High light induced changes in organization, protein profile and function of photosynthetic machinery in Chlamydomonas reinhardtii

    High light Induced Changes in Organization, Protein Profile and Function of Photosynthetic machinery in Chlamydomonas reinhardtii Srilatha Nama, Sai Kiran Madireddi, Elsin Raju Devadasu, RajagopalSubramanyam PII: DOI: Reference:

S1011-1344(15)00275-4 doi: 10.1016/j.jphotobiol.2015.08.025 JPB 10121

To appear in: Received date: Revised date: Accepted date:

29 June 2015 26 August 2015 27 August 2015

Please cite this article as: Srilatha Nama, Sai Kiran Madireddi, Elsin Raju Devadasu, RajagopalSubramanyam, High light Induced Changes in Organization, Protein Profile and Function of Photosynthetic machinery in Chlamydomonas reinhardtii, (2015), doi: 10.1016/j.jphotobiol.2015.08.025

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ACCEPTED MANUSCRIPT High light Induced Changes in Organization, Protein Profile and Function of Photosynthetic machinery in Chlamydomonas reinhardtii

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Srilatha Nama, Sai Kiran Madireddi, Elsin Raju Devadasu, RajagopalSubramanyam*

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Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad,

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500046, India

Corresponding author.

RajagopalSubramanyam

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Department of Plant Sciences, School of Life Sciences,

Tel. No.+91-40-2313 4572 Fax No. +91-40-2301 0120

E-mail: [email protected]

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University of Hyderabad, Hyderabad, 500046 India

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ACCEPTED MANUSCRIPT ABSTRACT The green alga Chlamydomonas (C.) reinhardtii is used as a model organism to understand

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the efficiency of photosynthesis along with the organization and protein profile of

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photosynthetic apparatus under various intensities of high light exposure for one hour. Chlorophyll (Chl) a fluorescence induction, OJIPSMT transient was decreased with increase

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in light intensity indicating the reduction in photochemical efficiency. Further, circular dichroism studies of isolated thylakoids from high light exposed cells showed considerable

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change in the pigment-pigment interactions and pigment-proteins interactions. Furthermore, the organization of supercomplexes from thylakoids is studied, in which, one of the heterotrimer of light harvesting complex (LHC) II is affected significantly in comparison to other

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complexes of LHC’s monomers. Also, other supercomplexes, PSII reaction center dimer and PSI complexes are reduced. Additionally, immunoblot analysis of thylakoid proteins revealed

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that PSII core proteins D1 and D2 were significantly decreased during high light treatment. Similarly, the PSI core proteins PsaC, PsaD and PsaG were drastically changed. Further, the

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LHC antenna proteins of PSI and PSII were differentially affected. From our results it is clear

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that LHC’s are damaged significantly, consequently the excitation energy is not efficiently transferred to the reaction center. Thus, the photochemical energy transfer from PSII to PSI is reduced. The inference of the study deciphers the structural and functional changes driven by light influence, it may therefore provide plants/alga to regulate the light harvesting capacity in excess light conditions.

Graphical abstract

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ACCEPTED MANUSCRIPT

Highlights

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 Gradual decrease in photochemical yield with increased light intensity.  Significant differences in pigment-protein interactions were observed. PSII core proteins (D1&2) are more reduced than core antenna proteins (CP43&47).

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

 Two of LHCII hetero-trimers were affected by excess light which is also observed in

High light induced decrease in protein content of PsaC and PsaD which is crucial for

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

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BN-PAGE.

function of PSI.

Key words Chlamydomonas reinhardtii, chlorophyll a fluorescence, high light intensity, light harvesting complexes, pigment-pigment interactions, supercomplexes, thylakoids

1. INTRODUCTION

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ACCEPTED MANUSCRIPT Photosynthesis is a primary process that provides energy source to the biosphere in the form of reduced carbon. The oxygenic photosynthetic organisms like cyanobacteria, algae and

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plants are often exposed to various abiotic stress factors, majorly by high light. Optimum

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light intensity is essential for photosynthesis to convert solar energy into chemical energy. As the environment is changing fast, hence, plants/algae are forced to deal with sudden high light

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conditions, which leads to damage of photosystems, particularly photosystem II [1]. These conditions are characterized by those light intensities, where the additional energy cannot be

