Photoinhibition of photosystem I in a pea mutant with altered LHCII organization

Journal of Photochemistry and Photobiology B: Biology xxx (2015) xxx–xxx

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Photoinhibition of photosystem I in a pea mutant with altered LHCII organization A.G. Ivanov a,⇑, R.M. Morgan-Kiss b, M. Krol a, S.I. Allakhverdiev c,d,e, Yu. Zanev f, P.V. Sane g, N.P.A. Huner a,⇑ a

Department of Biology and the Biotron Centre for Experimental Climate Change Research, University of Western Ontario, 1151 Richmond Street, N., London, Ontario N6A 5B7, Canada Department of Microbiology, Miami University, 700 E. High Street, Oxford, OH 45045, USA c Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russia d Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia e Department of Plant Physiology, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow 119991, Russia f Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria g Jain Irrigation Systems Limited, Jain Hills, Jalgaon 425001, India b

a r t i c l e

i n f o

Article history: Received 31 May 2015 Received in revised form 10 August 2015 Accepted 13 August 2015 Available online xxxx Keywords: High light stress P700 PSI photochemistry Pisum sativum Thermoluminescence State transition

a b s t r a c t Comparative analysis of in vivo chlorophyll fluorescence imaging revealed that photosystem II (PSII) photochemical efficiency (Fv/Fm) of leaves of the Costata 2/133 pea mutant with altered pigment composition and decreased level of oligomerization of the light harvesting chlorophyll a/b-protein complexes (LHCII) of PSII (Dobrikova et al., 2000; Ivanov et al., 2005) did not differ from that of WT. In contrast, photosystem I (PSI) activity of the Costata 2/133 mutant measured by the far-red (FR) light inducible P700 (P700+) signal exhibited 39% lower steady state level of P700+, a 2.2-fold higher intersystem electron pool size (e
/P700) and higher rate of P700+ re-reduction, which indicate an increased capacity for PSI cyclic electron transfer (CET) in the Costata 2/133 mutant than WT. The mutant also exhibited a limited capacity for state transitions. The lower level of oxidizable P700 (P700+) is consistent with a lower amount of PSI related chlorophyll protein complexes and lower abundance of the PsaA/PsaB heterodimer, PsaD and Lhca1 polypeptides in Costata 2/133 mutant. Exposure of WT and the Costata 2/133 mutant to high light stress resulted in a comparable photoinhibition of PSII measured in vivo, although the decrease of Fv/Fm was modestly higher in the mutant plants. However, under the same photoinhibitory conditions PSI photochemistry (P700+) measured as DA820
860 was inhibited to a greater extent (50%) in the Costata 2/133 mutant than in the WT (22%). This was accompanied by a 50% faster re-reduction rate of P700+ in the dark indicating a higher capacity for CET around PSI in high light treated mutant leaves. The role of chloroplast thylakoid organization on the stability of the PSI complex and its susceptibility to high light stress is discussed. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction The exposure of photosynthetic organisms to various environmental stresses such as low and high temperatures, excess light

Abbreviations: AG, afterglow thermoluminescence band; DCMU, 3-(3,4-dichlor ophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichlorophenolindophenol; Fv/Fm, maximum photochemical efficiency of PSII in the dark adapted state; Fr, capacity for state transitions; LHCII a/b, light-harvesting chlorophyll a/b-protein complex of PSII; MV, methyl viologen; P700, reaction center chlorophyll of PSI; PQ, plastoquinone; PSI, photosystem I; PSII, photosystem II; qN, non-photochemical quenching; qP, photochemical quenching parameter; SDS, sodium dodecyl sulfate; TL, thermoluminescence; TM, thermoluminescence peak temperature; Tricine, N-(tris (hydroxymethyl)methyl)glycine. ⇑ Corresponding authors. E-mail addresses: [email protected] (A.G. Ivanov), [email protected] (N.P.A. Huner).

and water and nutrient stress may cause an imbalance between the capacity for harvesting light energy and the capacity to dissipate this energy through metabolic activity, resulting in excess PSII excitation pressure. Excess excitation pressure, measured as the relative redox state of QA, the first stable quinine electron acceptor of photosystem II (PSII) reaction centers, reflects the overall reduction state of the photosynthetic electron transport chain [3–5]. The imbalance between the reducing equivalents produced in excess that exceeds the capacity of the metabolic sinks to utilize the electrons generated from the absorbed energy may be caused by either exposure to an irradiance that exceed the light harvesting capacity or by any environmental constrains that may decrease the capacity of the metabolic pathways downstream of photochemistry (C, N, and S assimilation) to utilize photosynthetically generated reductants [4–6]. This imbalance, which is a prerequisite for any stress

