Brassinosteroids regulate the thylakoid membrane architecture and the photosystem II function

Page 1

Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Brassinosteroids regulate the thylakoid membrane architectureand the photosystem II function

1011-1344/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jphotobiol.2013.07.008

⇑ Corresponding author. Tel.: +359 29 79 26 08; fax: +359 87 23 787.E-mail address: [email protected] (S. Krumova).

1 Permanent address: Department of Plant Physiology, Sofia University, 8 DraganTzankov Blvd., 1164 Sofia, Bulgaria.

S. Krumova a,⇑, M. Zhiponova b,c,1, K. Dankov a, V. Velikova d, K. Balashev e, T. Andreeva a, E. Russinova b,c,S. Taneva a,f

a Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgariab Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgiumc Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent 9, Belgiumd Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, 1113 Sofia, Bulgariae Department of Physical Chemistry, Faculty of Chemistry and Pharmacy, Sofia University ‘‘St. Kliment Ohridski’’, 1 James Bourchier Blvd., 1164 Sofia, Bulgariaf Unidad de Biofísica (CSIC/UPV-EHU), Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, POB 644, 48080 Bilbao, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 April 2013Received in revised form 7 June 2013Accepted 2 July 2013Available online 10 July 2013

Keywords:BrassinosteroidsPhotosynthesisThylakoid membraneFluorescence spectroscopyPhotosystem II functionOxygen yield

Brassinosteroids (BRs) are plant steroid hormones known to positively affect photosynthesis. In this workwe investigated the architecture and function of photosynthetic membranes in mature Arabidopsisrosettes of BR gain-of-function (overexpressing the BR receptor BR INSENSITIVE 1 (BRI1), BRI1OE) andloss-of-function (bri1-116 with inactive BRI1 receptor, and constitutive photomorphogenesis and dwarfism(cpd) deficient in BR biosynthesis) mutants. Data from atomic force microscopy, circular dichroism, fluo-rescence spectroscopy and polarographic determination of oxygen yields revealed major structural(enlarged thylakoids, smaller photosystem II supercomplexes) and functional (strongly inhibited oxygenevolution, reduced photosystem II quantum yield) changes in all the mutants with altered BR responsecompared to the wild type plants. The recorded thermal dependences showed severe thermal instabilityof the oxygen yields in the BR mutant plants. Our results suggest that an optimal BR level is required forthe normal thylakoid structure and function.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Brassinosteroids (BRs) are a large class of plant steroid hor-mones that are essential for plant growth and development. Theireffects span over multiple physiological processes and responsessuch as cell division and differentiation, stomata function, photo-synthesis, respiration, ion transport, etc. [1–3].

BRs are perceived at the plasma membrane by the BR INSENSI-TIVE 1 (BRI1) receptor and their binding triggers a signaling cas-cade that modifies the expression of BR-related genes involved inplant response to developmental changes or environmental condi-tions [4]. The BR response is subjected to a feed-back control whenBR signaling represses BR biosynthesis, and in this way an optimalBR level is achieved which is important for normal plant growthand development [5,6]. The genetic manipulation of BR biosyn-thetic and perception pathways was recognized as economicallyimportant and is regarded as a biotechnological target for im-proved productivity [1–3].

BR loss-of-function mutants display severe leaf phenotypesincluding dark-green, small, round-shaped leaves and short peti-oles [7–10]. BR gain-of-function mutants show larger and elon-gated leaf blades and longer petioles [11,12]. Arabidopsis plantsoverexpressing BRI1 (BRI1 (Y831F)) and having increased BR level,were reported to have larger leaves, as well as higher rates of CO2

assimilation, maximum Rubisco carboxylation, maximum electrontransport, and higher photochemical quenching and electron trans-port rate relative to the control wild type BRI1 variant [12].

The effect of BR on the functionality of the photosynthetic appa-ratus is studied in more details on BR-treated higher plants. Whenexogenously applied to leaves and roots BRs improve stress toler-ance and thus stimulate crops’ growth and yield [3,13–15]. The de-creased photosynthetic rate and photosystem II (PSII) activityunder heat, cadmium and oxidative stresses are compensated bypre-treatment with 24-epibrassinolide that is the most active BR[16,17]. The positive effects of BRs are attributed mainly to in-creased Rubisco activity and carboxilation efficiency and enhancedantioxidant systems [12,16,18–20].

The photosynthetic apparatus of higher plants is embedded inthe chloroplast thylakoid membranes (the locus of photosyntheticlight reactions). It is a highly complex system consisting of two mul-tisubunit pigment–protein complexes – the laterally separated but

Page 2

98 S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104

functionally synchronized photosystem I (PSI) and II (PSII) – andother membrane-embedded peripheral (such as the oxygen-evolv-ing complex (OEC)) and soluble protein complexes [21,22]. In higherplants the thylakoid membrane is highly curved and segregated ingrana (stacked membrane regions) and stroma (unstacked) lamel-lae. Grana membranes accommodate the vast majority of PSIIsupercomplexes, constituted by PSII core dimers associated withvariable amount of antennae complexes [23], while PSI is localizedin the stroma lamellae and the end grana membranes [24].

In this work we explore the effects that BRs exert on the photo-synthetic apparatus by investigating thylakoid membranes iso-lated from Arabidopsis plants with enhanced BR signaling(overexpressing the BRI1 receptor, BRI1OE) and BR insensitive ordeficient mutants (bri1-116 with inactive BRI1 receptor, and consti-tutive photomorphogenesis and dwarfism (cpd) deficient in BR bio-synthesis, respectively). The overall structural organization andphotosynthetic performance of the BR modulated plants are com-pared to Columbia-0 (Col-0) wild type control.

2. Materials and methods

2.1. Plant material

The experiments were performed on detached leaves or isolatedthylakoid membranes from four week-old Arabidopsis thalianaplants: wild type ecotype Col-0, BRI1OE line [10], bri1-116 [10]and cpd [8] mutants. The plants were grown on soil with 12 h pho-toperiod (23 �C day/18 �C night temperature), controlled lightintensity of 100 lmol m�2 s�1 photon flux density and relativehumidity of 65%.

2.2. Thylakoid membranes preparation

Thylakoid membranes were prepared as in Ref. [25] and usednot later than 2 h after the isolation. Prior to isolation, leaves weredark and ice-cold adapted for 30 min. All isolation steps were per-formed on ice and in dim light. For all measurements thylakoidmembranes were suspended in a medium containing 20 mM tri-cine (pH 7.8), 5 mM KCl, 5 mM MgCl2 and 400 mM sucrose (sus-pension buffer). The chlorophyll (chl) concentration wasdetermined by the method of [26].