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used for increased carbon fixation and oxygen production and thus are potentially harmful. These high light intensities cause photoinhibition resulting in reduction of photosynthetic quantum yield [2]. During evolution, green plants and photosynthetic organisms have adapted

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different approaches to develop and survive in various environmental conditions characterized by high light, low light response [3] and intense fluctuating light conditions or

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the incoming light which is spectrally altered due to certain types of different stress conditions [4]. On the other hand, there have been reports about influence of different quality

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and quantity of light conditions for their acclimation and photoprotective mechanisms such

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as, in diatoms for acclimation to high light require perception of blue light [5]. Under surplus light condition beyond threshold limit for photosynthetic efficiency, oxidative damage to photosynthetic apparatus frequently occurs by impart of singlet oxygen radicals in the proximity of PSII which can attack and cause irreversible damage to D1 protein, whereas superoxide and hydroxyl radicals formation at the acceptor side of PSI leads to oxidative damage of chloroplast proteins and lipids [6]. It has been reported that PSII photodamage is associated with light absorption by manganese cluster of oxygen evolving complex [7]. To restore the function of PSII by repair cycle process which includes selective degradation of the damaged D1 protein and regeneration of complex by de novo synthesized D1 protein [8,9]. It is well known that repair process of damaged PSII can be inhibited by reactive

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ACCEPTED MANUSCRIPT oxygen species (ROS) which disseminates from oxidative stress [10,11]. Different photoprotection mechanisms have been studied to avoid photodamage of PSII and thus

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maintaining repair of damaged PSII [12]. Excess light absorbed is dissipated as heat through

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a mechanism called non-photochemical quenching [13]. Various types of quenching processes have been reported based on their time scale of induction and relaxation i.e., high

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energy excitation quenching which is the main component of non-photochemical quenching (NPQ) and is triggered by the formation of a ΔpH across the thylakoid membrane [14],

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zeaxanthin dependent quenching through xanthophyll cycle [15]. Further, quenching due to state transitions, which is a process to balance excitation energy between photosystems by dissociation of peripheral antenna proteins from PSII and then it associates to PSI which

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results in regulation of linear and cyclic electron flow in the chloroplast [16]. Photoinhibitory quenching occurs by strong light induction in course of hours to days.

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In higher plants as well as green algae, the PSII subunits PsbS and LHCSR3 proteins are critical key factors in quenching process [17-19]. Under elevated light condition,

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over expression of PsbS has been shown to play important role in remodeling of PSII-LHCII

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supercomplexes that regulate the energy balance and in safeguarding the innovatory coordination between energy excitation and dissipation [18]. Protonation of LHCSR3 protein along with formation of PSII-LHCII-LHCR3 supercomplex is capable to dissipate excess energy [19]. From the above mentioned reports, we inferred various photoprotective mechanisms under different stressful environmental conditions, especially under surplus light [20]. It was very well established that there are two kinds of photoinhibition mechanisms in PSII based mostly on in vitro experiments namely, acceptor-side and donor-side photoinhibition [21,22] in which ROS, in particular singlet oxygen molecules (1O2), are mainly responsible for the photoinactivation of PSII and photo-induced damage to the D1

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ACCEPTED MANUSCRIPT protein when acceptor-side photoinhibition is dominating. No cleavage products were detected in Synechocystissp.PCC6803 cells or thylakoids when subjected to strong light.

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Hence, the degradation mechanism of the D1 protein by specific proteases differs between

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higher plants and cyanobacteria [23]. There is no direct effect of ROS on D1 protein cleavage in the PSII from higher plants [24].

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Cationic radicals such as P680+ and TryZ+ are formed from the illumination of the PSII when its donor-side is impaired [1,11,25]. This was achieved from PSII particles which

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includes removal of the lumen exposed extrinsic protein PsbO and Mn from PSII by washing the thylakoids with high concentration of salts such as 1 M CaCl2 or with alkaline solutions upon exposure of strong light. Furthermore, the oxidation of chlorophylls would cause the donor-side photoinhibition and it can be easily monitored by photobleaching of chlorophylls

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in the samples having the impaired donor side of PSII. However, the 1O2 is not shown to be

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related to the donor-side photoinhibition in PSII [26]. Apart from D1, the other protein subunits of PSII including D2 and CP43 protein

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were also degraded to some extent under high light stress [27,28]. Also, long term treatment

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of light results in decreased antenna size of LHCII [29], which is called acclimative proteolysis. Destabilization of chlorophylls and degradation of the apoproteins should occur during this process.

