurs at the light intensities above 300  μmol
quanta∙m
−2
∙s
−1
, whereas in barley, this level is above
800 μmol quanta∙m
−2
∙s
−1
.
It has been suggested that HL inhibits STN7 ki-
nase activity, resulting in blocking plants in state 1, in
which all proteins of LHCII complexes remain bound
to PSII. However, the exact mechanism of STN7 inhi-
bition under HL remains unclear [13, 14, 16]. STN7 is
a serine/threonine protein kinase containing a single
transmembrane domain, with its N-terminus exposed
to the thylakoid lumen and its catalytic domain and
C-terminus located on the stromal side. Both termini
contain two conserved Cys residues [17, 18]. Notably,
one Cys residue at the N-terminus is directed towards
the thylakoid membrane, while the other is exposed to
the lumen. It was shown that STN7 interacts with the
cytochrome b6f complex via the Rieske protein [17],
and the site of STN7 interaction is located between the
residues 51 and 97 in the PetC protein of this complex
[19]. The binding of reduced plastoquinone (plastoqui-
nol) to the binding site in the cytochrome b6f com-
plex activates STN7 kinase [20, 21]: the conformational
changes in the binding pocket lead to the reposition-
ing and exposure of the N-terminal Cys residue to the
lumen. Under these conditions, the two Cys residues
are brought into proximity in the lumen, enabling for-
mation of an intramolecular disulfide bond and then
of an intermolecular disulfide bond, resulting in cova-
lently linked the STN7 dimer, which ultimately leads
to the kinase activation [19].
The available data on whether STN7 kinase is ac-
tive in its oxidized or reduced state remain contradic-
tory. Wu et al. [22] demonstrated that the conserved
luminal cysteine residues of STN7 are critical for
maintaining the oxidized state of this kinase, which is
necessary for its enzymatic activity. In contrast, Singh
et al. [23] reported that the STN7 kinase remains ac-
tive in the presence of a reducing agent, indicating
that the reduced state of cysteines is required for the
catalytic function. However, it remains unclear wheth-
er stromal or luminal cysteine residues are responsi-
ble for regulating the STN7 activity. Using site-directed
mutagenesis, Shapiguzov et al. [19] showed that sub-
stitution of the stromal cysteines in STN7 kinase did
not impair the ST process, whereas substitution of its
luminal cysteines resulted in inhibition of ST.
Several studies have suggested that thioredoxins
play a key role in the inhibition of STN7 kinase under
HL conditions by reducing stromal Cys residues [24,
25]. For example, in plants overexpressing thioredox-
in m, phosphorylation of Lhcb1 and Lhcb2 proteins
was suppressed even under LL [26]. So far, the in-
volvement of thioredoxins in the STN7 regulation still
has to be proven. It was also proposed that superoxide
anion radical, which is potentially produced during
plastoquinol oxidation at the quinol oxidation site of
the cytochrome b6f complex, may influence the ac-
tivity of Chlamydomonas reinhardtii STT7 kinase, an
equivalent of STN7 from higher plants [23]. Coordi-
nated oxidation of plastoquinol at this site is expected
to reduce the likelihood of superoxide anion radical
generation in the cytochrome b6f complex.
Hydrogen peroxide is known to oxidize Cys res-
idues in various enzymes [27, 28], including kinases
[29, 30]. In a number of studies, including those con-
ducted by our group, it has been proposed that hy-
drogen peroxide produced in chloroplasts under HL
conditions may affect the STN7 kinase activity [23, 31-
33]. Roach et al. [34] demonstrated the effect of hy-
drogen peroxide on the STT7 activity in C.  reinhardtii.
However, this hypothesis has yet to be experimentally
verified in the context of hydrogen peroxide effect on
the STN7 activity in higher plants. Therefore, the aim
of this study was to investigate the effect of hydrogen
peroxide on the ST process and activity of STN7 kinase
in the isolated thylakoids of A.  thaliana.
MATERIALS AND METHODS
Plant growth conditions. Wild-type A.  thaliana
plants (Columbia ecotype) were grown in soil under
EFFECT OF H
2
O
2
ON STN7 KINASE ACTIVITY 945
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
controlled conditions in a climate chamber at a light
intensity of 80-100 μmol quanta∙m
−2
∙s
−1
with a 10 h
light/14 h dark photoperiod at 21°C. Thylakoids were
isolated from 6-8-week-old plants.
Thylakoid isolation. Intact, photosynthetically ac-
tive thylakoids were isolated as described by Casazza
et al. [35] with modifications: bovine serum albumin
(BSA) was included only in the homogenization medium
and the first washing buffer. Thylakoids were extract-
ed from A.  thaliana leaves after the dark period. The
leaves were detached and incubated in ice-cold wa-
ter in the dark for 30 min. Homogenization was per-
formed in a buffer containing 20mM Tricine (pH 8.4),
5mM MgCl
2
, 5mM EDTA, 10mM NaCl, 0.4M sorbitol,
0.5% BSA, and 0.1% sodium ascorbate. The homoge-
nate was filtered through two layers of nylon mesh
and centrifuged at 3500g for 3 min. The supernatant
was discarded, and the pellet was resuspended in a
washing buffer containing 0.4  M sorbitol, 0.5%  BSA,
5  mM MgCl
2
, 5  mM  EDTA, 10  mM  NaCl, and 50  mM
Hepes (pH7.6), followed by the second centrifugation.
