Download TEF30 interacts with photosystem II monomers and is involved in the

Plant Physiology Preview. Published on December 7, 2015, as DOI:10.1104/pp.15.01458
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Running head: TEF30 is involved in PSII repair
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Michael Schroda
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Molekulare Biotechnologie & Systembiologie
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TU Kaiserslautern
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Erwin-Schrödinger Straße 70, D-67663 Kaiserslautern
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Germany
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Tel.: ++49 631 205 2697
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Fax: ++49 631 2015 2699
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e-mail: [email protected]
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Most appropriate journal research area: Cell Biology
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TEF30 interacts with photosystem II monomers and is involved in the repair of
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photodamaged photosystem II in Chlamydomonas reinhardtii
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Ligia Segatto Muranaka*1, Mark Rütgers*1, Sandrine Bujaldon2, Anja Heublein3, Stefan
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Geimer3, Francis-André Wollman2, and Michael Schroda1
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70, D-67663 Kaiserslautern, Germany
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Biologie Physico-Chimique, UMR CNRS/UPMC 7141, Paris, France
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Molekulare Biotechnologie & Systembiologie, TU Kaiserslautern, Erwin-Schrödinger
Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste, Institut de
Zellbiologie/Elektronenmikroskopie, Universität Bayreuth, D-95440 Bayreuth, Germany
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* Equal contribution
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One-sentence Summary:
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A protein conserved in the green lineage binds quantitatively to photosystem II
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monomers at the stromal side of thylakoid membranes and facilitates supercomplex
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assembly during photosystem II repair.
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Footnotes:
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This work was supported by the Deutsche Forschungsgemeinschaft [Schr 617/9-1], the
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DAAD/CNPq [290020/2010-7], and the Forschungsschwerpunkt BioComp.
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Corresponding author:
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Michael Schroda
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e-mail: [email protected]
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List of author contributions:
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L.S.M. and M.R. performed most of the experiments and were supervised by M.S; S.B.
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contributed to the biophysical experiments, which were supervised by F.A.W. A.H. and
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S.G. performed the electron microscopy work; all authors contributed to the design of
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the experiments and data analysis; M.S. conceived the project and wrote the article with
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contributions of all the authors.
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Abstract
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The remarkable capability of photosystem (PS) II to oxidize water comes along with its
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vulnerability to oxidative damage. Accordingly, organisms harboring PSII have
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developed strategies to protect PSII from oxidative damage and to repair damaged PSII.
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Here we report on the characterization of the TEF30 protein in Chlamydomonas
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reinhardtii that is conserved in the green lineage and induced by high light. Fractionation
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studies revealed that TEF30 is associated with the stromal side of thylakoid membranes.
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By using blue native / Deriphat-PAGE, sucrose density gradients, and isolated PSII
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particles, we found TEF30 to quantitatively interact with monomeric PSII complexes.
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Electron microscopy images revealed significantly reduced thylakoid membrane stacking
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in TEF30-underexpressing cells when compared with control cells. Biophysical and
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immunological data point to an impaired PSII repair cycle in TEF30-underexpressing
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cells and a reduced ability to form PSII supercomplexes after high light exposure. Taken
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together, our data suggest potential roles for TEF30 in facilitating the incorporation of a
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new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting
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repaired PSII monomers from undergoing repeated repair cycles, or facilitating the
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migration of repaired PSII monomers back to stacked regions for supercomplex
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reassembly.
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Introduction
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Oxygenic photosynthesis is essential for almost all life on earth as it provides the
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reduced carbon and the oxygen required for respiration. A key enzyme in oxygenic
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photosynthesis is photosystem (PS) II, which catalyzes the light-driven oxidation of
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water. The core of PSII in algae and land plants contains D1 (PsbA), D2 (PsbD), CP43
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(PsbC), CP47 (PsbB), the α- (PsbE) and β-subunits (PsbF) of cytochrome b559 as well
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as several intrinsic low molecular-mass subunits. The core monomer is associated with
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the extrinsic oxygen evolving complex (OEC) consisting of OEE1 (PSBO), OEE2
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(PSBP), and OEE3 (PSBQ) which stabilize the inorganic Mn4O5Ca cluster required for
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water oxidation (reviewed in Pagliano et al., 2013). PSII core monomers assemble into
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dimers to which at both sides light-harvesting proteins (LHCII) bind to form PSII
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supercomplexes. In land plants, each PSII dimer binds two each of the monomeric minor
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antenna proteins CP24, CP26, and CP29 in addition to up to four trimeric major antenna
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(Caffarri et al., 2009; Kouril et al., 2011). Biochemical evidence suggests that in the
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thylakoid membrane up to eight LHCII trimers can be present per PSII core dimer,
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presumably because the existence of a pool of “extra” LHCII (Kouril et al., 2013). In
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Chlamydomonas reinhardtii, lacking CP24, PSII dimers bind two each of CP26 and
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CP29 monomers as well as up to six trimeric major antenna (Tokutsu et al., 2012). The
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reaction center proteins D1 and D2 bind all the redox-active cofactors required for PSII
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electron transport (Umena et al., 2011). Light captured by the internal antenna proteins
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CP43 and CP47 and the outer antenna induces charge separation in PSII, which in turn
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enables the OEC to oxidize water and provide electrons to the electron transfer chain. In
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land plants and green algae PSII supercomplexes are localized to stacked regions of the
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thylakoid membranes, while the synthesis of PSII cores is considered to take place in
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stroma lamellae.
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A particular feature of PSII is its vulnerability to light, with the D1 protein being a
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target of light-induced damage and damage being proportional to the photon flux density
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(PFD) applied (Tyystjarvi and Aro, 1996). To cope with this damage, an elaborate highly
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conserved mechanism has evolved termed the PSII repair cycle during which damaged
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PSII complexes are partially disassembled and the defective D1 protein is replaced by a
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de novo synthesized copy (for recent reviews see Nixon et al., 2010; Komenda et al.,
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2012; Mulo et al., 2012; Nath et al., 2013a; Nickelsen and Rengstl, 2013; Tyystjarvi,
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2013; Jarvi et al., 2015). Photodamage occurs at all light intensities, but when the rate of
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damage exceeds the capacity for repair, photoinhibition is manifested as a decrease in
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photosynthetic activity and in the proportion of active PSII reaction centers (Aro et al.,
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1993). While PSII photodamage occurs in the supercomplexes in the stacked membrane
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regions, the replacement of damaged D1 takes place in stroma lamellae (Aro et al.,
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2005). Thus, the PSII repair cycle requires lateral migration of PSII complexes, which is
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impaired by the macromolecular crowding in stacked thylakoid membranes (Kirchhoff,
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2013). Lateral migration of damaged PSII complexes is facilitated by thylakoid
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membrane unfolding and PSII supercomplex disassembly. Both processes are
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enhanced by the phosphorylation of PSII core subunits D1, D2, CP43, and PsbH, which
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is mainly mediated by the protein kinase STN8 (Tikkanen et al., 2008; Fristedt et al.,
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2009; Herbstova et al., 2012; Nath et al., 2013b; Wunder et al., 2013). Efficient PSII
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supercomplex disassembly also requires the THF1/NYC4/Psb29 protein (Huang et al.,
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2013; Yamatani et al., 2013). After migration of PSII monomers to unstacked thylakoid
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regions, core subunits are dephosphorylated by the PSII core phosphatase PBCP
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(Samol et al., 2012), which is required for the efficient degradation of D1 (Koivuniemi et
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al., 1995; Rintamaki et al., 1996; Kato and Sakamoto, 2014). Degradation of D1 is
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subsequently realized by the membrane-integral FtsH protease (Lindahl et al., 2000;
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Silva et al., 2003) and by lumenal and stromal Deg proteases (Haussuhl et al., 2001;
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Kapri-Pardes et al., 2007; Sun et al., 2010). Degradation is assisted by the lumenal
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protein TLP18.3, presumably by its phosphatase activity and ability to interact with
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lumenal Deg1 (Sirpio et al., 2007; Wu et al., 2011; Zienkiewicz et al., 2012). D1
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proteolysis follows the partial disassembly of the PSII complex during which CP43 and
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low molecular-mass subunits are released to generate a CP43-free PSII monomer (Aro
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et al., 2005). Thereafter, a newly synthesized D1 copy is co-translationally inserted from
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a plastidial 70S ribosome into the thylakoid membrane and processed by the CtpA
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peptidase (Zhang et al., 1999, 2000; Che et al., 2013). In Arabidopsis thaliana, the D1
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synthesis rate appears to be negatively regulated by the PDI6 protein (Wittenberg et al.,
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2014). Moreover, yet unknown steps during PSII repair require the stromal cyclophilin
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ROC4 and stromal HSP70 (Schroda et al., 1999; Yokthongwattana et al., 2001; Cai et
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al., 2008). The PSII repair cycle is completed by the reassembly of the CP43 protein,
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ligation of the OEC, back-migration of PSII to stacked membrane regions and
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supercomplex formation. Except for CtpA all mentioned factors appear to be specific for
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PSII repair, while many more auxiliary factors play roles in PSII de novo synthesis and
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repair (reviewed in Jarvi et al., 2015).
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In this study we report on the functional characterization of the TEF30 protein (for
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thylakoid enriched fraction 30) in Chlamydomonas. In this organism, TEF30 was first
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identified in a proteomics study on isolated thylakoid membranes (Allmer et al., 2006). It
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attracted our attention because its abundance increased 1.7-fold in membrane-enriched
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fractions of Chlamydomonas cells that had been shifted from 41 to 145 µmol photons m-
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Arabidopsis (MET1) has been functionally characterized only recently (Bhuiyan et al.,
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2015). Both MET1 and TEF30 interact quantitatively with monomeric PSII core particles
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at the stroma side of the thylakoid membranes and play a role in the assembly of PSII
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monomers and/or their migration to stacked membrane regions for supercomplex
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assembly. While MET1 appears to exert this function during PSII de novo biogenesis
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and during the PSII repair cycle in Arabidopsis, TEF30 appears to function exclusively
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during PSII repair in Chlamydomonas.
s-1 for 8 h (Mettler et al., 2014) (Supplemental Figure S1). The TEF30 ortholog in
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Results
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TEF30 is an abundant chloroplast protein capable of self-oligomerization
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As shown in Supplemental Figure S2A, TEF30 proteins are well conserved in the green
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lineage. The N-termini of TEF30 proteins are predicted to qualify as chloroplast transit
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peptides and all TEF30 homologs contain PDZ and TPR domains known to mediate
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protein-protein interactions (Allan and Ratajczak, 2011; Ye and Zhang, 2013)
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(Supplemental Figure S2B). Following the 36-amino acid chloroplast transit peptide
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predicted by ChloroP (Emanuelsson et al., 1999), Chlamydomonas TEF30 (but not its
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homologs from Arabidopsis or moss) harbors sequence motifs characteristic for
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thylakoid TAT pathway targeting signals (i.e., a RRXXφ twin-arginine motif and an AXA
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processing site, with X standing for any amino acid and φ for Leu, Val, Phe, or Met)
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(Albiniak et al., 2012). The region between both motifs is supposed to be hydrophobic,
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which however is not the case in Chlamydomonas TEF30. The calculated molecular
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mass of Chlamydomonas TEF30 lacking the predicted 36-amino acids chloroplast transit
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peptide is 33.3 kDa, while it is 30.4 kDa if the putative TAT targeting signal was
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processed. As shown in Figure 1A, mature TEF30 on SDS-gels with Chlamydomonas
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whole-cell protein extracts migrated slightly above recombinant TEF30 having a
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calculated molecular mass of 31.4 kDa (in recombinant TEF30 the predicted chloroplast
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and TAT targeting signals were replaced with a hexahistine tag, see Supplemental
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Figure S2B). This indicates that the predicted chloroplast transit peptide is cleaved off in
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mature TEF30, while the predicted TAT signal is not. Perhaps the incomplete TAT signal
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mediates TEF30’s targeting to the thylakoid periphery? Mature TEF30 was detected by
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immunoblot analyses in isolated chloroplasts, but hardly in isolated mitochondria (Figure
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1B). Moreover, immunofluorescence microscopy revealed TEF30 to be confined to the
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cup-shaped chloroplast (Figure 1C), thus indicating that TEF30 is a chloroplast protein.
