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. 2018 Dec 11;179(4):1739–1753. doi: 10.1104/pp.18.01251

The Low Molecular Mass Photosystem II Protein PsbTn Is Important for Light Acclimation1,[OPEN]

Yang-Er Chen a,b,2, Shu Yuan d,2, Lina Lezhneva a, Jörg Meurer c, Serena Schwenkert c, Fikret Mamedov e, Wolfgang P Schröder a,3,4
PMCID: PMC6446760  PMID: 30538167

PsbTn is a low-molecular-mass protein and a component of PSII that is important for water oxidation and light acclimation.

Abstract

Photosystem II (PSII) is a supramolecular complex containing over 30 protein subunits and a large set of cofactors, including various pigments and quinones as well as Mn, Ca, Cl, and Fe ions. Eukaryotic PSII complexes contain many subunits not found in their bacterial counterparts, including the proteins PsbP (PSII), PsbQ, PsbS, and PsbW, as well as the highly homologous, low-molecular-mass subunits PsbTn1 and PsbTn2 whose function is currently unknown. To determine the function of PsbTn1 and PsbTn2, we generated single and double psbTn1 and psbTn2 knockout mutants in Arabidopsis (Arabidopsis thaliana). Cross linking and reciprocal coimmunoprecipitation experiments revealed that PsbTn is a lumenal PSII protein situated next to the cytochrome b559 subunit PsbE. The removal of the PsbTn proteins decreased the oxygen evolution rate and PSII core phosphorylation level but increased the susceptibility of PSII to photoinhibition and the production of reactive oxygen species. The assembly and stability of PSII were unaffected, indicating that the deficiencies of the psbTn1 psbTn2 double mutants are due to structural changes. Double mutants exhibited a higher rate of nonphotochemical quenching of excited states than the wild type and single mutants, as well as slower state transition kinetics and a lower quantum yield of PSII when grown in the field. Based on these results, we propose that the main function of the PsbTn proteins is to enable PSII to acclimate to light shifts or intense illumination.

The function, composition, and molecular dimensions of photosystem II (Psb; PSII) are highly conserved in cyanobacteria, algae, and vascular plants. The cyanobacterial PSII structure has been solved at 1.9 Å (Umena et al., 2011; Suga et al., 2015) and used as a blueprint for other PSII complexes as well as a starting point for studies on the function of PSII and differences between the cyanobacterial and eukaryotic complexes. The supramolecular organization of the eukaryotic PSII (Psb) appears to be more complex than that of the cyanobacterial complex due to the presence of several additional subunits, including several light-harvesting complexes (LHCs) and the proteins PsbP, PsbQ, PsbS, PsbW, and PsbTn. In addition, various biochemical analyses have revealed differences in protein function between eubacterial and eukaryotic PSII complexes. The structures of two eukaryotic PSII complexes were recently solved—that of the red alga Cyanidium caldarium (at 2.76 Å resolution) using traditional x-ray crystallization techniques (Ago et al., 2016) and that of spinach (Spinacia oleracea; at 3.2 Å resolution) using single-particle cryo-electron microscopy (Wei et al., 2016). Comparisons of these structures to that of the cyanobacterial PSII revealed some previously unrecognized differences. Notably, the lumenal side of the eukaryotic PSII core complex exposes the four extrinsic subunits PsbO, PsbP, PsbQ, and PsbTn. The first three of these extrinsic subunits form a triangular “hat” that covers the lumenal side of the CP43 and D1 proteins in the PSII core, while PsbTn was suggested to intercalate between CP47 and the C-terminal region of PsbE (a subunit of cytochrome b559; Wei et al., 2016).

Intriguingly, over half of the PSII subunits are low-molecular-mass (<10 kD) proteins. These proteins have diverse functions, affecting electron transport, the redox potentials of the primary and secondary quinone acceptors (QA and/or QB), the assembly and stability of the PSII complex, photosensitivity, recovery from photoinhibition, and phosphorylation patterns (Swiatek et al., 2003; Ohad et al., 2004; Shi and Schröder, 2004; Schwenkert et al., 2006; Umate et al., 2007, 2008; von Sydow et al., 2016). Phylogenetically, PsbTn is among the youngest proteins in the PSII complex because it has not been found in cyanobacteria nor in red or green algae and thus seems to have evolved first in land plants during endosymbiosis. PsbTn is a nuclear-encoded subunit (and accordingly has the suffix “n”) and should not be confused with PsbTc, which is an unrelated chloroplast-encoded protein (and thus bears the suffix “c”; Umate et al., 2008). The PsbTn precursor protein incorporates a bipartite transit peptide that apparently targets the protein to the thylakoid lumen. The mature Arabidopsis (Arabidopsis thaliana) protein comprises 31 amino acids and is the smallest known PSII subunit, having a molecular mass of only 3.0 kD (Shi and Schröder, 2004). The protein was initially isolated from spinach thylakoids and partly sequenced (Ljungberg et al., 1986; Ikeuchi and Inoue, 1988); the cDNA sequence from upland cotton (Gossypium hirsutum) encoding PsbTn was subsequently determined (Kapazoglou et al., 1995). However, structural biochemical and functional analyses of the protein are entirely lacking.

In Arabidopsis, two PsbTn proteins that differ in only two amino acid residues (position 18 Q→P and position 31 Y→N) are encoded by the genes PsbTn1 (At3g21055) and PsbTn2 (At1g51400). In this study, we used single mutants to generate a double psbTn1 psbTn2 knockout mutant that was studied using biochemical and biophysical approaches. We show that psbTn1 psbTn2 double mutants suffer from severe photodamage under both intense illumination and fluctuating illumination. We also demonstrate that the primary function of PsbTn is to maintain PSII activity and protect PSII against photodamage under intense and/or fluctuating illumination.

RESULTS

Expression, Structure, and Localization of the Two PsbTn Proteins

Both PsbTn genes were mainly expressed in young and mature leaves; only weak expression was observed in senescing cauline leaves, stems, and other tissues (https://apps.araport.org). The PsbTn genes were coexpressed with genes encoding several PSII components (such as PsbO, P, W, X, Y; Supplemental Fig. S1), plastocyanin, and some PSI components (Photosystem a [Psa] D1), E2, G, H1, H2, L; http://evolver.psc.riken.jp; http://string-db.org; http://apps.araport.org).