The washing procedure was repeated using the same
buffer without BSA. The pellet was then resuspend-
ed in a shock buffer without sorbitol (50  mM  Hepes,
pH  7.6; 5  mM  MgCl
2
; 5  mM  EDTA; and 10  mM  NaCl),
followed by centrifugation. The final pellet was resus-
pended in a small volume (0.5-1ml) of buffer (50  mM
Hepes, pH 7.6; 400 mM sorbitol; 5 mM MgCl
2
; 5  mM
EDTA; 10 mM NaCl). During the experimental proce-
dures, thylakoids were kept on ice and in the dark;
only freshly isolated thylakoids were used.
Chlorophyll content determination. The content
of chlorophyll (Chl) was determined in 96% ethanol
extracts as described in [36].
Quantification of Lhcb1-P, Lhcb2-P, and D1-P
proteins. The reaction mixture contained thylakoids
(Chl concentration, 50  μg/ml), 100  mM sucrose, 2 mM
ATP, 1 μM gramicidin D, and 50 mM Hepes (pH 7.6).
Where indicated, hydrogen peroxide (25 or 50μM) or
catalase (300U/ml) were added. To induce state 2 con-
ditions in the thylakoid PA and promote phosphoryla-
tion of Lhcb1 and Lhcb2 proteins, the thylakoid sus-
pension was illuminated for 5min with a low-intensity
red light (LED, λ  =  640 nm, 60 μmol quanta∙m
−2
∙s
−1
,
LL conditions). For the HL experiments, illumination
was performed using red light (LED, λ  =  640 nm) at
800μmol quanta∙m
−2
∙s
−1
. All incubations were carried
out with constant stirring. In experiments with the
dark-adapted thylakoids, two treatments were used:
the “dark” variant, when samples were frozen imme-
diately, and the “dark  +  ATP” variant, when samples
were frozen after 5-min incubation with ATP in the
dark. After the incubation, 5 mM NaF was added to
inhibit the activity of phosphatases and to preserve
protein phosphorylation levels. The resulting samples
were used for Western blot analysis.
Protein electrophoresis was conducted under de-
naturing conditions using 15% polyacrylamide gel in
a Mini-PROTEAN cell (Bio-Rad, USA). The samples of
thylakoid membranes were dissolved in the loading
buffer (pH 6.8) containing 200 mM Tris-HCl, 8% SDS,
0.4% bromophenol blue, and 400 mM DTT, heated at
97°C for 5 min, and centrifuged at 13,000g for 5 min
in a MiniSpin centrifuge (Eppendorf, Germany). The
resulting supernatants were loaded on the gel (1.0 μg
of Chl per lane). Precision Plus Protein Kaleidoscope
(10-250 kDa; Bio-Rad) were used as molecular weight
markers. After electrophoresis, the proteins were
transferred to PVDF membranes (Bio-Rad) using a
Mini Trans-Blot Cell wet blotting system (Bio-Rad).
Primary antibodies were rabbit polyclonal antibodies
against Lhcb1-P and Lhcb2-P proteins and D1-P (phos-
phorylated D1 protein). Secondary antibodies were
goat anti-rabbit IgG conjugated with alkaline phos-
phatase (Agrisera, Sweden). Visualization of the reac-
tion was performed using a substrate kit for alkaline
phosphatase conjugates (Bio-Rad). Immunodetection
results were scanned and analyzed using ImageJ and
OriginPro software.
Measurements of Chl a fluorescence spectra
at 77 K. The thylakoid membranes were incubated as
described previously (see section “Quantification of
Lhcb1-P, Lhcb2-P, and D1-P proteins”) prior to fluo-
rescence measurements.
Immediately after the dark adaptation (state 1)
or light treatment (state 2), the thylakoid suspensions
were frozen in liquid nitrogen, and low-temperature
Chl a fluorescence spectra were recorded. For the
dark-adapted thylakoids, two treatment variants were
applied: in the “dark” variant, the samples were fro-
zen immediately, while in the “dark  +  ATP” variant,
the samples were incubated for 5min with ATP in the
dark prior to freezing. Where indicated, 10 mM NaF
was added before illumination to inhibit the phospha-
tase activity. The excitation wavelength was 440 nm,
and fluorescence spectra were recorded in the range
of 650-800nm using an Ocean QePRO spectrofluorom-
eter (Ocean Optics, USA). The fluorescence maximum
at 745 nm (F745) indicated excitation energy transfer
to PSI, while the peak at 685nm (F685) corresponded
to PSII [37]. A decrease in F685 and a concomitant
increase in F745 indicated a transition from state 1
to state  2. The F745/F685 ratio (PSI/PSII chlorophyll a
fluorescence peaks ratio) was used as a relative indi-
cator of the ST extent.
