Complex lumenal immunophilin AtCYP38 influences thylakoid remodelling in Arabidopsis thaliana

Journal Pre-proof Complex lumenal immunophilin AtCYP38 influences thylakoid remodelling in Arabidopsis thaliana Lea Vojta, Ana Tomaˇsi´c Pai´c, Lucija Horvat, Anja Rac, Hrvoje Lepeduˇs, Hrvoje Fulgosi

PII:

S0176-1617(19)30171-3

DOI:

https://doi.org/10.1016/j.jplph.2019.153048

Reference:

JPLPH 153048

To appear in:

Journal of Plant Physiology

Received Date:

1 February 2019

Revised Date:

11 July 2019

Accepted Date:

3 August 2019

Please cite this article as: Vojta L, Tomaˇsi´c Pai´c A, Horvat L, Rac A, Lepeduˇs H, Fulgosi H, Complex lumenal immunophilin AtCYP38 influences thylakoid remodelling in Arabidopsis thaliana, Journal of Plant Physiology (2019), doi: https://doi.org/10.1016/j.jplph.2019.153048

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Complex lumenal immunophilin AtCYP38 influences thylakoid remodelling in Arabidopsis thaliana

Lea Vojta1, Ana Tomašić Paić1, Lucija Horvat1, Anja Rac1, Hrvoje Lepeduš2,3, Hrvoje Fulgosi1,*

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Laboratory for Molecular Plant Biology and Biotechnology, Divison of Molecular Biology, Institute Ruđer Bošković, Bijenička cesta 54, HR-10 000 Zagreb, Croatia

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Faculty of Humanities and Social Sciences, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia 3

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Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia *

To whom correspondence may be addressed: Dr.sc. Hrvoje Fulgosi, Laboratory for

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Molecular Plant Biology and Biotechnology, Division of Molecular Biology, Ruđer Bošković

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Institute, Bijenička cesta 54, HR-10 000 Zagreb, E-mail: [email protected], Phone: ++ 385 1 4680-238, Fax: ++ 385 1 4561-177

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Abstract

Investigations of the luminal immunophilin AtCYP38 (cyclophilin 38) in Arabidopsis

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thaliana (At), the orthologue of the complex immunophilin TLP40 from Spinacia oleracea,

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revealed its involvement in photosystem II (PSII) repair and assembly, biogenesis of PSII complex, and cellular signalling. However, the main physiological roles of AtCYP38 and TLP40 are related to regulation of thylakoid PP2A-type phosphatase involved in PSII core protein dephosphorylation, and chaperone function during protein folding. Here we further investigate physiological roles of AtCYP38 and analyse the ultrastructure of chloroplasts from cyp38-2 plants. Transmission electron microscopy followed by quantitative 1

micrography revealed modifications in thylakoid stacking. We also confirm that the depletion of AtCYP38 influences PSII performance, which leads to stunted phenotype of cyp38-2 plants.

Abbreviations AtCYP38 chloroplastic peptidyl-prolyl cis-trans isomerase of 38 kDa from A. thaliana, F0 minimum fluorescence level, Fm Maximum fluorescence level, Fv/Fm Maximum quantum

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yield of PSII (Fm-F0/Fm), LHCII light-harvestiong complex II, NPQ non-photochemical quenching, OEC oxygen-evolving complex, PPIase peptidyl-prolyl cis-trans isomerase, PSI photosystem I, PSII photosystem II, RC reaction center, TAKs thylakoid membrane-

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associated protein kinases, TLP40 chloroplastic peptidyl-prolyl cis-trans isomerase of 40

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kDa, WT wild type.

