The Low Molecular Mass Photosystem II Protein ... - Plant Physiology

Plant Physiology Preview. Published on December 11, 2018, as DOI:10.1104/pp.18.01251

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Short title: PsbTn protein regulates light adaptation

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Author for correspondence: Wolfgang P. Schröder ([email protected]) Department of Chemistry, University of Umeå, Umeå SE-901 87, Sweden

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The Low Molecular Mass Photosystem II Protein PsbTn

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is Important for Light Acclimation

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Yang-Er Chen1,2,6, Shu Yuan4,6, Lina Lezhneva1, Jörg Meurer3, Serena Schwenkert3,

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Fikret Mamedov5 and Wolfgang P. Schröder1,*

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Department of Chemistry, University of Umeå, Umeå SE-901 87, Sweden

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College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China

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Department Biology I, Plant Sciences, Ludwig-Maximilians-University, Munich D-82152 PlaneggMartinsried, Germany

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China

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Molecular Biomimetrics, Department of Chemistry – Ångström Laboratory, Box 523, Uppsala University, SE-751 20 Uppsala, Sweden

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College of Resources Science and Technology, Sichuan Agricultural University, Chengdu 611130,

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These authors contributed equally to this article.

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One sentence summary:

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PsbTn is a low molecular mass protein and a component of Photosystem II that is important for

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water oxidation and light acclimation.

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Authors contribution

1 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Copyright 2018 by the American Society of Plant Biologists

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Y.E.C. and W.P.S. designed the research, performed the experiments, and wrote the paper. S.Y., L.L.,

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and F.M. participated in the experiments, evaluated data and edited the paper. J.M. and S.S. prepared

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mutants, performed measurements, and participated in writing.

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Key words: PsbTn proteins, photosystem II, photoinhibition, protein phosphorylation, Arabidopsis

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Abstract

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Photosystem II (PSII) is a supramolecular complex containing over 30 protein subunits and a large

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set of cofactors including various pigments and quinones as well as Mn, Ca, Cl, and Fe ions.

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Eukaryotic PSII complexes contain many subunits not found in their bacterial counterparts, including

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the proteins PsbP, PsbQ, PsbS, and PsbW, as well as the highly homologous, low molecular mass

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subunits PsbTn1 and PsbTn2 whose function is currently unknown. To determine the function of

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PsbTn1 and PsbTn2, we generated single and double psbTn1 and psbTn2 knock-out mutants in

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Arabidopsis thaliana. Cross-linking and reciprocal co-immunoprecipitation experiments revealed

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that PsbTn is a lumenal PSII protein situated next to the cytochrome b559 subunit PsbE. The removal

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of the PsbTn proteins decreased the oxygen evolution rate and PSII core phosphorylation level but

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increased the susceptibility of PSII to photoinhibition and the production of reactive oxygen species.

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The assembly and stability of PSII were unaffected, indicating that the deficiencies of the psbTn1

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psbTn2 double mutants are due to structural changes. Double mutants exhibited a higher rate of non-

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photochemical quenching of excited states than the wild type and single mutants, as well as slower

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state transition kinetics and a lower quantum yield of PSII when grown in the field. Based on these

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results, we propose that the main function of the PsbTn proteins is to enable PSII to acclimate to

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light shifts or intense illumination.

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INTRODUCTION

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The function, composition, and molecular dimensions of photosystem II (PSII) are highly conserved

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in cyanobacteria, algae, and vascular plants. The cyanobacterial PSII structure has been solved at

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1.9 Å (Umena et al., 2011; Suga et al., 2015) and used as a blueprint for other PSII complexes as

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well as a starting point for studies on the function of PSII and differences between the cyanobacterial

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and eukaryotic complexes. The supramolecular organization of the eukaryotic PSII appears to be

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more complex than that of the cyanobacterial complex due to the presence of several additional

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subunits, including several light-harvesting complexes (LHCs) and the proteins PsbP, PsbQ, PsbS,

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PsbW, and PsbTn. In addition, various biochemical analyses have revealed differences in protein

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function between eubacterial and eukaryotic PSII complexes. The structures of two eukaryotic PSII 2 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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complexes were recently solved – that of the red alga Cyanidium caldarium (at 2.76 Å resolution)

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using traditional X-ray crystallization techniques (Ago et al., 2016), and that of spinach (Spinacia

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oleracea) (at 3.2 Å resolution) using single-particle cryo-electron microscopy (Wei et al., 2016).

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Comparisons of these structures to that of the cyanobacterial PSII revealed some previously

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unrecognized differences. Notably, the lumenal side of the eukaryotic PSII core complex exposes the

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four extrinsic subunits PsbO, PsbP, PsbQ, and PsbTn. The first three of these extrinsic subunits form

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a triangular “hat” that covers the lumenal side of the CP43 and D1 proteins in the PSII core, while

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PsbTn was suggested to intercalate between CP47 and the C-terminal region of PsbE (a subunit of

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cytochrome b559) (Wei at al., 2016).

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Intriguingly, over half of the PSII subunits are low molecular mass (< 10 kDa) proteins. These

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proteins have diverse functions, affecting electron transport, the redox potentials of QA and/or QB,

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the assembly and stability of the PSII complex, photosensitivity, recovery from photoinhibition, and

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phosphorylation patterns (Swiatek et al., 2003; Ohad et al., 2004; Shi and Schröder, 2004;

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Schwenkert et al., 2006; Umate et al., 2007, 2008; von Sydow et al., 2016). Phylogenetically, PsbTn

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is among the youngest proteins in the PSII complex because it has not been found in cyanobacteria

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nor in red or green algae, and thus seems to have evolved first in land plants during endosymbiosis.

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PsbTn is a nuclear encoded subunit (and accordingly has the suffix “n”) and should not be confused

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with PsbTc which is an unrelated chloroplast-encoded protein (and thus bears the suffix “c”) (Umate

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et al., 2008). The PsbTn precursor protein incorporates a bipartite transit peptide that apparently

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targets the protein to the thylakoid lumen. The mature Arabidopsis thaliana protein comprises 31

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amino acids and is the smallest known PSII subunit, having a molecular mass of only 3.0 kDa (Shi

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and Schröder, 2004). The protein was initially isolated from spinach thylakoids and partly sequenced

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(Luneburg et al., 1986; Ikeuchi et al., 1988); the cDNA sequence from upland cotton (Gossypium

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hirsutum) encoding PsbTn was subsequently determined (Kapazoglou et al., 1995). However,

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structural biochemical and functional analyses of the protein are entirely lacking.

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In Arabidopsis, two PsbTn proteins that differ in only two amino acid residues (position 18 Q→P

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and position 31 Y→N) are encoded by the genes PsbTn1 (At3g21055) and PsbTn2 (At1g51400). In

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this study, we used single mutants to generate a double psbTn1 psbTn2 knockout mutant that was

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studied using biochemical and biophysical approaches. We show that psbTn1 psbTn2 double mutants

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suffer from severe photodamage under both intense illumination and fluctuating illumination. We

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also demonstrate that the primary function of PsbTn is to maintain PSII activity and protect PSII

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against photodamage under intense and/or fluctuating illumination. 3 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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RESULTS

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Expression, Structure, and Localization of the Two PsbTn Proteins

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Both PsbTn genes were mainly expressed in young and mature leaves; only weak expression was

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observed in senescing cauline leaves, stems, and other tissues (https://apps.araport.org). The PsbTn

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genes were co-expressed with genes encoding several PSII components (such as PsbO, P, W, X, Y)

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(Supplemental Figure S1), plastocyanin (PetE1), and some PSI components (PsaD1, E2, G, H1, H2,

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L) (http://evolver.psc.riken.jp; http://string-db.org; http://apps.araport.org).