Measurement of oxygen uptake rate. The rate
of oxygen consumption was measured in a tempera-
ture-controlled glass chamber at 21°C using a Clark-
type pO
2
electrode. Illumination was provided by a
red LED light source (λ = 640nm) at the light intensity
of 60 μmol quanta∙m
−2
∙s
−1
. The incubation medium
contained thylakoids (Chl concentration, 20  μg/ml),
BALASHOV et al.946
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
100  mM sucrose, 2  mM ATP, 1  μM gramicidin  D, and
50  mM Hepes (pH  7.6). Where indicated, hydrogen
peroxide (25 or 50  μM) was added to the thylakoid
suspension.
Statistical analysis was performed with the Orig-
inPro software. Analysis of variance (ANOVA) was con-
ducted, followed by comparison of mean values using
the Holm–Bonferroni method. The differences were
considered statistically significant at p < 0.05.
RESULTS
Effect of hydrogen peroxide on the low-tem-
perature Chl  a fluorescence spectra. A classical
method for assessing the PA transition to state 2 is
recording of low-temperature (77  K) Chl  a fluores-
cence spectra. This approach enables simultaneous
monitoring of Chl fluorescence changes in both pho-
tosystems. Upon illumination, an increase in the fluo-
rescence maximum at 745 nm (F745) accompanied by
a decrease in the maximum at 685 nm (F685) rela-
tive to the dark-adapted spectrum, indicates migration
of the LHCII from PSII to PSI, i.e., proceeding of ST.
To quantify the extent of ST, we used the F745/F685
ratio.
As shown in Table  1, illumination of thylakoid
suspensions for 5  min with the red LL (60  μmol
quanta∙m
−2
∙s
−1
) resulted in a statistically significant
increase in the F745/F685 ratio from 3.4  ±  0.2 (in the
“dark  +  ATP” condition) to 4.1  ±  0.1, indicating induc-
tion of state 2. However, when samples were illumi-
nated in the presence of hydrogen peroxide at con-
centrations of 25μM or 50μM, no significant increase
in the F745/F685 ratio compare to dark was observed,
suggesting inhibition of the ST process by hydrogen
peroxide at used concentrations.
To detect the extent of PA transition to state 2
without possible reverse transition to state 1 under
our experimental conditions, NaF (phosphatase in-
hibitor) was used. Inhibition of TAP38/PPH1 phospha-
tase prevents Lhcb 1 and Lhcb 2 proteins dephos-
phorylation and return of the antenna complexes
containing these proteins to PSII, i.e., prevents transi-
tion back to state 1. However, under our conditions,
addition of NaF during illumination did not lead to
changes in the F745/F685 ratio, indicating that tran-
sition to state 2 had proceeded to its maximum. The
F745/F685 ratio after 5-min LL illumination of the
thylakoids in the presence of 50µM H
2
O
2
and NaF did
not differ from that observed in the absence of NaF.
These results confirm that H
2
O
2
inhibited PA transi-
tion to state 2 and did not affect its reverse transition
to state 1 under the given conditions.
We also observed that incubation of A. thaliana
thylakoids in the dark in the presence of ATP with
stirring resulted in a significant increase in the F745/
F685 ratio, which may reflect either the occurrence
of ST in the dark or the activation of other processes
altering the F745/F685 ratio.
The effect of hydrogen peroxide on the accu-
mulation of phosphorylated Lhcb1, Lhcb2, and D1
proteins. The first step in the PA transition to state
2 is activation of STN7 kinase and phosphorylation of
Lhcb1 and Lhcb2 proteins. Considering the effect of
H
2
O
2
on the F745/F685 ratio, we investigated how hy-
drogen peroxide affected accumulation of these phos-
phorylated proteins in A.  thaliana thylakoid mem-
branes under LL illumination (Fig. 1). Incubation of
thylakoids in the dark with stirring in the presence
of ATP led to a significant increase in the Lhcb1-P
and Lhcb2-P levels, which was consistent with the
results of the low-temperature recordings of Chl a
fluorescence spectra (Table 1). The accumulation was
more pronounced for Lhcb2-P (Fig. 1). These findings
suggest either spontaneous phosphorylation of Lhcb1
and Lhcb2 in the dark in the presence of ATP in the
medium or the possibility of STN7 activation in the
dark resulting in Lhcb1 and Lhcb2 phosphorylation.
Illumination resulted in a significant light-depen-
dent accumulation of Lhcb1-P and Lhcb2-P in iso-
lated thylakoids compared to the “dark  +  ATP” vari-
ant. Incubation of the thylakoid suspension under
Table 1. Effect of hydrogen peroxide on the F745/F685
ratio
Thylakoid incubation conditions F745/F685
Dark 2.0 ± 0.2 c
Dark  +  ATP 3.4 ± 0.2 b
LL 4.1 ± 0.1 a
LL  +  NaF 4.1 ± 0.1 a
LL  +  25 μM H
2
O
2
3.6 ± 0.2 ab
LL  +  50 μM H
2
O
2
3.6 ± 0.1 b
LL  +  50 μM H
2
O
2
  +  NaF 3.7 ± 0.1 b
Note. The incubation medium contained: thylakoids (Chl
concentration, 50µg/mL), 100 mM sucrose, 2 mM ATP, 1 µM
gramicidin D, and 50 mM HEPES (pH 7.6); where indicated,
hydrogen peroxide was added to the thylakoid suspension at
the concentrations of 25 and 50µM. To initiate the transition
to state 2, the suspension was illuminated for 5 min (LED,
λ  =  640 nm; light intensity, 60 µmol quanta∙m
−2
∙s
−1
), which
corresponded to the LL variant. The data are presented as
mean  ±  standard error of mean (SEM); statistically significant
differences were determined using ANOVA followed by com-
parison of mean values using the Holm–Bonferroni method
(p <0.05) and are indicated by different letters (a, b, and c),
values sharing the same letter are not significantly different
(p ≤ 0.05).