Introduction

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chloroplast ultrastructure

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Keywords: chloroplasts, thylakoids, AtCYP38, immunophilin, photosynthetic performance,

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Plants, as sessile organisms, are exposed to the numerous environmental stresses, like changes in illumination (i.e. light quality and quantity), which influence electron transport

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and photosynthetic energy conversion between the photosystems. In order to maintain optimal electron transport rates and to minimize light-induced oxidative damage of the thylakoid pigment-protein complexes, particularly of the photosystem II (PSII), repair processes regulated by the reversible phosphorylation of PSII components have evolved (Andersson and Aro, 1997). When the system is not poised, oxidative damage can lead to a 2

photo inactivation of the entire photosynthetic electron transport chain. The most affected component of PSII is the reaction centre (RC) protein D1 (Andersson and Aro, 2001). Turnover rates of D1 during repair cycles of PSII are considered the highest among all of the thylakoid proteins (Mattoo et al., 1984). In photosynthetic protein repair cycles, significant number of auxiliary and chaperone enzymes are involved, governing a number of short- and long-term acclimation and adaptation processes in order to evade damage. A redox-regulated phosphorylation of the thylakoid membrane proteins, mostly light-harvesting (LHCII) and

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PSII core complexes (D1, D2, CP43, and PsbH polypeptide), represent such short-term acclimation mechanisms (Allen, 1992; Andersson and Aro, 1997; Gal et al., 1997; Vener et

al., 1998). Two thylakoid membrane-associated protein kinases (TAKs) (Rochaix, 2013) that

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are conserved in vascular plants and algae are responsible for phosphorylation of LHCII and PSII core proteins. This tandem includes STN7 kinase, which is controlled by redox state of

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the plastoquinone (PQ) pool, and by binding of PQ to the Qo site of cytb6f complex (Vener et

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al., 1997; Zito et al., 1999; Bellafiore et al., 2005); and STN8 kinase, responsible for phosphorylation of PSII core proteins (Bonardi et al., 2005; Vainonen et al., 2005). LHCII phosphorylation is essential for the reversible processes of state transitions, which occur

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when the light is limiting and when conditions promote preferential excitation of PSII relative to PSI (Haldrup et al., 2001; Allen and Forsberg, 2001; Pribil et al., 2010). On the other hand,

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phosphorylation of PSII core proteins is necessary for efficient repair of damaged protein D1.

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Dephosphorylation of the phosphorylated antenna (pLHCII) is performed by a PP2C-type phosphatase TAP38 (Pribil et al., 2010; Pesaresi et al., 2011) or PPH1 (Shapiguzov et al., 2010). TAP38/PPH1 is activated when PQ pool becomes oxidized due to the light-saturation of PSI, resulting in LHCII relocation back to the stacked membrane areas (State 1). PBPC, also a PP2C-type phosphatase, is an antagonist of STN8 and thus involved in PSII repair. In mutants lacking functional STN8, and in stn7stn8 double mutant, degradation of D1 was 3

significantly retarded under the high light conditions (Tikkanen et al., 2008). Remarkably, folding of thylakoid membranes was affected in such a way that grana size increased, while grana stacks were reduced (Fristedt et al., 2009). Before the discovery of PP2C-type phosphatases, the activity of PP2A-type thylakoid associated phosphatase has been described. This phosphatase was found to be regulated by the complex immunophilin TLP40 (Fulgosi et al., 1998; Vener et al., 1999; Rokka et al., 2000). Immunophilins are well known for their PPIase (peptidyl-prolyl cis-trans isomerase,

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rotamase) and/or chaperone-like activities, thereby in regulating the protein conformation, promoting protein-protein interactions, refolding, sorting and in functionality of chloroplast imported proteins. Complex immunophilins are in many cases regulated by light and are

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responsive to various forms of environmental stresses (Tomašić Paić and Fulgosi, 2016). The structure of the complex immunophilin TLP40, its localization in thylakoid lumen, and its

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possible association with thylakoid phosphatase implicates its multiple functions and

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involvement in diverse signalling networks (Fulgosi et al., 1998). Two putative phosphatasebinding modules at the N-terminal portion of TLP40 were shown to participate in the binding of a membrane-associated phosphatase involved in dephosphorylating PSII core proteins

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(Vener et al., 1999; Rokka et al., 2000). In 2002, Baena-González and Aro and in 2005 Aro et al. further suggested that a damaged D1 repair cycle includes TLP40 Arabidopsis orthologue

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AtCYP38. AtCYP38 lacks peptidyl–prolyl cis-trans isomerase (PPIase) activity (Shapiguzov

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et al., 2006; Edvardsson et al., 2007) and is also involved in the assembly of oxygen evolving complex (OEC) (Sirpiö et al., 2008). In addition, in vivo role of AtCYP38 protein has been investigated in cyp38 Arabidopsis plants (Fu et al., 2007; Sirpiö et al., 2008; Lepeduš et al., 2009), suggesting its primary role in PSII biogenesis and repair.