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The routine detection of TEF30 in shotgun proteomics approaches on
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Chlamydomonas indicates that it is a well-expressed protein (Hemme et al., 2014;
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Mettler et al., 2014; Ramundo et al., 2014; Schmollinger et al., 2014). To get an estimate
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of its cellular abundance, we separated Chlamydomonas whole-cell proteins next to
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recombinant TEF30 and D2 proteins on SDS gels and quantified signals obtained from
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immunoblot analyses (Figure 1D). These analyses revealed TEF30 and D2 to represent
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0.05±0.007 ng/µg chlorophyll (n=4) and 0.59±0.016 ng/µg chlorophyll (n=3),
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respectively.
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The PDZ and TPR domains in TEF30 are likely to mediate protein-protein
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interactions. To test whether TEF30 is capable of self-oligomerization, recombinant
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TEF30 was subjected to EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-mediated
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zero-length crosslinking and separated by SDS-PAGE. As shown in Figure 1E,
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untreated recombinant TEF30 migrated with an apparent molecular mass of ~30 kDa,
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while EDC-treated TEF30 also migrated with apparent masses of ~60, ~90, ~120, and
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~150 kDa, thus indicating that TEF30 is indeed capable of self-oligomerization.
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TEF30 is localized to the stromal side of thylakoid membranes
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TEF30 was previously detected in Chlamydomonas thylakoid membrane fractions
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(Allmer et al., 2006). To verify TEF30’s thylakoid localization, Chlamydomonas cells
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were separated into soluble and membrane-enriched fractions by freeze-thawing and
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fractions were analyzed by immunoblotting. As shown in Figure 2A TEF30, like integral
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membrane protein cytochrome f, was only detected in the membrane-enriched pellet,
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while stromal CGE1 was only found in the soluble fraction. Cell lysis by sonication had
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no effect on CGE1’s exclusive localization to the soluble fraction, but resulted in a partial
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localization of TEF30 and cytochrome f to the soluble fraction, presumably because
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sonication created small thylakoid membrane vesicles that were not precipitated by
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centrifugation. Incubation of cells lysed by freeze-thawing in the presence of NaCl,
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Na2CO3, CaCl2, or urea had no effect on the exclusive localization of cytochrome f to the
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pellet and of CGE1 to the soluble fraction. However, NaCl led to a partial, and Na2CO3,
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CaCl2 and urea to a virtually complete shifting of TEF30 to the soluble fraction (Figure
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2A). Hence, TEF30 appears to be associated with thylakoid membranes rather than
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embedded into them, which is in agreement with the TMHMM algorithm (Sonnhammer
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et al., 1998) predicting TEF30 not to contain any transmembrane helices.
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To determine whether TEF30 is associated with the stromal or the lumenal side of
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thylakoid membranes, thylakoid membranes from cells lysed by freeze/thawing were
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purified by flotation on sucrose step gradients and incubated with trypsin (Figure 2B).
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While the CF1β subunit of the ATP synthase – situated at the stromal side of thylakoid
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membranes – was almost completely degraded by trypsin, lumenal plastocyanin was
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degraded only to a small extent and integral membrane protein cytochrome f was not
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degraded at all. TEF30 was degraded to a similar extent as CF1β, indicating that TEF30
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is associated to the stromal side of thylakoid membranes.
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To test, whether TEF30’s association to thylakoid membranes was due to its
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interaction with one of the major thylakoid membrane protein complexes, we fractionated
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Chlamydomonas mutants devoid or depleted of PSII, PSI, cytochrome b6/f, or the ATP
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synthase by freeze/thawing into soluble and membrane-enriched fractions and analyzed
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the localization of TEF30 by immunoblotting. Although cell-walled strains were used (in
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contrast to the experiments shown in Figure 2A, which were done with a cell wall-
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deficient strain), TEF30 in wild-type cells was again mainly detected in membrane-
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enriched fractions (Figure 2C). Also in PSI, ATP synthase, and cytochrome b6/f mutants
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most of the TEF30 protein was detected in membrane-enriched fractions, suggesting
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that TEF30 was not associated with one of these complexes. In contrast, virtually all
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TEF30 shifted to soluble fractions in mutants affected in the core subunits of PSII (PsbA-
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D), while TEF30 retained its location to membrane-enriched fractions in a mutant lacking
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the O subunit of the oxygen evolving complex (OEC) of PSII. As this mutant still
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accumulates considerable amounts of PSII core subunits (de Vitry et al., 1989), our
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combined data indicate that TEF30 is associated with (a) PSII core subunit(s) exposed
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to the stromal side of the thylakoid membranes.
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TEF30 co-purifies with PSII particles
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To verify the interaction of TEF30 with PSII, we took advantage of a Chlamydomonas
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strain (D2His) in which the D2 subunit was genetically engineered to contain a
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hexahistidine tag at its C-terminus (Sugiura et al., 1998). Detergent-solubilized thylakoid
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membranes prepared from wild-type and D2His cells were subjected to nickel-affinity
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chromatography and bound proteins were eluted with imidazole. Immunoblot analyses
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revealed TEF30 and D1 to be amply present, PSAD to be present in small amounts, and
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CF1β and cytochrome f not to be present in eluates from the D2His strain (Figure 3A).
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None of these proteins was detected in control eluates from the wild-type strain. To
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make sure that TEF30 was not interacting with PSI present as a contamination in the
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PSII preparation, we immunoprecipitated TEF30 from purified PSII particles derived from
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the D2His strain. As shown in Figure 3B and Figure 3C, several proteins co-precipitated
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with TEF30 but not with preimmune serum. Among the co-precipitating proteins was D1
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but not PSAD, indicating that TEF30 is tightly interacting with PSII particles and not with
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PSI.
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TEF30 co-migrates with PSII core subunits on sucrose density gradients and blue
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native gels
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To further substantiate our finding that TEF30 interacts with PSII core particles, we
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isolated thylakoid membranes from cell wall-deficient Chlamydomonas cells by flotation
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on sucrose step gradients and, following detergent solubilization, separated protein
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complexes on sucrose density gradients. Immunoblot analyses on fractions taken from
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these gradients revealed that sample processing resulted in the separation of PSII cores
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from LHCII and the OEC. This has been observed before when sucrose density gradient
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centrifugation was employed for analyzing PSII supercomplex compositions (Caffarri et
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al., 2009; Tokutsu et al., 2012) and in our hands for unknown reasons occurred
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predominantly with cells lacking cell walls. TEF30 clearly co-migrated with monomeric
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PSII core complexes (Drop et al., 2014) and not with LHCII, the OEC, PSI, the b6/f
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complex, or the ATP synthase (Figure 4A).
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Sucrose density gradients were also run with solubilized thylakoid membranes
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isolated from cell-walled cells of wild-type, PSII mutant and PSI mutant strains. Here,
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sample processing left most PSII supercomplexes intact (Figure 4B). On gradients run
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with wild-type thylakoids, TEF30 co-migrated again quantitatively with monomeric PSII
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core complexes and only traces of TEF30 co-migrated with larger PSII assemblies that
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were still smaller than fully assembled PSII supercomplexes. TEF30 also did not co-
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migrate with LHCII, PSI, the b6/f complex, or the ATP synthase. While the quantitative
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co-migration of TEF30 with monomeric PSII core complexes was not altered in gradients
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run with thylakoids from the F23 PSI mutant, TEF30 was found to migrate much less
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deeply into the gradient, to a position even above LHCII, in gradients run with thylakoids
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from the ΔpsbA/D PSII mutant (Figure 4B). Solubilized thylakoids from cell-walled wild-
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type and mutant strains were also separated by BN-PAGE and analyzed by
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immunoblotting. As shown in Figure 4C, TEF30 was again found to co-migrate
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quantitatively with PSII monomers, but not with PSII supercomplexes, in gel lanes
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containing solubilized thylakoids from wild-type and PSI mutant strains, while TEF30
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was not detected in lanes with thylakoids from the nac2 PSII mutant strain. In summary,
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these results confirm that TEF30 is specifically associated with monomeric PSII core
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complexes.
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PSII in TEF30-amiRNA cells is more susceptible to photoinhibition than PSII in
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control cells
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To get insights into the function of TEF30 in Chlamydomonas chloroplasts, we
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generated strains constitutively expressing artificial microRNAs (amiRNAs) targeting the
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coding region of TEF30 transcripts based on the pChlamiRNA2 vector (Molnar et al.,
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2009). Strains cw15-325 and cw15-302 were used as recipients, and in both we could
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routinely identify transformants in which TEF30 protein levels were reduced to less than
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20% of wild-type levels (Supplemental Figure S3). When comparing cells from control
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and TEF30-amiRNA strains we observed no obvious morphological differences (as
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judged from light microscopy) and no differences in growth under hetero-, mixo- or
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photoautotrophic conditions at various light and temperature regimes (Supplemental
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Figure S4).