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The PsbTn protein was originally purified and partially sequenced from PSII core complexes in

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spinach and wheat (Triticum sp.), and was referred to as the 5.0-kDa protein (Ljungberg et al., 1986,

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Ikeuchi et al., 1988). The mature protein has a mass of 3.2 kDa in Arabidopsis (Shi and Schröder,

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2004). PsbTn proteins in Arabidopsis and Gossypium hirsutum exhibit about 68% sequence identity

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and feature cysteines at positions C20 and C29 that are highly conserved in plants (Supplemental

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Figure S2). A protein fold prediction made using the I-Tasser algorithm indicated that the mature

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protein lacks transmembrane α-helices, suggesting that PsbTn is either a soluble protein or attached

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to the thylakoid membrane via electrostatic interactions. However, its association with PSII was not

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unambiguously established. To elucidate the localization and the topology of the PsbTn protein, we

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raised polyclonal antibodies against it using a mixture of two synthetic peptides representing the

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protein’s N- and C-terminal parts. This mixture was used because the single peptide was found to be

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insufficiently antigenic to yield useful sera. The resulting polyclonal antibody was used to detect the

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PsbTn protein in both thylakoids (column “T” in Figure 1A) and isolated PSII membrane fragments,

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or BBY particles (column “B” in Figure 1A). The PSII association of PsbTn was further established

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by analyzing isolated grana, stroma lamellae, and intermediate membrane fractions from thylakoids

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and comparing them to PSII membrane fragments (columns “G”, “I”, and “S” in Figure 1A). As

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expected, the PSII proteins PsbA and PsbO were detected in thylakoids, BBY particles, the grana

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fraction, and to a lesser extent in the intermediate fraction, but were absent in the stroma lamellae.

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Also as expected, the PSI protein PsaD was mainly located in the stroma lamellae, indicating that the

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fractionation process was successful. The distribution of PsbTn in this experiment resembled those of

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the PSII proteins PsbA and PsbO, suggesting that PsbTn is a component of PSII.

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To determine whether the PsbTn protein is exposed to the stroma or lumen, we digested sonicated

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(broken) and untreated (intact) thylakoid membranes with chymotrypsin and then performed

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immundecoration experiments (Figure 1B). The lumen-localized PsbO and PsbQ proteins were

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found to be only partly protected against chymotrypsin in the intact (untreated) thylakoids (Figure 1B, 5 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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left panel), showing that the thylakoids used in these experiments were not fully intact. However,

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these proteins were found to be fully degraded in the sonicated (broken) thylakoids (Figure 1B, right

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panel). The PsbTn protein behaved in the same way as the extrinsic PsbO and PsbQ proteins,

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confirming the conclusion that it is a lumenal PSII protein.

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To determine the PsbTn protein’s location within the PSII complex, we performed chemical

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crosslinking experiments using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which

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crosslinks amino and carboxyl groups. After incubation with EDC, two bands were detected using

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the PsbTn-specific antibody (Figure 2A). The lower band at 5 kDa corresponds to non-crosslinked

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PsbTn, while the 12 kDa band presumably corresponds to a crosslinked complex consisting of PsbTn

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and a crosslinking partner with a molecular mass of 5-8 kDa. We therefore subjected this band to

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immunoblotting analysis using antibodies against a set of known PSII proteins with molecular

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masses in this range, including PsbE, F, H, I, R, W, X, and Y (results for E and F are shown in

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Figure 2B). Only the antibodies against PsbE bound to the 12 kDa band. The interaction between the

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PsbTn protein and the PsbE subunit of cytochrome b559 was further confirmed by co-

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immunoprecipitation analysis. Solubilized PSII membrane fragments were incubated with the PsbTn

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serum, immunoprecipitated, and analyzed by SDS-PAGE followed by immunoblotting. As shown in

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Figure 2C, PsbE was co-purified with the PsbTn protein. PsbTn was also reciprocally co6 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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immunoprecipitated with the PsbE antisera, as shown in Figure 2D. This clearly established that the

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PsbTn protein is located on the lumenal side of PSII close to the PsbE protein (large subunit) of the

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Cytb559 subcomplex. This conclusion is consistent with the recently published structure of the

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spinach PSII complex (Wei et al., 2016).

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The next step was to investigate how strongly the PsbTn protein is bound to the lumenal side of PSII.

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To this end, PSII membrane fragments were washed with various chaotropic and alkaline salt

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solutions including 0.1 M NaOH, 1 M NaCl, 0.1 MNa2CO3, 3 M NaSCN, 1 M CaCl2 and 0.8 M Tris

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pH 8.4 (Figure 2E). As the stringency of the washing solutions was increased, the extrinsic PSII

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proteins PsbO, PsbP, and PsbQ were released into the soluble fraction, whereas the membrane-

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anchored CP43 protein remained in the pellet (membrane) fraction. None of these treatments 7 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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released PsbTn from the thylakoid membrane, suggesting that the protein is strongly bound to the

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PSII complex despite lacking a predicted transmembrane α-helix.

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Analyzing PsbTn Knock-Out Mutants in Arabidopsis

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Because the Arabidopsis genome contains two genes encoding PsbTn proteins, it was necessary to

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interrupt both of them to prevent PsbTn expression. This was done by crossing the T-DNA insertion

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lines psbTn1 (GABI_143D01) and psbTn2 (SAIL_1214_E09). The T-DNA insertion sites were

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confirmed by PCR and sequencing (Supplemental Figure S3). The absence of the PsbTn protein in

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the double mutant was confirmed by immunoblotting using the PsbTn antisera, which recognized

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both isoforms. No PsbTn protein was detected in the double mutant, but levels of PsbTn in the single

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mutants (psbTn1 and psbTn2) varied (Supplemental Figure S4A). The phenotypes of single mutant

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plants did not differ greatly from the wild type (WT), but double psbTn1 psbTn2 mutants were

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generally slightly smaller (Supplemental Figure S4B). Additionally, the chlorophyll content of the

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psbTn1 psbTn2 double mutants was around 20% lower than that in the WT on a fresh weight basis,

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and their chlorophyll a/b ratio was around 5% lower (Supplemental Table S1).

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PSII Composition and Assembly in the PsbTn Mutants

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To determine how the absence of the PsbTn protein affected the composition of PSII,

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immunoblotting experiments were performed using sera raised against various proteins of PSII and

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other thylakoid membrane complexes. As shown in Figure 3, levels of the major PSII proteins in the

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single and double mutants were identical to those in the WT. This was true for the reaction center

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proteins D1, D2, CP43, PsbE, and PsbY, the extrinsic PsbO, P, and Q proteins, and the antenna

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proteins Lhcb1, Lhcb2, CP29, and CP26. The abundance of the PsbS protein, which is suggested to

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be important in the regulation of non-photochemical quenching by incident light, was also

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unchanged in the double PsbTn mutant (Figure 3A last panel). In addition, we examined two PSI

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proteins (PsaD and Lhca4), the AtpB protein belonging to the ATP synthase complex, and the PetB

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protein belonging to the Cytb6/f complex, none of which exhibited any detectable change in

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abundance upon removal of either PsbTn protein. Consequently, the PSII/PSI ratios of all three

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mutants were very similar to that of the WT.

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Blue-native SDS-PAGE experiments were performed to determine whether the PsbTn protein might

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influence the assembly of the PSII complex (Figure 4). The assigned complexes were identified

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previously by mass spectrometry (Granvogl et al., 2006; Schwenkert et al., 2006, Chen et al. 2016).

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The native gels revealed no detectable differences in PSII supracomplex formation between the WT 8 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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and the three PsbTn mutants (Figure 4A). Further analyses of the second dimension showed that the

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loading of proteins and the structure of the complex were similar in the WT and mutants (Figure 4B).

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Immunoblot analyses of the 2D-gel using antisera against the PSII protein D1 and the PsaD protein

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from PSI confirmed the assignment of the complexes (Figure 4C). As expected, PsbTn was detected

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in all PSII complexes but absent in the double mutant sample, which nevertheless formed the same

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protein complexes as the wild type.