2.3. Atomic force microscopy

Atomic force microscopy (AFM) imaging was performed in tap-ping mode in air (NanoScopeV system, Bruker Inc.). Silicon cantile-vers (Tap300Al-G, Budget Sensors, Innovative solutions Ltd.,Bulgaria) with 30 nm thick aluminum reflex coating were used(cantilever spring constant 1.5–15 N/m; resonance frequency150 ± 75 kHz; tip radius <10 nm). The scan rate was set to0.2 Hz; the images (512 � 512 pixels) were captured in the heightand phase modes in JPEG format.

Isolated thylakoid membranes were fixed with 2% (v/v) glutar-aldehyde and spread on a freshly cleaved mica surface coveredwith 0.01% poly-L-lysine; muscovite mica plates (Structure ProbeInc./SPI Supplies, West Chester, PA) of sizes 10 � 10 mm glued tothe metal pads were used. After 20 min incubation, the mica sur-face was rinsed with the suspension buffer and gently blown witha flow of nitrogen gas to dry out.

Images from 6–23 independent experiments were analyzed byNanoScope 6.13R1 software in the following order: first order flat-tening; roughness analysis applying 0.00 nm peak threshold value;area and height determination. For area estimation, the objectswere approximated to sphere or ellipse. The height of the objectswas determined as the difference between the threshold valueand the highest point of the image.

2.4. Circular dichroism

Circular dichroism (CD) spectra were recorded on Jobin–YvonCD6 dichrograph in the range 400–800 nm (1 nm step, 0.2 s inte-gration time, 2 nm bandpass, 1 cm optical path length of the cell,5 cm distance of the sample from the photomultiplier). For thetemperature dependences thylakoid samples were incubated for5 min at defined temperatures in the range 20–80 �C, in 5 �C steps.The intensity of the 684/670 nm CD band was determined by thedifference in the intensities of the 684 and 670 nm CD signals.For the 650 nm CD band, the reference value at 610 nm was sub-tracted from the intensity at 650 nm. The transition temperature,Tm, for 684/670 nm and 650 nm CD bands was determined as thetemperature at which the intensity of the respective CD bandwas half its value at 20 �C. The chl concentration of the sampleswas 20 lg mL�1.

2.5. Merocyanine 540 fluorescence

Excitation spectra of merocyanine 540 (MC540) incorporated inthylakoid membranes were recorded on Jobin Yvon JY3 spectroflu-orometer. The emission was measured at 590 nm in 1 nm stepupon 450–575 nm excitation (emission and excitation slits wereset to 10 nm) at 20 �C. The chl concentration was 20 lg mL�1. Priormeasurements, an aliquot of MC540 stock solution (1 mM MC540dissolved in ethanol) was added to thylakoid membrane suspen-sion at final concentration of 0.2 lM and the samples were incu-bated for 5 min. Each excitation spectrum is corrected for the chlemission of thylakoid membranes without MC540. MC540 waspurchased from Sigma-Aldridge Co.

2.6. 77K steady-state fluorescence spectroscopy

Chlorophyll emission spectra of thylakoid membranes were re-corded at 77 K on Jobin Yvon JY3 spectrofluorimeter upon 436 nmand 472 nm excitation. The chl concentration of the samples was20 lg mL�1.

2.7. Modulated chl fluorescence

Modulated chl fluorescence was used to determine the maximalfluorescence level for dark-adapted (Fm) and light-adapted (F 0m)plants; the fluorescence level when applying actinic light (F) andthe dark level of fluorescence (F0). The difference between Fm andF0 yields the variable fluorescence, Fv. These parameters allow forthe determination of: (i) maximal quantum yield of PSII, Fv/Fm;(ii) effective quantum yield of PSII, UPSII = ðF 0m � FÞ/F 0m; (iii) non-photochemical quenching, related to the photoprotection abilityof plants, NPQ = ðFm � F 0mÞ/F

0m, (iv) photochemical quenching coef-

ficient, qP = ðF 0m � FÞ/ðF 0m � F 00Þ, determined by the light energy usedfor photosynthesis; and (v) non-cyclic electron transport rate (ETR)through PSII, ETR = I � Aleaf � fractionPSII �UPSII, where I is thephoton flux density incident on the leaf, Aleaf is the proportion ofincident photon flux density absorbed by the leaf, fractionPSII isthe fraction of absorbed photon flux density that is received by PSII[27].

Modulated chl fluorescence was measured on leaf discs using aFluorescence Monitoring System (FMS, Hansatech Instruments,UK). Detached leaves of four-week-old Col-0, BRI1OE, bri1-116and cpd plants were used. Prior to each measurement plants weredark adapted for 60 min at 20 �C. F0 level was determined at mea-suring beam of 0.05 lmol m�2 s�1 photon flux density. For evalu-ation of the maximal fluorescence level, saturating pulse of10,000 lmol m�2 s�1 photon flux density of 0.8 s duration was ap-plied. After the measurements in dark, the plants were adapted tothe growing light level for 2 h for the induction of photosynthesis

Page 3

S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104 99

and the steady state fluorescence level F during actinic light illumi-nation (200 lmol m�2 s�1 photon flux density) and the maximumfluorescence in light adapted state after application of saturatingpulse, F 0m, were determined.

2.8. Oxygen evolution measurements

Oxygen evolution was studied as a function of the number of con-sequent saturating flashes and upon continuous illumination. Theoxygen yield at the third flash (Y3) and the amplitude of the initialoxygen burst upon continuous illumination are used as measuresof the activity of the oxygen-evolving complexes (OECs) [28,29].

Home-built Joliot type polarographic oxygen rate electrode[30,31] was used for the determination of oxygen flash yieldsand initial oxygen burst of isolated thylakoid membranes. Beforeeach measurement the samples were preilluminated with 20flashes and then dark adapted for 5 min. Oxygen flash yields wereinduced by saturating (4 J) and short (t1/2 = 10 ls) periodic flash se-quences with 650 ms dark spacing between them. The initial oxy-gen burst was recorded after irradiation with continuous whitelight (450 lmol photons m�2 s�1). The chl concentration of the thy-lakoid samples was adjusted to 150 lg mL�1. For thermal inactiva-tion experiments, the samples were incubated 5 min at 30 �Cbefore measuring the oxygen yield parameters and were comparedto the corresponding values determined at 20 �C.