The PsbTn protein was originally purified and partially sequenced from PSII core complexes in spinach and wheat (Triticum sp.) and was referred to as the 5.0-kD protein (Ljungberg et al., 1986; Ikeuchi and Inoue, 1988). The mature protein has a mass of 3.2 kD in Arabidopsis (Shi and Schröder, 2004). PsbTn proteins in Arabidopsis and Gossypium hirsutum exhibit about 68% sequence identity and feature cysteines at positions C20 and C29 that are highly conserved in plants (Supplemental Fig. S2). A protein fold prediction made using the I-Tasser algorithm indicated that the mature protein lacks transmembrane α-helices, suggesting that PsbTn is either a soluble protein or attached to the thylakoid membrane via electrostatic interactions. However, its association with PSII was not unambiguously established. To elucidate the localization and the topology of the PsbTn protein, we raised polyclonal antibodies against it using a mixture of two synthetic peptides representing the protein’s N- and C-terminal parts. This mixture was used because the single peptide was found to be insufficiently antigenic to yield useful sera. The resulting polyclonal antibody was used to detect the PsbTn protein in both thylakoids (column “T” in Fig. 1A) and isolated PSII membrane fragments (BBY) particles (column “B” in Fig. 1A). The PSII association of PsbTn was further established by analyzing isolated grana, stroma lamellae, and intermediate membrane fractions from thylakoids and comparing them to PSII membrane fragments (columns “G”, “I”, and “S” in Fig. 1A). As expected, the PSII proteins PsbA and PsbO were detected in thylakoids, BBY particles, the grana fraction, and to a lesser extent in the intermediate fraction, but were absent in the stroma lamellae. Also as expected, the PSI protein PsaD was mainly located in the stroma lamellae, indicating that the fractionation process was successful. The distribution of PsbTn in this experiment resembled those of the PSII proteins PsbA and PsbO, suggesting that PsbTn is a component of PSII.

Figure 1.

Figure 1.

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Localization and topology of PsbTn in Arabidopsis thylakoid membranes. A, Thylakoid membranes (T) were fractionated into grana (G), intermediate membranes (I), and stroma lamellae (S). The fractions including BBY (B) were tested for the presence of PsbTn. Successful fractionation of grana and stroma lamellae was also confirmed by immunodetection of PsbA, PsbO, and PsaD. B, Protease protection assays reveal that PsbTn is a membrane protein. Untreated and sonicated thylakoid membranes were incubated with (+) and without (−) chymotrypsin and subjected to immunoblotting using anti-PsbTn, -PsbO, and -PsbQ antibodies.

To determine whether the PsbTn protein is exposed to the stroma or lumen, we digested sonicated (broken) and untreated (intact) thylakoid membranes with chymotrypsin and then performed immundecoration experiments (Fig. 1B). The lumen-localized PsbO and PsbQ proteins were found to be only partly protected against chymotrypsin in the intact (untreated) thylakoids (Fig. 1B, left), showing that the thylakoids used in these experiments were not fully intact. However, these proteins were found to be fully degraded in the sonicated (broken) thylakoids (Fig. 1B, right). The PsbTn protein behaved in the same way as the extrinsic PsbO and PsbQ proteins, confirming the conclusion that it is a lumenal PSII protein.

To determine the PsbTn protein’s location within the PSII complex, we performed chemical cross linking experiments using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which cross links amino and carboxyl groups. After incubation with EDC, two bands were detected using the PsbTn-specific antibody (Fig. 2A). The lower band at 5 kD corresponds to non-cross-linked PsbTn, while the 12-kD band presumably corresponds to a cross linked complex consisting of PsbTn and a cross linking partner with a molecular mass of 5 to 8 kD. We therefore subjected this band to immunoblotting analysis using antibodies against a set of known PSII proteins with molecular masses in this range, including PsbE, F, H, I, R, W, X, and Y (results for E and F are shown in Fig. 2B). Only the antibodies against PsbE bound to the 12-kD band. The interaction between the PsbTn protein and the PsbE subunit of cytochrome b559 was further confirmed by coimmunoprecipitation analysis. Solubilized PSII membrane fragments were incubated with the PsbTn serum, immunoprecipitated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. As shown in Figure 2C, PsbE was copurified with the PsbTn protein. PsbTn was also reciprocally coimmunoprecipitated with the PsbE antisera, as shown in Figure 2D. This clearly established that the PsbTn protein is located on the lumenal side of PSII close to the PsbE protein (large subunit) of the Cyt b559 subcomplex. This conclusion is consistent with the recently published structure of the spinach PSII complex (Wei et al., 2016).

Figure 2.

Figure 2.

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Assays of PsbTn protein interactions using PsbTn-specific antibodies. A, PsbTn protein cross linking. BBY fractions were treated with the cross linker EDC at 6% and 12% (w/v), and the proteins were separated with (+) or without (−) β-mercaptoethanol treatment (as indicated) on SDS-PAGE, immunoblotted, and incubated with PsbTn sera. B, Untreated (−) and cross linked (+) samples were separated on 15% SDS-PAGE and subsequently immunoblotted against PsbTn, PsbE, and PsbF. C, Coimmunoprecipitation assays showing interactions between PsbTn and PsbE. Solubilized BBY were incubated with preimmune serum (control) and an excess of PsbTn antiserum. The immunoprecipitates were probed with specific antibodies, as indicated on the left. A sample of BBY equivalent to 2 µg of chlorophyll was loaded in the first lane (input). D, PsbTn also can be reciprocally coimmunoprecipitated with anti-PsbE sera. Gels were loaded with a quantity of BBY equivalent to 2 µg of chlorophyll. E, BBY fractions were treated with the indicated chaotropic agents and fractionated into pellets (P) and supernatants (S). They were then separated by SDS-PAGE and analyzed immunologically using anti-PsbTn antibodies and anti-PsbO, -PsbP, and -PsbQ antibodies as control extrinsic lumenal PSII proteins. CP43 was used as a control integral membrane protein.

The next step was to investigate how strongly the PsbTn protein is bound to the lumenal side of PSII. To this end, PSII membrane fragments were washed with various chaotropic and alkaline salt solutions including 0.1 m NaOH, 1 m NaCl, 0.1 MNa2CO3, 3 m NaSCN, 1 m CaCl2, and 0.8 m Tris, pH 8.4 (Fig. 2E). As the stringency of the washing solutions was increased, the extrinsic PSII proteins PsbO, PsbP, and PsbQ were released into the soluble fraction, whereas the membrane-anchored CP43 protein remained in the pellet (membrane) fraction. None of these treatments released PsbTn from the thylakoid membrane, suggesting that the protein is strongly bound to the PSII complex despite lacking a predicted transmembrane α-helix.

Analyzing PsbTn Knockout Mutants in Arabidopsis

Because the Arabidopsis genome contains two genes encoding PsbTn proteins, it was necessary to interrupt both of them to prevent PsbTn expression. This was done by crossing the transfer DNA (T-DNA) insertion lines psbTn1 (GABI_143D01) and psbTn2 (SAIL_1214_E09). The T-DNA insertion sites were confirmed by PCR and sequencing (Supplemental Fig. S3). The absence of the PsbTn protein in the double mutant was confirmed by immunoblotting using the PsbTn antisera, which recognized both isoforms. No PsbTn protein was detected in the double mutant, but levels of PsbTn in the single mutants (psbTn1 and psbTn2) varied (Supplemental Fig. S4A). The phenotypes of single-mutant plants did not differ greatly from the wild type, but double psbTn1 psbTn2 mutants were generally slightly smaller (Supplemental Fig. S4B). Additionally, the chlorophyll content of the psbTn1 psbTn2 double mutants was around 20% lower than that in the wild type on a fresh weight basis, and their chlorophyll a/b ratio was around 5% lower (Supplemental Table S1).