Hence, the literature shows that PSII is prone to strong light over PSI. However, few reports have shown that low light at low temperature induces damage to the PSI due to formation of ROS [30,31]. High light exposure to the higher plants, alga and cyanobacteria exhibits damage of PSII much faster than that of PSI, however, in thylakoids or PSI submembrane, exposure to strong light could also cause damage to PSI [32-34]. In the present study, we have used green algae Chlamydomonas reinhardtii as a model organism for plants to study the impact of short term exposure of high light intensities

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ACCEPTED MANUSCRIPT on photosynthetic apparatus, particularly organization of their supercomplexes. As the photosynthetic organisms are constantly subjected to different light conditions which would

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lead to photoinhibition of PSII that occurs when the rate of photodamage exceeds that of the

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repair [2,12]. From the literature, it was inferred that, to avoid net photoinhibition the organisms developed diverse photoprotective mechanisms in green algae such as phototaxis,

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screening of photoradiation, ROS scavenging systems, dissipation of absorbed light energy as thermal energy (NPQ), cyclic electron transport around PSI, photorespiratory pathway and

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state transitions which helps in balancing of excitation energy between two photosystems [35]. Since, many reports focused on various different mechanisms for acclimatization along with efficient protection under different light conditions but there was no report about

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organization of photosynthetic apparatus under high light intensities. In this article, we have discussed about photochemical activity in case of green algae under short term exposure of

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different high light stress. Also, we have investigated the structural organization of

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condition.

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supercomplexes in terms of protein profiling of thylakoid membranes subjected to high light

2. MATERIALS AND METHODS 2.1. Growth conditions of C. reinhardtii Cells of C.reinhardtii (obtained from Chlymadomonas culture collections at Duke University) were grown in a Tris-acetate phosphate medium (TAP) under continuous illumination (30-40 µmol photons m-2 s-1) at 25±2 °C temperature. 2.2. Treatment of algal culture media at high light intensities (short term exposure) Cells of C.reinhardtii with optical density of 0.8-0.9 were treated for one hour with different light intensities (1000, 1500 and 2000 µmol m-2s-1) with halogen light source which contains

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ACCEPTED MANUSCRIPT fibre optic adapter and cable to control the temperature. The light intensity was measured by light meter obtained from Hanstech instruments, U.K.

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2.3. Isolation of thylakoids After the light treatment the thylakoid membranes were isolated as described earlier [36,37]. The final pellet was resuspended in 2.0 mL of thylakoid resuspension buffer containing 5mM

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Tris-HCl (pH 7.5), 0.2M Sorbitol, 5mM CaCl2 and stored at -20 oC.

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2.4. OJIPSMT fluorescence transient measurement

Chl fluorescence fast induction curves were measured using Chl fluorimeter (PEA, plant efficiency analyzer, Hansatech, King’s Lynn, Norfolk, UK) up to one minute with excitation

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light wavelength of 650nm which was focused on cells. Light intensity used as 3000 µmol m²s-¹ to generate maximal fluorescence (Fm) for all the samples. Fluorescence was detected by

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a PIN-photodiode after passing through a long-pass filter (50% transmission at 720nm). For

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energy pipeline models, Biolyzer HP3 software, a Chla fluorescence analysis program was used. Three independent experiments were measured and each time identical graph were

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observed.

2.5. Circular Dichroism measurements Visible CD spectra of thylakoid membranes isolated from control and high light treated cells were measured in a J-810 spectropolarimeter (Jasco Inc., Easton, MD, USA). The spectra were recorded within a visible wavelength range (400-800nm) by a quartz cell of optical path length of 1cm. Three scans were accumulated with a continuous scan mode and scan speed of 100nm\min is collected for every nanometre. By using the buffer as blank, the base line was corrected for every spectrum. Chlorophyll concentration was maintained at 25µg\ml for all the measurements. Three independent experiments were measured and each time identical graph were observed. 8

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2.6. Immunoblot analysis

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Thylakoids isolated from high light treated cells were separated by SDS-PAGE on 12% acrylamide gel with equal amount of chlorophyll content in each lane. To identify and

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quantify the polypeptides contained in the thylakoid membranes, immunoblotting was performed. Electrophoretic transfer of proteins to PVDF membranes were incubated with

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polyclonal antibodies (primary antibodies) developed in rabbits. Primary antibodies against LHCII, PSII and PSI complex proteins were purchased from Agrisera. Peptide tag antibodies of LHCI complexes were developed in our laboratory [38]. Subsequently, the secondary antibodies ligated to horseradish peroxidase were applied. Chemi-luminescence reagents

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were used to develop the signal on the PVDF membrane. The images were recorded on a

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Kodak 4000 MM pro-image station.