2.9. Statistical evaluation of the data

All average values are presented with the standard error of themean; the number of individual repeats is denoted by n. The exper-imental data, obtained for the mutant lines, were compared tothose determined for Col-0 wild type plants as a reference and sub-jected to two-tailed paired Student’s t-test. Values of P < 0.05 wereconsidered significantly different from those characteristic of Col-0and are marked with asterisk.

3. Results

3.1. Thylakoid macroorganization

The thylakoid membrane’s architecture of plants with modu-lated BR level was compared to wild type Col-0 plants at the fourthweek of their growth when the photosynthetic apparatus is devel-oped; we studied the overall morphology of the thylakoids and thestructural arrangement of its constituents – the protein and the li-pid matrix.

As previously reported there were strong phenotype changes inthe mutant lines, compared to the wild type plants – BRI1OE plantshad elongated leaf blades and long petioles [11,12], whereas bri1-116 and cpd had small round-shaped leaves and short petioles(Fig. 1).

Freshly isolated thylakoids were imaged by AFM and twoparameters, namely, thylakoid area (Fig. 2A) and height (Fig. 2B),

Col-0 brBRI1OE

Fig. 1. Images of Arabidopsis thaliana Col-0, BRI1OE, bri1-116 and

were determined. The thylakoid area in BRI1OE, bri1-116 and cpd(26–35 lm2) is about twice larger than that of Col-0 (12 lm2,Fig. 2A) while its height remains similar to Col-0, on the order of0.64–0.82 lm (Fig. 2B), value similar to the reported for dark-adapted thylakoids [32].

To find out whether this expansion of the photosynthetic mem-branes in the BRI1OE line and the BR mutants affects the lateralarrangement of the pigment–protein complexes, we applied CDspectroscopy. CD is widely used in photosynthesis research sinceit probes both the short- and the long-range pigment–pigmentinteractions (fingerprints of the supramolecular organization ofthe photosynthetic complexes in the thylakoid membrane) [33].The long-range chromophore interactions in large (ca. 200–400 nm [34,35]) protein macrodomains give rise to intensive psi-type CD bands at 684/670 nm and at 501 nm; these were shownto be characteristic of LHCII-only domains [36,37] and ordered ar-rays of LHCII–PSII supercomplexes [38]. The 650 nm CD band isdue to short-range pigment–pigment interactions involving LHCII[39] and reflects its conformational stability [40].

The 650 nm CD band was identical in all studied lines demon-strating that the amount and conformation of LHCII were not af-fected by the altered BR response at 20 �C (Fig. 3). The CD spectraof BRI1OE, bri1-116 and cpd thylakoids exhibited lower intensityof the 684/670 nm CD band than the one present in the spectraof Col-0, indicating modified organization of LHCII and PSII super-complexes (Fig. 3). The thermal destabilization patterns of 684/670 nm and 650 nm CD bands for BRI1OE, bri1-116, cpd and Col-0were very similar (data not shown); they were characterized withTm of about 58 �C and 60 �C, respectively. It is worth noting, but be-yond the scope of this paper, that the 684/670 nm CD band in Ara-bidopsis appears more stable than in barley and pea (where itdisassembled at about 48 �C [40]) suggesting species-dependentstructural stability of the PSII supercomplexes.

The 501 nm CD band in BRI1OE, bri1-116 and cpd had higherintensity than in Col-0 that might be due to increased scatteringby the enlarged thylakoids (as revealed by AFM) in those samples.

To check whether the physical state of the lipid phase is modi-fied, we investigated the incorporation of the lipophylic fluorescentprobe MC540. The excitation spectra of MC540 when embedded inthylakoids are characterized by a 565 nm emission peak originat-ing from MC540 monomers, and a 536 nm shoulder correspondingto MC540 dimers, their ratio, F565/536, being a marker for the lipidmatrix organization in both model and biomembranes ([41,42]and refs. there in). For Col-0 F565/536 was 1.77 ± 0.03 while in thecase of BRI1OE, bri1-116 and cpd it was lower and varied in therange 1.59–1.66. The results indicate slightly decreased membranefluidity when the BR response is modified.

3.2. Light energy distribution between the photosystems

The observed modifications in the thylakoid macroorganizationin plants with modified BR response could affect the function of thetwo photosystems, and therefore we investigated the light energy

i1-116 cpd

cpd plants at the time of harvesting (fourth week of growth).

Page 4

0

10

20

30

40

Are

a (µ

m2 )

A

0.0

0.2

0.4

0.6

0.8*

*

B

bri1-11

6 cpd

BRI1OE

Col-0

Hei

ght (

µm)*

bri1-11

6 cpd

BRI1OE

Col-0

Fig. 2. Parameters derived from tapping mode AFM images of thylakoid membranes. Average thylakoid area (A) and height (B), error bars indicate SE (n = 6–23), P value (t-test) ⁄ 60.003, relative to Col-0.

450 500 550 600 650 700-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5501 nm

CD

, A x

10-4

Wavelength (nm)

Col-0BRI1OEcpdbri1-116

684 nm

670 nm650 nm

Fig. 3. Circular dichroism spectra of Col-0, BRI1OE, bri1-116 and cpd thylakoids,recorded in the 400–800 nm range at 20 �C.

100 S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104

distribution between PSI and PSII by means of 77 K chl fluores-cence (Fig. 4). Thylakoid emission spectra of Col-0, BRI1OE, bri1-116 and cpd were recorded upon excitation at 436 nm (Fig. 4A)and 472 nm (spectra not shown), preferentially exciting chl a(mostly found in the photosystems core complexes) and chl b(bound to the antennae complexes), respectively.

The ratio of the fluorescence intensities at 733 nm (originatingfrom PSI) and at 686 nm (due to PSII components) emission max-ima, F733/686, is often used as a measure of the relative amount ofPSI and PSII centers. It was found to be higher by about 20% in

675 700 725 750 775

20

40

60

80

100Col-0 BRI1OE bri1-116 cpd

A

Fluo

resc

ence

inte

nsity

(r.u

.)

Wavelength (nm)

733 nm

686 nm

Fig. 4. 77 K chlorophyll emission of thylakoid membranes. (A) Typical emission spectr733 nm fluorescence and the characteristic emission maxima are labeled. (B) Average F7

(light gray columns); error bars indicate SE (n = 4–8), P value (t-test) ⁄ 60.03, relative to

BRI1OE, bri1-116 and cpd than in Col-0 upon chl a excitation(Fig. 4B) which suggests increased relative PSI amount. However,there was no difference in the chl a/b ratio between Col-0 andBRI1OE, bri1-116 and cpd and thus the observed reduction in therelative PSII emission must be due to changes in the function andnot to the decline of the amount of PSII centers.