PSII Composition and Assembly in the PsbTn Mutants

To determine how the absence of the PsbTn protein affected the composition of PSII, immunoblotting experiments were performed using sera raised against various proteins of PSII and other thylakoid membrane complexes. As shown in Figure 3, levels of the major PSII proteins in the single and double mutants were identical to those in the wild type. This was true for the reaction center proteins D1, D2, CP43, PsbE, and PsbY, the extrinsic PsbO, P, and Q proteins, and the antenna proteins Lhcb1, Lhcb2, CP29, and CP26. The abundance of the PsbS protein, which is suggested to be important in the regulation of nonphotochemical quenching by incident light, was also unchanged in the double PsbTn mutant (Fig. 3A, right). In addition, we examined two PSI proteins (PsaD and Lhca4), the AtpB protein belonging to the ATP synthase complex and the PetB protein belonging to the Cytb6/f complex, none of which exhibited any detectable change in abundance upon removal of either PsbTn protein. Consequently, the PSII/PSI ratios of all three mutants were very similar to that of the wild type.

Figure 3.

Figure 3.

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Accumulation of thylakoid membrane proteins from the wild type and mutants. A, Immunoblot analyses of thylakoid membrane proteins were performed using antibodies against PsbTn and representative subunits of the PSII, PSI, cytochrome b6f, and ATP synthase complexes. Protein-containing samples were loaded in quantities corresponding to 0.5, 1, and 2 µg chlorophyll/µL (1/4, 1/2, and 1, respectively) of wild type (WT) and 2 µg chlorophyll/µL of mutants psbTn1, psbTn2, and psbTn1 psbTn2. B, The SDS-PAGE gels were stained with Coomassie Blue to check loading.

Blue-native SDS-PAGE experiments were performed to determine whether the PsbTn protein might influence the assembly of the PSII complex (Fig. 4). The assigned complexes were identified previously by mass spectrometry (Aro et al., 2005; Granvogl et al., 2006; Schwenkert et al., 2006; Chen et al., 2016). The native gels revealed no detectable differences in PSII supracomplex formation between the wild type and the three PsbTn mutants (Fig. 4A). Further analyses of the second dimension showed that the loading of proteins and the structure of the complex were similar in the wild type and mutants (Fig. 4B). Immunoblot analyses of the 2D gel using antisera against the PSII protein D1 and the PsaD protein from PSI confirmed the assignment of the complexes (Fig. 4C). As expected, PsbTn was detected in all PSII complexes but absent in the double-mutant sample, which nevertheless formed the same protein complexes as the wild type.

Figure 4.

Figure 4.

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Composition of the thylakoid membrane protein complexes in the wild type and mutants. A, Thylakoid membrane complexes (20 µg chlorophyll) from the wild type (WT) and mutants (psbTn1, psbTn2, and psbTn1 psbTn2) were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside (DM) and subjected to BN-PAGE. The assignments of thylakoid membrane complexes shown on the left were based on the work of Chen et al. (2016). NDH, NAD(P)H dehydrogenase; PS, photosystem; LHC, light-harvesting complex; Cyt b6/f, cytochrome b6/f; mc, megacomplex; sc, supercomplex. B, 2D BN/SDS-PAGE fractionation of thylakoid membrane protein complexes. After separation along the first dimension in a nondenaturing gel, the proteins were separated by SDS-PAGE along the second dimension and stained with Coomassie Blue. C, Immunoblot analysis of thylakoid membrane proteins separated along the second dimension by BN/SDS-PAGE.

In addition, the ultrastructures of thylakoid membranes from leaves from wild-type and mutant plants exposed to normal growth light or high-light conditions were investigated by electron microscopy (Supplemental Fig. S5). As shown in Supplemental Figure S5, the overall thylakoid structure did not differ significantly between the wild type and mutants under normal light. However, the double mutant had slightly fewer and smaller grana stacks (Supplemental Fig. S5D). All of the high-light samples exhibited starch granule accumulation, and the psbTn1 psbTn2 thylakoids from plants grown under high-light conditions again appeared to have fewer and smaller grana stacks than the corresponding wild-type thylakoids (Supplemental Fig. S5H).

Electron Transfer Within PSII in PsbTn Mutants

To determine whether the absence of the PsbTn proteins affected the functionality of PSII, various photosynthetic parameters were analyzed (Supplemental Table S1). The oxygen evolution rates of thylakoids and BBY particles from the double mutant were 66% and 71% of those in the wild type, respectively; the single mutants exhibited less pronounced reductions. The reduced oxygen evolution rates of the double mutant clearly indicate that removing the two PsbTn proteins from PSII causes defects in PSII function.

Further analysis of overall PSII functionality by pulse-amplitude modulation (PAM) measurements revealed that the maximum and effective quantum yield of PSII (Fv/Fm and ΦPSII) were reduced from 0.79 and 0.38, respectively, in the wild type to 0.74 and 0.30, respectively, in the psbTn1 psbTn2 double mutant (Supplemental Table S1). The psbTn1 psbTn2 double mutant also exhibited a reduced electron transport rate (ETR; Supplemental Table S1), which is again consistent with impaired PSII functionality. The double mutant also exhibited increased nonphotochemical quenching (NPQ) of excited states under both moderate and high-light conditions (Supplemental Table S1), presumably because of the reduced efficiency of electron transfer through PSII. To determine the cause of the ETR reduction, the quantum yield of PSI, ΦPSI, and quantum yield of nonphotochemical dissipation due to the donor-side limitation of PSI (ΦPSI [ND]), were measured (Supplemental Table S1). Compared to the wild type, the double mutant had a slightly lower value of ΦPSI but an appreciably greater value of ΦPSI (ND). This suggests that the electron flow to PSI is rate limiting in the double mutant because the absence of PsbTn makes PSII unable to deliver electrons efficiently.

The effect of PsbTn deletion was further characterized by measuring flash-induced variable fluorescence, which reflects the redox state of the first quinone acceptor, QA, in PSII (Supplemental Fig. S6). The kinetics of the variable fluorescence decay thus provide information on electron transfer from QA− (Mamedov et al., 2000; von Sydow et al., 2016). However, no significant differences between the wild type and the mutants could be detected (Supplemental Fig. S6A). When the variable fluorescence decay is measured in the presence of dimethyl sulfoxide (3-(3,4-dichlorophenyl)-1,1-dimethylurea [DCMU]), an inhibitor of electron transfer between QA and QB, the fluorescence decay is dominated by the kinetics of recombination between QA− and the S2 state in PSII. However, even under these conditions, the traces for the wild type, the two single mutants, and the double mutant were all very similar (Supplemental Fig. S6B). The loss of PsbTn thus does not affect the acceptor side of PSII. A more detailed acceptor-side analysis of PSII based on thermoluminescence measurements also revealed no differences between the wild type, single mutants, and double mutant.

The electron transfer components of PSII were also analyzed by electron paramagnetic resonance (EPR) spectroscopy, which was used to study the S2 state multiline signal, the QA− Fe2+ interaction signal, and the redox state of Cyt b559 (Supplemental Fig. S7). The deconvolutions of the EPR data are summarized in Supplemental Table S2. These experiments indicated that there were no differences between the wild type and the double mutant with respect to the redox state of Cyt b559 or the intensities of the QA− Fe2+ and S2 state multiline signals. Interestingly, however, the TyrD signal was 10% to 20% less intense in the spectra of the double mutant than those of the wild type (Supplemental Table S2). In general, the EPR experiments indicated that the absence of PsbTn had no dramatic effect on the electron path through PSII in the psbTn1 psbTn2 double mutants.