EFFECT OF H
2
O
2
ON STN7 KINASE ACTIVITY 947
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig.  1. The effect of hydrogen peroxide on the accumulation of Lhcb1-P (a) and Lhcb2-P (b). The incubation medium
contained: thylakoids (Chl concentration, 50 µg/mL), 100 mM sucrose, 2 mM ATP, 1 µM gramicidin D, and 50 mM HEPES
(pH 7.6). Where indicated, hydrogen peroxide (25 or 50 µM) was added to the thylakoid suspension. To initiate the tran-
sition to state 2, the suspension was illuminated for 5 min (LED, λ = 640 nm; light intensity, 60 µmol quanta∙m
−2
∙s
−1
).
The figure shows representative Western blots and densitometric analysis of the corresponding bands obtained in at least
three independent experiments. For each individual membrane, the optical density of the band in the LL variant was taken
as 100%. The data are presented as mean ± SEM; statistically significant differences were determined using ANOVA followed
by comparison of mean values using the Holm–Bonferroni method (p< 0.05) and are indicated by different letters (a, b, c,
d, e), bars sharing the same letter are not significantly different (p < 0.05).
illumination in the presence of 25 or 50 µM H
2
O
2
led
to a significant suppression of Lhcb1-P and Lhcb2-P
accumulation vs. samples illuminated in the absence
hydrogen peroxide; this effect was more pronounced
at the higher H
2
O
2
concentration (50 µM) (Fig. 1). At
this concentration, H
2
O
2
fully suppressed the light-
dependent accumulation of Lhcb1-P that reached the
levels similar to those observed in the “dark  +  ATP”
variant with stirring. The changes in the Lhcb2-P con-
tent were less pronounced in the “dark  +  ATP” with
stirring comparing to those registered in the dark in
the absence of ATP and stirring. We also observed
more significant changes in the light-dependent accu-
mulation of Lhcb2-P than in the Lhcb1-P, as well as a
higher inhibitory effect of H
2
O
2
addition. The observed
differences between the accumulation of Lhcb1-P and
Lhcb2-P may be related to their distinct functional
roles in the ST process (see “Discussion” section).
The amounts of Lhcb1-P and Lhcb2-P detected in
the illuminated samples reflected the balance between
the two opposite processes: phosphorylation by STN7
kinase and dephosphorylation by TAP38/PPH1 phos-
phatase. The suppression of light-dependent accumu-
lation of phosphorylated proteins could result from
either decreased kinase activity or increased phos-
phatase activity. To exclude the influence of H
2
O
2
on
the activity of TAP38/PPH1 phosphatase, we assessed
the light-dependent accumulation of phosphorylated
Lhcb1-P and Lhcb2-P in the presence of NaF also. The
inhibitory effect of H
2
O
2
on the light-induced accumu-
lation of phosphorylated proteins was not affected by
NaF (data not shown). Based on these results, we con-
cluded that hydrogen peroxide at the tested concentra-
tions specifically reduces the activity of STN7 kinase.
During illumination of isolated thylakoids, elec-
trons derived from water photolysis in PSII are trans-
ferred from components of the photosynthetic electron
transport chain (PETC), primarily those belonging to
PSI, to molecular oxygen as the only available acceptor,
since no alternative electron acceptors were used in
our experiments. Electron transfer to oxygen produces
superoxide anion radicals, which then generate hydro-
gen peroxide either through spontaneous dismutation
of superoxide anion radicals or via reduction of super-
oxide anion radical by plastoquinol (see “Discussion”
section). An increase in the light intensity leads to the
activation of H
2
O
2
production in thylakoids, as more
electrons enter the PETC and reduce molecular oxygen
[38]. Therefore, as an alternative approach to evalu-
ating the effect of hydrogen peroxide on the ST pro-
ceeding, we employed the HL conditions that ensured
an elevated rate of H
2
O
2
generation in the thylakoids.
BALASHOV et al.948
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. The effect of catalase on the light-dependent accumulation of Lhcb1-P (a) and Lhcb2-P (b) under HL. The incuba-
tion medium contained thylakoids (Chl concentration, 50 µg/mL), 100 mM sucrose, 2 mM ATP, 1 µM gramicidin D, and
50 mM HEPES (pH 7.6). Where indicated, catalase (300 U/mL) was added to the thylakoid suspension prior to illumination.
The “dark  +  ATP” variant corresponds to thylakoids incubated for 5 min in the above-mentioned medium with stirring.
The samples were illuminated for 5 min (LED, λ  =  640 nm); LL, 60 µmol quanta∙m
−2
∙s
−1
; HL, 800 µmol quanta∙m
−2
∙s
−1
.