3.3. Photosystem II function

The functionality of PSII was assessed by means of modulatedchl fluorescence and oxygen evolution measurements, probing itsacceptor and donor side, respectively. The derived fluorescenceparameters for Col-0, BRI1OE, bri1-116 and cpd are summarizedin Table 1. Only bri1-116 differed from Col-0 – it exhibited slightlylower values for Fv/Fm (by 2%) and F 0v/F 0m (by 7%) ratios, strongly re-duced UPSR (by 21%), NPQ (by 61%) and ETR (by 65%) but there wasno difference in its ability to perform photochemical quenching(qP) in comparison to Col-0.

The PSII donor side that is involved in water splitting wasprobed by oxygen evolution experiments, whose kinetics wasstudied upon flash and continuous illumination. The flash-inducedoscillation patterns of the oxygen flash yields (Fig. 5A) and the timecourses of the initial oxygen burst upon continuous illumination(Fig. 5B) of Col-0, BRI1OE, bri1-116 and cpd thylakoids were usedto determine the characteristic parameters oxygen yield at thethird flash (Y3, Fig. 5C) and amplitude of the initial oxygen burstupon continuous illumination (Fig. 5D). The oxygen yield at Y3 isreduced significantly in the BR modulated plants – by 29% forBRI1OE, 45% for bri1-116 and 39% for cpd (Fig. 5C). The amplitudeof the initial oxygen burst recorded upon continuous illumination

cpd

bri1-11

6

BRI1OE

B

Col-0

0.5

1.0

1.5* *

F 733

/686

*

a recorded upon 436 nm excitation. For clarity the spectra are normalized to the33/686nm ratio, determined for excitation at 436 nm (dark gray columns) and 472 nm

Col-0.

Page 5

Table 1Modulated chlorophyll fluorescence parameters determined on leaves of Col-0,BRI1OE, bri1-116 and cpd. Average values ± SE, P value (t-test) ⁄ 60.05, relative toCol-0.

Parameter Col-0 BRI1OE Bri1-116 cpd

Fv/Fm 0.85 ± 0.002 0.84 ± 0.002⁄ 0.83 ± 0.004⁄ 0.83 ± 0.006F 0v/F 0m 0.76 ± 0.01 0.78 ± 0.01 0.71 ± 0.02⁄ 0.83 ± 0.03⁄

UPSR 0.29 ± 0.01 0.30 ± 0.02 0.23 ± 0.01⁄ 0.32 ± 0.05NPQ 1.29 ± 0.12 1.48 ± 0.24 0.50 ± 0.20⁄ 2.05 ± 0.46qP 0.40 ± 0.02 0.38 ± 0.03 0.32 ± 0.01 0.33 ± 0.04ETR 0.65 ± 0.04 0.73 ± 0.06 0.23 ± 0.01⁄ 0.72 ± 0.13

S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104 101

is also much lower, approximately 40% less for BRI1OE, bri1-116and cpd compared to Col-0 thylakoids (Fig. 5D).

The stability of OEC organization against thermal challenge wastested at 30 �C (Fig. 6). For Col-0 the oxygen yield dropped by about62%, while for BRI1OE, bri1-116 and cpd the decrease was more se-vere – 77–83% lower than the respective values at 20 �C (Fig. 6A).Very similar effect was observed for the amplitude of the initialoxygen burst at 30 �C which was reduced to 47% for Col-0 and to20–25% for the BR modulated plants, relative to 20 �C (Fig. 6B).These results reveal that BRs affect photosynthesis by regulatingthe stability of OEC.

4. Discussion

In this work we have used BR synthesis and perception mutantsin order to study the effects they exert on the photosyntheticmachinery. We have shown that although BRs are perceived atthe plasma, and not at the thylakoid membrane level, they indi-rectly regulate the assembly and functioning of the photosyntheticcomplexes and the lipid matrix in which they are embedded.Evidences for the structural and functional consequences of modi-fied BR response in BRI1OE line and bri1-116 and cpd mutants arediscussed below.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 140

100

200

300

400

500

Col-0BRI1OEbri1-116cpd

Oxi

gen

flash

yie

ld (m

V)

Flash number

A

0

100

200

300

400

500

*

*

C

Oxi

gen

yiel

d at

Y3 (

mV)

*

cpd

bri1-11

6

BRI1OE

Col-0

Fig. 5. Oxygen flash yields of thylakoid membranes isolated from Col-0, BRI1OE, bri1-116time courses of the initial oxygen burst upon continuous irradiation (the onset of illuminthe third flash, Y3. (D) Mean amplitudes of the initial oxygen burst. Error bars indicate S

4.1. Brassinosteroids control the thylakoid architecture and PSIImacroorganization

The thylakoid membrane is a highly dynamic system that reactsto environmental factors and takes part in adaptation responses, asan intrinsic element of plant’s plasticity. Its architecture is deter-mined by the size of grana and stroma lamellae, their relative ratioand the extent of grana stacking, which strongly depend on multi-ple protein–protein and protein–lipid interactions. We found thatthe thylakoid membrane’s architecture in plants with geneticallymodified BR response was affected by BRs on three levels – overallsize of the membrane system (determined by AFM, Fig. 2), PSIIsupercomplex organization (explored by CD spectroscopy, Fig. 3),and lipid matrix packing (probed by MC540).

In the last decade AFM emerged as a useful tool for studies ofthylakoids ultrastructure [22,32,43,44] and mechanical properties[45]. Our AFM data demonstrate significantly enlarged thylakoidsystem in BRI1OE, bri1-116 and cpd mutants, as compared to Col-0. Thylakoids elongation in the BR modulated plants cannot beattributed directly to grana unstacking, since, as observed duringstate transitions, this process induced decrease in the height ofthe thylakoid network and only slight increase in its area [22,32]which is not the case in our experiments. Taking into account thatgrana stacking and the arrangement of LHCII complexes and PSIIsupercomplexes in the membrane are interrelated (for a recent re-view see Ref. [22]), it can rather be suggested that the observedchanges in the overall morphology of thylakoids are due to themodified macroorganization of the photosynthetic complexes (asevidenced by CD spectroscopy).

Alternatively, the twofold increase in thylakoid area of BRI1OE,bri1-116 and cpd might be due to changes in chloroplast divisionmechanisms; for example giant chloroplasts were detected in Ara-bidopsis mutants of dynamin-related proteins [46–48]. Downregu-lation of genes related to chloroplast division and developmentwas already established in bri1-9 mutant [49].