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2.7. BN-PAGE

Thylakoid proteins were isolated from cells treated with high light intensities as explained

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above. First dimension of blue native gel separation was performed by solubilisation of thylakoids in 1% n-dodecyl β-d-maltoside (DM) (sigma) along with the protease inhibitors 1mM 6-Amino caproic acid (ACA),1mM benzmidine hydrochloride 1mM PMSF, the gel with 50mM ACA was run at 4oC with increasing voltage [39]. For second dimension BN gel strips were treated with solubilisation buffer; Lamelli buffer: 138mMTris-HCl (pH 6.8), 6M urea, 22.2% (v/v) glycerol, 4.3% (w/v) SDS, 5% (v/v) 2-β mercaptoethanol. Second dimension of the BN gel was run in 12.5% SDS gels. The protein spots on second dimension gels were visualized by colloidal Coomassie staining method.

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ACCEPTED MANUSCRIPT 3. RESULTS AND DISCUSSION 3.1. Effect of high light on Chla fluorescence transients

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In all oxygen evolving organisms under dark adapted conditions, the intensity of Chla fluorescence shows a characteristic variation in time known as fluorescence transient or

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induction. This induction curve represents a real signature of photosynthesis, especially photochemical activity of PSII and it exhibits two transient phases that are basically

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represented by using the observed inflection points i.e., a fast wave (up to hundreds of milliseconds) that is named as OJIP and a slow wave (seconds to tens of minutes) labeled as PSMT [40]. The OJIPSMT transients represented as follows: O, the minimal Chla fluorescence (FO); O-J is a photochemical phase which reflects the reduction of QA to QA-, J-I

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and I-P are thermal phases involved in reduction of the PQ pool as well as that of the electron

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acceptor side of PSI (Fig.1). At the P level, all the electron carriers are in the reduced state between PSII reaction centre and NADP+. A decline from P to S may reflects ∆pH change

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that induce non-photochemical quenching and SMT indicates several processes which includes state transitions [41]. An investigation about slow fluorescence rise (PSMT) has

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been done to evaluate the regulatory mechanism, as was already done in cyanobacteria [42,43]. This fluorescence study denotes the kinetic and heterogeneity involved in the filling up of the PQ pool with electrons which culminates to infer about donor side of PSII, accordingly, it can be employed as a sensitive tool to interpret the photosynthetic apparatus in vivo under different physiological conditions. Fig.1 demonstrates the OJIPSMT fluorescence transients of cells subjected to different high light intensities at different time intervals along with the control. With the increase of light intensities along with time duration of exposure, the rise in the fluorescence transients was remarkably altered, the increase of Fo whereas decrease of Fm was observed.

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ACCEPTED MANUSCRIPT At high light intensities (1500 and 2000 µmol m-2s-1), the Chla fluorescence transient drastically varied when compared to cells exposed to 1000 µmol m-2s-1 light intensity. The Fo

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level increased with the time of exposure to high light treatment while the Fm level decreased,

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resulting in significant decrease of photochemical yield (Fv/Fm) which might be due to partial reduction of PQ pool. After one hour of treatment with 1000µmol m-2s-1l ight intensity, the

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fluorescence was raised rapidly which might be due to recovery (Fig.1A) whereas in other treatments, no such recovery was observed (Fig.1B&C). Also, there is a rise of fluorescence

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at J phase with increase of time of exposure to high light intensities that might be due to largely reduction of QA to QA- and then decrease in fluorescence yield, possibly as a consequence of decreased electron transport beyond QA-. An increase in Fo has been

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attributed to physical separation of the PSII (RC) from associated pigment antenna, which results in blockage of PSII trapped energy transfer, even if a part of this circumstance could