The figure shows representative Western blots and densitometric analysis of the corresponding bands obtained in at least
three independent experiments. For each membrane, the optical density of the LL band was taken as 100%. The data are
presented as mean ± SEM. Statistically significant differences were determined using ANOVA followed by comparison of
mean values using the Holm–Bonferroni method (p < 0.05) and are indicated by different letters (a, b, c), bars sharing the
same letter are not significantly different (p < 0.05).
Experiments were conducted in the presence or ab-
sence of catalase, an enzyme that decomposes hydro-
gen peroxide into water and molecular oxygen. It is
impossible to use low-temperature Chl  a fluorescence
spectra upon HL illumination to assess the induction
or inhibition of ST, since we have previously demon-
strated that the HL illumination leads to a strong in-
crease in the F745/F685 ratio unrelated to the ST [15].
Therefore, we evaluated the effect of HL only on the
light-dependent accumulation of Lhcb1-P and Lhcb2-P
proteins.
Illumination of the thylakoid suspensions in HL
(800 µmol quanta∙m
−2
∙s
−1
) failed to induce a marked
increase in the Lhcb1-P levels, but caused a statisti-
cally significant accumulation of Lhcb2-P compared to
that under the “dark  +  ATP” conditions (Fig. 2). How-
ever, the light-dependent accumulation of Lhcb2-P
upon HL illumination was significantly lower than un-
der the LL conditions (Fig.2). The oxygen uptake rate
under HL was ~11µmol O
2
/mg Chl per hour, i.e., nearly
two times higher than under LL conditions. The ad-
dition of catalase under HL resulted in a significant
increase in the light-dependent accumulation levels of
both Lhcb1-P and Lhcb2-P comparable to those ob-
served under LL (Fig.2), suggesting that the inhibition
of Lhcb1-P and Lhcb2-P accumulation under HL is a
peroxide-dependent process. The inhibitory effect of
HL was less pronounced than that observed in the
presence of exogenous H
2
O
2
. It can be assumed that
under the used illumination conditions, the amount
of generated H
2
O
2
was lower than the amount of ex-
ogenously added H
2
O
2
, but the hydrogen peroxide
produced under HL was generated directly by PETC
components, i.e., in close proximity to STN7 kinase.
Several kinases have been identified in A.  thali-
ana thylakoid membranes, including STN8 (STN7 ki-
nase paralog), which phosphorylates the PSII core
protein D1 [39, 40]. STN7 and STN8 are the main con-
tributors to the thylakoid membrane phosphorylation
and are essential for the rapid response to changes in
the redox status of the chloroplast PETC. It is known
that the maximum accumulation of phosphorylated D1
protein (D1-P) occurs under HL, i.e., under conditions
when H
2
O
2
generation in chloroplasts is upregulated.
It can be assumed that the activity of STN8 invivo is
unlikely to be inhibited by hydrogen peroxide, which
supports the hypothesis that H
2
O
2
specifically affects
the STN7 kinase and does not influence the activity
of STN8. To examine this suggestion, we assessed the
effect of H
2
O
2
at the concentrations used in this study
on the STN8 activity by analyzing D1-P accumulation
in the thylakoid membranes (Fig. 3).
EFFECT OF H
2
O
2
ON STN7 KINASE ACTIVITY 949
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 3. The effect of varying light intensities (a) and hydrogen peroxide addition (b) on the D1-P protein accumulation.
The incubation medium contained thylakoids (Chl concentration, 50 µg/mL), 100 mM sucrose, 2 mM ATP, 1 µM gramicidin
D, and 50 mM HEPES (pH 7.6). Where indicated, hydrogen peroxide (25 or 50 µM) was added to the thylakoid suspension
prior to illumination. Illumination was performed for 5 min (LED, λ = 640nm); LL, 60 µmol quanta∙m
−2
∙s
−1
; HL, 800 µmol
quanta∙m
−2
∙s
−1
. Dark, dark-adapted thylakoids frozen without prior incubation; Dark  +  ATP, thylakoids incubated for 5min
in the above described medium with stirring. The figure shows representative Western blots membranes and densitometric
analysis of the corresponding bands obtained in at least three independent experiments. For each membrane, the optical
density of the LL band was taken as 100%. The data are presented as means ± SEM. Statistically significant differences
were determined using ANOVA followed by comparison of mean values using the Holm–Bonferroni method (p < 0.05) and
are indicated by different letters (a, b).
Dark-adapted thylakoids (dark in Fig.3) contained
significant amounts of D1-P, in contrast to Lhcb1-P and
Lhcb2-P proteins. The amounts of D1-P in the “dark”
samples with or without ATP did not differ. Illumi-
nation of the thylakoid suspension with LL caused
no statistically significant increase in the D1-P accu-
mulation; a noticeable elevation in the D1-P content
was detected only after exposure to HL (Fig. 3a). No
inhibition of D1-P accumulation was observed after
addition of H
2
O
2
(Fig. 3b). Interestingly, illumination
in the presence of 50 µM H
2
O
2
led even to a consid-
erable increase in the D1-P accumulation compared
to illumination in the absence of hydrogen peroxide.
