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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10 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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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.
304 13 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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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).
327
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.
328
the WT (Figure 7A), indicating that the double mutant suffered serious damage under high light/dark
329
shift conditions. Moreover, the psbTn1 psbTn2 plants exhibited significantly greater reductions in
330
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
15 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
377 17 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
378
18 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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
19 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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
22 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
598 25 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
27 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
28 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
29 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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.
30 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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
31 Downloaded from on December 11, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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
32 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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