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In addition, the ultrastructures of thylakoid membranes from leaves from WT and mutant plants

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exposed to normal growth light or high light conditions were investigated by electron microscopy

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(Supplemental Figure S5). As shown in Supplemental Figure S5, the overall thylakoid structure did

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not differ significantly between the WT and mutants under normal light. However, the double mutant

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had slightly fewer and smaller grana stacks (Supplemental Figure S5D). All of the high light samples

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exhibited starch granule accumulation, and the psbTn1 psbTn2 thylakoids from plants grown under

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high light conditions again appeared to have fewer and smaller grana stacks than the corresponding

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WT thylakoids (Supplemental Figure S5H).

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Electron Transfer Within PSII in PsbTn Mutants


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To determine whether the absence of the PsbTn proteins affected the functionality of PSII, various

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photosynthetic parameters were analyzed (Supplemental Table S1). The oxygen evolution rates of

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thylakoids and BBY particles from the double mutant were 66% and 71% of those in the WT,

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respectively; the single mutants exhibited less pronounced reductions. The reduced oxygen evolution

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rates of the double mutant clearly indicate that removing the two PsbTn proteins from PSII causes

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defects in PSII function.

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Further analysis of overall PSII functionality by Pulse-Amplitude-Modulation (PAM) measurements

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revealed that the maximum and effective quantum yield of PSII (Fv/Fm and ΦPSII) were reduced from

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0.79 and 0.38, respectively, in the WT to 0.74 and 0.30, respectively, in the psbTn1 psbTn2 double

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mutant (Supplemental Table S1). The psbTn1 psbTn2 double mutant also exhibited a reduced

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electron transport rate (ETR) (Supplemental Table S1), which is again consistent with impaired PSII

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functionality. The double mutant also exhibited increased non-photochemical quenching (NPQ) of

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excited states under both moderate and high light conditions (Supplemental Table S1), presumably

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because of the reduced efficiency of electron transfer through PSII. To determine the cause of the

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ETR reduction, the quantum yield of PSI, ΦPSI, and the donor-side limitation of PSI, ΦPSI (ND), were

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measured (Supplemental Table S1). Compared to the WT, the double mutant had a slightly lower

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value of ΦPSI but an appreciably greater value of ΦPSI (ND). This suggests that the electron flow to

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PSI is rate limiting in the double mutant because the absence of PsbTn makes PSII unable to deliver

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electrons efficiently.

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The effect of PsbTn deletion was further characterized by measuring flash-induced variable

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fluorescence, which reflects the redox state of the first quinone acceptor, QA, in PSII (Supplemental

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Figure S6). The kinetics of the variable fluorescence decay thus provide information on electron

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transfer from QA- (Mamedov et al., 2000, von Sydow et al., 2016). However, no significant

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differences between the WT and the mutants could be detected (Supplemental Figure S6A). When

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the variable fluorescence decay is measured in the presence of Dimethylsulfoxide (DCMU), an

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inhibitor of electron transfer between QA and QB, the fluorescence decay is dominated by the kinetics

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of recombination between QA- and the S2 state in PSII. However, even under these conditions, the

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traces for the WT, the two single mutants, and the double mutant were all very similar (Supplemental

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Figure S6B). The loss of PsbTn thus does not affect the acceptor side of PSII. A more detailed

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acceptor-side analysis of PSII based on thermoluminescence measurements also revealed no

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differences between the WT, single mutants, and double mutant.

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The electron transfer components of PSII were also analyzed by electron paramagnetic resonance

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(EPR) spectroscopy, which was used to study the S2-state multiline signal, the QA- Fe2+ interaction

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signal, and the redox state of Cytb559 (Supplemental Figure S7). The deconvolutions of the EPR data

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are summarized in Supplemental Table S2. These experiments indicated that there were no

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differences between the WT and the double mutant with respect to the redox state of Cytb559 or the

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intensities of the QA- Fe2+ and S2 state multiline signals. Interestingly, however, the TyrD signal was

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10-20% less intense in the spectra of the double mutant than those of the WT (Supplemental Table 11 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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S2). In general, the EPR experiments indicated that the absence of PsbTn had no dramatic effect on

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the electron path through PSII in the psbTn1 psbTn2 double mutants.

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PSII Photoinhibition Analysis

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Because the PAM analysis revealed some minor differences between the double mutant and the WT

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with respect to ΦPSII and the ETR (Supplemental Table S1), we investigated the sensitivity of PSII to

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photoinhibition in the mutants. Four-week-old WT and mutant plants were illuminated with

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heterochromatic light at 1,000 µmol photons m-2 s-1 for 4 h, after which the samples were

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illuminated at a low intensity (10 µmol photons m-2 s-1) and their recovery was monitored over 24 h.

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Changes in the Fv/Fm fluorescence ratio were recorded and the levels of the D1 and PsaD proteins

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were analyzed immunologically (Figure 5). During the four hours of high light, the Fv/Fm values for

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the WT and double mutant decreased from 0.79 to 0.42 and 0.74 to 0.27, respectively (Figure 5A).

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The larger decrease observed for the double mutant indicates that the absence of PsbTn increased

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sensitivity to high light. The rates of recovery from photoinhibition were similar in the WT and the

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double mutant (Figure 5B) despite their different initial levels of inhibition. The double mutant’s

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greater reduction in Fv/Fm can thus be attributed to elevated photosensitivity but not to a failure to

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recover from photodestruction. To strengthen this conclusion, the plants were treated with

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lincomycin to inhibit D1 protein turnover and thus the PSII repair process. As expected, samples

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treated in this way exhibited a greater degree of PSII inactivation than untreated control samples

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(Figure 5A, lower traces). The greatest sensitivity was observed in the psbTn1 psbTn2 double mutant,

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whose Fv/Fm value was one third that of the WT.

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To determine whether high light-induced PSII inactivation reduced steady-state levels of the PSII

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complex, we analyzed the accumulation of the D1 protein in leaves from WT and mutants under high

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light conditions (1000 µmol photons m-2 s-1) in the absence and presence of lincomycin. After 3 h of

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illumination, the abundance of the D1 protein was determined by immunoblotting. Levels of D1

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declined moderately under high light in WT plants and to a larger extent in the double mutant

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(Figure 5C). In the presence of lincomycin, this effect was even more pronounced. However, no

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significant changes in the abundance of the PSI protein PsaD were detected in any sample (Figure

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5C). These results show that while the absence of PsbTn proteins has only minor effects on the

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electron pathway in PSII, it has dramatic effects on photoinhibition.

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PSII Protein Phosphorylation in the psbTn1 psbTn2 Double Mutants


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The phosphorylation and dephosphorylation of specific PSII proteins plays an important regulatory

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role in the PSII repair cycle and the energy balance (Aro and Ohad, 2003; Vener, 2007). To

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determine whether the phosphorylation pattern mediated by the kinases STN7 and STN8 is changed

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by the absence of the PsbTn protein, four-week-old plants were incubated in darkness (D), under

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normal growth light (NL), or under high light (HL) for 30 minutes. Thylakoid proteins were

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separated by SDS-PAGE and immunodecorated using antiphosphothreonine antibodies (Figure 6).

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As expected, all samples incubated in darkness exhibited little phosphorylation of PSII reaction

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center proteins, with the lowest levels of PSII phosphorylation being observed in the psbTn1 psbTn2

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double mutant (Figure 6). All samples incubated under normal light conditions exhibited higher

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levels of phosphorylation of the PSII core proteins CP43, D1, and D2 but again, these proteins

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(especially D1) were less extensively phosphorylated in the psbTn1 psbTn2 double mutant. Under

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high light, the WT and the single psbTn mutants still showed relatively high phosphorylation levels,

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but phosphorylation of the PSII reaction center was reduced in the psbTn1 psbTn2 double mutant. In

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addition, we observed no significant differences in LHCII phosphorylation between the WT and

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psbTn mutants under any light conditions. These results indicate that the complete removal of the

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PsbTn proteins from PSII affected the phosphorylation of PSII core proteins.