0 2 4 6 8 10 12 14 16 18

0

250

500

750

1000

1250

1500

1750

cpdbri1-116BRI1OECol-0

B

Oxi

gen

yiel

d (m

V)

Time (sec)

0

200

400

600

800

1000

1200

**

*

D

cpd

bri1-11

6

BRI1OE

Col-0

Am

plitu

de o

f ini

tial

oxy

gen

burs

t (m

V)

and cpd. (A) Typical oscillation patterns of the oxygen flash yields. (B) Characteristication is marked with arrow for each trace). (C) Average oxygen yields recorded afterE (n = 4–11), P value (t-test) *

60.05, relative to Col-0.

Page 6

0

10

20

30

40

*

*

cpd

bri1-11

6

BRI1OE

Oxy

gen

yiel

d at

Y3 (

%)

Col-0

A

*

0

10

20

30

40

50

60

***

Am

plitu

de o

f the

initi

al

oxyg

en b

urst

(%)

B

cpd

bri1-11

6

BRI1OE

Col-0

Fig. 6. Thermal destabilization of the oxygen yield at 30 �C relative to 20 �C. (A) Average oxygen yields recorded after the third flash, Y3. (B) Mean amplitudes of the initialoxygen burst. All values are estimated in % of those obtained for samples incubated at 20 �C (100%). Error bars indicate SE (n = 3–9), P value (t-test) ⁄ 60.05, relative to Col-0.

102 S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104

To our knowledge there are no in-depth microscopic studies onthylakoids isolated from BR mutants. However, electronmicrographs of plant cells in cucumber seedlings treated with24-epibrassinolide and brassinazole reveal different thylakoidmorphology – in the control the grana and stroma regions are welldiscernible, whereas in 24-epibrassinolide and brassinazole-trea-ted thylakoids the grana formation is less well defined [50]. Thesedata and our AMF observations justify the need of furthermicroscopic research on the thylakoid system in BR responsemutants.

The increase in the thylakoid area might also be explained as acompensatory mechanism for the inhibiting photosynthetic effectsobserved in plants with modified BR level. Additional target for fur-ther studies on the thylakoid morphology is the PSII phosphoryla-tion which was demonstrated to be related to the stacking andfolding of this membrane system [22,51–54].

The 684/670 nm CD band in intact thylakoid membranes wasassigned to PSII supercomplex and was shown to depend on itsstructural integrity [38]. The reduction of this CD signal in BRI1OE,bri1-116 and cpd, compared to Col-0 resembles the effect detectedfor Arabidopsis CP24 knockout mutants having PSII supercomplex-es depleted in LHCII trimers [38] and hence most probably reflectsreduced size of the PSII supercomplexes in the BR modulatedplants. The smaller peripherall antenna might be the reason forthe observed reduced relative PSII quantum yield in the BR mu-tants as compared to the wild type.

The altered macroorganization of the photosynthetic proteincomponents in BRI1OE, bri1-116 and cpd is accompanied by a slightrigidification of the lipid matrix; more sensitive techniques such asP31 NMR spectroscopy [55], time-resolved MC540 emission [41]etc. might reveal further details about the physical state of thishighly complex membrane system. These might be related to amodified lipid composition and fatty acid unsaturation, alteredgene expression or are a consequence of the protein rearrangementin the thylakoid membranes of the BR modulated plants.

4.2. Brassinosteroids affect the PSII functionality

PSII is the most sensitive component of the photosyntheticmachinery, affected by a large variety of environmental stresses[56] and endogenous hormonal signaling [57]. PSII is a multisubunitcomplex playing three functions – light harvesting, utilization andoptimization. Light harvesting is optimized by the size and the com-position of the PSII peripheral antennae; light utilization is coupledwith the water splitting by the OEC, and light regulation is achievedvia energy distribution between PSI and PSII, and a functional net-work of dynamic processes involved in photoprotection.

Our measurements of modulated chl fluorescence did not revealany strong effect of the modification of the endogenous BR level(cpd) or signaling (BRI1OE) on PSII photochemistry, with the onlyexception of bri1-116 mutant which had reduced chl fluorescenceparameters (Table 1). However, the more sensitive 77 K fluores-cence spectroscopy showed reduced PSII quantum yield in all theBRI1OE line and the BR mutants. It was more pronounced uponchl a excitation (preferential for PSII core) than chl b (preferentialfor PSII peripheral antenna) and hence shows that altered BR re-sponse affects strongly the PSII functionality.

The PSII functional impairment was confirmed by the oxygenevolution experiments which revealed dramatic reduction of theoxygen yields in BRI1OE, bri1-116 and cpd and severe thermalinstability. Various factors might be responsible for these effects.For example a strong correlation between PSII assembly and OECactivity has been established – the OEC subunits PsbP, PsbQ andPsbO have been found to be essential for optimal positioning ofthe antennae complexes relative to the PSII core complex [58]and for PSII stability [59,60]. Thus changes in the expression pro-files of the OEC subunits and the assembly of the complex mightbe responsible for the decreased oxygen yields, the reducedperipheral antennae of the PSII supercomplex (as suggested byCD spectroscopy), and the lower PSII quantum yield in the BRmodulated plants, fully in line with the hypothesis of De Las Rivaset al. [61].

And vice versa, PSII supercomplex assembly affects the bindingof the OEC subunits. CP43, CP47, D2, PsbL and PsbJ are all neededfor proper association of OEC to the PSII core and efficient watersplitting [62,63]. Furthermore, stable association of the OEC andits ligands was suggested to require dimeric PSII core and periph-erally bound LHCII complexes [64].

Alternatively the observed effects might be attributed to im-paired PSII repair mechanism. The balance between PSII damageand repair is a subject of thorough investigation [65–67]. Althoughthose studies concern the photo- and heat-induced PSII damageand repair, two facts relate to our experimental results – retardedPSII repair correlates with lower oxygen evolution and the repairprocess is slowed down upon rigidification of the thylakoid lipidphase. Thus the reduced oxygen yields and membrane fluidity inthe BRI1OE, bri1-116 and cpd mutants might readily be explainedwith hampered PSII repair.