http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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can potentially result in generation of reactive oxygen species (ROS) such as 1O2 and O
2 , leading to photoinhibition and photooxidative damage of photosystem II (PSII) [7–13]. Apart from PSII, various environmental stress conditions can also cause photoinhibitory damage on photosystem I (PSI) [14–22]. This potential for photoinhibition makes it necessary for the plant to develop mechanisms for photoprotection of the photosynthetic apparatus. The major photoprotective mechanism playing a key role for de-excitation of excess light energy in green plants and algae is considered to be the DpH- and zeaxanthin (Zx)-dependent non-photochemical quenching (NPQ) occurring in the pigment bed of LHCII proteins [23–26], although alternative/supplementary photoprotective mechanisms for effective thermal deactivation of excess light energy have also been proposed [13,24,27–29]. In addition to its essential role in development of NPQ, lightharvesting Chl a/b-binding protein complex of PSII (LHCII) is the major component of the chloroplast thylakoid membranes, which mediates their macrostructural arrangement (granal stacking), and is believed to regulate the excitation energy transfer between photosystem I (PSI) and photosystem II (PSII) via redox-dependent reversible phosphorylation of its major component [13,29,30–34]. LHCII is also known to be involved in adaptation of plants to the light environment [35] and is dynamically regulated by short-term or long-term changes in the environmental growth conditions such as temperature, irradiance and nutrient availability [6,36]. Electron crystallography at 3.4 Å resolution of LHCII crystals from pea revealed that LHCII exists in the trimeric form, which is believed to predominate in the thylakoids in vivo [37,38]. The importance of high ordered oligomeric structural organization of LHCII for the dynamics and functioning of the photosynthetic membranes has been well established [39–43]. Indeed, light induced LHCII trimer to monomer transitions as well as LHCII trimer–trimer interactions in higher plants have been suggested to have significant impact on light harvesting/quenching capacity of thylakoid membranes in vivo [42,43]. It has been suggested that the xanthophylls cycle pigments loosely bound to the periphery of LHCII, are important element in stabilizing the structure of LHCII trimer aggregates [25,44,45]. More importantly, it has been demonstrated that trimeric organization of LHCII is better adapted for efficient light harvesting, exhibit enhanced protein stability and possess the optimal capacity for non-radiative energy dissipation [46,47]. However, the traditional view of the organization of PSII and PSI as separate entities has been challenged recently. New evidence indicates that PSII and PSI are energetically connected because they are embedded in a common lake of LHCII in Arabidopsis thaliana [13,29,48,49]. In view of these versatile and important roles of LHCII trimers in functioning of the photosynthetic apparatus, the impact of structural organization of the LHCII complexes was assessed in a Costata 2/133 pea mutant with altered pigment content and decreased level of LHCII oligomerization [1,2,50]. The response of the Costata mutant to high light stress at low temperature was compared to wild type plants by in vivo measurements of PSII and PSI photochemical performance and pigment and polypeptide composition of thylakoid membranes. We demonstrate that while exposure of the Costata 2/133 mutant to excess light caused stronger inhibition of both PSII and PSI photochemistry compared to wild type plants, PSI photochemistry is more severely affected. The higher susceptibility to photoinhibition of PSI correlates with lower abundance of PSI-related proteins in the mutant. 2. Materials and methods 2.1. Plant material Wild type (WT) (Pisum sativum L. cv. Borek) and Costata 2/133 mutant with altered pigment content and decreased level of LHCII

oligomerization [1,2,50] pea plants were germinated from seeds in coarse vermiculite with 16 h light/dark period in controlled environment growth chambers (Conviron, Winnipeg, MB, Canada). Fluorescent tubes (Cool White, 160 W, F72T12/CW/VHO, Sylvannia, Drummondville, QC, Canada) provided PAR which was adjusted to 250 lmol photons m
2 s
1 PPFD. Day/night temperatures were 20°C/16 °C. Relative humidity was 50%. Fully expanded leaves harvested 2–3 h after the beginning of the light cycle were used in all experiments. 2.2. Non-denaturating SDS–PAGE Isolation of thylakoid membranes for non-denaturating SDS– PAGE was performed as described previously [1]. Chloroplast membranes for electrophoretic separation of Chl–protein complexes were prepared according to [51]. Samples were resuspended in a deoxycholic acid (DOC):SDS:Chl ratio of 20:10:1 in a 0.3 M Tris–HCl (pH 8.0) solubilization buffer containing 13% (w/v) glycerol. Separation of the chlorophyll–protein complexes by non-denaturing SDS–PAGE was performed on an 8% (w/v) polyacrylamide resolving gel containing 150 mM Tris–HCl (pH 6.35) buffer and a 4% (w/v) stacking gel containing 40 mM Tris–HCl (pH 6.14) buffer. Samples were loaded with an equal amount of protein (20 lg per line). Protein concentration was determined using Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Thermo Scientific, USA). The excised lanes were scanned at 671 nm on a Beckman DU 640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, USA). 2.3. SDS–PAGE and immunoblotting Thylakoid membranes for SDS-PAGE WT type and Costata 2/133 mutant of pea were isolated as described earlier [52]. Benzamidine and aminocaproic acid were present in the homogenization buffer at concentrations of 2 mM. Samples containing equal amounts of protein were separated on a 15% (w/v) linear polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (0.2 lm pore size, Bio-Rad) at 5 °C for 2 h at 25 mA. Immunoblot analysis was performed as in [1] and the thylakoid proteins were detected with specific antibodies at the following dilutions: PsbA (D1), 1:5000; Lhcb2, 1:5000, Lhcb3, 1:5000; PsaA/B, 1:500; PsaD, 1:1000; Lhca1, 1:2000. All antibodies, except the antibody against PsaA/B heterodimer, were obtained from AgriSera AB (Vanas, Sweden). PsaA/B antibody was acquired as described in [51]. After incubation with anti-rabbit horseradish peroxidase-conjugated secondary antisera (Sigma–Aldrich, St. Louis, 1:20,000 dilution), the antibody complexes were visualized by incubation of the blots in ECLTM chemiluminescent detection reagents (Amersham Biosciences, GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and developed on Cronex 4 X-ray film (Kodak). Immunoblots were performed on samples from at least three independent replicate experiments. Densitometric scanning and analysis of X-ray films from each replicate immunoblot was performed with a Hewlett Packard ScanJet 4200C desktop scanner and ImageJ 1.41o densitometry software (Wayne Rosband, National Institute of Health, USA, http://rsb.info.nih.gov.ij). 2.4. Modulated chlorophyll fluorescence Chlorophyll a fluorescence of a dark adapted (30 min) leaves of WT and Costata 2/133 pea mutant plants was measured under ambient CO2 conditions using a PAM 101 chlorophyll fluorescence measuring system (Heinz Walz GmbH, Effeltrich, Germany) as described in [18,53]. Alternatively, a modulated imaging fluorometer (IMAGING-PAM, Heinz Walz GmbH, Effeltrich, Germany) was used for capturing the chlorophyll fluorescence images and