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Reactive Oxygen Species Production and Oxidative Stress in psbTn1 psbTn2 Double Mutants

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Photoinhibition leads to imbalances in electron fluxes through the photosynthetic electron transport

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chain, which in turn cause reactive oxygen species (ROS) production. The increased photoinhibition

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in the psbTn1 psbTn2 double mutant therefore prompted us to analyze its ROS production. Three

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independent methods were used to detect oxidative stress: (I) direct color staining with NBT (blue),

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(II) DAB (orange) staining of plant seedlings, and (III) confocal microscopy imaging using the

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singlet oxygen sensor green (SOSG) reagent (Supplemental Figure S8). NBT staining indicated that

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formazan formation under high light conditions was more extensive in the psbTn1 psbTn2 mutant

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than in the WT, indicating greater O2˙ˉ accumulation in the double mutant (Supplemental Figure

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S8A). Double mutant plants subjected to high light were also stained more intensely with DAB,

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indicating that they accumulated more H2O2 (Supplemental Figure S8B). The SOSG fluorescence

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indicator was used to detect singlet oxygen (1O2) in detached younger leaves. Leaves of double

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mutants subjected to high light generated a stronger fluorescence signal than WT leaves under the

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same conditions, suggesting that the psbTn1 psbTn2 mutants also accumulated more 1O2 than the

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WT (Supplemental Figure S8C). The psbTn1 psbTn2 double mutant thus accumulated higher levels

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of three different ROS than the WT, indicating increased oxidative stress.

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To determine whether detectable photobleaching occured in leaves of psbTn1 psbTn2 double mutants

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under changing illumination, a modification of a previously reported light-to-dark shifting protocol

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was used (Tikkanen et al., 2010). To specifically induce the production of 1O2, WT and mutant plants

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were placed in darkness for 30 min (and not low light as in Tikkanen et al., 2010) and then exposed

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to high light for 30 min; this cycle was repeated for 4 hours (Wagner et al., 2004; Lee et al., 2007).

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Under these conditions, white spots formed in the leaves of psbTn1 psbTn2 double mutants but not in 14 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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the WT (Figure 7A), indicating that the double mutant suffered serious damage under high light/dark

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shift conditions. Moreover, the psbTn1 psbTn2 plants exhibited significantly greater reductions in

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Fv/Fm and NPQ than the WT plants (Figure 7B). This suggests that PsbTn is essential for proper

331

acclimation of PSII to artificially high light/dark shift conditions. In addition, both cell death

332

(detected by trypan blue staining) and ROS accumulation were observed in seedlings of the double

333

mutant (Figure 7C, D) under the light shift conditions but not in the WT. These results further

334

demonstrate that the double mutants are more sensitive to oxidative stress than the WT.

335 336

PSII Protein Phosphorylation and Complexes Under high light/dark shift conditions

337

To investigate the double mutants’ sensitivity to high light/dark shift conditions, we studied the

338

phosphorylation patterns of PSII and thylakoid membrane protein complexes in the WT and mutants

339

exposed to alternating 30 minute periods of darkness and intense illumination for 4 hours. The

340

differences in phosphorylation between the WT and mutants previously observed under constant

341

light conditions (Figure 6) became even more pronounced under high light/dark shift conditions


342

(Figure 8A). As was also observed when studying PSII phosphorylation under constant light

343

conditions, phosphorylation of the D1 and D2 proteins under high light/dark shift conditions was

344

reduced in the double mutants when compared to than in the WT, but no such effect was observed on

345

the phosphorylation of CP43 and LHCII. The high light/dark shift illumination also made the PSII-

346

LHCII supercomplex and PSII dimer less abundant in the double mutants than in the WT (Figure 8C).

347 348

Chlorophyll Fluorescence Analysis

349

Because the psbTn1 psbTn2 double mutants were more sensitive to photoinhibition than the WT,

350

their energy dissipation and state transitions were investigated by chlorophyll fluorimetry (Figure 9).

351

An important mechanism that protects against excessive excitation in plants is thermal energy

352

dissipation, which contributes to NPQ (de Bianchi et al., 2011). Therefore, WT and mutant plants

353

were illuminated with saturating light (1,000 μmol photons m-2 s-1) and the changes in their NPQ

354

were measured over a period of 1000-5000 seconds. Under the high light periods, both the mutants

355

and the WT displayed the typical rapid increase in NPQ followed by a slower increase (Figure 9A,

356

B), but the double mutant exhibited a significantly slower and weaker increase than the WT.

357

However, the mutants and the WT achieved similar rates of recovery in darkness after intense

358

illumination (Figure 9A). When a second illumination period was applied (Figure 9B), the increase

359

in NPQ was again slower in the double mutants than in the WT, implying that the absence of PsbTn

360

increases sensitivity to photoinhibition and favors ROS production under high light conditions. 16 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

361 362

State transitions are a good indicator of the rate of energy dissipation (de Bianchi et al., 2011).

363

Therefore, state transitions in the WT and mutant plants were investigated by monitoring changes in

364

chlorophyll fluorescence characteristics. The amplitude of the state transitions was the same in WT

365

and single mutant plants when plants were grown in a greenhouse under constant conditions

366

(Supplemental Figure S9). However, when grown in the field, significant differences in state

367

transitions were observed between the double mutant and WT plants (Figure 9C). Specifically, the

368

final amplitudes of the state transitions in the double mutants were lower than those in the WT.

369

These results show that the lack of PsbTn affected state transitions under natural conditions. The

370

rapid decrease in the kinetics of state transition may be due to the light intensity in the field, which

371

may be as high as 1,000 μmol photons m-2 s-1 at noon under natural conditions. Moreover, the

372

effective quantum yield of PSII (ΦPSII) was substantially lower in double mutants than in the WT

373

(Figure 9D), indicating that PsbTn is required for PSII activity under natural conditions. These

374

differences in state transitions and ΦPSII between the WT and mutants when grown in the field or in

375

climate chambers indicate that PsbTn is important for acclimation to intense and fluctuating

376

illumination under natural conditions.


378


379

DISCUSSION

380

PsbTn is a PSII Subunit Located on the Lumenal Side

381

The PsbTn protein was detected in thylakoids, enriched in grana stacks and isolated PSII membrane

382

fragments (BBY-particles) (Figure 1), and found to comigrate with PSII complexes in BN-PAGE

383

experiments (Figure 4). These results suggest that it is a bona fide component of the PSII complex.

384

Chymotrypsin digestion of thylakoid membranes with and without sonication showed that the PsbTn

385

protein is located on the lumenal side of the membrane (Figure 1). We next investigated its location

386

within the PSII complex. Chemical crosslinking experiments and co-immunoprecipitation

387

experiments using PsbTn or PsbE antisera revealed that PsbTn is located close to the PsbE subunit of

388

cytochrome b559 (Figure 2). A protein structure prediction using the I-Tasser algorithm and a

389

hydrophobicity analysis identified no putative transmembrane helices in the small PsbTn protein

390

(Supplemental Figure S2), suggesting that it is an extrinsic membrane protein that is only bound to

391

the PSII complex by electrostatic interactions. However, washing with various chaotropic reagents

392

did not release PsbTn from either the thylakoid membrane or the PSII fragments even under

393

conditions that triggered the release of extrinsic proteins such as PsbO (Figure 2). This suggests that

394

PsbTn is not a typical transmembrane protein but is nevertheless strongly bound to the PSII complex.

395

These results are consistent with the recently published structure of the spinach PSII complex

396

obtained by cryo-EM, which suggests that PsbTn intercalates between CP47 and the C-terminal part

397

of the PsbE protein (Wei et al., 2016). We thus conclude that the PsbTn protein is an intrinsic

398

lumenal PSII subunit in Arabidopsis.