PSII Photoinhibition Analysis

Because the PAM analysis revealed some minor differences between the double mutant and the wild type with respect to ΦPSII and the ETR (Supplemental Table S1), we investigated the sensitivity of PSII to photoinhibition in the mutants. Four-week-old wild-type and mutant plants were illuminated with heterochromatic light at 1,000 µmol photons m−2 s−1 for 4 h, after which the samples were illuminated at a low intensity (10 µmol photons m−2 s−1), and their recovery was monitored over 24 h. Changes in the Fv/Fm fluorescence ratio were recorded and the levels of the D1, and PsaD proteins were analyzed immunologically (Fig. 5). During the 4 h of high light, the Fv/Fm values for the wild type and double mutant decreased from 0.79 to 0.42 and 0.74 to 0.27, respectively (Fig. 5A). The larger decrease observed for the double mutant indicates that the absence of PsbTn increased sensitivity to high light. The rates of recovery from photoinhibition were similar in the wild type and the double mutant (Fig. 5B) despite their different initial levels of inhibition. The double mutant’s greater reduction in Fv/Fm can thus be attributed to elevated photosensitivity, but not to a failure to recover from photodestruction. To strengthen this conclusion, the plants were treated with lincomycin to inhibit D1 protein turnover and thus the PSII repair process. As expected, samples treated in this way exhibited a greater degree of PSII inactivation than untreated control samples (Fig. 5A, lower traces). The greatest sensitivity was observed in the psbTn1 psbTn2 double mutant, whose Fv/Fm value was one-third that of the wild type.

Figure 5.

Figure 5.

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PSII photosensitivity of mutants during high-light illumination. (A) Untreated (−) and lincomycin-treated (+) wild-type (WT) and mutant leaves were subjected to high-light conditions (1,000 µmol photons m−2 s−1) for 4 h and (B) then the photoinhibited samples were allowed to recover at low-light intensity (10 µmol photons m−2 s−1) for up to 24 h with regular measurement of their Fv/Fm values. Values are means ± se of three replicates. (C) Immunoblot analysis of the wild type and psbTn1, psbTn2, and psbTn1 psbTn2 mutants with D1 and PsaD antibodies before (−) and after (+) photoinhibition using a light intensity of 1,000 µmol photons m−2 s−1 for 3 h. The PsaD protein was used as a loading control.

To determine whether high-light-induced PSII inactivation reduced steady-state levels of the PSII complex, we analyzed the accumulation of the D1 protein in leaves from wild type and mutants under high-light conditions (1,000 µmol photons m−2 s−1) in the absence and presence of lincomycin. After 3 h of illumination, the abundance of the D1 protein was determined by immunoblotting. Levels of D1 declined moderately under high light in wild-type plants and to a larger extent in the double mutant (Fig. 5C). In the presence of lincomycin, this effect was even more pronounced. However, no significant changes in the abundance of the PSI protein PsaD were detected in any sample (Fig. 5C). These results show that while the absence of PsbTn proteins has only minor effects on the electron pathway in PSII, it has dramatic effects on photoinhibition.

PSII Protein Phosphorylation in the psbTn1 psbTn2 Double Mutants

The phosphorylation and dephosphorylation of specific PSII proteins plays an important regulatory role in the PSII repair cycle and the energy balance (Aro and Ohad, 2003; Vener, 2007). To determine whether the phosphorylation pattern mediated by the kinases STN7 and STN8 is changed by the absence of the PsbTn protein, 4-week-old plants were incubated in darkness, under normal growth light, or under high light for 30 min. Thylakoid proteins were separated by SDS-PAGE and immunodecorated using antiphospho-Thr antibodies (Fig. 6). As expected, all samples incubated in darkness exhibited little phosphorylation of PSII reaction center proteins, with the lowest levels of PSII phosphorylation being observed in the psbTn1 psbTn2 double mutant (Fig. 6). All samples incubated under normal-light conditions exhibited higher levels of phosphorylation of the PSII core proteins CP43, D1, and D2 but again, these proteins (especially D1) were less extensively phosphorylated in the psbTn1 psbTn2 double mutant. Under high light, the wild type and the single psbTn mutants still showed relatively high phosphorylation levels, but phosphorylation of the PSII reaction center was reduced in the psbTn1 psbTn2 double mutant. In addition, we observed no significant differences in LHCII phosphorylation between the wild type and psbTn mutants under any light conditions. These results indicate that the complete removal of the PsbTn proteins from PSII affected the phosphorylation of PSII core proteins.

Figure 6.

Figure 6.

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Light-dependent phosphorylation of thylakoid membrane proteins. Immunodetection of isolated thylakoid membrane proteins on a 12% SDS gel was performed using antiphospho-Thr antibodies from Cell Signaling. Four-week-old plants were dark-adapted for 16 h and then kept in darkness (D), exposed to normal light (120 μmol photons m−2 s−1) for 30 min (NL), or exposed to high light (1,000 μmol photons m−2 s−1) for 30 min (HL). Samples were loaded at a level corresponding to 1 μg of chlorophyll. The positions of the major phosphorylated proteins and the molecular markers are indicated in the figure. Loading was checked by Coomassie Brilliant Blue (CBB) staining of wild-type (WT) and mutant proteins.

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

Photoinhibition leads to imbalances in electron fluxes through the photosynthetic electron transport chain, which in turn cause reactive oxygen species (ROS) production. The increased photoinhibition in the psbTn1 psbTn2 double mutant therefore prompted us to analyze its ROS production. Three independent methods were used to detect oxidative stress: (1) direct color staining with NBT (blue), (2) 3,3′-diaminobenzidine (DAB; orange) staining of plant seedlings, and (3) confocal microscopy imaging using the singlet oxygen sensor green reagent (Supplemental Fig. S8). NBT staining indicated that formazan formation under high-light conditions was more extensive in the psbTn1 psbTn2 mutant than in the wild type, indicating greater O2˙− accumulation in the double mutant (Supplemental Fig. S8A). Double-mutant plants subjected to high light were also stained more intensely with DAB, indicating that they accumulated more H2O2 (Supplemental Fig. S8B). The singlet oxygen sensor green fluorescence indicator was used to detect singlet oxygen (1O2) in detached younger leaves. Leaves of double mutants subjected to high light generated a stronger fluorescence signal than wild-type leaves under the same conditions, suggesting that the psbTn1 psbTn2 mutants also accumulated more 1O2 than the wild type (Supplemental Fig. S8C). The psbTn1 psbTn2 double mutant thus accumulated higher levels of three different ROS than the wild type, indicating increased oxidative stress.