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In this work, we have investigated the involvement of AtCYP38 and its regulatory functions in morphogenesis of chloroplast and thylakoid stacking. We further revisit the influence of AtCYP38 depletion on PSII performance.

Materials and methods Plant material and growth conditions Wild-type (WT) Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0) and A. thaliana

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insertional mutant line SALK_029448 (cyp38-2) (Alonso et al., 2003) were grown in growth chamber RK-900 CH (Kambič, Slovenija) under 80 mol PHOTONS m-2 s-1 growth light (GL) in 16-h light/8-h dark cycles at 22C and a relative air humidity of 60%-light/70%-dark. The

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insertional mutant line in SALK background was obtained from ABRC (The Ohio State

Electron microscopy

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At3g01480 gene (Lepeduš et al., 2009).

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University, USA) (Scholl et al., 2000) in order to determine a homozygous line for

For ultrastructural studies, plant tissue was fixed with 2% glutaraldehyde in 0.05 M Na-

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cacodylate buffer (pH 7.2) for 30 min at 4 oC. After washing, the tissue was further fixed with 1% osmium tetroxide in the same buffer for 1 h at 4 oC. Dehydration was performed by a

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graded series of ethanol followed by embedding the tissue in Spurr´s resin. Ultrathin sections

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of the fixed tissue were stained by uranyl acetate and lead citrate and examined using Zeiss EM 10 electron microscope (Oberkochen, Germany). Image analysis was performed by using Optimas 6.5.1 program (BioScan, Inc., Edmonds, WA, USA).

Chlorophyll a fluorescence measurements

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Chlorophyll a fluorescence parameters were measured by MINI-PAM portable chlorophyll fluorimeter (Walz, Germany) in vivo, using 4-week-old intact leaves of WT and cyp38-2 mutant plants, grown on potting substrate (Stender, Germany) under 80 mol PHOTONS m-2 s-1. Plants were dark adapted for 30 min before measuring a minimum fluorescence level (F0). Maximum fluorescence level (Fm) was calculated following saturating flash of light in darkadapted state. Maximum quantum yield (Fv/Fm) of PSII was defined as (Fm-F0/Fm). PSIIdriven relative electron transport rate (rel. ETR) and non-photochemical quenching of

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chlorophyll fluorescence (NPQ) were calculated as PPFD x 0.5 x ΦPSII and (Fm - Fm’)/ Fm’, where PPFD is photosynthetic photon flux density, ΦPSII was calculated using following

expression: (Fm’ – Fs)/ Fm’, Fm is maximum fluorescence level in dark adapted material, Fm’

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is a maximum fluorescence level in the light, and Fs designates steady-state fluorescence

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level.

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Data analyses

Image analyses and morphometry (n=26) performed with Optimas program were statistically evaluated by unpaired t-test (two-tailed). Error bars represent SD (** P(t)<0,01; ***

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P(t)<0,001). Chlorophyll a fluorescence in situ measurements data were also subjected to unpaired t-test (two-tailed) for small samples. Error bars represent SD (n=5; * P(t)<0,05; **

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P(t)<0,01; *** P(t)<0,001 relative to WT). All tests were performed using GraphPad Prism

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version 4.0 for Windows (GraphPad Software, San Diego, CA, USA).