399 400

The Structure of PSII is Unaltered in psbTn Mutants

401

Both genomic PCR (Supplemental Figure S3) and immunoblotting analyses using a polyclonal

402

PsbTn antibody were used to verify that the PsbTn protein was absent in the psbTn1 psbTn2 double

403

mutant (Supplemental Figure S3 and Figure 4). Aside from being somewhat smaller than WT plants,

404

the single and double psbTn mutants exhibited no readily apparent phenotypic abnormalities under

405

normal laboratory growth conditions (Supplemental Figure S4B). However, the chlorophyll content

406

and chlorophyll a/b ratio in the psbTn1 psbTn2 double mutant were slightly lower than in the WT

407

(Supplemental Table S1). This bleaching may indicate that the double mutant is subject to increased

408

stress. However, measurements of the plants’ contents of PSI, PSII, ATP synthase, the cytochrome

409

b6f complex, and various antenna proteins revealed no compositional changes that could explain the

410

reduced chlorophyll content of the psbTn1 psbTn2 double mutant. Furthermore, no changes in the

411

PSII assembly were observed in any psbTn mutant (Figure 4). This clearly shows that the PsbTn


412

protein is not directly required for the assembly and stability of the PSII complex even though

413

structural changes are likely to occur in the mutants and account for the observed deficiencies.

414 415

The PsbTn Protein Does Not Seem to Be Directly Involved in Electron Transport Within PSII

416

We performed many experiments to probe the functional status of PSII in the psbTn mutants,

417

including PAM measurements (Supplemental Table S1), variable chlorophyll fluorescence analyses

418

(Supplemental Figure S6), and analyses of selected EPR signals (Supplemental Table S2 and

419

Supplemental Figure S7). None of these experiments revealed any major differences between the

420

WT and psbTn1 psbTn2 double mutants with respect to electron transfer through PSII. However,

421

there were some differences in the rates of oxygen evolution and total photosynthetic electron

422

transfer rates (ETR) (Supplemental Table S1). This seems to exclude the possibility that PsbTn

423

might be directly involved in the electron pathway through the PSII complex.

424 425

These findings could only partially explain the increased photoinhibition and ROS production

426

observed in the psbTn1 psbTn2 double mutant (Figure 5, Figure 7, and Supplemental Figure S8), and

427

it was not clear how the low molecular mass PsbTn protein could influence oxygen evolution and the

428

ETR without affecting the normal electron pathway through PSII. Our biochemical results and the

429

recently published structure of the spinach PSII complex (Wei et al., 2016) clearly indicate that the

430

PsbTn protein is located in very close proximity to TyrD and Cytb559 in the PSII complex

431

(Supplemental Figure S10). This may indicate that its function is similar to that of the PsbY protein,

432

which was suggested to be a Cyt b559 redox regulator (von Sydow et al., 2016). However, EPR

433

studies indicated that the redox state of Cyt b559 in the psbTn1 psbTn2 double mutant was identical to

434

that in the WT (Supplemental Table S2), ruling out a Cyt b559-related function of PsbTn. Another

435

possible function of PsbTn is based on its suggested location between CP47 and PsbE close to the

436

TyrD residue of the D2-protein (Wei et al., 2016), where it could influence the functionality and

437

redox state of TyrD.

438 439

Removal of the PsbTn Protein Increased Sensitivity to Photoinhibition

440

Photosynthetic organisms must cope with ever changing light and temperature conditions via flexible

441

regulatory mechanisms that adjust the light absorption and photosynthetic processes based on

442

metabolic and environmental cues. Plants have evolved diverse mechanisms to optimize

443

photosynthesis by regulating the absorption or distribution of light energy by the light-absorbing

444

LHC proteins. Two of these mechanisms are well documented: (1) quenching of excited states of the

445

light-harvesting antennae by NPQ (Ruban and Murchie, 2012; Niyogi and Truong, 2013); and (2) 20 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

446

state transitions in which phosphorylated LHCII is detached from PSII and PSI excitation is

447

increased to balance the energy distribution, and thus the electron transfer rate, through the two

448

photosynthetic complexes (Depège et al., 2003). Interestingly, both these regulatory mechanisms

449

were affected in the psbTn1 psbTn2 double mutant (Figure 9). The double mutant exhibited a

450

delayed onset of NPQ upon prolonged illumination, which was probably due to the changes in the

451

trans-thylakoid pH gradient (de Bianchi et al., 2011), changes in the relative abundance of protein

452

subunits hosting quenching sites (Bonente et al., 2008), or changes in the level or function of the pH

453

sensor PsbS (Li et al., 2004). The occurrence of state transitions also depends on the phosphorylation

454

of the LHCII proteins (Bellafiore et al., 2005) and their association with the PSI proteins, particularly

455

PSI-H (Lunde et al., 2000). Indeed, the psbTn1 psbTn2 double mutant also showed a general

456

decrease in the phosphorylation of the PSII core proteins (Figure 6 and Figure 8). The STN7 kinase

457

plays a pivotal role in LHCII phosphorylation and state transition (Bellafiore et al., 2005). Under low

458

light, the phosphorylation pattern of LHCII in the STN7 knock-out mutant resembles that seen in the

459

WT under high light conditions, causing PSII excitation to be favored over PSI excitation. This

460

greatly reduces intersystem photosynthetic electron transfer (Bellafiore et al., 2005; Tikkanen et al.,

461

2006; Tikkanen et al., 2010). The STN7 knock-out mutant has a stunted phenotype when grown

462

under fluctuating light conditions but develops similarly to the WT under continuous illumination

463

(Tikkanen et al., 2010). The psbTn1 psbTn2 double mutant behaved similarly to the STN7 knock-out

464

mutant even though the degree of LHCII phosphorylation in psbTn mutants was very similar to that

465

in the WT (Figure 6). However, phosphorylation of CP43, D2, and D1 was severely affected in the

466

psbTn1 psbTn2 double mutants. These findings suggest that the STN8 kinase is inactivated in these

467

mutants, which may indicate that the PsbTn protein is involved in regulating STN8, which has been

468

implicated in PSII repair.

469 470

The removal of PsbTn presumably induced structural changes in the PSII complex that reduced its

471

accessibility to kinases. The ability of the psbTn1 psbTn2 double mutant to regulate photosynthetic

472

imbalances via phosphorylation changes, NPQ, and/or state transition is impaired, leading to

473

increased photoinhibition, which in turn causes increased ROS accumulation under high light

474

conditions (Supplemental Figure S8). This hypothesis is supported by the photobleached phenotype

475

of the double mutant under high light/dark shift conditions (Figure 7). Although the composition of

476

the PSII supramolecular complex in the psbTn1 psbTn2 double mutant was unchanged (Figure 3), its

477

overall abundance was lower than in the WT (Figure 8C). Previous studies have indicated that high

478

light conditions facilitate the disassembly of PSII complexes (Tikkanen et al., 2008; Chen et al.,

479

2017). Therefore, the reduced abundance of PSII complexes in the psbTn1 psbTn2 double mutant 21 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

480

may be due to more severe oxidative damage. Given that our findings indicate that PsbTn is a low

481

molecular mass protein tightly bound to the lumenal side of PSII next to the essential PsbE protein

482

(Swiatek et al., 2003), the effects of its removal on photoinhibition, PSII phosphorylation, state

483

transition, and ROS production are not straightforward to explain. One interesting possibility is that

484

PsbTn may interact with TyrD and could function as a redox sensor/regulator for the STN8 kinase,

485

which is involved in the phosphorylation of the PSII core proteins and thereby influences

486

photoinhibition.

487 488

Conclusions

489

PsbTn is a lumenal PSII protein, situated next to the cytochrome b559 subunit PsbE. Its removal

490

decreased the oxygen evolution rate and PSII core phosphorylation level but increased the

491

susceptibility of PSII to photoinhibition and the formation of ROS. We propose that the main

492

function of the PsbTn proteins is to enable PSII to acclimate to light/dark shift conditions and high

493

light intensity conditions.