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As we found membrane-associated TEF30 to accumulate in response to higher
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light intensities and to be associated with PSII core complexes (Supplemental Figure S1;
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Figure 2, Figure 3, and Figure 4), we reasoned that TEF30 might play a role in the
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protection/repair of PSII from photoinhibition. To test this idea, we monitored PSII
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maximum quantum efficiency (Fv/Fm) in cells from control and TEF30-amiRNA strains
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grown at ~30 µmol photons m-2 s-1 that had been shifted to high light (HL) at a PFD of
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1800 µmol photons m-2 s-1 for 60 min and allowed to recover at ~30 µmol photons m-2 s-1
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for 3 h. As shown in Figure 5A, PSII maximum quantum efficiency declined more
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strongly and recovered more slowly in TEF30-amiRNA cells as compared to control
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cells. The same was observed by 77K fluorescence emission spectroscopy
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(Supplemental Figure S5). To test, whether this apparent higher sensitivity of PSII to HL
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in TEF30-amiRNA cells was also reflected at the level of PSII subunit accumulation, we
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shifted cells from control and TEF30-amiRNA strains grown at ~30 µmol photons m-2 s-1
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to HL of 800 µmol photons m-2 s-1 for 6 h and monitored the accumulation of subunits of
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all four major thylakoid membrane complexes, as well as of TEF30, VIPP1, and
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LHCSR3 by immunoblotting (Figure 5B). VIPP1 and LHCSR3 were monitored as
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controls because they are strongly induced by HL (Supplemental Figure S1; Naumann
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et al., 2005; Nordhues et al., 2012). VIPP1 was suggested to play a role in the
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organization of lipid microdomains in thylakoid membranes (Rutgers and Schroda,
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2013), whereas LHCSR3 was shown to be essential for the induction of non-
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photochemical quenching (NPQ) in Chlamydomonas (Peers et al., 2009).
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While TEF30 was virtually absent in the TEF30-amiRNA strain used, it
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accumulated during HL exposure in cells of the control strain, thus confirming the
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proteomics data (Supplemental Figure S1). The exposure to HL in neither strain affected
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the accumulation of cytochrome f, CF1β, LHCII, or the CP26/CP29 minor antenna. In
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contrast, levels of OEE1/2 proteins declined only in TEF30-amiRNA cells. Moreover,
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levels of D1, D2, and CP43 declined in control and TEF30-amiRNA cells, but to a much
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larger extent in the latter, indeed indicating a role of TEF30 in the protection/repair of
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PSII from photoinhibition. We noted elevated levels of PsaA in TEF30-amiRNA cells that
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declined during HL treatment to similar levels as did PsaA in control cells. This
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observation appears to be specific to the strain used here, as we did not detect elevated
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PsaA levels in other TEF30-amiRNA strains.
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Significant photoinhibition of PSII is known to occur also at low light intensities
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under chilling conditions (Greer et al., 1988; Li et al., 2004). This is because
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temperature-dependent reactions during the PSII repair cycle occur with reduced pace
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at low temperatures, while photochemical reactions are temperature-independent
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(Tyystjarvi, 2013). To test, whether TEF30-amiRNA cells are also more prone to
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photoinhibition under chilling conditions, we shifted control and TEF30-amiRNA cells
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grown at ~30 µmol photons m-2 s-1 from 25°C to 10°C for 18 h and monitored levels of
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TEF30 and of marker subunits of the four major thylakoid membrane complexes by
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immunoblotting. As shown in Figure 5C, chilling led to a slight increase in TEF30 protein
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levels in control cells, but in cells of neither strain did chilling affect levels of PSAD,
374
cytochrome f, and CF1β. However, chilling did induce a decline of D1 levels in TEF30-
375
amiRNA cells, while it did not affect D1 levels in control cells, therefore corroborating the
376
notion that TEF30 plays a role in the protection/repair of PSII from photoinhibition.
377
We made the interesting observation that the accumulation of the LHCSR3
378
protein in HL was impaired in TEF30-amiRNA cells when compared to control cells,
379
while that of VIPP1 was unaffected (Figure 5B). The reduced accumulation of the
380
LHCSR3 protein in TEF30-amiRNA cells was only observed at time points later than 1 h
381
of HL exposure and, as revealed by RT-qPCR, was due to a reduced induction of
382
LHCSR3 gene expression (Figure 6A and Supplemental Figure S6A). Moreover, this
383
effect was only observed in cells grown under mixotrophic conditions with ammonium as
384
nitrogen source (TAP-NH4 medium), while it was not observed in cells grown
385
mixotrophically on nitrate (TAP-NO3) or photoautotrophically on ammonium (TMP-NH4).
386
Accordingly, the induction of NPQ was similar in photoautotrophically grown control and
387
TEF30-amiRNA cells (Supplemental Figure S6B).
388
The induction of LHCSR3 gene expression was shown to correlate with the PFD
389
applied and to be abolished in the presence of photosystem II inhibitor 3-(3,4-
390
dichlorophenyl)1,1-dimethylurea (DCMU) and therefore appears to correlate with the
391
rate of linear electron flow (LEF) (Naumann et al., 2007; Petroutsos et al., 2011;
392
Maruyama et al., 2014). Therefore, a smaller LEF in TEF30-amiRNA cells versus control
393
cells grown mixotrophically in the presence of ammonium and exposed to HL could
13
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394
provide an explanation for the reduced induction of LHCSR3 gene expression in TEF30-
395
amiRNA cells. This decrease in LEF might be a consequence of an increased fraction of
396
photodamaged PSII in TEF30-amiRNA cells. To test this idea, we measured oxygen
397
evolution as a direct measure for LEF rates on thylakoid membranes isolated from
398
TEF30-amiRNA and control cells grown at LL (~30 µmol photons m-2 s-1) or HL
399
(~800 µmol photons m-2 s-1) for 4 h. As shown in Figure 6B, the oxygen evolution
400
capacity at 600 µmol photons m-2 s-1 was indeed ~25% lower in HL-grown TEF30-
401
amiRNA cells compared with control cells, while no difference was seen in LL-grown
402
cells.
403
404
Neither PSII de novo synthesis nor PSII stability, but efficient repair of
405
photodamaged PSII is impaired in TEF30-amiRNA cells
406
We observed that levels of the D1 protein of PSII were sometimes slightly lower in LL-
407
grown TEF30-amiRNA cells than in control cells (Figure 5B and Figure 5C). We
408
envisaged two explanations for this finding, namely that TEF30 plays a role in D1
409
synthesis during de novo biogenesis of PSII reaction centers, or that a role of TEF30 in
410
counteracting photoinhibition leads to increased photodamage of PSII in TEF30-amiRNA
411
cells also under LL conditions. To distinguish between these two possibilities, we
412
induced PSII degradation in control and TEF30-amiRNA cells by sulfur starvation for 48
413
h and monitored the re-accumulation of PSII subunits through de novo synthesis upon
414
sulfur repletion for another 24 h (Kosourov et al., 2005; Malnoe et al., 2014). As shown
415
in Figure 7A and Figure 7B, levels of D1, D2, and CP43 declined faster during sulfur
416
starvation in TEF30-amiRNA cells when compared to control cells, but recovery kinetics
417
were the same in both strains. As PSII subunits re-accumulating during sulfur repletion
418
may not necessarily be incorporated into PSII supercomplexes, we analyzed PSII
419
supercomplexes by BN-PAGE during sulfur starvation (for 24 h) and sulfur repletion (for
420
6 h) (Figure 7C and Figure 7D). As judged from D1 and CP43 signals from immunoblots
421
on native gels, PSII supercomplexes in TEF30-amiRNA cells declined and re-
422
accumulated during sulfur starvation and repletion as fast as those in control cells.
423
Interestingly, PSII dimers declined faster in TEF30-amiRNA cells during sulfur
424
starvation, but during sulfur repletion re-accumulated with the same kinetics as in control
14
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425
cells. Moreover, levels of unassembled CP43 were higher in TEF30-amiRNA cells than
426
in control cells under control conditions and during sulfur repletion (Figure 7C).
427
Although these data do not support a role of TEF30 in PSII de novo biosynthesis,
428
it was still possible that the absence of TEF30 during PSII de novo synthesis rendered
429
PSII more unstable. To test this, we grew TEF30-amiRNA and control cells under LL
430
conditions, added chloramphenicol (CAP) to inhibit de novo synthesis of plastid-encoded
431
proteins, and left the cultures at LL, placed them into the dark, or exposed them to 200
432
µmol photons m-2 s-1 and monitored levels of PSII subunits. We found D1 levels to
433
decline faster in TEF30-amiRNA cells than in control cells only if CAP-treated cultures
434
were placed in the light (Figure 8). Hence, PSII complexes assembled in TEF30-
435
amiRNA cells are not generally more unstable than those assembled in control cells.
436
We could, however, still not rule out the possibility that improper de novo
437
assembly of PSII in TEF30-amiRNA cells increased the sensitivity of PSII to
438
photodamage. To address this possibility, we exposed cells from control and TEF30-
439
amiRNA strains to HL of 1800 µmol photons m-2 s-1 in the presence of CAP for 1 h. CAP
440
was added to prevent the repair of photodamaged PSII during HL exposure, and the
441
exposure time of 1 h was chosen because after 1 h HL treatment LHCSR3 levels were
442
still comparable in both strains (Figure 6A). After HL exposure, CAP was washed out to
443
allow PSII repair to proceed. As there was no significant difference regarding the decline
444
in PSII maximum quantum efficiency (Fv/Fm) and D1 protein levels between both strains
445
after HL exposure, we can conclude that PSII is equally light sensitive in TEF30-amiRNA
446
and control cells (Figure 9). However, Fv/Fm values and D1 protein levels recovered
447
significantly faster in control than in TEF30-amiRNA cells, strongly suggesting that
448
TEF30 plays a role in PSII repair from photodamage.
449
450
TEF30-amiRNA cells appear to be impaired in PSII supercomplex formation during
451
PSII repair
452
To get an idea at which step of the PSII repair cycle TEF30 might be involved, we
453
analyzed the partitioning of the D1 protein into PSII core and supercomplexes in cells
454
from control and TEF30-amiRNA cells by native Deriphat-PAGE (Formighieri et al.,
455
2012). As shown in Figure 10, there was no difference in the partitioning of D1 in cells of
456
both strains grown under LL conditions. However, much less D1 was found to
15
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
457
accumulate in PSII supercomplexes than in core complexes in TEF30-amiRNA cells
458
compared to control cells when cells had been exposed to HL of 800 µmol photons m-2
459
s-1 for 4 h. TEF30 in native extracts from both strains co-migrated quantitatively with PSII
460
monomers, again corroborating that TEF30 specifically interacts with PSII monomers,
461
but not with PSII supercomplexes. While TEF30 levels were clearly reduced in TEF30-
462
amiRNA cells, HL inducibility and co-migration of TEF30 with monomers were retained
463
in mutant cells. Over all, these data indicate a possible role of TEF30 in redirecting
464
repaired PSII monomers from stroma exposed regions back to appressed regions for
465
PSII supercomplex reassembly after repair.