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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estimation of the maximal photochemical efficiency of PSII (Fv/Fm), quantum yield of PSII photochemistry (UPSII) and nonphotochemical (qN) fluorescence quenching parameters [20]. Fluorescence images were captured by a CCD camera (IMAG-K, Allied Vision Technologies) featuring 640  480 pixel CCD chip size and CCTV camera lens (Cosmicar/Pentax F1.2, f = 12 mm). Light emitting diode ring array (IMAG-L) consisting of 96 blue LEDs (470 nm) provided standard modulated excitation intensity of 0.5 lmol quanta m
2 s
1 (modulation frequency 1–8 Hz) for measuring the basal (Fo) chlorophyll fluorescence and a saturation pulse of 2400 lmol quanta m
2 s
1 PAR for measuring the maximal chlorophyll fluorescence (Fm). The fluorescence characteristics were evaluated when the steady state Fs level was reached. The nomenclature of van Kooten and Snel [54] was used for calculating the quantum yield of PSII photochemistry (UPSII), photochemical (qP), and non-photochemical (qN) fluorescence quenching parameters. All measurements were performed at the growth temperature of 20 °C. Electron transport rates (ETR) were calculated as: ETR = PAR  0.5  UPSII  A [55], where A is the actual measured leaf absorptance before performing the fluorescence measurement. The apparent leaf absorptance (A) was measured following the procedure described by the manufacturer of the IMAGING-PAM (imagm-series0e_k.doc, Heinz Walz GmbH, Efeltrich, Germany) and was calculated pixel by pixel from the red (R)- and near infra-red (NIR)images using the formula: A = 1
R/NIR. Reduction state of the plastoquinone (PQ) pool was assessed following the post-illumination transient increase of chlorophyll fluorescence at the Fo’ level [21,56,57].

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far red light (FR) provided by a FL-101 light source (kmax = 715 nm, 10 W m
2, Schott filter RG 715). The relative capacity for state transition (Fr) was calculated as: Fr = [(FI0
FI)
(FII0
FII)]/(FI’
FI), where FI and FII designate fluorescence in the presence of PSI light (far-red) in state 1 and state 2, respectively, while FI’ and FII’ designate fluorescence in the absence of PSI light in state 1 and state 2, respectively [20,32]. 2.7. P700 Measurements The reduction–oxidation (redox) state of P700 was determined in vivo under ambient O2 and CO2 conditions using a PAM-101 modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany) equipped with a dual wavelength emitter-detector ED-P700DW unit and PAM-102 units [62] as described in detail by Ivanov et al. [18]. Far red light (FR; kmax = 715 nm, 10 W m
2, Schott filter RG 715) was provided by a FL-101 light source. The redox state of P700 was evaluated as the absorbance change around 820 nm (DA820
860) in a custom designed cuvette. Multiple turnover (MT, 50 ms) and single turnover (ST, half peak 14 ls) saturating flashes were applied with XMT-103 and XST-103 (Heinz Walz GmbH, Effeltrich, Germany) power/control units, respectively. The absorbance transients of P700+ signal after application of single (ST), multiple (MT) turnover flashes of white saturating light and actinic light (AL) were used for estimation of the intersystem electron (e
) pool size [18,56] and electron flow to the intersystem chain from stromal components [56]. 2.8. Electron transport measurements

2.5. HPLC pigment analysis Pigments from pea leaves were extracted with 100% acetone at 4 °C under dim light. Pigments were separated and quantified by high-performance liquid chromatography (HPLC) as described previously in [58] with some modifications. The system contained a Beckman System Gold programmable solvent module 126, diode array detector module 168 (Beckman Instruments, San Ramon, California, USA), CSC-Spherisorb ODS-1 reverse-phase column (5 lm particle size, 25  0.46 cm I.D.) with an Upchurch Perisorb A guard column (both columns from Chromatographic Specialties Inc., Concord, Ontario, Canada). Pigments were eluted isocratically for 6 min with a solvent system acetonitrile: methanol: 0.1 M Tris–HCl (pH 8.0), (72:8:3.5, v/v/v), followed by a 2 min linear gradient to 100% methanol:hexane (75:25, v/v) which continued isocratically for 4 min. Total run time was 12 min. Flow rate was 2 cm3 min
1. Absorbance was detected at 440 nm and peak areas were integrated by Beckman System Gold software. Retention times and response factors of lutein and ß-carotene were determined by injection of known amounts of pure standards purchased from Sigma (St. Louis, MO, USA). The retention times of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) and neoxanthin were determined by using pigments purified by thin-layer chromatography as described in [59]. The epoxidation state of the xanthophylls pool (EPS) was estimated following the methods of Thayer and Björkman [60] and calculated as: EPS = (V + 0.5A)/(V + A + Z), where V, A, and Z correspond to the concentrations of the xanthophyll carotenoids violaxanthin, antheraxanthin, and zeaxanthin, respectively. 2.6. State transitions State 1 and state 2 transitions in leaves of WT and Costata 2/133 mutant pea plants were estimated at the growth temperature of 23 °C as described by Ivanov et al. [20,21,61], using the PAM-101 chlorophyll fluorescence measuring system equipped with a blue light source consisting of a lamp with a Corning 4-96 filter and a