494 495 496


497

METHODS

498

Plant Materials and Growth Conditions

499

The Arabidopsis thaliana (Accession Columbia) T-DNA insertion lines psbTn1 (GABI_143D01)

500

and psbTn2 (SAIL_1214_E09) were obtained from the GABI-KAT and ABRC collections,

501

respectively. The T-DNA insertion sites were confirmed by sequencing (Supplemental Figure S3A

502

and Supplemental Table S3). Double mutant plants were obtained by crossing single mutant plants to

503

generate the F1 progeny. To obtain homozygous mutants, F1 plants were selfed and homozygous

504

plants in the F2 progeny were screened by genotyping via PCR (Supplemental Figure S3B) and

505

confirmed by protein blotting using a monospecific PsbTn antibody (Supplemental Figure S4A).

506

Plants were grown in a growth chamber with an 8 h light/16 h dark cycle with a light intensity of 120

507

μmol of photons m-2 s-1 (Fluorescent Philips master 930) and day/night temperatures of 22/15°C.

508

After four weeks, the plants were harvested for the isolation of thylakoid membrane proteins. For the

509

light-to-dark shifting experiment, four-week-old plants were alternately exposed to 1,000 μmol of

510

photons m-2 s-1 for 30 min and then incubated in full darkness for 30 min over a period of 4 h.

511 512

Homology Searches, Co-Expression and Structure Analyses

513

Plant homologs of PsbTn were identified by a BLAST search using the web service provided by the

514

National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Amino acid

515

sequences of different land plants were aligned in Jalview using ClustalW. Structural homology

516

models of Arabidopsis PsbTn1 (At3g21055) and PsbTn2 (At1g51400) were generated using I-Tasser

517

(http//:zhanglab.ccmb.med.umich.edu/) (Roy et al., 2010). Co-expression correlation coefficients

518

were performed according to the method of Granlund et al. (2009).

519 520

Measurements of Photosynthetic Parameters and Chlorophyll Fluorescence Measurements In

521

Vivo

522

Chlorophyll a fluorescence induction kinetics and changes in PSI absorbance at 820 nm in the WT

523

and mutant plants were measured using a Dual-PAM-100 fluorometer (Heinz Walz) on whole leaves

524

at room temperature according to Ohad et al. (2004). ΦPSI and ΦPSI (ND) were expressed as described

525

previously (Klughammer and Schreiber, 1994). Photochemical and non-photochemical parameters

526

were calculated as described by Maxwell and Johnson (2000).

527 528

State transition experiments were performed using whole plants according to established protocols

529

(Pietrzykowska et al., 2014). Arabidopsis plants were subjected to a red/far-red light treatment using

530

a customized LED light source (SL 3500-R-D) from Photon System Instruments as previously 23 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

531

described by Leoni et al. (2013). Plants were dark-adapted for 1 h prior to measurements. NPQ was

532

measured using a WALZ Dual PAM-100 fluorometer according to Bianchi et al. (2011).

533 534

During light-to-dark shifts, chlorophyll fluorescence images were obtained using a modulated

535

imaging fluorometer (the Imaging PAM M-Series Chlorophyll Fluorescence system, Heinz-Walz

536

Instruments, Effeltrich, Germany) according to the manufacturer’s instructions. The image data

537

averaged in each experiment were normalized to a false color scale.

538 539

Electron Microscopy

540

Chloroplast morphology was investigated by transmission electron microscopy of the WT and

541

mutants using leaves before and after HL illumination at 1,000 μmol photons m-2 s-1 for 2 h as

542

described (Brouwer et al., 2012).

543 544

Isolation of Thylakoids, PSII Membranes and Pigment and Protein Analyses

545

Thylakoid membranes were isolated according to Chen et al. (2016) from fresh or frozen material in

546

liquid nitrogen. All of the extraction buffers contained 10 mM NaF to inhibit phosphatase activity.

547

PSII-enriched membranes (BBY) were prepared according to Arellano et al. (1994) with some

548

modifications. The pellet containing the thylakoid membranes was resuspended in 1.5 mL BBY-

549

Stacking (20 mM MES-NaOH pH 6.3, 5 mM MgCl2 and 15 mM NaCl) and while gently stirring at

550

4°C, 10% Triton X-100 (w/v) was added to the thylakoid membranes to a final Triton X-100 to

551

chlorophyll ratio of 10:1 (w/w). Exactly 18 min after addition of the detergent the solubilized

552

thylakoid membranes were collected and centrifuged at 1,000 g for 2 min, then the supernatant was

553

transferred to a new tube and centrifuged at 40,000 g for 30 min at 4°C. The resulting pellet was

554

resuspended in BBY-storage buffer (20 mM MES-NaOH pH 6.3, 400 mM sorbitol, 5 mM MgCl2, 10

555

mM CaCl2, and 15 mM NaCl). Chlorophyll concentrations were measured after extraction with 80%

556

acetone (v/v).

557 558

Immunoblot analysis was performed on thylakoid membranes or BBY according to Chen et al.

559

(2016). PsbTn antisera were generated using a mixture of two synthetic peptide sequences

560

representing the protein’s C-terminal (CVTMPTAKI) and N-terminal (EPKRGTEAAKKKYAQ)

561

regions (Agrisera). Other monospecific antibodies used in the experiments were obtained from

562

Agrisera (Umea, Sweden). Loading was determined by staining with Coomassie Brilliant Blue prior

563

to western blotting. The immunodecoration was visualized using Western Bright Quantum

564

(Advansta), and signals were detected using a LAS-3000 cooled CCD camera (Fujifilm). 24 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

565

Quantification of the immunoblots of thylakoid membrane proteins was done using Quantity One

566

software.

567 568

BN-PAGE was performed as described (Chen et al., 2016). Thylakoid membranes containing 20 µg

569

of chlorophyll were solubilized with 1% (w/v) n-dodecyl-β–D–maltoside (DM) and separated on a

570

gradient of 5 - 12.5% acrylamide in the separation gel. For 2D electrophoresis, the lanes of the first

571

dimension were cut out and incubated in Laemmli buffer containing 5% (v/v) β-mercaptoethanol

572

(Laemmli, 1970) for 1 h at room temperature. After this, the gel strips were subjected to SDS-PAGE

573

with 15% acrylamide (v/v) and 6 M urea. Gels were either stained with Coomassie Brilliant Blue R

574

or used for immunoblotting.

575 576

Localization and Topology of PsbTn

577

Thylakoid membranes and BBY from WT plants were treated with different salt-containing buffers

578

as described previously (Torabi et al., 2014). Thylakoid membranes were treated with the protease

579

chymotrypsin (200 µg/mL) for 10 min at room temperature with or without sonication before

580

immunoblot analysis. Subfractionation of grana-enriched and stroma lamellae-enriched thylakoids

581

was performed as described by Torabi et al. (2014).

582 583

Cross-Linking and Coimmunoprecipitation

584

EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) cross-linking of PsbTn was

585

performed according to the method of Hansson et al. (2007). Coimmunoprecipitation was performed

586

according to the manufacturer’s instructions (Molecular Probes Co-IP Kit 26149). The eluted

587

proteins were separated on a 15% polyacrylamide gel and then subjected to immunoblot analysis.

588 589

Analysis of PSII Photoinhibition and Recovery

590

The sensitivity of PSII to light stress, measured in terms of changes in Fv/Fm as a function of

591

exposure time, was determined using leaves of mutant and WT plants in the presence/absence of

592

lincomycin. For photoinactivation, the detached leaves were exposed to 1,000 µmol photons m-2 s-1

593

heterochromatic light for 4 h. The photoinactivation of PSII is expressed as a function of the

594

exposure time. Recovery from photoinhibition was determined under low light conditions (10 µmol

595

photons m-2 s-1) for 24 h. To assess the accumulation of D1 proteins under intense illumination,

596

proteins were extracted from leaves, separated by SDS-PAGE, and subjected to immunoblotting

597

using D1 antibodies.


599

Trypan-Blue Staining

600

Four-week-old Arabidopsis seedlings were alternately exposed to 1,000 μmol photons m-2 s-1 light

601

and complete darkness for 30 min each over a total period of 4 h. Photobleaching (cell death) was

602

then visually detected by trypan-blue staining (1.25 mg/mL) (Lam et al., 2004).