4.3. Optimal BR level is required for the proper assembly and activity ofthe photosynthetic apparatus

BRs are known to support the growth homeostasis, and there-fore a drift from the optimal BR limits would result in growth

Page 7

S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104 103

inhibition. This was demonstrated for Arabidopsis roots and leaveswhere both low and high BR levels exerted inhibitory effect on celldivision progression [5,6]. In red cabbage cotyledons treatmentwith 0.001 lM epi-brassinolide had positive effect on their growth(as deduced from the increased fresh weight, pigment and antho-cyanine content and peroxidase activity), whereas 10 lM concen-tration had inhibiting action [68], which confirms therequirement for an optimal BR level for proper plant growth anddevelopment. Taking into account the complexity of the BR action,we suggest that the similarity in the changes of the photosyntheticapparatus in BRI1OE, bri1-116, and cpd leaves, could be due to thelack of an optimal BR level. The enhanced BR signaling in BRI1OEplants triggers a feedback mechanism that consequently sup-presses the BR biosynthesis. As a result of the suboptimal BR level,BRI1OE thylakoids are modified structurally and functionally as theones in the BR loss-of-function mutants.

5. Conclusion

Our work reveals a novel control role of BR in the assembly andfunction of the photosynthetic apparatus as a part of the wide net-work of developmental and physiological processes affected by BRsignaling. We detected modified overall architecture of thylakoidmembranes and putative defect in chloroplast division in BR mod-ulated plants. In functional aspect we revealed an impaired PSIIfunction and oxygen evolution in BR mutants as a consequenceof altered OEC and PSII supercomplex assembly. This work demon-strates that the biophysical approach might complement the ge-netic and molecular investigations on BR action and signalingand improve our understanding of the structural organizationand the function of photosynthetic membranes.

6. Abbreviations

A

amplitude of the initial oxygen burst uponcontinuous illumination

AFM

atomic force microscopy BR brassinosteroid BRI1 BR INSENSITIVE 1 receptor bri1-

116

mutant with inactive BRI1 receptor

chl

chlorophyll CD circular dichroism Col-0 Arabidopsis thaliana ecotype Columbia-0 cpd constitutive photomorphogenesis and dwarfism

mutant

ETR non-cyclic electron-transport rate F fluorescence level detected upon application of

actinic light

Fm maximal fluorescence level for dark-adapted plants F 0m maximal fluorescence level for light-adapted plants F0 dark level of fluorescence Fv variable fluorescence LHCII major light-harvesting complex of photosystem II MC540 merocyanine 540 NPQ non-photochemical quenching OEC oxygen-evolving complex PSI photosystem I PSII photosystem II qP photochemical quenching coefficient Y3 oxygen yield at the third flash

Acknowledgments

The authors are grateful to Prof. Tsonko Tsonev for the technicalhelp and useful discussions. This work was supported by the Bul-garian National Science Fund [Grant Number DMU 02-7].

References

[1] U.K. Divi, P. Krishna, Brassinosteroid: a biotechnological target for enhancingcrop yield and stress tolerance, Nat. Biotechnol. 26 (2009) 131–136.

[2] V. Khripach, V. Zhabinskii, A. de Groot, Twenty years of brassinosteroids:steroidal plant hormones warrant better crops for the XXI century, Ann. Bot. 86(2000) 441–447.

[3] C. Vriet, E. Russinova, C. Reuzeau, Boosting crop yields with plant steroids,Plant Cell 24 (2012) 842–857.

[4] T. Kinoshita, A. Cano-Delgado, H. Seto, S. Hiranuma, S. Fujuika, J. Chory, Bindingof brassinosteroids to the extracellular domain of plant receptor kinase BRI1,Nature 433 (2005) 167–171.

[5] M.P. González-García, J. Vilarrasa-Blasi, M. Zhiponova, F. Divol, S. Mora-García,E. Russinova, A.I. Caño-Delgado, Brassinosteroids control meristem size bypromoting cell cycle progression in Arabidopsis roots, Development 138(2011) 849–859.

[6] M.K. Zhiponova, I. Vanhoutte, V. Boudolf, C. Betti, S. Dhondt, F. Coppens, E.Mylle, S. Maes, M.-P. González-García, A.I. Caño-Delgado, D. Inzé, G.T.S.Beemster, L. De Veylder, E. Russinova, Brassinosteroid production andsignaling differentially control cell division and expansion in the leaf, NewPhytol. 197 (2013) 490–502.

[7] S.D. Clouse, M. Langford, T.C. McMorris, A brassinosteroid-insensitive mutantin Arabidopsis thaliana exhibits multiple defects in growth and development,Plant Physiol. 11 (1996) 671–678.

[8] M. Szekeres, K. Németh, Z. Koncz-Kálmán, J. Mathur, A. Kauschmann, T.Altmann, G.P. Rédei, F. Nagy, J. Schell, C. Koncz, Brassinosteroids rescue thedeficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis, Cell 85 (1996) 171–182.

[9] S. Choe, B.P. Dilkes, S. Fujioka, S. Takatsuto, A. Sakurai, K.A. Feldmann, TheDWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple22alpha-hydroxylation steps in brassinosteroid biosynthesis, Plant Cell 10(1998) 231–243.

[10] D.M. Friedrichsen, C.A.P. Joazeiro, J. Li, T. Hunter, J. Chory, Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase, Plant Physiol. 123 (2000) 1247–1255.

[11] S. Choe, S. Fujioka, T. Noguchi, S. Takatsuto, S. Yoshida, K.A. Feldmann,Overexpression of DWARF4 in the brassinosteroid biosynthetic pathwayresults in increased vegetative growth and seed yield in Arabidopsis, Plant J.26 (2001) 573–582.

[12] M.-H. Oh, J. Sun, D.H. Oh, R.E. Zielinski, S.D. Clouse, S.C. Huber, EnhancingArabidopsis leaf growth by engineering the BRASSINOSTEROID INSENSITIVE1receptor kinase, Plant Physiol. 157 (2011) 120–131.

[13] A. Bajguz, S. Hayat, Effects of brassinosteroids on the plant responses toenvironmental stresses, Plant Physiol. Biochem. 47 (2009) 1–8.

[14] X.J. Xia, Z. Chen, J.-Q. Yu, ROS mediate brassinosteroids-induced plant stressresponses, Plant Signal. Behav. 5 (2010) 532–534.

[15] I. Sharma, P.K. Pati, R. Bhardwaj, Effect of 24-epibrassinolide on oxidativestress markers induced by nickel-ion in Raphanus sativus L, Acta Physiol. Plant33 (2012) 1723–1735.

[16] J.O. Ogweno, X.S. Song, K. Shi, W.H. Hu, W.H. Mao, Y.H. Zhou, J.Q. Yu, S. Nogues,Brassinosteroids alleviate heat-induced inhibition of photosynthesis byincreasing carboxylation efficiency and enhancing antioxidant systems inLycopersicon esculentum, J. Plant Growth Regul. 27 (2008) 49–57.