466
467
Thylakoid membrane stacks in TEF30-amiRNA cells contain fewer lamellae than
468
control cells
469
Finally, to investigate whether depletion of TEF30 had any phenotypes on thylakoid
470
membrane ultrastructure, we analyzed a total of 500 images by transmission electron
471
microscopy from cells of two control and three independent TEF30-amiRNA strains
472
grown at LL or at HL of ~800 µmol photons m-2 s-1 for 4 h. In ~66% of cw15-325 control
473
cells grown in LL we found stacks containing at least 10 thylakoid lamellae (Figure 11)
474
(Nordhues et al., 2012). However, only ~22% of LL-grown TEF30-amiRNA cells
475
contained stacks with more than 10 lamellae, whereas thylakoids were much more
476
loosely organized in ~78% of the mutant cells (Figure 11). While the fraction of cells
477
containing thylakoid stacks with more than 10 lamellae remained unchanged in HL-
478
exposed cells (~61% in control and ~21% in TEF30-amiRNA cells), 26% of control and
479
mutant cells contained swollen thylakoids. The fraction of cells with loosely organized
480
thylakoids declined in control and mutant cells (to 13% and 53%, respectively). These
481
data suggest that the depletion of TEF30 results in less pronounced thylakoid
482
membrane stacking in cells grown in both, LL and HL intensities.
483
484
Discussion
485
We report here on the functional characterization of the TEF30 protein in
486
Chlamydomonas reinhardtii. As a protein present in the green algal, flowering and
487
nonflowering plant lineages, but not detected in nonphotosynthetic organisms, TEF30 is
488
one out of 597 GreenCut proteins. Moreover, because TEF30 is not present in
16
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
489
cyanobacteria, C. merolae, P. tricornutum, or T. pseudonana, it is part of the ViridiCut
490
subcategory comprising 312 proteins (Supplemental Figure S1A; Merchant et al., 2007;
491
Heinnickel and Grossman, 2013). ViridiCut proteins are likely enriched in green lineage-
492
specific functions, such as mechanisms of chlorophyll a/b protein regulation (Karpowicz
493
et al., 2011) – a prediction that according to the results of this study turned out to be
494
correct.
495
496
TEF30 is a chloroplast protein associated with PSII monomers at the stromal side
497
of thylakoid membranes
498
Immunological data on isolated organelles and immunofluorescence data clearly
499
indicate that TEF30 is localized in the chloroplast (Figure 1B and Figure 1C). Cell
500
fractionation and trypsin digestion experiments revealed TEF30 to be largely associated
501
with the stromal side of thylakoid membranes, although no transmembrane helices were
502
predicted in TEF30. Accordingly, fractionations done in the presence of (chaotropic)
503
salts indicated that the membrane association of TEF30 is realized via electrostatic
504
interactions (Figure 2). The following evidence indicates that TEF30’s association with
505
thylakoid membranes is realized by its direct interaction with PSII monomers: (i) TEF30
506
was largely recovered in soluble fractions when mutant cells deficient in D1, D2, CP43,
507
or CP47 proteins were fractionated into membrane and soluble fractions (Figure 2C). (ii)
508
TEF30 co-eluted with affinity-purified PSII particles (Figure 3). (iii) TEF30 co-migrated
509
quantitatively with PSII monomers on sucrose density gradients, on blue-native gels,
510
and on native Deriphat gels (Figure 4 and Figure 10). (iv) Co-migration of TEF30 with
511
PSII monomers was abolished if thylakoid membrane proteins were derived from PSII
512
mutants (Figure 4B and Figure 4C).
513
With TEF30 and D2 representing ~0.05 and ~0.59 ng protein per ng chlorophyll
514
(Figure 1D) and exhibiting molecular masses of 33.3 and 39.45 kDa, respectively,
515
TEF30 on a molar basis is ~10 times less abundant than D2. Given that some TEF30
516
was also recovered in soluble fractions and that TEF30 was found to be able to form
517
oligomers (Figure 1E, Figure 2A and Figure 2C), the stoichiometry of TEF30
518
(oligomers?) to PSII is likely below 1:10. PSII monomers are not the main active form of
519
PSII and are mainly located to stroma thylakoids (Danielsson et al., 2006). Therefore,
520
TEF30’s association with PSII monomers is in agreement with the calculated
17
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
521
stoichiometries between TEF30 and D2. A localization of TEF30 to stroma lamellae was
522
also indicated by the finding that TEF30 and CF1β with a known location to stroma
523
lamellae shared the same sensitivity to trypsin digestion of isolated thylakoid
524
membranes. Consistently, proteomics approaches on thylakoid membranes separated
525
into grana and stroma lamellae identified the Arabidopsis TEF30 homolog (AT1G55480)
526
exclusively in stroma-lamellae (Tomizioli et al., 2014).
527
528
TEF30 is involved in PSII repair
529
TEF30 shares its ability to associate with PSII components at the stroma side with a
530
number of other stromal proteins, including Deg7 (Sun et al., 2010), THF1/Psb29/NYC4
531
(Wang et al., 2004; Huang et al., 2013), AtPDI6 (Wittenberg et al., 2014), LPA3 (Cai et
532
al., 2010), and ROC4 (Lippuner et al., 1994; Cai et al., 2008). While Deg7,
533
THF1/Psb29/NYC4, AtPDI6, and ROC4 play roles in the PSII repair cycle (Keren et al.,
534
2005; Cai et al., 2008; Sun et al., 2010; Huang et al., 2013; Yamatani et al., 2013;
535
Wittenberg et al., 2014), LPA3 appears to be required for coordinating the membrane
536
insertion of de novo synthesized CP43 (Cai et al., 2010).
537
Of these possible functions of TEF30 (PSII de novo synthesis or repair), our data
538
argue against a role of TEF30 in PSII de novo synthesis. This is because control and
539
TEF30-amiRNA cells, in which PSII degradation was induced by sulfur starvation
540
(Kosourov et al., 2005; Malnoe et al., 2014), re-accumulated de novo synthesized PSII
541
core components, dimers and supercomplexes with the same kinetics after sulfur
542
repletion (Figure 7). As judged from blue native and native Deriphat gels, PSII
543
(super)complexes in TEF30-underexpressing cells also appear to be correctly
544
assembled (Figure 7C and Figure 10). This is supported by the observation that in the
545
presence of chloroplast protein synthesis inhibitors, PSII stability in the dark and PSII
546
sensitivity to HL exposure were comparable with those of control cells (Figure 8C and
547
Figure 9). Moreover, O2 evolution capacities of LL-grown cells were the same in control
548
and TEF30-underexpressing strains (Figure 6B).
549
Rather, our results point to a role of TEF30 in the PSII repair cycle: most
550
importantly, PSII activity in the absence of protein synthesis inhibitors was more
551
sensitive to HL and recovered more slowly in TEF30-underexpressing cells than in
552
control cells (Figure 5A; Supplemental Figure S5). This was reflected by the
18
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
553
accumulation kinetics of PSII core subunits, which after HL exposure or in LL under
554
chilling conditions (known to enhance photoinhibition (Greer et al., 1988; Li et al., 2004))
555
declined more pronouncedly in TEF30-underexpressing than in control cells, while minor
556
and major antenna were unaffected (Figure 5B and Figure 5C). Moreover, PSII stability
557
in the presence of chloroplast protein synthesis inhibitors was impaired in TEF30-
558
underexpressing cells only when they were grown in the light, but not when they were
559
grown in the dark (Figure 8). Finally, like TEF30-underexpressing cells, mutants in
560
factors known to be involved in PSII repair hardly display any phenotypes if not grown
561
under high / fluctuating light (Supplemental Figure S4) (Keren et al., 2005; Sirpio et al.,
562
2007; Cai et al., 2008; Sun et al., 2010).
563
At which step of the PSII repair cycle may TEF30 play a role? Native Deriphat
564
gels indicated a strongly reduced accumulation of PSII supercomplexes in TEF30-
565
underexpressing versus control cells following an exposure to HL for 4 h (Figure 10).
566
This observation, combined with the finding that TEF30 did not co-migrate with PSII-
567
LHCII supercomplexes on sucrose density gradients, Deriphat- and BN-PAGE (Figure 4
568
and Figure 10), points to a role of TEF30 in directing repaired PSII monomers from
569
stroma-exposed regions back to stacked regions for PSII dimer/supercomplex re-
570
assembly, and/or in stabilizing/protecting repaired PSII monomers (Figure 12). In
571
Arabidopsis, grana margins (when compared with grana cores and stroma membranes)
572
were shown to represent the membrane regions in which PSII core subunits are mainly
573
dephosphorylated, D1 levels are lowest and FtsH levels highest and were therefore
574
identified as the main thylakoid subdomain for proteolytic degradation of D1 (Tietz et al.,
575
2015). Hence, if unprotected PSII monomers in TEF30-underexpressing cells would
576
linger too long in such proteolytically active domains after repair, they may undergo
577
further cycles of D1 degradation and replacement, thus explaining the inefficient PSII
578
repair in TEF30-underexpressing cells. However, TEF30 may also facilitate complex re-
579
assembly (e.g. of temporally released CP43) or the incorporation of de novo synthesized
580
D1 during repair (Figure 12). In the absence of de novo D1 synthesis during PSII repair,
581
PSII core monomer complexes completely disappeared from stroma membranes (Aro et
582
al., 2005). PSII monomers whose re-assembly is delayed during repair may therefore be
583
more prone to proteolytic attack. It is important to note that TEF30 apparently facilitates
584
the PSII repair cycle but is not essential for it. The PSII repair cycle exists in all
19
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
585
organisms capable of performing oxygenic photosynthesis but TEF30 is found only in
586
the Viridiplantae. Therefore, its role in facilitating PSII repair may have evolved to help
587
overcome constraints to the PSII repair cycle that are specific to the Viridiplantae.
588
589
Reduced accumulation of LHCSR3 and reduced thylakoid membrane stacking
590
may be pleiotrophic effects of impaired PSII repair in TEF30-underexpressing cells
591
Upon HL exposure, the LHCSR3 protein accumulated to lower levels in TEF30-
592
underexpressing cells than in control cells (Figure 5B). LHCSR3 becomes activated by
593
an increased acidification of the thylakoid lumen and mediates the dissipation of excess
594
light energy by a protonation-induced interaction with PSII supercomplexes which is
595
dependent on the PSBR protein (Bonente et al., 2011; Tokutsu and Minagawa, 2013;
596
Xue et al., 2015). The reduced accumulation of LHCSR3 in TEF30-underexpressing
597
cells was observed only when cells were exposed for more than 1 h to HL and was
598
realized at the level of gene expression (Figure 6A and Supplemental Figure S6A).