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probably indicates the accumulation of the reduced form of QA-. Also, rise in Fo may be release pigments in the media as a result of cellular damage due to exposure to excessive

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light. A slow fluorescence rise (PSMT) has shown in the Fig.1D to F, is plotted on a linear

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scale in which SMT rise is distinguished at all-time intervals in cells treated to 1000µmol ms light intensity while in cells subjected to 1500 µmol m-2s-1 light intensity distinctly shows

SMT rise only at 15 min, however, the intensity is lower than that of control. Additionally, the P to S phase is significantly increased while exposure of cells to high light indicate the ∆pH change that induces non-photochemical quenching is increased (Fig.1 D to F). The energy pipeline models of the photosynthetic apparatus was generated from Biolyzer software using handy PEA data. It is a dynamic model in which the value of each energy flux, either changing as a function of time or modified by the manipulated physiological conditions is expressed by the appropriately adjusted width of the corresponding arrow [44,45]. This model gives the information about the efficiency of flow 11

ACCEPTED MANUSCRIPT of energy from the antennae to the electron transport chain component through the RC of PSII. As shown in the Fig.2, the area of the arrows form each of the parameters, ABS ⁄ CS o,

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TRo ⁄ CSo, ETo ⁄ CSo and DIo ⁄ CSo, indicates the efficiency of light absorption, trapping,

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electron transport and dissipation per cross-section of PSII, respectively [46]. The open circles indicates active RC of PSII to trap photons for efficient electron transport system

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while dark circles indicates inactive RC. A decrease in the density of open circles and an increase in the density of dark circles with efficient absorption of energy at certain time

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interval of different high light conditions were observed. In 1000 µmol m-2s-1 intensity of light treated cells show increased inactive RC up to 30 min and then later the system shows recovery of active RC whereas at 1500 µmol m-2s-1 intensity of light, the system shows slight

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recovery after 45 min of treatment but in high light stress i.e. 2000µmol photons m-2s-1 the closed circles were increased with the time of exposure suggesting no recovery of active RC

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to trap the photons for efficient electron transport system (Fig.2). The result of this model demonstrates that at different time intervals of different light intensities treated cells were

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showing different time points for acclimatizing to high light stress.

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3.2. Visible CD Spectra of thylakoid membranes CD spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. It is a sensitive technique which is done in visible region of spectrum to monitor the excitonic pigment-pigment interactions and pigment-protein interactions. CD spectra indicates the difference in the absorption of left-handed circularly polarized light and right-handed circularly polarized light and occurs when a molecule contains one or more chiral chromophores.

Photosynthetic membranes and isolated

complexes, despite their remarkable diversity, exhibits strong anisotropic and chiral pigment organization. This originates in the reaction center complex as well as the antenna- reaction center supercomplexes. In chloroplasts, the Chl molecules are bound to different pigment– 12

ACCEPTED MANUSCRIPT protein complexes, in which the distances between the pigment molecules and their mutual orientation are well-defined and the differences in the distances between the molecules that

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arise due to change in structure or arrangement of the pigment protein complexes may lead to

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the changes in the CD spectra. Visible region (400–700 nm) in terms of CD studies was divided into Soret and Qy regions [47]. A typical CD spectrum of thylakoids isolated from

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plant/C. reinhardtii will have the following peaks. In the Qy region, there are two negative peaks at 640 and 672 nm and one positive peak at 656 nm in agreement with our previous

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report [48]. The two major bands at 656 and 672 nm are due to Chl dimers caused by the excitonic interaction of Chla in thylakoids, whereas the negative peak at 640 nm is characteristic of Chl b. In the soret region, the positive peak at 443 nm originates from Chl a,

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while the negative peak at 460 nm is characteristic of Chl b [49]. Here, we showed the visible CD spectrum of isolated thylakoid membranes of C.reinhardtii which were treated with

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different high light intensities (1000, 1500 and 2000µmol m-2s-1) (Fig.3). The calculated values of 656/672 nm excitonic band at different light conditions the excitonic band altered in

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1000 and 1500 µmol m-2s-1 significantly, however a marginal change was observed in 2000