The level of D1-P after illumination with LL in the
presence of 50 µM H
2
O
2
was comparable to its lev-
el observed after exposure to HL (Fig. 3). Hence, no
inhibition of STN8 kinase by hydrogen peroxide was
detected at the concentrations used in our study.
Assessment of H
2
O
2
impact on the electron
transport rate (ETR) in isolated thylakoids. The
above-described effects of H
2
O
2
may be related not
only to its direct action on the STN7 kinase but also to
the indirect influence of H
2
O
2
on the ETR. To examine
whether hydrogen peroxide at the applied concentra-
tions affected ETR in the thylakoid membranes, we
measured the oxygen uptake rate in a suspension of
isolated thylakoids under the same conditions as those
used for evaluating ST and accumulation of phosphor-
ylated proteins.
H
2
O
2
at the concentrations used in the study did
not decrease the rate of oxygen uptake, i.e., had no
negative effect on the ETR in the PETC of isolated
thylakoids (Table 2). Therefore, the observed H
2
O
2
-
induced inhibition of ST was not associated with the
effect of hydrogen peroxide on the ETR.
Table 2. Effect of hydrogen peroxide addition on the
oxygen uptake rate
Variant of thylakoid
incubation
Oxygen uptake rate,
µmol O
2
per mg Chl
per hour
LL 6.9 ± 0.4
LL  +  25 μM H
2
O
2
7.5 ± 0.6
LL  +  50 μM H
2
O
2
7.1 ± 0.5
Note. The incubation medium contained thylakoids (Chl
concentration, 20µg/mL), 100 mM sucrose, 2 mM ATP, 1 µM
gramicidin D, and 50 mM HEPES (pH 7.6). Where indicated,
hydrogen peroxide (25 or 50µM) was added to the thylakoid
suspension prior illumination. The light source was a red
LED (λ  =  640 nm); light intensity, 60 µmol quanta∙m
−2
∙s
−1
).
The data are presented as mean ± SEM.
BALASHOV et al.950
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
DISCUSSION
Redistribution of light-harvesting complexes is
considered to serve as a mechanism of adaptation
to changes in spectral composition of light. However,
a recent study by Hommel et al. [41] demonstrated
that light that predominantly excites one of the pho-
tosystems, as well as the low-intensity white light,
which excites both photosystems, exert a similar in-
fluence on ST and LHCII protein phosphorylation.
Furthermore, it was recently shown that the optimal
electron flow ratio through PSII and PSI (~1.02) at LL
is achieved by PA transition into state 2 [42]. These
findings highlight the significance of this mechanism
not only in response to changes in the light spectral
composition, but also upon exposure to light after
a dark period.
Previous studies conducted in whole leaves have
reported accumulation of Lhcb1-P and Lhcb2-P pro-
teins, as well as the possibility of state 2 formation
in the dark under stress conditions, such as iron de-
ficiency or heat stress [43, 44]. In this study, we found
that incubation of thylakoid membranes isolated from
A.  thaliana plants grown under normal conditions,
in the presence of ATP with stirring in the dark re-
sulted in the accumulation of Lhcb1-P and Lhcb2-P
proteins (Fig. 1), and partial PA transition to state 2
(Table 1), which could be attributed to spontaneous
phosphorylation of Lhcb1 and Lhcb2 or activation
of STN7 kinase in the dark in the presence of ATP
by light-independent manner. These findings must be
taken into account when assessing the extent of ST
in isolated thylakoids. We further evaluated the effect
of hydrogen peroxide specifically on the light-depen-
dent accumulation of Lhcb1-P and Lhcb2-P, since it is
assumed that the invivo influence of H
2
O
2
is mainly
relevant upon illumination, when hydrogen peroxide
is generated by components of the PETC.
Inhibition of ST under HL conditions has been re-
peatedly demonstrated, although its underlying mech-
anisms are still debated (see section “Introduction”).
In this study, two independent approaches – recording
of low-temperature fluorescence spectra and analysis
of Lhcb1-P and Lhcb2-P proteins accumulation – were
used to demonstrate a role of hydrogen peroxide in
the inhibition of ST. Illumination of thylakoid suspen-
sions in the presence of 25 and 50 µM H
2
O
2
did not
provide a significant increase in the F745/F685 ratio
compared to the dark conditions (Table 1), indicating
inhibition of the thylakoid PA transition to state  2
upon illumination in the presence of H
2
O
2
. The tran-
sition from state 1 to state 2 can be divided into two
stages: (i)activation of STN7 kinase and phosphoryla-
tion of Lhcb1 and Lhcb2 proteins and (ii) LHCII mi-
gration from PSII to PSI. The observed inhibition of
state 2 transition may occur at either of these stages.
To determine whether hydrogen peroxide affects
the activity of STN7 kinase namely, we assessed the
light-dependent accumulation of Lhcb1-P and Lhcb2-P
in the absence or presence of exogenous H
2
O
2
. Illu-
mination of thylakoid suspension in the presence of
25 and 50 µM H
2
O
2
led to a significantly lower light-
dependent accumulation of Lhcb1-P and Lhcb2-P
compared to that under control conditions (Fig. 1).