599
Moreover, the effect was only observed in cells grown mixotrophically with ammonium
600
as nitrogen source and not in cells grown under photoautotrophic conditions with
601
ammonium, or grown mixotrophically with nitrate as nitrogen source (Figure 6A). Hence,
602
it appear as if the impaired induction of the LHCSR3 gene in TEF30-underexpressing
603
cells is limited to conditions in which sinks for products of the light reactions are limiting
604
(nitrate reduction and carbon fixation are highly ATP- and NADPH-demanding).
605
LHCSR3 gene expression was shown to correlate with the PFD applied and thus with
606
linear electron flow (LEF) rates (Naumann et al., 2007; Peers et al., 2009; Petroutsos et
607
al., 2011; Maruyama et al., 2014). Accordingly, in cells grown mixotrophically in the
608
presence of ammonium and exposed to HL for >1 h, LEF rates are supposed to be
609
lower if TEF30 levels are low. This indeed appeared to be the case, as the oxygen
610
evolution capacity was ~25% lower in HL-exposed TEF30-underexpressing cells than in
611
control cells, but unaltered in LL-grown cells (Figure 6B). Therefore, we propose that the
612
reduced accumulation of LHCSR3 in TEF30-underexpressing cells grown under
613
apparently sink-limited conditions is a consequence of increased PSII photoinhibition
614
and thus of reduced LEF under such conditions rather than the cause for increased
615
photoinhibition. In support of this idea, blocking the Calvin-Benson cycle was shown to
20
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
616
inhibit
the
repair
of
photodamaged
PSII,
617
Chlamydomonas (Takahashi and Murata, 2005).
thus
enhancing
photoinhibition
in
618
Using transmission electron microscopy we observed that ~66% of the cells of
619
TEF30-underexpressing strains contained fewer than 10 lamellae in thylakoid
620
membrane stacks, whereas this was true for only ~22% of the cells in control strains
621
(Figure 11). Therefore, the thylakoid organization in control cells under our growth
622
conditions (LL of ~30 µmol photons m-2 s-1) resembles that of shade leaves with many
623
thylakoids per stack, while that of TEF30-underexpressing cells resembles the
624
organization found in sun leaves, with fewer lamellae per stack (Guillot-Salomon et al.,
625
1978; Lichtenthaler et al., 1981). One possible explanation for this phenotype is that
626
TEF30 directly affects thylakoid stacking and impaired PSII repair is a consequence of
627
this. The number of membrane layers in grana stacks was shown to correlate with the
628
abundance of the CURT1 protein, which is located to grana margins and induces
629
membrane curvature (Armbruster et al., 2013). Hence, TEF30 might positively modulate
630
the function of CURT1, leading to reduced membrane stacking in TEF30-
631
underexpressing cells. An argument against this idea is that reduced thylakoid
632
membrane stacking was shown to improve PSII repair by facilitating lateral migration of
633
damaged PSII supercomplexes and therefore increasing their accessibility to the FtsH
634
protease (Tikkanen et al., 2008; Fristedt et al., 2009; Kirchhoff et al., 2013; Nath et al.,
635
2013b). Indeed, reduced membrane stacking may account for the faster degradation
636
rates of PSII dimers and supercomplexes upon sulfur starvation in TEF30-
637
underexpressing cells (Figure 7), but conflicts with their impaired capacity for PSII repair.
638
A second, more plausible explanation for the reduced thylakoid membrane stacking in
639
TEF30-underexpressing cells is that it represents a countermeasure of these cells to
640
improve PSII repair, e.g. by reducing CURT1 function at the levels of CURT1 protein
641
accumulation or phosphorylation state, or by increasing the phosphorylation state of PSII
642
core subunits (Tikkanen et al., 2008; Fristedt et al., 2009; Goral et al., 2010; Samol et
643
al., 2012; Armbruster et al., 2013; Kirchhoff et al., 2013; Tietz et al., 2015).
644
645
Did an additional function for TEF30 in supercomplex assembly of de novo
646
synthesized PSII evolve in land plants?
21
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
647
The Arabidopsis TEF30 homolog (AT1G55480) was first described by Ishikawa et al.
648
(2005), who termed it ZKT for protein containing a PDZ domain, a K-box region, and a
649
TPR region and proposed a role for the protein in wounding responses. Very recently,
650
the same protein was named MET1, for M-enriched thylakoid protein 1 (where M stands
651
for mesophyll cells) (Bhuiyan et al., 2015). Our results on Chlamydomonas TEF30 are
652
largely congruent with those from the excellent study by Bhuiyan et al. with respect to
653
the inducibility of TEF30/MET1 by HL, the interaction of the proteins with the stroma side
654
of PSII complexes in thylakoid membranes, the lack of obvious phenotypes in mutants
655
under standard growth conditions, as well as the increased sensitivity of PSII and the
656
reduced accumulation of PSII supercomplexes in TEF30/MET1-depleted mutants
657
compared to controls upon HL exposure. Therefore we agree with Bhuiyan et al. that a
658
function of TEF30/MET1 in wounding is unlikely.
659
However, there are also differences between traits observed for TEF30/MET1 in
660
Chlamydomonas and Arabidopsis, which led to slightly different conclusions regarding
661
the function of this protein. By immunoblot analysis of Arabidopsis thylakoid membrane
662
protein complexes separated by BN-PAGE, Bhuiyan et al. found MET1 to co-migrate
663
with several PSII (sub)complexes including dimers, monomers, CP43-less monomers,
664
reaction centers, and unassembled proteins. With the yeast 2-hybrid system they also
665
found MET1 to directly interact with stromal loops of CP43 and CP47. Based on these
666
data Bhuiyan et al. concluded that MET1 is involved in the folding and/or stabilization of
667
these stroma-exposed CP43/CP47 domains, thereby ensuring their correct configuration
668
upon assembly of the PSII core during de novo PSII biogenesis and during the PSII
669
repair cycle. By analyzing Chlamydomonas thylakoid membrane protein complexes on
670
sucrose density gradients, BN-PAGE and Deriphat-PAGE, we observed TEF30 to
671
quantitatively interact with PSII monomers and observed only traces of TEF30 to co-
672
migrate with larger PSII assemblies (sucrose gradients, Figure 4B) or smaller ones
673
(Deriphat-PAGE, Figure 10). Moreover, based on the finding that PSII supercomplex
674
formation was only impaired in TEF30-underexpressing cells pushed towards PSII repair
675
by HL exposure (Figure 10) and not in cells pushed towards de novo PSII synthesis
676
during recovery from sulfur starvation (Figure 7), we concluded that TEF30 is rather
677
involved in PSII repair and not in the assembly of de novo synthesized PSII complexes.
678
Whether these differences are due to experimental setups or whether MET1 has
22
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
679
acquired an additional role in PSII de novo assembly during land plant evolution remains
680
to be elucidated. The latter idea may be supported by the finding that PSII de novo
681
synthesis and repair appear to be spatially separated in Chlamydomonas (Uniacke and
682
Zerges, 2007), while both processes appear to be only temporally separated during cell
683
differentiation in land plants (Nickelsen and Rengstl, 2013).
684
685
Materials and Methods
686
Chlamydomonas strains and culture conditions
687
Chlamydomonas strain cw15-325 (cwd, mt+, arg7-) and cw15-302 (cwd, mt+, arg7-, nit-),
688
kindly provided by R. Matagne, University of Liège, Belgium, were used as recipient
689
strains for transformation with plasmid pCB412 (containing only the ARG7 gene)
690
(Schroda et al., 1999) to generate control strains, or with pMS632 (containing the ARG7
691
gene and the TEF30-amiRNA construct) to downregulate TEF30. Transformations were
692
carried out using 1 µg of HindIII-linearized plasmids and vortexing with glass beads
693
(Kindle, 1990). Photosynthesis mutants used were F23 (lacking PsaA) (Girard et al.,
694
1980), FUD34 (lacking CP43) (Rochaix et al., 1989), FUD44 (lacking OEE1) (Mayfield et
695
al., 1987), ΔbB/ ΔpsbB (lacking CP47) (Minai et al., 2006), FUD47 and nac2 (both
696
lacking D2) (Erickson et al., 1986; Kuchka et al., 1989), FUD50 (lacking CF1β)
697
(Woessner et al., 1986), ΔpetA (lacking cyt f) (Rimbault et al., 2000), and a double
698
mutant ΔpsbA/ΔpsbD (lacking D1 and D2). Strain cc124 (mt-) was employed as a wild-
699
type control for experiments with photosynthesis mutants. Strain D2-H (Sugiura et al.,
700
1998) was a gift from J. Minagawa, National Institute for Basic Biology, Japan. Unless
701
specified otherwise, all strains were grown mixotrophically in Tris-acetate-phosphate
702
(TAP) medium (Harris, 2008) supplemented with 7.5 mM NH4Cl and a revised
703
micronutrient composition (Kropat et al., 2011) on a rotatory shaker at 25°C and
704
~30 μmol m-2 s-1. Minimal medium (TMP) was prepared in the same way, but lacking
705
acetate. Cultures were diluted the day before experiments were conducted and grown to
706
mid-log phase. Cell densities were determined using a Z2 Coulter Counter (Beckman
707
Coulter).
708
709
Phylogenetic analyses
23
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
710
Multiple
sequence
alignments
711
Chlamydomonas
712
(XP_001416302),
Micromonas
713
(XP_002955399),
Physcomitrella
714
moellendorffii
715
(AP004670.4),
716
(XP_003629926), and Glycine max (XP_001235451) using the ClustalW2.1 program
717
((Larkin et al., 2007). Transit peptide sequences were predicted with ChloroP
718
(Emanuelsson et al., 1999) or TargetP (Emanuelsson et al., 2000) programs. The
719
phylogenetic tree was inferred by the neighbor-joining method using the MEGA5.1
720
program (Tamura et al., 2011) and statistical support was assessed with 1000 bootstrap
721
replicates.
reinhardtii
were
with
(Cre01.g031100.t1.2),
(XP_002988351),
Arabidopsis
performed
patens
thaliana
mays
sequences
Ostreococcus
(XP_003059792),
pusilla
Zea
protein
(XP_008652074),
(AT1G55480.1),
lucimarinus
Volvox
(Phpat.019G052300.1),
carteri
Selaginella
Oryza
Medicago
from
sativa
truncatula
722
723
Cloning, expression and purification of recombinant TEF30
724
The TEF30 coding sequence was amplified from Chlamydomonas cDNA with primers 5’-
725
tactggatccgccgaatacatcgagctgga-3’
726
The resulting 817-bp PCR product was digested with BamHI and HindIII and cloned in
727
frame into the PetDuet vector (Novagen) cleaved with the same enzymes, yielding
728
pMS649. TEF30 was overexpressed in E. coli BL21 and purified by nickel-nitrilotriacetic
729
acid agarose (Ni-NTA) according to the manufacturer’s instructions (Qiagen). Imidazole
730
was largely removed by three successive runs of concentration/dilution with KMH buffer
731
(20 mM HEPES-KOH pH 7.2, 80 mM KCl, 2.5 mM MgCl2) using Amicon Ultra-4 tubes
732
(Millipore). After that, proteins were supplemented with 10% glycerol and 1 mM
733
dithiothreitol (DTT). For antibody generation TEF30 was purified under denaturing
734
conditions with guanidine-HCl and eluted with 200 mM imidazole in 6 M urea. 1 mg of
735
purified protein was used for a 3-months rabbit immunization protocol (SeqLab) for
736
antibody generation.
and
5’-gcaacatgttcaacaaggagaagtaagcttactca-3’.