[17] S.A. Hasan, S. Hayat, A. Ahmad, Brassinosteroids protect photosyntheticmachinery against the cadmium induced oxidative stress in two tomatocultivars, Chemosphere 84 (2011) 1446–1451.

[18] X.-J. Xia, L.-F. Huang, Y.-H. Zhou, W.-H. Mao, K. Shi, J.-X. Wu, T. Asami, Z. Chen,J.-Q. Yu, Brassinosteroids promote photosynthesis and growth by enhancingactivation of Rubisco and expression of photosynthetic genes in Cucumissativus, Planta 230 (2009) 1185–1196.

[19] C.-Y. Wu, A. Trieu, P. Radhakrishnan, S.F. Kwok, S. Harris, K. Zhang, J. Wang, J.Wan, H. Zhai, S. Takatsuto, S. Matsumoto, S. Fujioka, K.A. Feldmann, R.I.Pennella, Brassinosteroids regulate grain filling in rice, Plant Cell 20 (2008)2130–2145.

[20] Y.-P. Jiang, F. Cheng, Y.-H. Zhou, X.-J. Xia, W.-H. Mao, K. Shi, Z. Chen, J.-Q. Yu,Cellular glutathione redox homeostasis plays an important role in thebrassinosteroid-induced increase in CO2 assimilation in Cucumis sativus, NewPhytol. 194 (2012) 932–943.

[21] J. Nield, J. Barber, Refinement of the structural model for the Photosystem IIsupercomplex of higher plants, Biochim. Biophys. Acta 1757 (2006) 353–361.

[22] R. Nevo, D. Charuvi, O. Tsabari, Z. Reich, Composition, architecture anddynamics of the photosynthetic apparatus in higher plants, Plant J. 70 (2012)157–176.

[23] E.J. Boekema, H. van Roon, F. Calkoen, R. Bassi, J.P. Dekker, Multiple types ofassociation of photosystem II and its light-harvesting antenna in partiallysolubilized photosystem II membranes, Biochemistry 38 (2009) 2233–2239.

Page 8

104 S. Krumova et al. / Journal of Photochemistry and Photobiology B: Biology 126 (2013) 97–104

[24] J.P. Dekker, E.J. Boekema, Supramolecular organization of thylakoid membraneproteins in green plants, Biochim. Biophys. Acta 1706 (2005) 12–39.

[25] M.A. Harrison, A. Melis, Organization and stability of polypeptides associatedwith the chlorophyll a-b light-harvesting complex of photosystem-II, PlantCell Physiol. 33 (1992) 627–637.

[26] D. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in betavulgaris, Plant Physiol. 24 (1949) 1–15.

[27] N.R. Baker, Chlorophyll fluorescence: a probe of photosynthesis in vivo, Ann.Rev. Plant Biol. 59 (2008) 89–113.

[28] B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2

evolution. I. A linear four step mechanism, Photochem. Photobiol. 11 (1970)457–475.

[29] P. Joliot, B. Kok, Oxygen evolution in photosynthesis, in: Govinjee (Ed.),Bioenergetics of Photosynthesis, Academic Press, New York, 1975, pp. 387–412.

[30] P. Joliot, A. Joliot, A polarographic method for detection of oxygen productionand reduction of Hill reagent by isolated chloroplasts, Biochim. Biophys. Acta153 (1968) 625–634.

[31] Y. Zeinalov, Equipment for investigations of photosynthetic oxygen productionreactions, Bulg. J. Plant Physiol. 28 (2002) 57–67.

[32] S.G. Chuartzman, R. Nevo, E. Shimoni, D. Charuvi, V. Kiss, I. Ohad, V. Brumfeld,Z. Reich, Thylakoid membrane remodeling during state transitions inArabidopsis, Plant Cell 20 (2008) 1029–1039.

[33] G. Garab, H. van Amerongen, Linear dichroism and circular dichroism inphotosynthesis research, Photosynth. Res. 101 (2009) 135–146.

[34] G. Garab, S. Wells, L. Finzi, C. Bustamante, Helically organized macroaggregatesof pigment-protein complexes in chloroplasts: evidence from circularintensity differential scattering, Biochemistry 27 (1988) 5839–5843.

[35] L. Finzi, C. Bustamante, G. Garab, C.B. Juang, Direct observation of large chiraldomains in chloroplast thylakoid membranes by differential polarizationmicroscopy, Proc. Natl. Acad. Sci. USA 86 (1989) 8748–8752.

[36] G. Garab, L. Mustárdy, Role of LHCII-containing macrodomains in thestructure, function and dynamics of grana, Aust. J. Plant Physiol. 26 (2009)649–658.

[37] J.K. Holm, Z. Várkonyi, L. Kovács, D. Posselt, G. Garab, Thermooptically inducedreorganizations in the main light harvesting antenna of plants. II. Indicationsfor the role of LHCII-only macrodomains in thylakoids, Photosynth. Res. 86(2005) 275–282.

[38] L. Kovács, J. Damkjær, S. Kereïche, C. Ilioaia, A.V. Ruban, E.J. Boekema, S.Jansson, P. Horton, Lack of the light-harvesting complex CP24 affects thestructure and function of the grana membranes of higher plant chloroplasts,Plant Cell 18 (2006) 3106–3120.

[39] S. Georgakopoulou, G. van der Zwan, R. Bassi, R. van Grondelle, H. vanAmerongen, R. Croce, Understanding the changes in the circular dichroism oflight harvesting complex II upon varying its pigment composition andorganization, Biochemistry 46 (2007) 4745–4754.

[40] A.G. Dobrikova, Z. Várkonyi, S.B. Krumova, L. Kovács, G.K. Kostov, S.J. Todinova,M.C. Busheva, S.G. Taneva, G. Garab, Structural rearrangements in chloroplastthylakoid membranes revealed by differential scanning calorimetry andcircular dichroism spectroscopy. Thermo-optic effect, Biochemistry 42(2003) 11272–11280.

[41] S.B. Krumova, R.B.M. Koehorst, A. Bóta, T. Páli, A. van Hoek, G. Garab, H. vanAmerongen, Temperature dependence of the lipid packing in thylakoidmembranes studied by time- and spectrally resolved fluorescence ofMerocyanine 540, Biochim. Biophys. Acta 1778 (2008) 2823–2833.

[42] C. Kuo, R.M. Hochstrasser, Super-resolution microscopy of lipid bilayer phases,J. Am. Chem. Soc. 133 (2011) 4664–4667.