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µmol m-2s-1, but we could see the shift in 676 nm (data not shown). This indicates the change in the structure of protein-pigment complexes. However, the 640 nm peak intensity is significantly changed in high light exposure. Also, the soret band peaks at 443 nm reveals no drastic change whereas at 460 nm shows decrease in the amplitude indicates the impact of high light is attributed more to Chlb when compared to Chla. These results reveals that pigment-pigment interactions have been disturbed and consequently may lead to change in interaction between protein-pigments. The results of CD changes indicate the following modifications either all or combinational effect under high light conditions: structural changes in the LHCII trimers that alter the interactions between chlorophylls and carotenoids; macrostructural changes (protein-protein interactions) that affect excitonic interactions

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ACCEPTED MANUSCRIPT between pigments belonging to different complexes; and intermolecular lipid-protein environment of pigments which causes spectral alterations.

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3.3. Protein profile analysis of PSII core subunits and LHCII polypeptides The isolated thylakoid membranes from different high light intensities were used for our

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study. The proteins were separated using SDS-PAGE and immunoblotting was carried out by probing with specific antibodies to determine the protein content. Reaction centres of PSII

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core includes D1and D2 as intrinsic core proteins along with chlorophyll containing inner antenna subunits of CP43 and CP47, which are located within the thylakoid membrane [50]. Under high light stress, PSII core proteins i.e. D1 and D2 were reduced while no significant change was observed in CP43 and CP47, the extrinsic antenna proteins (Fig.4). The protein

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content of oxygen evolving complex (OEC) was reduced at all light intensities. These core antenna proteins transfers energy through chlorophyll molecules to the RC. This result

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implies that high light intensities shows more impact on D1 and D2 core proteins as already

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mentioned in the earlier reports [11,25]. In C. reinhardtii, there are six trimers per one dimeric core in thylakoid membrane.

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Two minor antenna CP29 and CP26 exists as monomers, while CP24 is not found in the genome of this algae [51]. The light harvesting complexes, the Lhcb1 and Lhcb3 content in thylakoids are affected with increase in light intensity but there is no noteworthy change in Lhcb2.The minor subunits Lhcb4 (CP29) protein content is drastically reduced while Lhcb5 (CP26) content is unchanged with increased light intensity (Fig.4). This result suggests that PSII-LHCII protein complexes organization were partially destabilized in high light exposure. The differential degradation of Lhcb proteins could be due to differential arrangement of these subunits in PSII-LHCII supercomplexes. Interestingly, the Lhcb1 and Lhcb3 are more prone to high light exposure which means that they may be the primary subunits to harvest the light, as they are peripherally arranged. Similarly, Lhcb4 (CP29) is 14

ACCEPTED MANUSCRIPT also susceptible to high light since this subunit is also arranged peripherally to the core. Thus, one should anticipate that these subunits may be prone to high light stress in comparison to

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intrinsically arranged Lhc subunits that transfer the excitation energy to the core. Other

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explanation could be that generation of ROS during high light treatment could damage the peripherally arranged Lhcb subunits. It is well characterized that 1O2 is responsible for the

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damage of the D1 protein in the acceptor side of PSII under photoinhibitory light [1,11]. Other report shows that 1O2 produced through lipid peroxidation probably damages the D1

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protein and the LHCII subunits as well. Hence, our results are in agreement with the reports published earlier [52].

3.4. Immunoblots of PSI core and LHCI polypeptides

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It is a well known phenomenon that PSI is more stable to photoinhibition in higher plants and cyanobacteria. However, in vitro studies illustrate that PSI can also be damaged by strong

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light [32,33]. The PSI structural arrangements in C. reinhardtii are different than higher plants or cyanobacteria where it has more copies of Lhc genes [53]. In high light condition,

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no remarkable changes were observed from the core subuntis of PsaA and PsaB which are the

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central core proteins that binds all cofactors of the electron transport chain except the cluster of ferredoxin. Other RC subunit PsaC was decreased by 50% and 30% on exposure of 1500 and 2000 µmolm-2s-1 light respectively, while the PsaD content was reduced to 25% in 2000 µmol m-2s-1 (Fig.5). The result infers serious decay of the stromal subunits in C. reinhardtii under high light, while the core subunits PsaA and PsaB were unaffected. In the absence of PsaC, there is an assembly of core proteins but prevents the binding of PsaD and PsaE, possibly this might be responsible for stability of core proteins under high light [54]. The degradation of PsaC finally terminates the ETC; as it binds the terminal electron acceptor side of ferredoxin which is necessary for the function of PSI [55]. Interestingly, we found out the stability of PsaH by impact of excess light; as it is known to be the docking site for

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ACCEPTED MANUSCRIPT association with LHCII during state transitions which results in enhancing the light harvesting capacity [56]. Surprisingly, PsaG is sensitive to high light as it is physically

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associated with LHCI [53]. The degradation of PsaG can lead to the dissociation of a large

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part of the antenna [57] indicating its role in stable association of LHCI with PSI and prevention of further damage due to over excitation of PSI core.