737
738
Protein analyses and thylakoid membrane isolation
739
Protein extractions, SDS–PAGE, semi-dry blotting, and immunodetections were carried
740
out as described before (Liu et al., 2005; Schulz-Raffelt et al., 2007). Proteins on SDS-
741
polyacrylamide gels were loaded on the basis of chlorophyll concentrations or protein
24
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
742
concentrations determined according to (Lowry et al., 1951). Immunodetection was done
743
using enhanced chemiluminescence (ECL) and the FUSION-FX7 Advance™ imaging
744
system (PEQLAB). Antisera described previously were against mitochondrial carbonic
745
anhydrase (Agrisera AS11 1737), HSP70B (Schroda et al., 1999), HSP70A (Schulz-
746
Raffelt et al., 2007), VIPP1 (Liu et al., 2005), LHCSR3 (Naumann et al., 2007), CF1β
747
(Lemaire and Wollman, 1989), PSAD (Naumann et al., 2005), LHCII (Vallon et al.,
748
1986), CP43 (Vallon et al., 1985), Cyt f (Pierre and Popot, 1993), D1 (Agrisera AS05
749
084), OEE33 (Agrisera AS06 142-33), OEE23 (Agrisera AS06 167), PsaA (Agrisera
750
AS06 172), PsaB (Agrisera AS10 695), CP26 (Agrisera AS09 407), CP29 (Agrisera
751
AS06 117), mtCA (Agrisera AS11 1737), D2 (Agrisera AS06 146), and CGE1 (Schroda
752
et al., 2001). Densitometric quantification of the bands after immunodetection was
753
calculated by the FUSIONCapt Advance program for image capture and analysis
754
(PEQLAB). Thylakoids were isolated as described previously (Chua and Bennoun,
755
1975). Briefly, cells were lysed using a BioNebulizer (Glas-Col) with an operating N2
756
pressure of 1.5-2 bar and lysates centrifuged through a sucrose step gradient (0.6, 1.3,
757
and 1.8 M sucrose) at 40,000 rpm for 1 h. Thylakoids floating between the 1.8 and 1.3 M
758
transition were collected, diluted in a buffer containing 5 mM HEPES-KOH pH 7.5 and
759
10 mM EDTA, and pelleted by a centrifugation at 40,000 rpm for 15 min.
760
761
EDC-crosslinking of recombinant TEF30
762
100 ng of recombinant TEF30 was incubated in KMH buffer with 2.5 mM EDC (1-Ethyl-
763
3[3-dimethylaminopropyl]carbodiimid hydrochlorid) and the double concentration of NHS
764
(N-hydroxysuccinimid). The mixture was incubated for 30 min at 25°C. The crosslinking
765
reaction was stopped by the addition of 0.2 M ammonium acetate and samples were
766
analyzed by immunoblotting.
767
768
Cell fractionations
769
Isolation of chloroplasts was performed as described previously (Zerges and Rochaix,
770
1998). Mitochondria were isolated according to (Eriksson et al., 1995), but using a
771
BioNebulizer (Glas-Col) for cell disruption. For the separation of soluble and membrane
772
enriched fractions, 2 x 107 cells were harvested by a 2-min centrifugation at 1300 g and
773
4°C and resuspended in lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 x
25
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
774
protease inhibitor cocktail (Roche)). An aliquot was taken for whole-cell proteins. The
775
remainder was frozen in liquid nitrogen and thawed at 25°C. This cycle was repeated
776
two times. Disrupted cells were centrifuged for 30 min at 18000 g and 4°C. The
777
supernatant was collected as soluble protein fraction. Pellets were resuspended by brief
778
sonication with the same volume of lysis buffer. When indicated, a final concentration of
779
250 mM NaCl, 200 mM Na2CO3, 1 M CaCl2, or 6 M urea was added to the lysis buffer
780
before freeze/thawing the cells. For localization experiments, isolated thylakoids were
781
incubated on ice with 0.1 mg/mL trypsin. After the addition of 0.25 x proteinase inhibitor
782
cocktail (Roche), proteins were precipitated with 80% acetone. Pellets were
783
resuspended in Laemmli buffer for SDS-PAGE (Laemmli, 1970).
784
785
Vector construction and screening for TEF30-underexpressing transformants
786
The amiRNA targeting TEF30 was designed according to (Molnar et al., 2009) using the
787
WMD2 web platform (http://wmd2.weigelworld.org) (Ossowski et al., 2008). The resulting
788
oligonucleotides
789
ctagtAAGTTCGCTCGCGGTGGCGAAtctcgctgatcggcaccatgggggtggtggtgatcagcgctaTTC
790
GTTACCGCGAGCGAACTTg-3’
791
ctagcAAGTTCGCTCGCGGTAACGAAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTT
792
CGCCACCGCGAGCGAACTTa -3’ were annealed by a gradual temperature reduction
793
from 100°C to 60°C (0.3°C/min) in a thermocycler and ligated into the pChlamiRNA2
794
(Molnar et al., 2009), yielding pMS632. Correct constructs were screened as described
795
by Molnar et al. and sequenced. After transformation of this construct into
796
Chlamydomonas cells, 50 arginine-prototrophic transformants of each recipient strain
797
(cw15-325 or cw15-302) were screened by immunoblotting.
5’and
5’-
798
799
Cold stress, high light, photoinhibition and sulfur starvation experiments
800
For cold stress, 150 mL of 2 x106 cells grown at 25°C were incubated under continuous
801
agitation in a water bath at 10°C and ~30 μmol photons m−2 s−1 for 18 h. High light
802
experiments were performed with 150 mL of 2 x106 cells in 400-mL beakers placed onto
803
spots on a rotary shaker that were illuminated (~800 μmol photons m−2 s−1) by Osram
804
HLX 250W 64663 Xenophot bulbs. A glass plate non-permissive to infrared and UV
805
irradiation was placed between light sources and beakers. The position of the beakers
26
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
806
was altered in regular intervals and the cell culture temperature maintained at 25°C. The
807
same setup was used for photoinhibition, but cultures were exposed to ~1800 μmol
808
photons m−2 s−1. For recovery after photoinhibition, cultures were placed on a rotatory
809
shaker at 25°C and ~30 μmol photons m–2 s–1. When indicated, 500 μg mL−1 lincomycin
810
and/or 100 μg mL−1 chloramphenicol were added before photoinhibiton or in the
811
beginning of the recovery phase. To wash out the inhibitors, cells were centrifuged for
812
2 min at 956 g and 25°C and cell pellets were resuspended in fresh TAP-NH4 medium.
813
Photon flux densities (PFD) were determined with a light meter (LI-COR LI-250A). Sulfur
814
starvation experiments were performed in TAP-NH4 according to (Malnoe et al., 2014),
815
but cultures were maintained at ~50 μmol photons m–2 s–1. To follow de novo synthesis
816
of photosystem II, cells were centrifuged and resuspended in TAP-NH4 medium and
817
kept in the light (~30 μmol photons m-2 s–1) for 24 hours.
818
819
Isolation of PSII particles and immunoprecipitation
820
Isolation of PSII particles was performed with ca. 2 L of 3 x106 cells/mL of the D2His
821
strain according to Cullen et al. (2007) with a ProBondTM resin column (Invitrogen). After
822
their elution, PSII particles were mixed with an equal volume of 20 mM MES, pH 6.3,
823
15 mM NaCl, 20% (w/v) PEG 8000, 10 mM ascorbic acid and 10 mM EDTA, and
824
centrifuged at 11,000 g for 10 min. The pellet was resuspended in 20 mM MES pH 6.3,
825
15 mM NaCl, 10 mM ascorbic acid, 10% PEG, 10 mM CaCl2 and centrifuged as before
826
to decrease the imidazole concentration. The pellet containing isolated PSII particles
827
was resuspended in 250 µL of buffer A (20 mM MES, pH 6.3, 15 mM NaCl, 333 mM
828
sucrose, and 10 mM CaCl2). The same procedure was followed with cc124 cells as
829
control. The coupling of antibodies against TEF30 and of preimmune serum to protein A-
830
Sepharose beads for immunoprecipitation was carried out as described previously
831
(Schroda et al., 2001; Liu et al., 2005). Coupled beads were washed twice in buffer A
832
and incubated with 250 µL of isolated PSII particles for one hour at 4°C. Beads were
833
then washed three times in buffer B (buffer A with the addition of 0.1% (v/v) of Triton
834
X-100) and two times in 10 mM Tris-HCl, pH 7.5. Immunopreciptated proteins were
835
collect by boiling the beads in 2% SDS and 12% sucrose for 40 sec. After addition of 0.1
836
M DTT, eluates were subjected to SDS-PAGE.
837
27
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
838
Blue native-PAGE, Deriphat-PAGE and sucrose density centrifugation
839
Blue native-PAGE (BN-PAGE) was performed with isolated thylakoids, crude membrane
840
fractions or whole-cell proteins according to (Schagger and von Jagow, 1991; Schagger
841
et al., 1994). Native Deriphat-PAGE was performed according to (Peter and Thornber,
842
1991) with a 4% acrylamide stacking gel poured upon the 7.5% acrylamide running gel.
843
Thylakoid membranes (0.25 mg chlorophyll/mL) were solubilized for 20 min on ice in 5
844
mM HEPES-KOH pH 7.5 and 1 mM EDTA containing 1% α-dodecyl maltoside (α-DDM).
845
For sucrose density centrifugation, isolated thylakoids were solubilized with 1% β-
846
dodecyl maltoside (β-DDM) and loaded onto a linear 0.2 to 0.5 M sucrose gradient
847
containing 10 mM HEPES-KOH pH 7.5 and 0.05% β-DDM. On the bottom of the
848
gradient a 2 M sucrose cushion was present. The gradient was centrifuged at 205,000 g
849
for 16 h at 4°C and 25-26 fractions were collected from bottom to top.