[43] J.-W. Liou, X. Mulet, D.R. Klug, Absolute measurement of phosphorylationlevels in a biological membrane using atomic force microscopy: the creation ofphosphorylation maps, Biochemistry 41 (2002) 8535–8539.

[44] C.C. Gradinaru, P. Martinsson, T.J. Aartsma, T. Schmidt, Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo,Ultramicroscopy 99 (2004) 235–245.

[45] T. Yamada, H. Arakawa, T. Okajima, T. Shimada, A. Ikai, Use of AFM for imagingand measurement of the mechanical properties of light-convertible organellesin plants, Ultramicroscopy 91 (2002) 261–268.

[46] J. Austin II, A.N. Webber, Photosynthesis in Arabidopsis thaliana mutants withreduced chloroplast number, Photosynth. Res. 85 (2005) 373–384.

[47] H. Gao, T.L. Sage, K.W. Osteryoung, FZL, an FZO-like protein in plants, is adeterminant of thylakoid and chloroplast morphology, Proc. Natl. Acad. Sci.103 (2006) 6759–6764.

[48] J.M. Glynn, S.-Y. Miyagishima, D.W. Yoder, K.W. Osteryoung, S. Vitha,Chloroplast division, Traffic 8 (2007) 451–461.

[49] S.Y. Kim, B.H. Kim, K.H. Nam, Reduced expression of the genes encodingchloroplast-localized proteins in a cold-resistant bri1 (brassinosteroid-insensitive 1) mutant, Plant Signal. Behav. 5 (2010) 458–463.

[50] X.-J. Xia, Y.-J. Wang, Y.-H. Zhou, Y. Tao, W.-H. Mao, K. Shi, T. Asami, Z. Chen, J.-Q. Yu, Reactive oxygen species are involved in brassinosteroid-induced stresstolerance in cucumber, Plant Physiol. 150 (2009) 801–814.

[51] R. Fristedt, A. Willig, P. Granath, M. Crevecoeur, J.D. Rochaix, A.V. Vener,Phosphorylation of photosystem II controls functional macroscopic folding ofphotosynthetic membranes in Arabidopsis, Plant Cell 21 (2009) 3950–3964.

[52] R. Fristedt, P. Granath, A.V. Vener, A protein phosphorylation threshold forfunctional stacking of plant photosynthetic membranes, PLoS ONE 5 (2010)(2010) e10963.

[53] M. Tikkanen, E.-M. Aro, Thylakoid protein phosphorylation in dynamicregulation of photosystem II in higher plants, Biochim. Biophys. Acta 2012(1817) 232–238.

[54] L. Dietzel, K. Brautigam, S. Steiner, K. Schuffler, B. Lepetit, B. Grimm, M.A.Schottler, T. Pfannschmidt, Photosystem II supercomplex remodeling serves asan entry mechanism for state transitions in Arabidopsis, Plant Cell 23 (2011)2964–2977.

[55] S.B. Krumova, C. Dijkema, P. de Waard, H.V. As, G. Garab, H. van Amerongen,Phase behavior of phosphatidylglycerol in spinach thylakoid membranes asrevealed by 31P-NMR, Biochim. Biophys. Acta 1778 (2008) 997–1003.

[56] M. Havaux, Stress tolerance of photosystem II in vivo: antagonistic effects ofwater, heat, and photoinhibition stresses, Plant Physiol. 100 (1992) 424–432.

[57] J. Cela, L. Arrom, S. Munné-Bosch, Diurnal changes in photosystem IIphotochemistry, photoprotective compounds and stress-relatedphytohormones in the CAM plant, Aptenia cordifolia, Plant Sci. 177 (2009)404–410.

[58] E.J. Boekema, J.F.L. van Breemen, H. van Roon, J.P. Dekker, Conformationalchanges in photosystem II supercomplexes upon removal of extrinsic subunits,Biochemistry 39 (2000) 12907–12915.

[59] K. Ifuku, Y. Yamamoto, T.A. Ono, S. Ishihara, F. Sato, PsbP protein, but not PsbQprotein, is essential for the regulation and stabilization of photosystem II inhigher plants, Plant Physiol. 139 (2005) 1175–1184.

[60] X. Yi, S.R. Hargett, L.K. Frankel, T.M. Bricker, X. Yi, S.R. Hargett, L.K. Frankel, T.M.Bricker, The PsbP protein, but not the PsbQ protein, is required for normalthylakoid architecture in Arabidopsis thaliana, FEBS Lett. 583 (2009) 2142–2147.

[61] J. De Las Rivas, P. Heredia, A. Roman, Oxygen-evolving extrinsic proteins (PsbO,P, Q, R): bioinformatic and functional analysis, Biochim. Biophys. Acta 1767(2007) 575–582.

[62] J. Nield, O. Kruse, J. Ruprecht, P. da Fonseca, C. Büchel, J. Barber, Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcuselongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization, J. Biol. Chem. 275 (2000) 27940–27946.

[63] M. Suorsa, R.E. Regel, V. Paakkarinen, N. Battchikova, R.G. Herrmann, E.-M. Aro,Protein assembly of photosystem II and accumulation of subcomplexes in theabsence of low molecular mass subunits PsbL and PsbJ, Eur. J. Biochem. 271(2004) 96–107.

[64] A. Hashimoto, W.F. Ettinger, Y. Yamamoto, S.M. Theg, Assembly of newlyincorporated oxygen-evolving complex subunits in isolated chloroplasts: sitesof assembly and mechanism of binding, Plant Cell 9 (1997) 441–452.

[65] S.I. Allakhverdiev, N. Murata, Environmental stress inhibits the synthesis denovo of proteins involved in the photodamage-repair cycle of Photosystem IIin Synechocystis sp. PCC 6803, Biochim. Biophys. Acta 1657 (2004) 23–32.

[66] P. Mohanty, S.I. Allakhverdiev, N. Murata, Application of low temperaturesduring photoinhibition allows characterization of individual steps inphotodamage and the repair of photosystem II, Photosynth. Res. 94 (2007)217–224.

[67] S.I. Allakhverdiev, V.D. Kreslavski, V.V. Klimov, D.A. Los, R. Carpentier, P.Mohanty, Heat stress: an overview of molecular responses in photosynthesis,Photosynth. Res. 98 (2008) 541–550.

[68] S. Çag, N. Gören-Saglam, Ç. Çıngıl-Barıs�, E. Kaplan, The effect of differentconcentration of epibrassinolide on chlorophyll, protein and anthocyanincontent and peroxidase activity in excised red cabbage (Brassica oleraceae L.)cotyledons, Biotechnol. Biotechnol. Eq. 21 (2007) 422–425.