To determine whether detectable photobleaching occurred in leaves of psbTn1 psbTn2 double mutants under changing illumination, a modification of a previously reported light-to-dark shifting protocol was used (Tikkanen et al., 2010). To specifically induce the production of 1O2, wild type and mutant plants were placed in darkness for 30 min (and not low light as in Tikkanen et al., 2010) and then exposed to high light for 30 min; this cycle was repeated for 4 h (Wagner et al., 2004; Lee et al., 2007). Under these conditions, white spots formed in the leaves of psbTn1 psbTn2 double mutants, but not in the wild type (Fig. 7A), indicating that the double mutant suffered serious damage under high-light/dark-shift conditions. Moreover, the psbTn1 psbTn2 plants exhibited significantly greater reductions in Fv/Fm and NPQ than the wild-type plants (Fig. 7B). This suggests that PsbTn is essential for proper acclimation of PSII to artificially high-light/dark-shift conditions. In addition, both cell death (detected by trypan blue staining) and ROS accumulation were observed in seedlings of the double mutant (Fig. 7, C and D) under the light-shift conditions but not in the wild type. These results further demonstrate that the double mutants are more sensitive to oxidative stress than the wild type.

Figure 7.

Figure 7.

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Analysis of photobleaching lesions in the wild type and mutants under high-light/dark-shift conditions. A, Phenotypes of single leaves from 4-week-old plants subjected to high-light/dark-shift conditions (alternating between 1,000 μmol photons m−2 s−1 and complete darkness at 30-min intervals for 4 h in total). B, False-color images showing Fv/Fm and NPQ after the light-/dark-shift treatment of 4-week-old wild-type and mutant plants. Quantitative values (±sd) are shown below the individual fluorescence images. C, Trypan blue staining of 4-week-old seedlings grown under light-/dark-shift conditions. Significant microscopic mesophyll cell death (shown as blue stained cells) was observed in double mutants. Bars, 50 µm. D, Confocal microscopy images of 1O2 levels in 4-week-old seedlings grown under normal light and then exposed to light/dark shift conditions for 4 h. After the fluctuating light treatment, detached leaves were incubated for 2 h in singlet oxygen sensor green reagent. 1O2 was visualized by excitation at 488 nm and emission at 535 to 590 nm. Chlorophyll autofluorescence (red) was visualized by excitation at 488 nm and detected at 650 to 710 nm. Bars, 100 µm.

PSII Protein Phosphorylation and Complexes under High-Light/Dark-Shift Conditions

To investigate the double mutants’ sensitivity to high-light/dark-shift conditions, we studied the phosphorylation patterns of PSII and thylakoid membrane protein complexes in the wild type and mutants exposed to alternating 30-min periods of darkness and intense illumination for 4 h. The differences in phosphorylation between the wild type and mutants previously observed under constant light conditions (Fig. 6) became even more pronounced under high-light/dark-shift conditions (Fig. 8A). As was also observed when studying PSII phosphorylation under constant light conditions, phosphorylation of the D1 and D2 proteins under high-light/dark-shift conditions was reduced in the double mutants when compared to the wild type, but no such effect was observed on the phosphorylation of CP43 and LHCII. The high-light/dark-shift illumination also made the PSII-LHCII supercomplex and PSII dimer less abundant in the double mutants than in the wild type (Fig. 8C).

Figure 8.

Figure 8.

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Analysis of thylakoid membrane protein phosphorylation and complexes in the wild type and mutants under light-/dark-shift conditions. A, Immunodetection of isolated thylakoid membrane proteins on a 12% SDS gel was performed using antiphospho-Thr antibodies. Four-week-old plants were exposed to light-/dark-shift (alternating between 1,000 μmol photons m−2 s−1 and complete darkness at 30-min intervals) for 4 h in total. Samples were loaded at levels corresponding to 1 μg chlorophyll. The positions of the major phosphorylated proteins and the molecular markers are indicated in the figure. B, Loading was checked by Coomassie Brilliant Blue (CBB) staining of wild-type and mutant proteins. C, Thylakoid membrane complexes (20 µg chlorophyll) from the wild type and mutants (psbTn1, psbTn2, and psbTn1 psbTn2) grown under light/dark shift conditions were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside (DM) and subjected to BN-PAGE. The assignments of thylakoid membrane complexes indicated at the left were identified according to Chen et al. (2016). NDH, NAD(P)H dehydrogenase; PS, photosystem; LHC, light-harvesting complex; Cyt b6/f, cytochrome b6/f; mc, megacomplex; sc, supercomplex.

Chlorophyll Fluorescence Analysis

Because the psbTn1 psbTn2 double mutants were more sensitive to photoinhibition than the wild type, their energy dissipation and state transitions were investigated by chlorophyll fluorimetry (Fig. 9). An important mechanism that protects against excessive excitation in plants is thermal energy dissipation, which contributes to NPQ (de Bianchi et al., 2011). Therefore, wild-type and mutant plants were illuminated with saturating light (1,000 μmol photons m−2 s−1), and the changes in their NPQ were measured over a period of 1,000 to 5,000 s. Under the high-light periods, both the mutants and the wild type displayed the typical rapid increase in NPQ followed by a slower increase (Fig. 9, A and B), but the double mutant exhibited a significantly slower and weaker increase than the wild type. However, the mutants and the wild type achieved similar rates of recovery in darkness after intense illumination (Fig. 9A). When a second illumination period was applied (Fig. 9B), the increase in NPQ was again slower in the double mutants than in the wild type, implying that the absence of PsbTn increases sensitivity to photoinhibition and favors ROS production under high-light conditions.

Figure 9.

Figure 9.

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Assays of nonphotochemical quenching and photosynthetic functions of the wild type and mutants. A, Measurements of NPQ kinetics in wild type (WT), psbTn1, psbTn2, and psbTn1 psbTn2 leaves grown in the greenhouse. Bars on top, white bar (light on) and black bar (dark). B, NPQ kinetics of wild-type and psbTn1 psbTn2 plants grown in the greenhouse during two consecutive periods of illumination with 1,000 µmol photons m−2 s−1 for 25 min with a 15 min period of darkness in between, as indicated by the white (light on) and black (dark) bars on top of figure. C, Pulse amplitude-modulated fluorescence traces after shifts from state 1 to state 2 light and back for wild-type and psbTn1 psbTn2 plants growing in the field. The bars at the bottom indicate illumination with red (shown in red) and far-red (dark red) light. Fluorescence is shown in arbitrary units. D, PSII quantum yield (ΦPSII) of the wild-type and psbTn1 psbTn2 plants growing in the field under fluctuating light conditions. For A, B, and D, white bar on top (light on) and black bar (dark), data represent means ± se from three independent measurements. In C, a typical representative trace is shown.

State transitions are a good indicator of the rate of energy dissipation (de Bianchi et al., 2011). Therefore, state transitions in the wild type and mutant plants were investigated by monitoring changes in chlorophyll fluorescence characteristics. The amplitude of the state transitions was the same in wild-type and single-mutant plants when plants were grown in a greenhouse under constant conditions (Supplemental Fig. S9). However, when grown in the field, significant differences in state transitions were observed between the double-mutant and wild-type plants (Fig. 9C). Specifically, the final amplitudes of the state transitions in the double mutants were lower than those in the wild type. These results show that the lack of PsbTn affected state transitions under natural conditions. The rapid decrease in the kinetics of state transition may be due to the light intensity in the field, which may be as high as 1,000 μmol photons m−2 s−1 at noon under natural conditions. Moreover, the effective quantum yield of PSII (ΦPSII) was substantially lower in double mutants than in the wild type (Fig. 9D), indicating that PsbTn is required for PSII activity under natural conditions. These differences in state transitions and ΦPSII between the wild type and mutants when grown in the field or in climate chambers indicate that PsbTn is important for acclimation to intense and fluctuating illumination under natural conditions.