Thylakoid membranes from WT and the Costata 2/133 mutant pea plants were isolated as described earlier [63] and resuspended in 10 mM Tricine-NaOH buffer (pH 8.0, 330 mM sucrose, 5 mM MgCI2, and 10 mM KCl to a final chlorophyll concentration of about 3 mg/ml. Total chlorophyll concentration was determined by the method of Arnon [64]. Photosystem I (PSI)-mediated electron transport rates (DCPIP. H2 to methyl viologen (MV)-induced O2 uptake) were measured polarographycally in a medium containing: 66 mM phosphate buffer (pH 7.5), 330 mM sucrose, 130 mM KCl, 0.4 mM DCPIP, 0.1 mM MV, 0.04 mM DCMU, 4 mM ascorbate, 0.04 mM sodium azide and 25 lg Chl ml
1 using a Clark type O2 electrode and a CB1D oxygen electrode control box (Hansatech Ltd., Kings Lynn, England) assembly at 20 °C [65]. Actinic light of 250 lmol photons m
2 s
1, corresponding to the growth light, was provided by LS2 light source control box (Hansatech Ltd., Kings Lynn, England). Photosystem II (PSII)-supported electron transport (H2O to DCPIP) rates were monitored by measuring DCPIP photoreduction at 590 nm using a Beckman DUÒ640 spectrophotometer (Beckman Instruments, San Ramon, California) as described in [66]. Reaction mixture contained 10 mM Tricine (pH 8.0), 330 mM sucrose, 5 mM MgCl2, 0.03 mM DCPIP and chloroplast membranes corresponding to 25 lg Chl ml
1 was used. Red actinic light was supplied by the same light source as in the PSI measurements except that a red cut-off filter (Schott RG 11) was fitted. 2.9. Thermoluminescence Far-red (FR) light induced thermoluminescence (TL) emission measurements of control and high-light treated WT and Costata 2/133 mutant pea leaf discs (diameter, 15 mm) were performed on a personal-computer-based TL data acquisition and analysis system [67] with some modifications [61,68] essentially as described in [69]. The samples were dark adapted for 15 min at 2 °C and illuminated at the same temperature for 60 s with FR light (kmax = 715 nm, 10 W m
2, Schott filter RG-715) provided by an

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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FL-101 (Heinz Walz GmbH, Effeltrich, Germany) light source. A drop of 50% (v/v) glycerol in water (100 ll) was placed on the center of the aluminum plate to ensure good thermal contact with the leaf. Immediately after switching off the far-red light, temperature was increased and the luminescence emission by the sample during heating was measured by a red-sensitive photomultiplier tube (Hamamatsu R943-02, Hamamatsu Photonics, Shizuoka-ken, Japan) equipped with a high voltage power supply (Model C3350, Hamamatsu Photonics, Shizuoka-ken, Japan). Experiments were performed at a heating rate of 0.6 °C s
1. The output signal from the photomultiplier, which is proportional to the TL photon emission, and the output signal from a bridge amplifier (Model YZ6, Bulgarian Academy of Sciences, Sofia, Bulgaria) which is proportional to the temperature of the sample holder, were recorded in two synchronized channels of an A/D converter data acquisition card and a personal computer [67]. The nomenclature of Sane et al. [70] was used for characterization of the TL glow peaks.

3. Results In agreement with an earlier report [2], the photochemical efficiency of PSII measured as Fv/Fm in the Costata 2/133 mutant was identical to that in WT plants (Fig. 1A, Table 1). In addition, the values of photochemical quenching (qP) and the yield of PSII photochemistry (UPSII), measured at the growth light intensity of 250 lmol photons m
2 s
1 were marginally affected (Fig. 1A), compared to WT plants. However, the mutant plants exhibited consistently lower qP values within a wide range of light intensities tested (Fig. 1B). In parallel, non-photochemical quenching (qN) was substantially increased in the mutant plants within the same light intensity range (Fig. 1C). Despite these rather minor differences the light response curves of photosynthetic electron transport rates (ETR) in control Costata 2/133 mutant was only marginally lower compared to control WT plants (Fig. 1E, open symbols). Since the non-photochemical quenching (qN) originating from the LHCII is believed to be DpH and/or zeaxanthin-dependent [23,71], a quantitative analysis of the zeaxanthin cycle pigments was performed. The carotenoid composition and content analyses demonstrated a decreased total amount of the three components of the xanthophyll cycle, i.e. violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) (V + A + Z) in Costata 2/133 mutant compared to control WT leaves (Table 2). However, despite the initially lower level of the xanthophyll pool size (V + A + Z) [2] (Table 2), a 3-fold larger fraction of A + Z pool corresponded to 12% lower epoxidation state (EPS) in the mutant compared to WT plant (Table 2). This may explain the higher capacity for non-photochemical quenching in Costata 2/133 plants. Exposure of either WT or Costata 2/133 mutant plants to high light stress resulted in much lower, but comparable EPS values (Table 2). This indicates that the WT and the Costata mutant exhibited equally operational and effective xanthophyll cycle activity under photoinhibitory conditions. The effects of exposure to high light treatment at low temperature (5 °C) on the photochemical efficiency of PSII measured as Fv/Fm in WT and the Costata 2/133 mutants plants are presented in Fig. 1D. As expected high light stress resulted in a similar gradual decrease of PSII photochemistry (Fv/Fm) in both WT and 2/133 mutant plants, although the mutant exhibited slightly higher (10%) susceptibility to PSII photoinhibition (Fig. 1D). However, the mutant pants exhibited much higher sensitivity of the photosynthetic electron transport rates (ETR) to high light stress compared to WT plants (Fig. 1E, closed symbols). Far red (FR) light-induced absorbance transients at 820 nm (DA820
860), which reflects the oxidation of P700 to P700+ [18,20–22,62] were used to estimate the functional performance