603 604

In Situ Detection of ROS

605

ROS measurements were performed in situ as previously described by Lu et al. (2011). H2O2 and

606

superoxide accumulation were detected by incubation with 3,3'-diaminobenzidine (DAB) (Sigma-

607

Aldrich) or nitroblue tetrazolium (NBT) (Sigma-Aldrich), respectively, for 2 h in darkness followed

608

by high light (1,000 μmol photons m-2 s-1) for 2 h. Stained leaves were boiled in acetic acid: glycerol:

609

ethanol (1:1:3 [v/v/v]) and photographed. Singlet oxygen accumulation was visualized by confocal

610

laser scanning microscopy on leaves incubated in 500 nM Singlet Oxygen Sensor Green (Molecular

611

Probes) for 2 h and then exposed to high light for 2 h as described previously (Lu et al., 2011).

612 613

Oxygen Evolution Measurements

614

The oxygen evolving activities of thylakoid membrane and BBY preparations from mutant and WT

615

plants were measured in assay media containing (at final concentrations) 25 mM HEPES pH 7.6

616

(KOH), 0.2 M sucrose, 10 mM NaCl, and 5 mM CaCl2 supplemented with the artificial electron

617

acceptor phenyl-p-benzoquinone (PPBQ) at a final concentration of 0.5 mM. Oxygen evolution rates

618

were measured at 20°C under saturating light using a Clark-type electrode (Hansatech UK) (García-

619

Cerdan et al., 2009).

620 621

Flash-Induced Fluorescence Decay Kinetics and Thermoluminescence Measurements

622

Flash-induced increase and subsequent relaxation of the chlorophyll fluorescence yield (variable

623

fluorescence decay kinetics) were measured as described in von Sydow et al., 2016 and García-

624

Cerdán et al., 2011 with a FL3300 dual-modulation fluorometer (Photon System Instruments, Brno,

625

Czech Republic) in the 150 μs - 100 s time range. The actinic flash duration was 30 μs. PSII

626

membranes at a concentration of 10 μg Chl mL−1 were dark-adapted for 5 min before fluorescence

627

detection. The measurements were performed in the absence or presence of 20 μM DCMU. The

628

kinetics were analysed in terms of several exponential components (fast, intermediate, and slow

629

phases) as described by von Sydow et al., (2016) and García-Cerdán et al., (2011).

630 631

Thermoluminescence signals were measured with a TL200/PMT thermoluminescence system

632

(Photon System Instruments, Brno, Czech Republic) (von Sydow et al., 2016, García-Cerdán et al., 26 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.

633

2011). PSII membranes at a concentration of 150 μg Chl mL−1 were dark-adapted for 5 min at 20°C

634

then cooled to -10°C and excited by an actinic flash of 50 μs duration. The sample was then heated to

635

60°C at a heating rate of 1°C/sec. The measurements were performed in the absence or presence of

636

20 μM DCMU.

637 638

EPR Spectroscopy

639

EPR experiments were performed using a Bruker ELEXYS E500 spectrometer with a SuperX

640

EPR049 microwave bridge and a SHQ4122 cavity, equipped with an ESR 900 liquid helium cryostat

641

and ITC 503 temperature controller from Oxford Instruments, UK. EPR samples at a concentration

642

of ∼3 mg Chl mL−1 were illuminated by ambient laboratory light for 1 min and then dark adapted for

643

5 min before freezing in liquid N2 to ensure full oxidation of YD•. The S2 state multiline signal was

644

induced by illumination at 200 K for 6 min and complete oxidation of cytochrome b559 was induced

645

by illumination at 77 K for 6 min. The reduction of the QA- Fe2+ semiquinone iron complex was

646

induced by subsequent incubation with 15 mM formate and 50 mM dithionite for 15 min each at

647

room temperature (Chen et al., 2011).

648 649

Field Experiments

650

The field experiment was performed at the experimental garden in Umeå University as described

651

previously (Mishra et al., 2012). The WT and double mutant plants were first grown under short day

652

conditions in a climate chamber and then transferred to the outdoor field once they had developed

653

three to four leaves. The plants were shaded on the first day to allow for some acclimation. After four

654

weeks outside, the state transition kinetics and ΦPSII were measured in the WT and double mutant

655

plants.

656 657

Accession Numbers

658

Sequence data from this article can be found in the GenBank/EMBL libraries under the following

659

accession numbers: PsbTn1 (At3g21055), PsbTn2 (At1g51400), and ACTIN1 (At2g37620).

660 661

Supplemental Data

662

Supplemental Figure S1. Hierarchical clustering of co-expression coefficients.

663

Supplemental Figure S2. Sequence and structure of PsbTn.

664

Supplemental Figure S3. Identification of the psbTn mutants.

665

Supplemental Figure S4. Identification of psbTn Mutants and Phenotypes.


666

Supplemental Figure S5. Transmission electron micrographs of chloroplasts from the leaves of the

667

wild type and the mutants.

668

Supplemental Figure S6. Variable fluorescence relaxation kinetics in the PSII membrane

669

preparations from Arabidopsis thaliana

670

Supplemental Figure S7. EPR signals from the donor and acceptor sides of PSII from PSII

671

membrane preparation

672

Supplemental Figure S8. Analysis of oxidative stress in the WT and mutants.

673

Supplemental Figure S9. Assays of state transitions in the WT and mutants.

674

Supplemental Figure 10. A detailed structure of the region of PsbTn based on spinach PSII

675

structure.

676

Supplemental Table S1. Photosynthetic parameters of Arabidopsis thaliana WT, psbTn1, psbTn2

677

and double mutant plants.

678

Supplemental Table S2. Comparison of EPR signals in PSII membranes from A. thaliana WT,

679

psbTn1, psbTn2, and psbTn1 psbTn2 mutants.

680 681 682

Figure text

683

Figure 1. Localization and Topology of PsbTn in Arabidopsis Thylakoid Membranes.

684

(A) Thylakoid membranes (T) were fractionated into grana (G), intermediate membranes (I), and

685

stroma lamellae (S). The fractions including BBY (B) were tested for the presence of PsbTn.

686

Successful fractionation of grana and stroma lamellae was also confirmed by immunodetection of

687

PsbA, PsbO, and PsaD.

688

(B) Protease protection assays reveal that PsbTn is a membrane protein. Untreated and sonicated

689

thylakoid membranes were incubated with (+) and without (-) chymotrypsin and subjected to

690

immunoblotting using anti-PsbTn, -PsbO, and -PsbQ antibodies.

691 692

Figure 2. Assays of PsbTn Protein Interactions Using PsbTn-Specific Antibodies.

693

(A) PsbTn protein crosslinking. BBY fractions were treated with the crosslinker EDC at 6% and 12%

694

(w/v) and the proteins were separated with (+) or without (-) β-mercaptoethanol treatment (as

695

indicated) on SDS-PAGE, immunoblotted, and incubated with PsbTn sera.

696

(B) Untreated (-) and cross-linked (+) samples were separated on 15% SDS-PAGE and subsequently

697

immunoblotted against PsbTn, PsbE, and PsbF.


698

(C) Coimmunoprecipitation assays showing interactions between PsbTn and PsbE. Solubilized BBY

699

were incubated with preimmune serum (control) and an excess of PsbTn antiserum. The

700

immunoprecipitates were probed with specific antibodies, as indicated on the left. A sample of BBY

701

equivalent to 2 µg of chlorophyll was loaded in the first lane (input).

702

(D) PsbTn also can be reciprocally coimmunoprecipitated with anti-PsbE sera. Gels were loaded

703

with a quantity of BBY equivalent to 2 µg of chlorophyll.

704

(E) BBY fractions were treated with the indicated chaotropic agents and fractionated into pellets (P)

705

and supernatants (S). They were then separated by SDS-PAGE and analyzed immunologically using

706

anti-PsbTn antibodies and anti-PsbO, -PsbP, and -PsbQ antibodies as control extrinsic lumenal PSII

707

proteins. CP43 was used as a control integral membrane protein.