DISCUSSION

PsbTn Is a PSII Subunit Located on the Lumenal Side

The PsbTn protein was detected in thylakoids, enriched in grana stacks and isolated PSII membrane fragments (BBY particles; Fig. 1), and found to comigrate with PSII complexes in blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments (Fig. 4). These results suggest that it is a bona fide component of the PSII complex. Chymotrypsin digestion of thylakoid membranes with and without sonication showed that the PsbTn protein is located on the lumenal side of the membrane (Fig. 1). We next investigated its location within the PSII complex. Chemical cross linking experiments and coimmunoprecipitation experiments using PsbTn or PsbE antisera revealed that PsbTn is located close to the PsbE subunit of cytochrome b559 (Fig. 2). A protein structure prediction using the I-Tasser algorithm and a hydrophobicity analysis identified no putative transmembrane helices in the small PsbTn protein (Supplemental Fig. S2), suggesting that it is an extrinsic membrane protein that is only bound to the PSII complex by electrostatic interactions. However, washing with various chaotropic reagents did not release PsbTn from either the thylakoid membrane or the PSII fragments even under conditions that triggered the release of extrinsic proteins such as PsbO (Fig. 2). This suggests that PsbTn is not a typical transmembrane protein but is nevertheless strongly bound to the PSII complex. These results are consistent with the recently published structure of the spinach PSII complex obtained by cryo-electron microscopy, which suggests that PsbTn intercalates between CP47 and the C-terminal part of the PsbE protein (Wei et al., 2016). We thus conclude that the PsbTn protein is an intrinsic lumenal PSII subunit in Arabidopsis.

The Structure of PSII Is Unaltered in psbTn Mutants

Both genomic PCR (Supplemental Fig. S3) and immunoblotting analyses using a polyclonal PsbTn antibody were used to verify that the PsbTn protein was absent in the psbTn1 psbTn2 double mutant (Supplemental Fig. S3; Fig. 4). Aside from being somewhat smaller than wild-type plants, the single and double psbTn mutants exhibited no readily apparent phenotypic abnormalities under normal laboratory growth conditions (Supplemental Fig. S4B). However, the chlorophyll content and chlorophyll a/b ratio in the psbTn1 psbTn2 double mutant were slightly lower than in the wild type (Supplemental Table S1). This bleaching may indicate that the double mutant is subject to increased stress. However, measurements of the plants’ contents of PSI, PSII, ATP synthase, the cytochrome b6f complex, and various antenna proteins revealed no compositional changes that could explain the reduced chlorophyll content of the psbTn1 psbTn2 double mutant. Furthermore, no changes in the PSII assembly were observed in any psbTn mutant (Fig. 4). This clearly shows that the PsbTn protein is not directly required for the assembly and stability of the PSII complex even though structural changes are likely to occur in the mutants and account for the observed deficiencies.

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

We performed many experiments to probe the functional status of PSII in the psbTn mutants, including PAM measurements (Supplemental Table S1), variable chlorophyll fluorescence analyses (Supplemental Fig. S6), and analyses of selected EPR signals (Supplemental Table S2; Supplemental Fig. S7). None of these experiments revealed any major differences between the wild type and psbTn1 psbTn2 double mutants with respect to electron transfer through PSII. However, there were some differences in the rates of oxygen evolution and total photosynthetic ETRs (Supplemental Table S1). This seems to exclude the possibility that PsbTn might be directly involved in the electron pathway through the PSII complex.

These findings could only partially explain the increased photoinhibition and ROS production observed in the psbTn1 psbTn2 double mutant (Figs. 5 and 7; Supplemental Fig. S8), and it was not clear how the low-molecular-mass PsbTn protein could influence oxygen evolution and the ETR without affecting the normal electron pathway through PSII. Our biochemical results and the recently published structure of the spinach PSII complex (Wei et al., 2016) clearly indicate that the PsbTn protein is located in very close proximity to TyrD and Cyt b559 in the PSII complex (Supplemental Fig. S10). This may indicate that its function is similar to that of the PsbY protein, which was suggested to be a Cyt b559 redox regulator (von Sydow et al., 2016). However, EPR studies indicated that the redox state of Cyt b559 in the psbTn1 psbTn2 double mutant was identical to that in the wild type (Supplemental Table S2), ruling out a Cyt b559-related function of PsbTn. Another possible function of PsbTn is based on its suggested location between CP47 and PsbE close to the TyrD residue of the D2 protein (Wei et al., 2016), where it could influence the functionality and redox state of TyrD.

Removal of the PsbTn Protein Increased Sensitivity to Photoinhibition

Photosynthetic organisms must cope with ever-changing light and temperature conditions via flexible regulatory mechanisms that adjust the light absorption and photosynthetic processes based on metabolic and environmental cues. Plants have evolved diverse mechanisms to optimize photosynthesis by regulating the absorption or distribution of light energy by the light-absorbing LHC proteins. Two of these mechanisms are well documented: (1) quenching of excited states of the light-harvesting antennae by NPQ (Ruban and Murchie, 2012; Niyogi and Truong, 2013) and (2) state transitions in which phosphorylated LHCII is detached from PSII and PSI excitation being increased to balance the energy distribution, and thus the electron transfer rate, through the two photosynthetic complexes (Depège et al., 2003). Interestingly, both these regulatory mechanisms were affected in the psbTn1 psbTn2 double mutant (Fig. 9). The double mutant exhibited a delayed onset of NPQ upon prolonged illumination, which was probably due to the changes in the transthylakoid pH gradient (de Bianchi et al., 2011), changes in the relative abundance of protein subunits hosting quenching sites (Bonente et al., 2008), or changes in the level or function of the pH sensor PsbS (Li et al., 2004). The occurrence of state transitions also depends on the phosphorylation of the LHCII proteins (Bellafiore et al., 2005) and their association with the PSI proteins, particularly PSI-H (Lunde et al., 2000). Indeed, the psbTn1 psbTn2 double mutant also showed a general decrease in the phosphorylation of the PSII core proteins (Figs. 6 and 8). The STN7 kinase plays a pivotal role in LHCII phosphorylation and state transition (Bellafiore et al., 2005). Under low light, the phosphorylation pattern of LHCII in the STN7 knockout mutant resembles that seen in the wild type under high-light conditions, causing PSII excitation to be favored over PSI excitation. This greatly reduces intersystem photosynthetic electron transfer (Bellafiore et al., 2005; Tikkanen et al., 2006, 2010). The STN7 knockout mutant has a stunted phenotype when grown under fluctuating light conditions (Pesaresi et al., 2009) but develops similarly to the wild type under continuous illumination (Tikkanen et al., 2010). The psbTn1 psbTn2 double mutant behaved similarly to the STN7 knockout mutant even though the degree of LHCII phosphorylation in psbTn mutants was very similar to that in the wild type (Fig. 6). However, phosphorylation of CP43, D2, and D1 was severely affected in the psbTn1 psbTn2 double mutants. These findings suggest that the STN8 kinase is inactivated in these mutants, which may indicate that the PsbTn protein is involved in regulating STN8, which has been implicated in PSII repair.