of PSI in WT and the Costata 2/133 mutant pea leaves in vivo. The typical traces illustrating the FR light-induced oxidation of P700 to P700+ presented in Fig. 2 indicate a much lower (64%) capacity for P700 photo-oxidation (P700+) measured as DA820
860 in the mutant compared with the P700+ values in WT leaves under control conditions (Fig. 2B, Table 1). Concomitantly, kinetic measurements of dark re-reduction of P700+ after turning off the FR light (is thought to reflect the extent of cyclic electron flow (CEF) around PSI [18,72,73] and/or the interaction of stromal components with the intersystem electron transport chain [74]. The dark rereduction of P700+ indicated a significantly accelerated rereduction of P700+ in the Costata 2/133 mutant compared to WT plants (Fig. 2, Table 1). In addition, the apparent electron donor pool size to PSI was assessed by measuring single (ST) and multiple (MT)-turnover flash induced DA820
860 under steady state oxidation of PSI by FR light [18,56,74]. Mutant plants exhibited 2.2fold higher electron pool size compared to WT (Table 1). Thus, in contrast to PSII photochemistry, in vivo measurements clearly demonstrate that PSI photochemical activity measured as P700 photooxidation is significantly lower in Costata 2/133 mutant plants compared to WT. In vitro measurements of partial photochemical activities of PSII and PSII in isolated thylakoid membranes also demonstrated that while both WT and the Costata 2/133 mutant exhibited comparable PSII activities, PSI-dependent electron transport was reduced to 66% of that in WT plants (Table 3). These results are consistent with the in vivo measurements and suggest that the differences in PSI photochemistry reflect the intrinsic properties and differences in the photosynthetic apparatus between WT and the Costata 2/133 mutant plants. Selective inhibition of PSI-related photochemical activities under low temperatures and either high light or moderate/weak illumination has also been reported in various plant species [14– 19,21,22]. Concomitant with the lower level of oxidized P700 (P700+) (Fig. 2B, Table 1), exposure of Costata 2/133 mutant leaves to high light at low temperature caused much greater loss of PSI activity measured as P700 photo-oxidation (P700+) during the 2h measuring period than in wild-type leaves (Fig. 2D, Fig. 3A). In addition, the rates of P700+ dark re-reduction (tP700+decay ) in high 1/2 light treated mutants plants were significantly faster than in WT during the same measuring period (Fig. 3B). This suggests that despite the initially elevated rates of P700+ dark re-reduction, the mutant plants can further develop higher capacity for PSIdependent cyclic electron flow (CEF) in response to high light stress than WT pea pants. The composition of thylakoid membranes isolated from WT and the Costata 2/133 pea mutant leaves were examined by nondenaturating SDS–PAGE to separate the major Chl–protein complexes. Typical densitograms of non-denaturating SDS–PAGE gels of thylakoid membranes from WT (Fig. 4A) and the Costata mutant (Fig. 4B) plants resolved six distinct bands previously identified and designated to: LHCI – the major Chl a/b protein complex of PSI, CP1 – the major Chl a core protein complex of PSI containing the reaction center P700, LHCII1, LHCII2 and LHCII3 – the oligomeric, dimeric and monomeric forms respectively of the major Chl a/b light-harvesting protein complex of PSII, and CPa – the Chl a protein complex containing the reaction center of PSII (P680) and its associated core antennae [1,2,20]. The quantitative densitometric analysis of the gel scans of the Chl–protein complexes confirmed the previously reported decrease of LHCII1/LHCII3 ratio in the Costata 2/133 mutant [1] (Table 4). However, the relative abundance of PSI-related LHC1–CP1 and CP1 bands were reduced to 44% and 79%, respectively in the Costata 2/133 mutant compared to the WT plants (Fig. 4B, Table 4), whereas the abundance of PSII-associated complex containing the reaction center P680 (CPa) exhibited minimal differences (Table 4). The lower

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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Fig. 1. (A) Chlorophyll fluorescence imaging analysis of control and high light treated WT and Costata 2/133 mutant leaves representing the maximum photochemical efficiency of PSII measured as Fv/Fm (a, b, c, d), photochemical (qP) chlorophyll fluorescence quenching (e, f, g, h) and quantum yield of PSII photochemistry – UPSII (i, j, k, l). (B) and (C) Light intensity dependence of photochemical (qP) and non-photochemical (qN) chlorophyll fluorescence quenching, respectively in control WT and 2/133 mutant leaves. (D) Effects of high light treatment on the photochemical efficiency of PSII (Fv/Fm). (E) Light intensity dependence of ETR in control (open symbols) and high light treated (closed symbols) leaves of WT and Costata 2/133 mutant. The values in panels B–E represent means ± SE calculated from 3 to 5 independent measurements.

relative abundance of LHC1–CP1 and CP1 bands suggests an alteration in the stoichiometry of PSI (CP1) and PSII (CPa) chlorophyll– protein complexes in the Costata 2/133 mutant relative to the WT control. Indeed, an 18% lower CP1/CPa ratio was detected in the mutant plants (Table 4).

The relative abundance of the major constituents of PSI and PSII was further analyzed by immuno-detection of key polypeptides of these pigment–protein complexes (Fig. 4C). Representative immunoblots of the reaction center heterodimer polypeptide of PSI (PsaA/B), PsaD and PSI-related light harvesting (Lhca1) proteins

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Table 1 Photosystem II (PSII) photochemistry – and photosystem I (PSI) photosynthetic performance measured in vivo as Fv/Fm and photo-oxidation of P700 (DA820
860), respectively in WT and Costata 2/133 mutant of pea. The intersystem electron pool size (e
/P700) and kinetics of P700+ re-reduction (t1/P700red ) were also measured in vivo. The capacity for state 2 transitions (Fr) was estimated as described in [32]. Mean values ± SE were calculated from 8 to 10 measurements in 3–4 independent experiments. Parameters

Wild type (WT)

Costata 2/133 mutant

% of WT

Fv/Fm DA820
860 (P700+, mV) e
/P700 (MT/ST) tP700red (s) 1/2 State transition (Fr)

0.82 ± 0.01 100 ± 5.09 10.6 ± 1.0 1.72 ± 0.16 0.68 ± 0.05

0.81 ± 0.01 61.16 ± 3.52 23.8 ± 2.2 1.27 ± 0.07 0.42 ± 0.06

98.8 61.1 225.7 73.8 61.76

Table 2 Effects of high light (HL) stress (1000 lmol photons m
2 s
1, 120 min, 5 °C) on pigment composition and epoxidation state (EPS) of the xanthophylls pool (V + A + Z) in WT and Costata 2/133 mutant pea leaves. V – violaxanthin, A – antheraxanthin, Z – zeaxanthin. Epoxidation state was calculated as: EPS = (V + 0.5A)/(V + A + Z). Mean values ± SE are calculated from 3 to 4 independent measurements. Pigments

WT control

WT + HL

2/133 mutant Control

2/133 mutant + HL

A + V + Z (lg gFW
1) V (% of A + V + Z) A (% of A + V + Z) Z (% of A + V + Z) EPS