The inability of exogenous H
2
O
2
to fully block the
light-dependent accumulation of Lhcb1-P and Lhcb2-P
may result from the insufficient H
2
O
2
concentrations
used in the experiment. However, higher H
2
O
2
con-
centrations inhibited electron transport in the PETC
(data not shown), which made interpretation of the
obtained results ambiguous. Another possible ex-
planation for the partial inhibition of STN7 kinase
is a limited accessibility of its Cys residues to H
2
O
2
,
which is especially relevant if the inhibition mecha-
nism involves Cys residues exposed to the lumen [19].
Furthermore, it is possible that inhibition of STN7 ki-
nase proceeds via multiple mechanisms. Considering
that STN7 is thought to be activated through plastoqui-
nol binding to the quinol-oxidizing site in the cyto-
chrome b6f complex [20, 45], it can be assumed that
the electron transfer rate and, in particular, the rate
of plastoquinol oxidation at this site, also directly reg-
ulate the kinase activity. In addition, STN7 inhibition
may be induced by its dephosphorylation. In A.  thali-
ana, STN7 kinase contains four phosphorylated resi-
dues (Ser and Thr) near the C-terminus [46], some of
which undergo autophosphorylation. Rapid inhibition
under HL is associated with a partial dephosphoryla-
tion of STN7. Exposure to excessive light intensity can
caused complete STN7 dephosphorylation, which has
been shown to correlate with its degradation [47, 48].
Inhibition of STN7 kinase by H
2
O
2
may be regarded
as a necessary step, if the kinase has already been
activated at the cytochrome complex level but not yet
dephosphorylated for subsequent proteolysis.
Interesting results have been obtained by com-
paring accumulation of phosphorylated Lhcb1 and
Lhcb2 proteins. We found a significant phosphory-
lation of Lhcb1 in the dark in the presence of ATP,
which was considerably greater than phosphorylation
of Lhcb2. In contrast, Lhcb2-P was characterized by a
clearly manifested light-dependent accumulation and,
correspondingly, a more pronounced inhibitory effect
of hydrogen peroxide on this accumulation. Previous
studies have shown that only ~30% of total Lhcb1
undergoes phosphorylation, which might potentially
explain its near-maximum phosphorylation levels in
the darkness (Fig. 1), whereas the extent of Lhcb2
phosphorylation can reach 70% [9]. These findings
may also reflect the functional differences between
Lhcb1 and Lhcb2 in ST, as Lhcb2-P has been shown
to play a key role in LHCII docking to PSI [10,  49].
EFFECT OF H
2
O
2
ON STN7 KINASE ACTIVITY 951
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Moreover, it was recently demonstrated that replace-
ment of native Lhcb1 with a protein with eliminated
phosphorylation site (Thr  →  Val) had no effect on ST,
whereas the same substitution in Lhcb2 abolished
ST entirely [50]. A more pronounced inhibition of
Lhcb2-P accumulation observed in this study was
consistent with the earlier reported essential role
of Lhcb2 in ST.
Previously, it was shown that increasing illumina-
tion leads to the increase of hydrogen peroxide pro-
duction in isolated thylakoid membranes, both under
conditions when oxygen is the only electron acceptor
and in the presence of methyl viologen (an artificial
electron acceptor which is more efficient than mo-
lecular oxygen) [31, 51, 52]. Illumination of isolated
thylakoids with HL (800μmol quanta∙m
−2
∙s
−1
) induced
a complete inhibition of the light-dependent accumu-
lation of Lhcb1-P and partial suppression of Lhcb2-P
accumulation compared to the LL conditions (Fig. 2).
Apparently, illumination for 5 min was insufficient
for the accumulated H
2
O
2
to fully inhibit the light-de-
pendent accumulation of Lhcb2-P (Fig. 2). Another
possible explanation for the incomplete inhibition of
Lhcb2-P accumulation may be that the complete in-
hibition of STN7 requires additionally the thioredox-
in activity in the stroma, since it was suggested that
thioredoxins participate in STN7 inhibition [24, 25].
To verify the role of H
2
O
2
in the inhibition of
Lhcb1-P and Lhcb2-P accumulation in isolated thyla-
koids under HL, we illuminated these preparations in
the presence of catalase, an enzyme that decomposes
H
2
O
2
. The addition of catalase significantly enhanced
Lhcb1-P and Lhcb2-P accumulation upon HL illumi-
nation compared to the control (in the absence of
catalase). Removal of H
2
O
2
molecules prevented STN7
inhibition upon HL illumination of isolated thylakoids,
while addition of H
2
O
2
in the above-described experi-
ments in LL mimicked the increase in the H
2
O
2
levels
as in the case of HL inhibiting STN7 kinase. It cannot
be excluded that under the LL conditions, we could
nor observe the maximum accumulation of Lhcb1-P
and Lhcb2-P and complete transition from state 1 to
state 2, considering that in this case, H
2
O
2
was also
generated by PETC components, albeit at a lower rate
than under HL.
We also demonstrated that the H
2
O
2
-mediated in-
hibition of STN7 kinase was specific. The genome of
A.  thaliana contains a STN7 paralog – STN8 kinase,
which is responsible for phosphorylating PSII core
proteins, in particular, D1 protein [39, 40]. Under HL
conditions, accumulation of D1-P protein is upregu-
lated, which facilitates partial grana unstacking and
promotes faster turnover of core proteins [39, 40].