Results

Homozygous cyp38-2 mutant line shows stunted plant phenotype and defects in PSII functionThe cyp38-2 homozygous mutant line described in this work has been previously characterized by using gene- and T-DNA-specific PCR primers (Lepeduš et al., 2009) and 6

differs from the line described by Sirpiö et al. (2008). Four-week-old cyp38-2 null-mutant plants exhibit retarded growth, small leaves and low survival of seedlings, even under growth light (GL) conditions (80 mol PHOTONS m-2 s-1) (Lepeduš et al., 2009). To further investigate observed developmental defects, chlorophyll a fluorescence measurements were conducted on the leaves of cyp38-2 mutants and compared to WT plants. Maximum PSII quantum yield of primary photochemistry (Fv/Fm) measured in dark-adapted leaves of the WT was approximately 0.8 (Fig. 1A). In cyp38-2, maximum efficiency of PSII value was slightly

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decreased (0.68), possibly indicating photoinhibition or damage of PSII RCs. According to Schreiber et al. (1994), optimal Fv/Fm value for healthy plants is about 0.8-0.83 with lower values indicating stress. Further, non-photochemical quenching (NPQ) of chlorophyll a

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fluorescence (Bilger and Björkman, 1990) was measured. NPQ is involved in protection of photosynthetic apparatus from oxidative damage (Li et al., 2002). At light intensity of 100

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mol PHOTONS m-2 s-1, cyp38-2 had a significantly higher NPQ value (0.350.05) than the WT

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(0.210.12), (p<0.05) (Fig. 1B). At high light intensities of 250 mol PHOTONS m-2 s-1 or 500 mol PHOTONS m-2 s-1, NPQ was not significantly decreased in cyp38-2, in comparison to the

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WT (Figure 1B). Most conspicuously, cyp38-2 showed considerably lower photosynthetic electron transport rates (ETR) under all tested light conditions, (17.351.73) for 100 mol m-2 s-1, (22.253.53) for 250 mol PHOTONS m-2 s-1, and (18.558.14) for 500 mol

PHOTONS

m-2 s-1 (Fig. 1C). These declines suggest damage to the PSII and consequently lower

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PHOTONS

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Figure 1. Chlorophyll a fluorescence analyses of cyp38-2 compared to the WT plants. Plants were grown under 80 mol PHOTONS m-2 s-1 for 4 weeks. a. Fv/Fm, the maximum efficiency of PSII photochemistry; b. NPQ, non-photochemical quenching parameter; c. ETR, relative electron transport rate. Error bars represent SD (n=5); * P(t)<0.05; ** P(t)<0.01; *** P(t)<0.001 relative to WT.

photosynthetic capacity in cyp38-2 plants, irrespective of the light intensity. 8

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Figure 2. Electron microscopy analyses of chloroplast ultrastructure and organization of thylakoid membranes. Chloroplast ultrastructure analyses of the: a. WT and b. cyp38-2 plants grown under growth light conditions (80 mol PHOTONS m-2 s-1) are shown. Scale bar = 1 m. Morphometric analyses of chloroplasts and thylakoid membranes. c. Thylakoid content (%). d. Mean of the appressed stack height (m). e. Mean of the plastid area (m2) of the WT and cyp38-2 plants, quantified by the Optimas image analysis software. Results were subjected to unpaired t-test (two-tailed). Error bars represent SD; ** P(t)<0.01; *** P(t)<0.001.

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Ultrastructural analyses and plastid size determination-To investigate the influence of AtCYP38 depletion on chloroplast fine structure and to establish whether interactions of

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AtCYP38 with a thylakoid phosphatase play a role in thylakoid remodelling, we have analysed ultrastructures of cyp38-2 chloroplasts. In 4-week-old Arabidopsis WT leaves (Fig. 2A), chloroplasts showed well-developed thylakoid system and arrangement of grana typical for normal chloroplasts. In cyp38-2, chloroplasts subtly differed from those in the WT (Fig. 2B). Cyp38-2 chloroplasts contained on average 5% more thylakoids (Fig. 2C) than the WT 9

chloroplasts. Further, in chloroplasts of cyp38-2, statistically significant differences were recorded in plastid area and height of the appressed membranes. Overall increased height of appressed thylakoid regions (Fig. 2D) and plastid area of the cyp38-2 plastids (Fig. 2E) can be observed. AtCYP38 deficiency likely leads to the constitutive activation of the PSII core phosphatase, thus leading to drastically decreased phosphorylation of PSII core (Sirpiö et al., 2008). This decreased phosphorilation consequently leads to lower repulsion between thylakoid membrane stacks and, accordingly, to higher thylakoid stacking, as depicted in Fig.