70.2 ± 0.5 94.6 ± 1.0 4.3 ± 0.1 1.3 ± 1.0 1.0 ± 0.01

73.5 ± 0.5 24.6 ± 1.2 16.2 ± 0.3 59.1 ± 1.4 0.3 ± 0.02

55.9 ± 1.4 82.5 ± 1.8 10.2 ± 0.3 7.3 ± 1.9 0.9 ± 0.02

62.8 ± 1.8 19.6 ± 0.9 19.4 ± 0.7 60.9 ± 1.5 0.3 ± 0.01

Fig. 2. Typical traces of in vivo measurements of far-red light (FR) induced P700 oxidation (P700+) in control (A, B) and high light (1000 lmol photons m
2 s
1, 1 h) stressed (C, D) fully expanded leaves of WT (A, C) and Costata 2/133 mutant (B, D) pea leaves. After reaching a steady state level of P700+ by FR light, single turnover (ST) and multiple turnover (MT) flashes of white light were applied. Arrows indicate application of ST, MT, or FR light sources. The measurements were performed at the growth temperature and ambient O2 and CO2 concentrations.

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Table 3 Photosystem II (PSII)-and photosystem I (PSI) – dependent photosynthetic electron transport rates in isolated chloroplast membranes of wild type (WT) and Costata 2/133 mutant pea plants. Means and standard errors were calculated from 5 to 7 independent experiments. Sample

PSII activity (lmol DCPIPred mg Chl
1 h
1)

%

PSI activity (lmoles O2 mg Chl
1 h
1)

%

Wild type control Costata 2/133 mutant

62.8 ± 1.2 58.2 ± 3.2

100 92.8

518.6 ± 3.8 344.4 ± 4.9

100 66.4

indicated that the relative amounts of all of the examined PSIrelated proteins was reduced significantly in the Costata 2/133 mutant plants. The quantitative densitometric analysis demonstrated that the abundance of PsaA/B, PsaD and Lhca1 was reduced to 64%, 54%, and 57% respectively in the mutant plants compared to WT controls (Fig. 4D). In contrast to PSI-related proteins, the relative abundance of PSII reaction center protein, PsbA (D1) and the light harvesting proteins associated with PSII (Lhcb2 and Lhcb3) in the Costata 2/133 mutant did not exhibit any significant differences from the WT plants (Fig. 4D). Hence, the quantitative analysis of Chl–protein complexes and their major constituent polypeptides clearly demonstrate a significantly lower abundance and altered structure and composition of PSI in the Costata 2/133 mutant. The lower abundance of PSI-related components and altered stoichiometry of PSI and PSII complexes in the mutant plants could affect the state transitions (State 1–State 2 transitions), representing the mechanism for short term regulation of the excitation energy distribution between PSII and PSI [31,32,75,76]. As expected, Chl fluorescence transients (Fig. 1SA) in response to the presence and absence of PSI light in WT plants indicated relatively high capacity for state transitions (Table 1). In contrast, mutant plants exhibited 38% lower capacity for state transitions (Fig. 1SB, Table 1) indicating restricted ability for regulation of the excitation energy distribution between PSII and PSI compared with control WT plants. In addition to the lower abundance of LHC1–PS1 complex and the PSI-reaction center heterodimer protein psaA/B (Fig. 4, Table 4), the apparent lower oxidation level of P700 under FR and the larger intersystem electron donor pool size in the Costata 2/133 mutant leaves might also be due to a rapid donation of electrons to P700+ from stromal or cytosolic substrates [56,74,77]. The extent of the post-illumination fluorescence transient increase of Fo’ after turning off the AL (Fig. 5) was used as an estimate of the dark reduction of the PQ pool by stromal reductants [21,57,78,79]. A moderate post-illumination transient increase of Fo’ level over a period of 40 s followed by a gradual decrease was observed in control WT pea leaves under ambient atmospheric conditions after pre-illuminating the leaves with 300 lmol photons m
2 s
1 of white actinic light (Fig. 5A). In contrast, Costata 2/133 mutant leaves exhibited a substantial increase of Fo’ reaching the level of Fs over the same measuring time range under the same conditions (Fig. 5B). This indicates that the mutants plants have a lower capacity to keep the PQ pool oxidized in the dark compared to WT. Exposure to high light stress resulted in an even higher levels of Fo’ in both WT and Costata mutant plants (Fig. 5C and D). However, regardless of the lack of a rapid transient and the gradual increase of Fo’ during the entire measuring time range, the increase of Fo’ in the mutant leaves was much higher reaching values 2-fold higher than Fs (Fig. 5D) than in WT (Fig. 5C). This suggests that the re-oxidation process in the mutant plants under photoinhibitory conditions is much slower compared to WT and could not counterbalance to the same extent the introduction of reducing equivalents into the PQ pool [78]. Alternatively, far-red (FR) light induced afterglow thermoluminescence (TL) emission (AG band), which is attributed to a back flow of electrons originating from reducing compounds in the stroma to the PQ pool and the quinone acceptors of PSII [80] was

used to assess the electron flow to P700. In addition, the AG band has been suggested and used as a sensitive measure of the cyclic electron flow (CEF) around PSI [69,81–83]. As expected, exposure of dark adapted control WT leaves to FR light at low temperature

Fig. 3. Time courses of the steady state level of FR light induced P700 photooxidation (P700+) measured by DA820–860 (A), electron pool size (B) and half-times P700+decay of P700+ re-reduction (t1/2 ) after turning off the FR light illumination (C) during exposure of WT (open circles) and Costata 2/133 mutant (closed circles) leaves to high light (1000 lmol photons m
2 s
1) treatments at 5 °C. The results for P700+ are presented as percentage of the P700+ values in non-high light-treated leaves. Mean values ± SE were calculated from 5 to 14 measurements in 3–5 independent experiments.