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The LHCI subunits show varying susceptibility under high light stress. No significant change in polypeptides such as Lhca1, Lhca2, Lhca4, and Lhca9 from the PSI-LHCI

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supercomplexes was distinguished. While Lhca5 and Lhca7 were degraded in all treated samples, but Lhca3 and Lhca8 subunits were slightly reduced in samples treated with 2000µmol photons m-2s-1(Fig.6).The structure of PSI-LHCI in higher plants has been

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determined which declared the location of four Lhca subunits on one side of core proteins [58,59], where as in green algae, it was identified as nine Lhca protein subunits which are

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arranged in two rows possibly due to the presence of additional subunits i.e. Lhca5 to Lhca9 that will help in more efficient harvesting of light energy to increase the production of

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biomass [53,60]. The arrangement of LHCI subunits along with the core has been reported

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from our lab. The LHCI is arranged in a crescent shape which includes Lhca5, Lhca6, Lhca8, Lhca7, Lhca3 as inner half ring and Lhca1, Lhca4, Lhca2, Lhca9 compose the outer half ring [53]. It is interesting to reveal that outer half ring subunits are less susceptible whereas inner subunits shows sensitivity to high light. The blue native gel analysis also supports that PSI complexes including LHC’s and PSI core are degraded under high light illumination (Fig.7). Hence, we report here that, PSI is also equally sensitive to high light stress. 3.5. Identification of Supercomplexes by BN-PAGE and its protein profile of thylakoids Further, we have checked the supercomplexes of both PSII and PSI from isolated thylakoids. Blue native- PAGE is the method used for separation of hydrophobic membrane protein complexes. Hundreds of proteins were identified by BN-PAGE which has an enormous

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ACCEPTED MANUSCRIPT impact on the investigation of the respiratory chains and photosynthetic complexes [61].When combined with SDS-PAGE, BN-PAGE allows an assignment of proteins to their

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protein complexes and displays highly hydrophobic proteins in two dimensions. Here, the

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isolated thylakoids from different light intensities (1000, 1500 and 2000 µmol m-2s-1) for one hour were taken to separate the supercomplexes using mild nonionic detergent like

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dodecylmaltoside, for solubilization of the thylakoid membranes (Fig.7). The analysis revealed that the PSII-LHCII, PSI-LHCI complex and PSII dimerization organization

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changed in 2000µmol m-2s-1 light intensity. Also the PSI and PSII core monomer were severely affected by excess light (2000µmol m-2s-1) (Fig.7A).There are two forms of LHCII trimers which we have reported recently [62], in which one of the trimer is almost diminished

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in high light condition. Further, the monomer content was also decreased in high light conditions (Fig.7A-B). Hence, alteration of LHCII trimer is in agreement with the data

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obtained from CD spectra. It was noticed that Chl b peaks at 640 and 460 nm (Fig. 3) were diminished in high light exposure indicating that Chl b is more abundant in LHCII is affected.

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These results suggest that the organization of PSII as well as PSI is disrupted or disorganized

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under high light intensity (2000µmol m-2s-1) which leads to changes in photochemical yield. Particulary, the protein profile obtained from 2-D blue native gel (Fig.7B) shows that PSII RC dimer is prone to high light condition indicating that D1 and D2 are affected which correlates with the western blot data (Fig.4).Also, one of the LHCII trimer (named as Lhc3b) complex proteins is severely degraded apart from moderate effect of high light on other trimer and monomer of LHCII complexes, which would illustrate the less efficiency of excitation energy transfer to the PSII core. Hence, the change in fluorescence, protein complexes and pigments may be due to disruption in supercomplexes in high light. 4. CONCLUSION