The removal of PsbTn presumably induced structural changes in the PSII complex that reduced its accessibility to kinases. The ability of the psbTn1 psbTn2 double mutant to regulate photosynthetic imbalances via phosphorylation changes, NPQ, and/or state transition is impaired, leading to increased photoinhibition, which in turn causes increased ROS accumulation under high-light conditions (Supplemental Fig. S8). This hypothesis is supported by the photobleached phenotype of the double mutant under high-light/dark-shift conditions (Fig. 7). Although the composition of the PSII supramolecular complex in the psbTn1 psbTn2 double mutant was unchanged (Fig. 3), its overall abundance was lower than in the wild type (Fig. 8C). Previous studies have indicated that high-light conditions facilitate the disassembly of PSII complexes (Tikkanen et al., 2008; Chen et al., 2017). Therefore, the reduced abundance of PSII complexes in the psbTn1 psbTn2 double mutant may be due to more severe oxidative damage. Given that our findings indicate that PsbTn is a low-molecular-mass protein tightly bound to the lumenal side of PSII next to the essential PsbE protein (Swiatek et al., 2003), the effects of its removal on photoinhibition, PSII phosphorylation, state transition, and ROS production are not straightforward to explain. One interesting possibility is that PsbTn may interact with TyrD and could function as a redox sensor/regulator for the STN8 kinase, which is involved in the phosphorylation of the PSII core proteins and thereby influences photoinhibition.

CONCLUSION

PsbTn is a lumenal PSII protein, situated next to the cytochrome b559 subunit PsbE. Its removal decreased the oxygen evolution rate and PSII core phosphorylation level but increased the susceptibility of PSII to photoinhibition and the formation of ROS. We propose that the main function of the PsbTn proteins is to enable PSII to acclimate to light-/dark-shift conditions and high-light-intensity conditions.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana; accession Columbia) T-DNA insertion lines psbTn1 (GABI_143D01) and psbTn2 (SAIL_1214_E09) were obtained from the German plant genomics research program Kölner Arabidopsis T-DNA lines and Arabidopsis Biological Resource Center collections, respectively. The T-DNA insertion sites were confirmed by sequencing (Supplemental Fig. S3A; Supplemental Table S3). Double-mutant plants were obtained by crossing single-mutant plants to generate the F1 progeny. To obtain homozygous mutants, F1 plants were selfed and homozygous plants in the F2 progeny were screened by genotyping via PCR (Supplemental Fig. S3B) and confirmed by protein blotting using a monospecific PsbTn antibody (Supplemental Fig. S4A). Plants were grown in a growth chamber with an 8-h light/16-h dark cycle with a light intensity of 120 μmol of photons m−2 s−1 (Fluorescent Philips master 930) and day/night temperatures of 22°C /15°C. After 4 weeks, the plants were harvested for the isolation of thylakoid membrane proteins. For the light-to-dark shifting experiment, 4-week-old plants were alternately exposed to 1,000 μmol of photons m−2 s−1 for 30 min and then incubated in full darkness for 30 min over a period of 4 h.

Homology Searches, Coexpression, and Structure Analyses

Plant homologs of PsbTn were identified by a BLAST search using the web service provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Amino acid sequences of different land plants were aligned in Jalview using ClustalW. Structural homology models of Arabidopsis PsbTn1 (At3g21055) and PsbTn2 (At1g51400) were generated using I-Tasser (http//:zhanglab.ccmb.med.umich.edu/; Roy et al., 2010). Coexpression correlation coefficients were performed according to the method of Granlund et al. (2009).

Measurements of Photosynthetic Parameters and Chlorophyll Fluorescence Measurements In Vivo

Chlorophyll a fluorescence induction kinetics and changes in PSI A820 in the wild-type and mutant plants were measured using a Dual-PAM-100 fluorometer (Heinz Walz) on whole leaves at room temperature according to Ohad et al. (2004). ΦPSI and ΦPSI (ND) were expressed as described previously (Klüghammer and Schreiber, 1994). Photochemical and nonphotochemical parameters were calculated as described by Maxwell and Johnson (2000).

State transition experiments were performed using whole plants according to established protocols (Pietrzykowska et al., 2014). Arabidopsis plants were subjected to a red/far-red light treatment using a customized LED light source (SL 3500-R-d) from Photon System Instruments as previously described by Leoni et al. (2013). Plants were dark adapted for 1 h prior to measurements. NPQ was measured using a WALZ Dual PAM-100 fluorometer according to de Bianchi et al. (2011).

During light-to-dark shifts, chlorophyll fluorescence images were obtained using a modulated imaging fluorometer (the Imaging PAM M-Series Chlorophyll Fluorescence system, Heinz-Walz Instruments) according to the manufacturer’s instructions. The image data averaged in each experiment were normalized to a false color scale.

Electron Microscopy

Chloroplast morphology was investigated by transmission electron microscopy of the wild type and mutants using leaves before and after high-light illumination at 1,000 μmol photons m−2 s−1 for 2 h as described (Brouwer et al., 2012).

Isolation of Thylakoids, PSII Membranes, and Pigment and Protein Analyses

Thylakoid membranes were isolated according to Chen et al. (2016) from fresh or frozen material in liquid nitrogen. All of the extraction buffers contained 10 mm NaF to inhibit phosphatase activity. PSII-enriched membranes (BBY) were prepared according to Arellano et al. (1994) with some modifications. The pellet containing the thylakoid membranes was resuspended in 1.5 mL BBY-stacking (20 mm MES-NaOH pH 6.3, 5 mm MgCl2 and 15 mm NaCl) and, while gently stirring at 4°C, 10% Triton X-100 (w/v) was added to the thylakoid membranes to a final Triton X-100 to chlorophyll ratio of 10:1 (w/w). Exactly 18 min after addition of the detergent, the solubilized thylakoid membranes were collected and centrifuged at 1,000g for 2 min, then the supernatant was transferred to a new tube and centrifuged at 40,000g for 30 min at 4°C. The resulting pellet was resuspended in BBY storage buffer (20 mm MES-NaOH pH 6.3, 400 mm sorbitol, 5 mm MgCl2, 10 mm CaCl2, and 15 mm NaCl). Chlorophyll concentrations were measured after extraction with 80% acetone (v/v).

Immunoblot analysis was performed on thylakoid membranes or BBY according to Chen et al. (2016). PsbTn antisera were generated using a mixture of two synthetic peptide sequences representing the protein’s C-terminal (CVTMPTAKI) and N-terminal (EPKRGTEAAKKKYAQ) regions (Agrisera). Other monospecific antibodies used in the experiments were obtained from Agrisera. Loading was determined by staining with Coomassie Brilliant Blue prior to western blotting. The immunodecoration was visualized using western Bright Quantum (Advansta), and signals were detected using a LAS-3000 cooled CCD camera (Fujifilm). Quantification of the immunoblots of thylakoid membrane proteins was done using Quantity One software.