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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Fig. 4. Typical densitograms of non-denaturating SDS–PAGE profiles of the major chlorophyll–protein complexes from WT (A) and Costata 2/133 mutant, (B) pea thylakoid membranes. LHC1–CP1 – light-harvesting Chl–protein complex of PSI; CP1 – major Chl a protein complex of PSI containing the reaction center P700; LHCII1, LHCII2 and LHCII3 – the oligomeric, dimeric and monomeric forms of the major Chl a/b light-harvesting protein complex associated with PSII, respectively; CPa – the Chl a protein complex containing P680 and the associated core antennae of PSII; FP – free pigments. The gels were scanned at 671 nm and the densitograms were normalized to the LHCII1 peak intensities. C – Representative Western blots and densitometric analysis (D, E) of polypeptides probed with antibodies raised against PsaA/B, PsaD, Lhca1, PsbA(D1), Lhcb2 and Lhcb3 polypeptides in thylakoid membranes isolated from WT and 2/133 mutant. Mean values ± SE were calculated from 3 independent experiments. The presented data were normalized to the relative abundance of PSI- and PSII-related proteins in WT plants.

Table 4 Relative amounts of the Chl–protein complexes (LHC1–CP1, CP1 and CPa) and the ratios of LHCII1/LHCII3 Chl–protein complexes in thylakoid membranes isolated from WT and Costata 2/133 mutant pea leaves. The relative amount of each Chl–protein complex represented by the corresponding peak area was expressed as percentage of the total area. LHC1–CP1 – supercomplex of light-harvesting Chl–protein complex of PS I (LHCI) and the major Chl a protein complex of PSI containing the reaction center P700 (CP1); CP1 – major Chl a protein complex of PSI containing the reaction center P700; LHCII1, LHCII3 – the oligomeric and monomeric forms, respectively, of the major Chl a/b light-harvesting protein complex associated with PSII; CPa – the Chl a protein complex containing P680 and the associated core antennae of PSII. Mean values ± SE were calculated from 3 independent measurements including isolation of thylakoids. Parameters

WT (control)

2/133 mutant

% of control

LHCI–CP1 CP1 CPa LHCII1/LHCII3 CP1/CPa

14.22 ± 0.44 18.31 ± 0.74 12.74 ± 0.33 5.02 ± 0.43 1.44 ± 0.09

6.29 ± 0.64 14.55 ± 0.61 12.33 ± 0.49 3.43 ± 0.27 1.18 ± 0.08

44.23 79.46 96.78 68.32 81.94

(2 °C) exhibited the typical for all C3 higher plants [70,81,84] TL glow curve (Fig. 6A) consisting of downshifted S2QB-band at around 16 °C and an AG band with a characteristic temperature peak (TM) at 41.1 °C (41.3 ± 0.8 °C, n = 4). In control Costata 2/133 mutant leaves the AG band was downshifted (Fig. 6B) peaking at 37.5 °C (37.5 °C ± 0.3 °C, n = 4). High light treatment of WT leaves induced almost 5 °C downshift of the AG band (TM = 36.6 ± 0.8 °C, n = 5), while in high light treated Costata mutant plants the peak position of AG band was even lower (TM = 33.3 ± 0.8 °C, n = 5), (Fig. 6D). 4. Discussion Characterization of the chlorophyll–protein complexes in thylakoid membranes of the Costata 2/133 pea mutant confirmed previous observations reporting lower relative ratio of oligomeric (LHCII1) to monomeric (LHCII3) forms of the light-harvesting Chl a/b complex (LHCII) of PSII (Table 4) and lower amount of the xanthophylls pigments pool in 2/133 mutant (Table 2) than in WT [1,2]. More detailed analysis of the chlorophyll protein complexes and polypeptide abundance provided in this study revealed that, in spite of the altered oligomerization sate of LHCII, only marginal

differences in the abundance of PSII polypeptides were observed in the mutant compared to WT plants (Fig. 4E). In contrast, PSI related chlorophyll protein complexes LHC1–CP1 and CP1 (Fig. 4B, Table 4), as well as, the key PSI-related polypeptides PsaA/B, PsaD and Lhca1 were greatly reduced (Fig. 4C and D) in the mutant. This clearly indicates that Costata 2/133 exhibits typical phenotypic characteristics of a PSI-defective mutant. Decreased stability and a lower abundance of PSI-related chlorophyll–protein complexes and PSI polypeptides were reported in dgd1 mutant of Arabidopsis [20,85] and ascribed to a lower amount of digalactosyl-diacylglycerol (DGDG) within the thylakoid membranes of the dgd1 mutant. Furthermore, oligomerization of the LHCII complex into trimers, which is a prerequisite for efficient development of non-radiative energy dissipation (qN) [45–47] has been suggested to depend on, and stabilized in the presence of the two dominating neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) [86,87]. Indeed, lower levels of these two galactolipids were reported in the Costata 2/133 mutant [88], which may explain the lower oligomerization of LHCII as well as the lower abundance of PSI components. The lower oligomerization state of LHCII seems to contradict with the higher non-photochemical quenching (qN)

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A.G. Ivanov et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2015) xxx–xxx

9

Fig. 5. Post-illumination chlorophyll fluorescence transients from Fs to Fo’ after the actinic light (AL, 5 min) was turned off in control (A, C) and high light treated (1000 lmol photons m
2 s
1, 1 h, 5 °C) (B, D) WT (A, B) and Costata 2/133 mutant (C, D). The measurements were undertaken under ambient CO2 and O2 at 20 °C and white actinic light (AL) of 300 lmol photons m
2 s
1. All traces are averages from 3 independent experiments.