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2B. Similar phenotype was observed in stn8 and stn7stn8 kinase inactivation mutants, due to deficiency in light-induced phosphorylation (Fristedt et al., 2010, see Figure 8, left panel therein). Further, the height of grana stacks is increased in cyp38-2 mutant (Fig. 2D)

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approximately 2 times, indicating that grana contain more thylakoid membranes than in the

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WT chloroplasts.

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Discussion

Cyp38-2 plants have small leaves, low seed production, and retarded growth when compared to the WT plants grown at long day cycle and 80 mol PHOTONS m-2 s-1, confirming that

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AtCYP38 protein has a strong influence on photosynthetic processes and plant viability in general. Similar physiological properties of AtCYP38 depleted plants were noticed earlier (Fu

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et al., 2007; Sirpiö et al., 2008). Further, ultrastructural characteristics of chloroplasts showed

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subtle difference in the thylakoid system and grana arrangements between WT and cyp38-2 plants. However, plastid size measurements revealed significant increase of appressed membranes in cyp38-2 mutants. This observation suggests a link between the reversible phosphorylation and membrane dynamics, caused by the lack of the AtCYP38. Chuartzman et al. (2008) reported that phosphorylation of PSII core proteins affects thylakoid ultrastructure. Similar remodelling of thylakoid membrane stacking, as observed on chloroplast 10

ultrastructures of cyp38-2 mutant, has been observed in stn8 and stn7stn8 mutant plants (Fristedt et al., 2010), where most of the PSII and LHCII phosphorylation was lost. In cyp382 plants, the phosphorylation of PSII proteins was significantly reduced (Sirpiö et al., 2008), exerting the same impact on the cyp38-2 ultrastructure, as demonstrated in earlier studies (Fristedt et al., 2009). Moreover, in AtCYP38-deficient plants, the migration of damaged D1 from grana to stroma lamellae could be postponed, causing the retarded proteolysis and de novo synthesis, and consequently affecting the overall membrane architecture.

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In cyp38-2 plants, phosphatase is very likely constantly being activated as there is no auxiliary control. Sirpiö et al. (2008) demonstrated that phosphorylation process in cyp38-2 mutant is also affected, influencing the rate of proteolytic degradation of the damaged D1

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protein. According to Sirpiö et al. (2008), phosphorylation of the PSII core proteins in AtCYP38 mutant thylakoids is reduced, as compared to the WT plants. Additionally,

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phosphorylation of LHCII proteins was also diminished.

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Maximum quantum yield (Fv/Fm ratio) of PSII is slightly reduced in cyp38-2 plants (0.68) when compared to WT value (~0.8). This is in accord with polyphasic chlorophyll a transient measurements that were performed in our previous study (Lepeduš et al., 2009). Decreased

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Fv/Fm parameter indicates the extent of photoinhibition in cyp38-2 plants, possibly influenced by damage or disassembly of PSII core components even under growth light

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intensities. Non-photochemical quenching (NPQ) of chlorophyll fluorescence, which protects

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PSII and PSI from a photodamage and excess excitation energy, was found to be higher in mutants than in the WT plants at light intensity of 100 mol PHOTONS m-2 s-1. However, at 250 and 500 mol PHOTONS m-2 s-1 NPQ values measured in cyp38-2 were slightly reduced, as compared to the WT. Further, the electron transport rate (ETR) through PSII measured in cyp38-2 mutants, is significantly reduced when compared to the WT. This decrease in ETR could be attributed to altered biogenesis of PSII core in cyp38-2 mutant. 11

Author Contributions: Hrvoje Fulgosi designed experiments, analysed results and wrote the paper. Lea Vojta discussed results and wrote the paper. Ana Tomašić Paić wrote the paper. Lucija Horvat performed electron microscopy ultrastructural studies. Anja Rac edited the manuscript. Hrvoje Lepeduš performed chlorophyll a fluorescence experiments. All authors reviewed the results and approved the final version of the manuscript.

Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had

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no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Croatian Science Foundation to Hrvoje Fulgosi.

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Acknowledgments: This work has been funded by the Grant IP-2014-09-1173 of the

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