850
851
Immunofluorescence staining and transmission electron microscopy
852
Immunofluorescence microscopy was performed with control transformants in the cw15-
853
325 strain background. Cells were fixed and stained as described previously (Uniacke et
854
al., 2011). Primary antibodies were against TEF30, HSP70B and HSP70A, used in
855
1:300, 1:8000, 1:4000 dilutions, respectively. As a secondary antibody we used a
856
fluorescein isothiocyanate–labeled goat anti-rabbit antibody (Sigma- Aldrich) in 1:500
857
dilution. After incubation with the secondary antibody, slides were washed in PBS and a
858
drop of mounting solution containing DAPI (Vectashield, Vector Laboratories, United
859
Kingdom) was applied at the center of each the slide. Images were obtained with an
860
OLYMPUS BX53 microscope with a violet filter for DAPI and a green filter for FITC and
861
an OLYMPUS DP26 color camera. For transmission electron microscopy, cells were
862
collected, fixed and embedded as described previously (Nordhues et al., 2012). Images
863
were analyzed with a JEM-2100 (Jeol) or EM 902A (Carl Zeiss) transmission electron
864
microscope (both operated at 80 kV). Micrographs were taken using a 4080x4080 pixel
865
or 1350x1040 pixel charge-coupled device camera (UltraScan 4000 or Erlangshen
866
ES500W, respectively; Gatan) and the Gatan Digital Micrograph software (version
867
1.70.16).
868
869
RNA extraction and qRT-PCR
28
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
870
2 x107 cells were collected for RNA extraction with RNeasy Plant Mini kit (Qiagen)
871
following the manufacturer’s instructions. DNA contaminations were removed with
872
DNAse (RNase-free Turbo DNase, Ambion). cDNA synthesis was done with the MULV
873
reverse transcriptase (Promega), deoxynucleotide triphosphate and oligo(dT)18 primers.
874
qRT-PCR was performed using the StepOnePlus RT-PCR system (Applied Biosystems)
875
and the Maxima SYBR Green kit from Fermentas as described in (Strenkert et al.,
876
2011). Primers for LHCSR3.1 were the same as described in (Peers et al., 2009).
877
878
Biophysical measurements
879
Fluorescence induction measurements were performed at 25°C with a DeepGreen
880
Fluorometer (JBeamBio). The maximum quantum efficiency of PSII, Fv/Fm, was
881
determined after incubating cells in the dark for 1 min according to the equation Fv = Fm -
882
F0, where F0 represents the fluorescence when the electron acceptor of PSII is fully
883
oxidized and Fm the maximum PSII fluorescence. Fm was measured during 250 ms
884
saturating flash of light or in the presence of 10 mM DCMU. Low-temperature
885
fluorescence emission spectra were performed with a laboratory built setup. Samples
886
were submerged in liquid nitrogen and excited with a LED source (LS-450, Ocean Optics
887
- blue LED, 450 nm). The emission spectra were measured by a CCD spectrophotometer
888
(QE6500, Ocean Optics). The device was calibrated by the manufacturer and calibration
889
was verified using standard band-pass filters. For the fluorescence quenching
890
measurements, cells were grown mixotrophically in TAP-NH4 medium at 50 µmol photons
891
m-2 s-1, harvested in the exponential phase and resuspended in TMP-NH4 at a
892
concentration of 2 x 107 cells per mL. Cells were agitated and exposed to HL intensities of
893
500 µmol photons m-2 s-1 provided by a WH1200 Phyto (JBeambio) to induce the
894
accumulation of LHCSR3. The NPQ response was measured using a chlorophyll
895
fluorescence imaging system (Johnson et al., 2009). Samples were incubated in the dark
896
under strong agitation for 15 min before each measurement. NPQ was calculated as (Fm
897
- Fm’) / Fm’, where Fm’ is the maximum PSII fluorescence in light (after 10 sec at
898
700 μmol photons m-2 s-1). Fm and Fm’ were measured after exposure to a brief
899
saturating pulse of 2100 μmol photons m-2 s-1 for 250 ms. Oxygen evolution was
900
measured with a Clark-type oxygen electrode in the presence of 0.5 mM 2,6-
901
dichlorobenzoquinone
(2,6-DCBQ)
and
2 mM
potassium
hexacyanoferrate
29
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
(III)
902
(K3Fe(CN)6) as electron acceptors. The measurements were carried out with crude
903
membrane fractions in TMP-NH4 medium at 25°C.
904
905
Supplemental Figure S1. TEF30 protein accumulates in Chlamydomonas cells
906
exposed to high light.
907
908
Supplemental Figure S2. Phylogenetic tree and alignment of TEF30 homologs.
909
910
Supplemental Figure S3. Screening of TEF30-amiRNA transformants.
911
912
Supplemental Figure S4. TEF30-underexpressing transformants exhibit no
913
obvious growth phenotype under a variety of growth conditions.
914
915
Supplemental Figure S5. PSII fluorescence is more affected by high light in
916
TEF30-amiRNA cells than in control cells.
917
918
Supplemental Figure S6. Reduced induction of LHCSR3 expression in TEF30-
919
amiRNA cells is realized at the transcriptional level and NPQ is not affected under
920
photoautotrophic conditions.
921
922
Acknowledgments
923
We would like to thank Jun Minagawa for providing the D2His strain, Michael Hippler for
924
providing antisera against LHCSR3 and PSAD, and Fabrice Rappaport for helping with
925
the biophysical measurements.
926
927
Figure legends
928
Figure 1. Antibody characterization and analysis of TEF30 oligomer formation,
929
cellular abundance and intracellular localization.
930
(A) Immunodetection of TEF30 in cw15-325 whole-cell proteins (WC) corresponding to 2
931
µg chlorophyll compared with 0.25 ng recombinant TEF30 protein.
932
(B) Subcellular localization of TEF30 by immunoblotting. 10 or 3 µg (depending on the
933
antiserum used) protein from whole cells (WC), chloroplasts (cp) and mitochondria (mt)
30
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
934
isolated from strain cw15-302 were separated by SDS-PAGE and immunodetected with
935
antisera against TEF30, mitochondrial carboanhydrase (mtCA), and stromal HSP70B.
936
(C) Microscopy images taken from strain cw15-325. Shown are from left to right: DIC
937
images, DAPI staining, immunofluorescence, merge of DAPI and immunofluorescence,
938
and a schematic drawing that combines information from the images. Antisera used for
939
immunofluorescence were against HSP70B and HSP70A as markers for stroma and
940
cytosol, respectively, and against TEF30. Chloroplast – cp; pyrenoid – P; cytosol – C;
941
flagella – f; immunofluorescence signal – green.
942
(D) 1 to 8 ng of recombinant proteins TEF30 (purified from E. coli) and D2 (purchased
943
from Agrisera) were separated by SDS-PAGE together with cw15-325 whole-cell
944
proteins corresponding to 0.125-2 µg chlorophyll and immunodetected with antibodies
945
against TEF30 and D2.
946
(E) 100 ng of recombinant TEF30 protein was incubated with EDC/NHS, separated on a
947
SDS-polyacrylamide gel, and immunodetected with antibodies against TEF30.
948
949
Figure 2. Localization of TEF30 in thylakoids of Chlamydomonas wild-type and
950
mutant cells.
951
(A) Salt treatments during cell fractionation. Whole cells (WC) of strain cw15-325 were
952
resuspended in lysis buffer (LB) containing the indicated salts, subjected to
953
freeze/thawing, and separated into soluble (S) and pellet (P) fractions. One sample
954
resuspended in LB only was subjected to sonication instead of freeze/thawing. Proteins
955
were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and
956
against integral membrane protein cytochrome f (Cytf) and stromal CGE1 as controls.
957
(B) Trypsin treatment of thylakoid membranes. Isolated thylakoids from strain cw15-325
958
were incubated on ice with trypsin, separated by SDS-PAGE and immunodetected with
959
antibodies against TEF30 and the peripheral CF1β subunit of the ATPase complex,
960
lumenal plastocyanin (PCY1) and integral membrane protein cytochrome f (Cytf) as
961
controls.
962
(C) Cell fractionation of various photosynthesis mutants. Whole cells (WC) of wild type
963
(WT) and thylakoid membrane protein mutants were separated into soluble (S) and
964
pellet (P) fractions via freeze/thawing. Proteins were separated by SDS-PAGE and
31
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
965
immunodetected with antibodies against TEF30 and against integral membrane protein
966
cytochrome f (Cytf) or D1 and stromal CGE1 as controls.
967
968
Figure 3. Co-purification of TEF30 with PSII particles.
969
(A) Nickel-affinity purification of PSII particles. Detergent-solubilized thylakoid
970
membranes from a Chlamydomonas strain harboring a hexahistidine-tagged D2 protein
971
(D2His) and from wild-type control strain cc124 (WT) were subjected to nickel-affinity
972
purification. Thylakoid membrane input proteins corresponding to 1 µg chlorophyll and
973
4% of the imidazole eluate were separated by SDS-PAGE and immunodetected with
974
antibodies against TEF30 and proteins representative for the major thylakoid membrane
975
complexes.
976
(B) Silver staining of proteins immunoprecipitated from hexahistidine-tagged PSII
977
particles with preimmune serum (Pre) and antibodies against TEF30.
978
(C) Immunodetection of proteins immunopreciptated from PSII particles. Purified PSII
979
particles equivalent to ~1 µg chlorophyll (Input) and 20% of the immunoprecipitates
980
obtained with preimmune serum and antibodies against TEF30 (IP) were separated by
981
SDS-PAGE and immunodecorated with the antisera indicated.
982
983
Figure 4. Co-migration of TEF30 with PSII core subunits as revealed by sucrose
984
density gradient centrifugation and BN-PAGE.
985
(A) Thylakoid membranes from strain cw15-325 (1 mg chlorophyll/mL) were solubilized
986
with 1% β-DDM and separated on a linear 0.2 to 0.5 M sucrose gradient at 205,000 g for
987
16 h. Proteins were collected in 25 fractions, separated by SDS-PAGE and detected
988
with colloidal blue staining (upper panel) or immunologically with the antisera indicated
989
(lower panels).
990
(B) Analysis of thylakoid membrane protein complexes by sucrose density gradient
991
centrifugation as described in (A) was done on wild-type strain cc124, PSI mutant F23,
992
and PSII mutant ΔpsbA/D.
993
(C) Analysis of thylakoid membrane protein complexes by BN-PAGE. Thylakoids from
994
wild-type strain cc124, PSI mutant F23, and PSII mutant nac2 were solubilized with 1%
995
β-DDM and proteins corresponding to 5 µg chlorophyll separated on a 5-15%
996
polyacrylamide BN gradient gel, followed by Coomassie staining (left panel) and
32
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
997
immunodetection with antibodies against TEF30 (right panel). Protein bands were
998
assigned to LHCII monomers (#1), LHCII trimers (#2), PSII monomers (#3), PSII II
999
dimers (#4), PSI supercomplexes (#5), and PSII supercomplexes (#6) according to Jarvi
1000
et al. (2011).