The addition of H
2
O
2
to the isolated thylakoid suspen-
sions did not inhibit the STN8 activity (in contrast to
STN7), i.e., did not reduce D1-P accumulation (Fig. 3),
which confirmed differential regulation of these ki-
nases and highlighted the specificity of H
2
O
2
ac-
tion on STN7. Interestingly, addition of 50 μM H
2
O
2
during illumination significantly increased D1-P ac-
cumulation (Fig. 3) up to levels comparable to those
observed under HL. This is consistent with recent
findings of the H
2
O
2
-mediated inhibition of PBCP
phosphatase [53], which dephosphorylates D1-P. It
was proposed that the inhibition of PBCP by hydro-
gen peroxide under HL conditions may account for
the observed increase in the phosphorylation levels of
PSII core proteins, which results from the inhibition
of phosphatase rather than upregulation of the STN8
activity [53].
It has been previously shown that formation of
H
2
O
2
in chloroplasts occurs not only in the stroma
(the so-called stromal H
2
O
2
) as a result of superoxide
anion radical dismutation, but also takes place in the
vicinity of thylakoid membrane (membrane-associated
H
2
O
2
) due to the reaction of superoxide anion radi-
cal with plastoquinol present in the membrane lipid
phase [38, 51]. Increased illumination enhances the
overall H
2
O
2
production rate in chloroplasts, how-
ever, mainly due to the elevated formation of mem-
brane-associated H
2
O
2
rather than stromal H
2
O
2
[52].
Consequently, the proportion of H
2
O
2
generated near
the PETC components, including the transmembrane
STN7 kinase, increases with an increase in light in-
tensity. Hence, it can be assumed that it is the mem-
brane-associated H
2
O
2
that plays a key role in the inhi-
bition of STN7 activity invivo. This suggestion offers a
new interpretation of the ambiguous role of plastoqui-
none pool in ST: although reduced plastoquinone mol-
ecules are required for the STN7 activation, illumina-
tion with HL (when plastoquinone is mostly reduced)
results in the ST inhibition and decreased phosphory-
lation levels of LHCII proteins [25, 54]. Therefore, the
regulatory role of plastoquinone pool redox state in
the control of STN7 activity should be considered as a
complex system that involves not only plastoquinone
reduction level but also H
2
O
2
production.
CONCLUSION
In this work, we demonstrated suppression of
the PA transition to state 2, specifically, inhibition
of STN7 kinase, upon illumination of isolated thyla-
koid suspensions with LL in the presence of hydro-
gen peroxide. H
2
O
2
removal caused an increase in
the light-dependent accumulation of STN7 kinase
products (Lhcb1-P and Lhcb2-P) upon HL illumina-
tion of isolated thylakoid suspensions. Inhibition of
STN7 kinase upon increased illumination may be
an important mechanism regulating PA functioning.
The involvement of STN7 kinase in the long-term
BALASHOV et al.952
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
regulation of the PSII antenna size under increased
illumination was shown in [54], which may include
changes in its activity and redox or phosphorylation
states. Inhibition of STN7 kinase during abrupt chang-
es in light conditions could represent a critical step in
triggering the retrograde signaling for the regulation
of PSII antenna size by suppressing the biosynthe-
sis of Lhcb proteins. Along with MAP kinases, STN7
acts as a key participant in the reversible phosphor-
ylation in chloroplasts and influences not only the
activity of these organelles, but the functioning of
the entire cell. Inhibition of STN7 kinase may thus
serve as a crucial signal for the adaptive remodeling
of reversible protein phosphorylation in plant cells.
Several studies have reported STN7 inhibition in re-
sponse to other abiotic stress factors, such as low
temperature [55, 56], NaCl addition, or treatment with
H
2
O
2
, in cultured A.  thaliana cells [57]. All these abi-
otic stresses also induce accumulation of reactive ox-
ygen species, e.g., hydrogen peroxide, suggesting that
the H
2
O
2
-mediated mechanism of STN7 inhibition de-
scribed here may represent a universal response to
various stress factors.
Abbreviations. Chl, chlorophyll; HL, high-in-
tensity light; Lhcb1-P and Lhcb2-P, phosphorylated
Lhcb1 and Lhcb2 proteins; LHCII, light-harvesting
complex II; LL, low-intensity light; PA, photosynthet-
ic apparatus; PSI, photosystem I; PSII, photosystem II;
PETC, photosynthetic electron transport chain; ST,
state transition.
Acknowledgments. The study was carried out
using the equipment of the Shared Research Facil-
ities Center of the Pushchino Scientific Center for
Biological Research, Russian Academy of Sciences
(https://www.ckp-rf.ru/ckp/670266/).
Contributions. N.V.B., D.V.V. , and M.M.B.-M. con-
ducted the experiments and discussed the results;
D.V.V. prepared the draft of the manuscript; N.V.B. and
M.M.B.-M. edited the manuscript.
Fundings. This work was supported by the Rus-
sian Science Foundation (project no. 22-74-10088,
https://rscf.ru/project/22-74-10088/ [in Russian]).
Ethics approval and consent to participate.
This work does not describe any studies involving hu-
mans or animals as subjects performed by any of the
authors.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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O
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