708 709

Figure 3. Accumulation of Thylakoid Membrane Proteins from the WT and Mutants.

710

(A) Immunoblot analyses of thylakoid membrane proteins were performed using antibodies against

711

PsbTn and representative subunits of the PSII, PSI, cytochrome b6f, and ATP synthase complexes.

712

Protein-containing samples were loaded in quantities corresponding to 0.5, 1, and 2 µg

713

chlorophyll/µL (1/4, 1/2, and 1, respectively) of WT, and 2 µg chlorophyll/µL of mutants psbTn1,

714

psbTn2, and psbTn1 psbTn2.

715

(B) The SDS-PAGE gels were stained with Coomassie blue to check loading.

716 717

Figure 4. Composition of the Thylakoid Membrane Protein Complexes in the WT and Mutants.

718

(A) Thylakoid membrane complexes (20 µg chlorophyll) from the WT and mutants (psbTn1, psbTn2,

719

and psbTn1 psbTn2) were solubilized with 1% (w/v) DM and subjected to BN-PAGE. The

720

assignments of thylakoid membrane complexes shown on the left were based on the work of Chen et

721

al. (2016). NDH, NAD(P)H dehydrogenase; PS, photosystem; LHC, light-harvesting complex; Cyt

722

b6/f, cytochrome b6/f; mc, megacomplex; sc, supercomplex.

723

(B) 2D BN/SDS-PAGE fractionation of thylakoid membrane protein complexes. After separation

724

along the first dimension in a nondenaturing gel, the proteins were separated by SDS-PAGE along

725

the second dimension and stained with Coomassie blue.

726

(C) Immunoblot analysis of thylakoid membrane proteins separated along the second dimension by

727

BN/SDS-PAGE.

728 729

Figure 5. PSII Photosensitivity of Mutants during high light Illumination.


730

(A) Untreated (-) and lincomycin-treated (+) WT and mutant leaves were subjected to high light

731

conditions (1,000 µmol photons m-2 s-1) for 4 h and (B) then the photoinhibited samples were

732

allowed to recover at low light intensity (10 µmol photons m-2 s-1) for up to 24 h, with regular

733

measurement of their Fv/Fm values. Values are means ± SE of three replicates.

734

(C) Immunoblot analysis of the WT and psbTn1, psbTn2, and psbTn1 psbTn2 mutants with D1 and

735

PsaD antibodies before (-) and after (+) photoinhibition using a light intensity of 1,000 µmol photons

736

m-2 s-1 for 3 h. The PsaD protein was used as a loading control.

737 738

Figure 6. Light-Dependent Phosphorylation of Thylakoid Membrane Proteins.

739

Immunodetection of isolated thylakoid membrane proteins on a 12% SDS gel was performed using

740

antiphosphothreonine antibodies from Cell Signaling. Four-week-old plants were dark-adapted for

741

16 h and then kept in darkness (D), exposed to normal light (120 μmol photons m-2 s-1) for 30 min

742

(NL), or exposed to high light (1000 μmol photons m-2 s-1) for 30 min (HL). Samples were loaded at

743

a level corresponding to 1 μg of chlorophyll. The positions of the major phosphorylated proteins and

744

the molecular markers are indicated in the figure. Loading was checked by Coomassie Brilliant Blue

745

(CBB) staining of WT and mutant proteins.

746 747

Figure 7. Analysis of Photobleaching Lesions in the WT and Mutants under high light/dark

748

shift conditions.

749

(A) Phenotypes of single leaves from four-week-old plants subjected to high light/dark shift

750

conditions (alternating between 1,000 μmol photons m-2 s-1 and complete darkness at 30 min

751

intervals for 4 h in total).

752

(B) False-color images showing Fv/Fm and NPQ after the light/dark shift treatment of four-week-old

753

WT and mutant plants. Quantitative values (± SD) are shown below the individual ﬂuorescence

754

images.

755

(C) Trypan-blue-staining of four-week-old seedlings grown under light/dark shift conditions.

756

Significant microscopic mesophyll cell death (shown as blue stained cells) was observed in double

757

mutants. Bars = 50 µm.

758

(D) Confocal microscopy images of 1O2 levels in four-week-old seedlings grown under normal light

759

and then exposed to light/dark shift conditions for 4 h. After the fluctuating light treatment, detached

760

leaves were incubated for 2 h in Singlet Oxygen Sensor Green reagent. 1O2 was visualized by

761

excitation at 488 nm and emission at 535 to 590 nm. Chlorophyll auto-fluorescence (red) was

762

visualized by excitation at 488 nm and detected at 650 to 710 nm. Bars = 100 µm.


763 764

Figure 8. Analysis of Thylakoid Membrane Protein Phosphorylation and Complexes in the WT

765

and Mutants under light/dark shift conditions.

766

(A) Immunodetection of isolated thylakoid membrane proteins on a 12% SDS gel was performed

767

using antiphosphothreonine antibodies. Four-week-old plants were exposed to light/dark shift

768

(alternating between 1,000 μmol photons m-2 s-1 and complete darkness at 30 min intervals) for 4 h in

769

total. Samples were loaded at levels corresponding to 1 μg chlorophyll. The positions of the major

770

phosphorylated proteins and the molecular markers are indicated in the figure.

771

(B) Loading was checked by Coomassie Brilliant Blue (CBB) staining of WT and mutant proteins.

772

(C) Thylakoid membrane complexes (20 µg chlorophyll) from the WT and mutants (psbTn1, psbTn2,

773

and psbTn1 psbTn2) grown under light/dark shift conditions were solubilized with 1% (w/v) DM and

774

subjected to BN-PAGE. The assignments of thylakoid membrane complexes indicated at the left

775

were identified according to Chen et al. (2016). NDH, NAD(P)H dehydrogenase; PS, photosystem;

776

LHC, light-harvesting complex; Cyt b6/f, cytochrome b6/f; mc, megacomplex; sc, supercomplex.

777 778

Figure 9. Assays of Nonphotochemical Quenching and Photosynthetic Functions of the WT and

779

Mutants.

780

(A) Measurements of NPQ kinetics in WT, psbTn1, psbTn2, and psbTn1 psbTn2 leaves grown in the

781

greenhouse. Bars on top; white bar (light on) and black bar (dark).

782

(B) NPQ kinetics of WT and psbTn1 psbTn2 plants grown in the greenhouse during two consecutive

783

periods of illumination with 1,000 µmol photons m-2 s-1 for 25 min with a 15 min period of darkness

784

in between, as indicated by the white (light on) and black (dark) bars on top of figure.

785

(C) Pulse amplitude-modulated fluorescence traces after shifts from state 1 to state 2 light and back

786

for WT and psbTn1 psbTn2 plants growing in the field. The bars at the bottom indicate illumination

787

with red (shown in red) and far-red (dark red) light. Fluorescence is shown in arbitrary units.

788

(D) PSII quantum yield (ΦPSII) of the WT and psbTn1 psbTn2 plants growing in the field under

789

fluctuating light conditions. For A, B, and D, white bar on top (light on) and black bar (dark), data

790

represent means ± SE from three independent measurements. In C a typical representative trace is

791

shown.

792 793

ACKNOWLEDGMENTS

794

We thank Malgorzata Pietrzykowska, Kim Sungyong, Kati Mielke and Lotta von Sydow for

795

discussions and help during this project. The authors acknowledge the facilities and technical


796

assistance of the Umeå Core Facility Electron Microscopy (UCEM) at the Chemical Biological

797

Centre (KBC), Umeå University, part of the National Microscopy Infrastructure, NMI (VR-RFI

798

2016-00968). This work was supported by Sven and Lilly Lawski Foundation, Carl Tryggers

799

Foundation, and by the German Science Foundation (Deutsche Forschungsgemeinschaft; ME1794/7

800

and TRR 175 TP A03 to J.M. and TRR175 TP B06 to S.S.).

801 802


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