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ACCEPTED MANUSCRIPT The above experimental results demonstrate that photochemical yield is decreased with increased light exposure.Interestingly, CD data depicts significant difference in the peak

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intensities from light exposed samples indicating the changes in pigment-pigment interactions

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which mainly affects the LHC complexes. Two of the LHCII hetero-trimer complexes, monomer LHC, PSII RC dimer and PSI were decreased with exposure of high light.Further,

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PSII core proteins are more reduced than core antenna proteins. Moreover, by the impact of light shows decrease in protein content of PsaC and PsaD which are known to be essential for

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function of PSI. Also, differential degradation of LHCI proteins were observed. Most of the LHCI polypeptides are coordinately regulated to adjust the high light in short time scale. We conclude that over saturation of light induced changes in the organisation of photosynthetic

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apparatus,hence, disrupting the protein profile which leads to decrease of photosynthetic

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quantam yield.

ACKNOWLEDGMENTS

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RS thanks Council of Scientific and Industrial Research No.38 (1381)/14/EMR-II, and

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Department of Biotechnology (BT-BRB-TF-1-2012), Department of Science and Technology (DST/INT/JSPS/P-159/2013) and DST-FIST, Govt. of India, for financial support. We thank Ms. Shreya Dubey, Department of Plant Sciences, University of Hyderabad for critical reading of the manuscript. Also SN thanks CSIR for JRF and SM thanks CSIR-UGC for JRF and SRF.

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Figures Legends

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Fig.1: Changes in Chla fluorescence induction curves of light treated cells of C.reinhardtiistrain CC125 of different high light intensities (1000, 1500 and 2000µmol m-2 s1

) labeled as (A), (B), (C) respectively of different time intervals (15, 30, 45 and 60 min) for

(D), (E), (F) respectively.

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each intensities, which were plotted on a logarithm scale while on a linear scale labeled as

for each of the

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Fig.2:The Energy pipeline model shows the area of the arrows form

parameters, ABS ⁄ CSo, TRo ⁄ CSo, ETo ⁄ CSo and DIo ⁄ CSo, indicate the efficiency of light

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absorption, trapping, electron transport and dissipation per cross-section of PSII, respectively.

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Energy pipeline models of algal cells subjected to different high light intensities (1000, 1500 and 2000µmol m-2 s-1) of different time intervals which were consequently labelled as (A), (B), (C) in the figure.

Fig.3: Visible CD spectra of thylakoid membranes isolated from Chlymodomonasreinhardtii treated with different high light intensities (1000, 1500 and 2000µmol m-2 s-1) along with control. Fig.4: Immunoblots of PSII core proteins (D1, D2, Cp43, and Cp47), OEC and PSII-LHCII polypeptides (Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5) of thylakoids isolated from control (WT) and the cells subjected to high light of different intensities. The adjacent bar diagrams which

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ACCEPTED MANUSCRIPT were developed by Image J software to show the quantity of protein present. The wild type thylakoids were loaded with different dilutions (25, 50 and 100%) as a positive control.

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Fig.5: Immunoblots of PSI core proteins (PsaA, PsaB, PsaC, PsaD, PsaG, PsaH) of

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thylakoids isolated from control and the cells subjected to high light with different intensities and the adjacent bar diagrams which were developed by using Image J software to represents

dilutions (25, 50 and 100%) as a positive control.

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the quantity of the respective proteins.The wild type thylakoids were loaded with different

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Fig.6: Immunoblots of PSI-LHCI Polypeptides (Lhca1-Lhca9) of thylakoids isolated from control and the cells subjected to high light with different intensities and the adjacent bar diagrams represents the quantity of the respective protein measured using Image J software.

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The wild type thylakoids were loaded with different dilutions (25, 50 and 100%) as a positive control.

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Fig.7: (A)Blue Native PAGE of thylakoid membranes isolated from cells treated with different light intensities (1000, 1500, 2000µmol photons m-2s-1)for one hour along with

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control. (B) 2nd dimension of BN PAGE labeled as: I( PS I) II (PSIIRCC Monomer), IL (PSI

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LHC I Complex) , IIL (PSII LHC II Complex), II2 ( PSII Dimer), A (ATP Synthase), R(RUBISCO), C (Cytochrome b6f) L3a L3b (LHC Trimers), L(LHC Monomers).

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Graphical Abstract:

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