BN-PAGE was performed as described (Chen et al., 2016). Thylakoid membranes containing 20 µg of chlorophyll were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside and separated on a gradient of 5% to 12.5% acrylamide in the separation gel. For 2D electrophoresis, the lanes of the first dimension were cut out and incubated in Laemmli buffer containing 5% (v/v) β-mercaptoethanol (Laemmli, 1970) for 1 h at room temperature. After this, the gel strips were subjected to SDS-PAGE with 15% acrylamide (v/v) and 6 m urea. Gels were either stained with Coomassie Brilliant Blue R or used for immunoblotting.

Localization and Topology of PsbTn

Thylakoid membranes and BBY from wild-type plants were treated with different salt-containing buffers as described previously (Torabi et al., 2014). Thylakoid membranes were treated with the protease chymotrypsin (200 µg/mL) for 10 min at room temperature with or without sonication before immunoblot analysis. Subfractionation of grana-enriched and stroma lamellae-enriched thylakoids was performed as described by Torabi et al. (2014).

Cross Linking and Coimmunoprecipitation

EDC cross linking of PsbTn was performed according to the method of Hansson et al. (2007). Coimmunoprecipitation was performed according to the manufacturer’s instructions (Molecular Probes Co-IP Kit 26149). The eluted proteins were separated on a 15% polyacrylamide gel and then subjected to immunoblot analysis.

Analysis of PSII Photoinhibition and Recovery

The sensitivity of PSII to light stress, measured in terms of changes in Fv/Fm as a function of exposure time, was determined using leaves of mutant and wild-type plants in the presence/absence of lincomycin. For photoinactivation, the detached leaves were exposed to 1,000 µmol photons m−2 s−1 heterochromatic light for 4 h. The photoinactivation of PSII is expressed as a function of the exposure time. Recovery from photoinhibition was determined under low-light conditions (10 µmol photons m−2 s−1) for 24 h. To assess the accumulation of D1 proteins under intense illumination, proteins were extracted from leaves, separated by SDS-PAGE, and subjected to immunoblotting using D1 antibodies.

Trypan-Blue Staining

Four-week-old Arabidopsis seedlings were alternately exposed to 1,000 μmol photons m−2 s−1 light and complete darkness for 30 min each over a total period of 4 h. Photobleaching (cell death) was then visually detected by trypan blue staining (1.25 mg/mL; Lam, 2004).

In Situ Detection of ROS

ROS measurements were performed in situ as previously described by Lu et al. (2011). H2O2 and superoxide accumulation were detected by incubation with DAB (Sigma-Aldrich) or NBT (Sigma-Aldrich), respectively, for 2 h in darkness followed by high light (1,000 μmol photons m−2 s−1) for 2 h. Stained leaves were boiled in acetic acid:glycerol:ethanol (1:1:3 [v/v/v]) and photographed. Singlet oxygen accumulation was visualized by confocal laser scanning microscopy on leaves incubated in 500 nm singlet oxygen sensor green (Molecular Probes) for 2 h and then exposed to high light for 2 h as described previously (Lu et al., 2011).

Oxygen Evolution Measurements

The oxygen-evolving activities of thylakoid membrane and BBY preparations from mutant and wild-type plants were measured in assay media containing (at final concentrations) 25 mm HEPES pH 7.6 (KOH), 0.2 m Suc, 10 mm NaCl, and 5 mm CaCl2 supplemented with the artificial electron acceptor phenyl-p-benzoquinone at a final concentration of 0.5 mm. Oxygen evolution rates were measured at 20°C under saturating light using a Clark-type electrode (García-Cerdán et al., 2009).

Flash-Induced Fluorescence Decay Kinetics and Thermoluminescence Measurements

Flash-induced increase and subsequent relaxation of the chlorophyll fluorescence yield (variable fluorescence decay kinetics) were measured as described in von Sydow et al. (2016) and García-Cerdán et al. (2011) with a FL3300 dual-modulation fluorometer (Photon System Instruments, Brno, Czech Republic) in the 150 μs to 100 s time range. The actinic flash duration was 30 μs. PSII membranes at a concentration of 10 μg Chl mL−1 were dark-adapted for 5 min before fluorescence detection. The measurements were performed in the absence or presence of 20 μm DCMU. The kinetics were analyzed in terms of several exponential components (fast, intermediate, and slow phases) as described by von Sydow et al. (2016) and García-Cerdán et al. (2011).

Thermoluminescence signals were measured with a TL200/PMT thermoluminescence system (Photon System Instruments, Brno, Czech Republic; García-Cerdán et al., 2011; von Sydow et al., 2016). PSII membranes at a concentration of 150 μg Chl mL−1 were dark adapted for 5 min at 20°C then cooled to −10°C and excited by an actinic flash of 50 μs duration. The sample was then heated to 60°C at a heating rate of 1°C/s. The measurements were performed in the absence or presence of 20 μm DCMU.

EPR Spectroscopy

EPR experiments were performed using a Bruker ELEXYS E500 spectrometer with a SuperX EPR049 microwave bridge and a SHQ4122 cavity, equipped with an ESR 900 liquid helium cryostat and ITC 503 temperature controller from Oxford Instruments. EPR samples at a concentration of ∼3 mg Chl mL−1 were illuminated by ambient laboratory light for 1 min and then dark adapted for 5 min before freezing in liquid N2 to ensure full oxidation of YD•. The S2 state multiline signal was induced by illumination at 200 K for 6 min and complete oxidation of cytochrome b559 was induced by illumination at 77 K for 6 min. The reduction of the QA− Fe2+ semiquinone iron complex was induced by subsequent incubation with 15 mm formate and 50 mm dithionite for 15 min each at room temperature (Chen et al., 2011).

Field Experiments

The field experiment was performed at the experimental garden in Umeå University as described previously (Mishra et al., 2012). The wild-type and double-mutant plants were first grown under short-day conditions in a climate chamber and then transferred to the outdoor field once they had developed three to four leaves. The plants were shaded on the first day to allow for some acclimation. After 4 weeks outside, the state transition kinetics and ΦPSII were measured in the wild-type and double-mutant plants.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: PsbTn1 (At3g21055), PsbTn2 (At1g51400), and ACTIN1 (At2g37620).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

The authors thank Malgorzata Pietrzykowska, Kim Sungyong, Kati Mielke, and Lotta von Sydow for discussions and help during this project. The authors acknowledge the facilities and technical assistance of the Umeå Core Facility Electron Microscopy (UCEM) at the Chemical Biological Centre (KBC), Umeå University, part of the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).

Footnotes

1

This work was supported by Sven and Lilly Lawski Foundation, Carl Tryggers Foundation, and by the German Science Foundation (Deutsche Forschungsgemeinschaft; ME1794/7 and TRR 175 TP A03 to J.M. and TRR175 TP B06 to S.S.).

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