observed in the mutant. However, it is worth reminding that qN is considered to be an integral parameter including not only energy de-excitation within the pigment bed (LHCII) of PSII, but also other quenching processes depending on redistribution of excitation energy, photoinhibitory damage and constitutive quenching processes occurring within the reaction center of PSII [24,28]. Thus, the higher qN values in Costata 2/133 mutant might not be entirely related to quenching processes depending on the oligomerization state of LHCII [45–47], but rather to excitation energy quenching occurring within the reaction center PSII [28] as suggested earlier [2]. Although previous studies on the Costata 2/133 mutant demonstrated decreased photochemical quenching (qP) corresponding to higher excitation pressure, i.e. a higher reduction state of QA for a given irradiance [2], altered energy redistribution between the two photosystems and the ratio of functionally active PSIIa to PSIIb

centers, as well as decay kinetics of the oxygen bursts [89], they did not address the composition and functional activity of photosystem I. The mutant plants exposed to high light stress exhibited much greater inhibition (22%) of the photosynthetic electron transport rates (ETR) regardless of the measuring light intensities, compared to WT plants under the same photoinhibitory and measuring conditions (Fig. 1E). The higher susceptibility of ETR to high light stress in the mutant plants could not be attributed exclusively to the moderate decrease of PSII photochemistry (Fig. 1D) and clearly suggests certain limitations beyond PSII. The functional analysis of PSI photochemistry demonstrated lower FR light induced steady state P700 oxidation (P700+), higher capacity for PSI-dependent cyclic electron transport (CET) (Table 1) and the much higher sensitivity of PSI to photoinhibition (Fig. 3A) than that of PSII (Fig. 1D) in Costata 2/133 plants. These effects can

Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018

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A.G. Ivanov et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2015) xxx–xxx

Fig. 6. Thermoluminescence (TL) glow curves of control (solid lines) and high light treated (dashed lines) WT (A) and Costata 2/133 mutant, (B) pea leaf discs illuminated for 60 s with FR light at 2 °C. The dotted lines represent the TL signal from non-FR illuminated samples. High light treatment was performed at 5 °C for 1 h and illumination of 1000 lmol photons m
2 s
1. All experimental curves represent averaged scans of 4–5 independent experiments.

be explained by either limitations at the acceptor side of PSI [90] or lower abundance of specific components of PSI [20]. Indeed, severe photoinhibition was reported in A. thaliana plants lacking the PsaD subunit of PSI [91]. Furthermore, the enhancement of CET in the mutant plants could be attributed to a PSI acceptor-side limitation [92,93] or to elevated excitation pressure to the photosystems occurring as a result of high light treatment [15,18]. The lower abundance of PsaD in the Costata 2/133 mutant (Fig. 4D) clearly suggests that the mutant plants are PSI acceptor-side limited. Moreover, Costata 2/133 plants exhibited higher excitation pressure [2] and 30% faster kinetics (t1/2) for the dark reduction of P+700 (Table 1), which is also consistent with the suggestion that PSI is acceptor-side limited under the growth light conditions. This is supported by the 2.2-fold higher electron pool size (Table 1), as well as the more pronounced transient rise in Fo’ fluorescence in the dark (Fig. 5B), suggesting that the PQ pool in the Costata 2/133 mutant exhibits a higher reduction state than the WT under comparable conditions due to PSI limitations. The restriction in electron transport observed in Costata 2/133 mutant was also associated with a restricted capacity for state transitions (Fig. 1S, Table 1), thus indicating a severe impairment in the regulation of excitation energy distribution between the photosystems. This could not be due to lower proportion of reduced PQ, one of the major prerequisites for effective state transitions [31,75]. Our data show that, in fact, the intersystem electron transport chain is more reduced in the Costata 2/133 mutant than WT (Table 1, Fig. 5). The observed lower abundance (64%,

Fig. 4D) of PSI reaction center polypeptides (PsaA/B) may easily explain the restricted capacity for state transitions. In addition, the mutant exhibited even higher reduction of PsaD to only 54% of that in WT (Fig. 4D) may also account for the inhibition of state transitions. Indeed, similar reduction of the state transitions was also observed in PsaD mutant of Arabidopsis [94] suggesting that limitations at the ferredoxin docking site of PSI [95] or the acceptor side of PSI in general might also be involved in controlling the energy distribution between PSII and PSI. More recently, the phosphorylation of LHCII trimers has been shown to increase the affinity between LHCII and PSI [29]. Evidently, the observed lower proportion of LHCII trimers in the Costata 2/133 mutant would decrease the probability for LHCII–PSI interaction and may also account, at least partially, for the lower capacity for state transitions in the mutant plants. The dynamic acclimation to fluctuating growth light via state transitions provides effective short term mechanism for lowering the excitation pressure on PSII relative to PSI [13,34]. Consequently, this physiological adjustment may well serve as an important protective mechanism for preventing over-reduction of chloroplast stroma components, thus limiting possible photoinhibition of PSI [34]. Our results are in full agreement with this suggestion and demonstrate that the lower levels of LHCII trimers and the restricted capacity for state transition correlates with the observed higher sensitivity of PSI to photoinhibition in the Costata 2/133 mutant plants. It has been also suggested that PSI with an intact antenna system is more resistant to high light stress and this is due to lower production of reactive oxygen species [96]. Furthermore, the same authors imply that in contrast to PSII photoinhibition, where the reaction center protein D1 is the first target, PSI antenna chlorophyll–proteins, but not the reaction centers (PsaA/B heterodimer) are the first target of high light damage in PSI. Thus, the reduced amount of Lhca1 polypeptides (Fig. 4D) can also contribute to the preferential photoinhibition of PSI in Costata 2/133 mutant plants. The lower capacity for state transitions caused by the lower levels of LHCII trimers and lower abundance of PSI chlorophyll protein complexes and key PSI polypeptides indicate that Costata 2/133 mutant plants might experience chronic down-regulation of PSI and are predisposed to higher sensitivity of PSI to high light stress even at the growth light intensities. Acknowledgements We thank Prof. N. Naidenova (Institute of Genetics, Bulgarian Academy of Sciences) for providing us with the Costata 2/133 mutant seeds. SIA was supported by grants from the Russian Foundation for Basic Research (Nos: 14-04-01549 and 14-04-92690) and by the Molecular and Cell Biology Programs of the Russian Academy of Sciences.

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Please cite this article in press as: A.G. Ivanov et al., Photoinhibition of photosystem I in a pea mutant with altered LHCII organization, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.08.018