1001
1002
Figure 5. PSII activity and levels of PSII core subunits are more sensitive to high
1003
light intensities or low temperatures in TEF30-amiRNA cells than in control cells.
1004
(A) cw15-325 control (Con) and TEF30-amiRNA strains were photoinhibited (PI) for 60
1005
min at 1800 μmol photons m–2 s–1, and allowed to recover at 30 μmol photons m–2 s–1.
1006
PSII fluorescence was measured by pulse amplitude modulated fluorometry. Values
1007
represent the mean from three biological replicates, error bars indicate SD.
1008
(B) Control (Con) and TEF30-amiRNA #1.48 cells in the cw15-325 strain background
1009
were exposed to high PFDs (HL) of ~800 µmol photons m-2 s-1 for 6 h. Whole-cell
1010
proteins corresponding to 0.2, 1 or 2 µg chlorophyll (depending on the antiserum used)
1011
were separated on 10 or 14% SDS-polyacrylamide gels and analyzed by
1012
immunoblotting.
1013
(C) Control (Con) and TEF30-amiRNA #2.48 cells in the cw15-325 strain background
1014
were grown at a PFD of ~30 μmol photons m–2 s–1 and shifted from a growth
1015
temperature of 25°C to 10°C for 18 h. Samples were analyzed as in (B).
1016
1017
Figure 6. Comparison of LHCSR3 expression and oxygen evolution in control and
1018
TEF30-amiRNA cells.
1019
(A) Time course of LHCSR3 accumulation at 600 μmol photons m–2 s–1 (HL) in cw15-
1020
325 control and TEF30-amiRNA cells grown in TAP-NH4, TAP-NO3, or TMP-NH4.
1021
Whole-cell proteins were separated by SDS-PAGE und immunodetected with antibodies
1022
against LHCSR3 and CF1β as loading control.
1023
(B) Oxygen evolution from thylakoid membranes isolated from cw15-325 control and
1024
TEF30-amiRNA cells grown in TAP-NH4 medium at LL of ~30 µmol photons m-2 s-1 or
1025
HL of ~800 µmol photons m-2 s-1 for 4 h. Values represent the mean from experiments
1026
on two independent control and TEF30-amiRNA lines. Error bars indicate SD. (Note that
1027
the same thylakoid preparation was used for all PFDs applied, thus explaining the
1028
decline in O2 evolution at 1600 µmol photons m-2 s-1).
33
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1029
1030
Figure 7. PSII de novo synthesis is not impaired in TEF30-amiRNA strains.
1031
(A) TEF30-amiRNA and control cells in the cw15-325 background were grown in TAP-
1032
NH4 at ~30 µmol photons m-2 s-1 to exponential phase. Cells were then harvested,
1033
washed and resuspended in TAP medium lacking sulfur (-S). After 48 h under –S
1034
conditions, cells were shifted back to regular TAP medium (+S) for another 24 h. Whole-
1035
cell proteins from samples taken during this time course were separated by SDS-PAGE
1036
and PSII polypeptide abundance measured via immunoblotting using the antibodies
1037
indicated and CF1β as loading control.
1038
(B) D1 signal intensities from (A) were quantified using the FUSIONCapt Advance
1039
program. Signals were normalized to the initial D1 levels in each strain. Error bars
1040
represent SD, n = 2.
1041
(C) S-depletion was done as in (A), but time on –S and +S reduced to 24 h and 6 h,
1042
respectively. Thylakoids were solubilized with 1% α-DDM and proteins corresponding to
1043
5 µg chlorophyll separated on a 5-15% polyacrylamide BN gradient gel, followed by
1044
Coomassie staining (upper panel) and immunodetection with antibodies against D1 and
1045
CP43 (lower panels). Protein bands were assigned as in Figure 4C, the asterisk
1046
designates free CP43.
1047
(D) D1 signal intensities from protein bands #6 (PSII supercomplexes) and #4 (PSII
1048
dimers) from (C) were quantified using the FUSIONCapt Advance program. Signals
1049
were normalized to the initial D1 levels in each strain. Error bars represent SD, n = 4.
1050
1051
Figure 8. PSII stability is not impaired in TEF30-amiRNA strains.
1052
(A) TEF30-amiRNA and control cells in the cw15-325 background were grown at ~30
1053
µmol photons m-2 s-1 in TAP-medium in the presence or absence of 100 µg/mL CAP for
1054
12 h. Proteins from whole cells harvested during the time course corresponding to 0.5 or
1055
1 µg chlorophyll (depending on the antiserum used) were analyzed by immunoblotting.
1056
D1 levels were quantified with the FUSIONCapt Advance program. D1 levels were
1057
normalized to the values before the addition of CAP (at 0 h) and the ratio between D1
1058
levels from TEF30-amiRNA strains and controls calculated. Values represent the mean
1059
from four independent replicates, errors bars indicate SD.
34
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1060
(B) TEF30-amiRNA and control cells in the cw15-325 background were grown in TMP
1061
minimal medium at 200 μmol photons m–2 s–1 in the presence of 100 µg/mL CAP for 12
1062
h. Analyses were done as in (A) with two biological replicates.
1063
(C) TEF30-amiRNA and control cells in the cw15-325 background were grown in TAP-
1064
medium in the dark in the presence of 100 µg/mL CAP for 72 h. Analyses were done as
1065
in (A) with two biological replicates.
1066
1067
Figure 9. Not PSII sensitivity to high light, but PSII repair is impaired in TEF30-
1068
amiRNA strains.
1069
(A) Cells of cw15-325 control (Con) and TEF30-amiRNA strains were photoinhibited (PI)
1070
for 60 min at 1800 μmol photons m–2 s–1 in the presence of chloroplast translation
1071
inhibitors chloramphenicol and lincomycin, and allowed to recover at 30 μmol photons
1072
m–2 s–1 (LL) without inhibitors. PSII fluorescence was measured by pulse amplitude
1073
modulated fluorometry. Values represent the mean from two biological replicates, error
1074
bars indicate SD.
1075
(B) Immunoblot analysis of whole-cell proteins collected from the experiments described
1076
in (A). Results from a typical experiment are shown in the upper panel, the mean from
1077
quantified D1 signals (normalized relative to the initial D1 signal) are shown in the lower
1078
panel. Values represent the mean from five biological replicates, error bars indicate SD.
1079
1080
Figure 10. PSII supercomplex accumulation is impaired in TEF30-amiRNA strains
1081
exposed to high light.
1082
cw15-325 control (Con) and TEF30-amiRNA cells were grown at 30 µmol photons m-2 s-1
1083
(LL) and exposed to 800 µmol photons m-2 s-1 for 4 h (HL). Thylakoid membranes were
1084
isolated,
1085
electrophoretically on a 7.5% native Deriphat-polyacrylamide gel. The gel was then
1086
photographed (left panels) and processed for immunoblotting using antibodies against
1087
TEF30 and the D1 protein (right panels). Bands were assigned to free pigments (#0),
1088
antenna
1089
supercomplexes (#4) according to Formighieri et al. (2012).
solubilized
monomers
with
(#1),
1%
LHCII
α-DDM
trimers
and
(#2),
protein
PSI/II
complexes
cores
1090
35
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
(#3),
separated
and
PSI/II
1091
Figure 11. Cells of TEF30-amiRNA strains contain fewer thylakoid membranes per
1092
stack
1093
(A) Electron microscopy images of cw15-325 cells characteristic for the categories
1094
normally stacked, reduced stacking, and swollen. Overview images are shown on the
1095
left and a zoom-in of the region demarcated by the black box is shown on the right. N,
1096
nucleus; P, pyrenoid; S, starch. Bars in overview images correspond to 1 µm and those
1097
in zoom-ins to 0.2 µm.
1098
(B) cw15-325 control (Con) and TEF30-amiRNA cells were grown in TAP-medium at
1099
~30 µmol photons m-2 s-1 (LL) and exposed to ~800 µmol photons m-2 s-1 (HL) for 4 h. 50
1100
electron micrographs each for two control strains and three independent TEF30-amiRNA
1101
strains were taken for each condition and thylakoid phenotypes assessed according to
1102
the categories shown in (A). Values are given in percent and represent mean values
1103
from the two control and three TEF30-amiRNA strains. Error bars indicate SD.
1104
Significance was tested using a t-test (*P < 0.05 and **P < 0.01).
1105
1106
Figure 12. A model for TEF30 function during PSII repair
1107
When PSII is damaged e.g. at high light intensities, the PSII complex enters the repair
1108
cycle. For this, PSII supercomplexes are disassembled and PSII monomers migrate
1109
from stacked into stroma-exposed membrane regions. After PSII monomers are partially
1110
disassembled to generate CP43-less forms, damaged D1 is degraded by FTSH and
1111
DEG proteases and a newly synthesized D1 copy is inserted into PSII monomers. At this
1112
step TEF30 may facilitate incorporation of the new D1 protein and/or reassembly of
1113
CP43, bind to repaired PSII monomers to protect them from undergoing repeated repair
1114
cycles, or facilitate the migration of repaired PSII monomers back to stacked regions for
1115
supercomplex reassembly. These functions are not mutually exclusive.
1116
1117
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Figure 1. Antibody characterization and analysis of TEF30 oligomer formation, cellular abundance and
intracellular localization.
(A) Immunodetection of TEF30 in cw15-325 whole-cell proteins (WC) corresponding to 2 µg chlorophyll compared
with 0.25 ng recombinant TEF30 protein.
(B) Subcellular localization of TEF30 by immunoblotting. 10 or 3 µg (depending on the antiserum used) protein from
whole cells (WC), chloroplasts (cp) and mitochondria (mt) isolated from strain cw15-302 were separated by SDSPAGE and immunodetected with antisera against TEF30, mitochondrial carboanhydrase (mtCA), and stromal
HSP70B.
(C) Microscopy images taken from strain cw15-325. Shown are from left to right: DIC images, DAPI staining,
immunofluorescence, merge of DAPI and immunofluorescence, and a schematic drawing that combines information
from the images. Antisera used for immunofluorescence were against HSP70B and HSP70A as markers for stroma
and cytosol, respectively, and against TEF30. Chloroplast – cp; pyrenoid – P; cytosol – C; flagella – f;
immunofluorescence signal – green.
(D) 1 to 8 ng of recombinant proteins TEF30 (purified from E. coli) and D2 (purchased from Agrisera) were
separated by SDS-PAGE together with cw15-325 whole-cell proteins corresponding to 0.125-2 µg chlorophyll and
immunodetected with antibodies against TEF30 and D2.
(E) 100 ng of recombinant TEF30 protein was incubated with EDC/NHS, separated on a SDS-polyacrylamide gel,
and immunodetected with antibodies against TEF30.
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Figure 2. Localization of TEF30 in thylakoids of Chlamydomonas wild-type and
mutant cells.
(A) Salt treatments during cell fractionation. Whole cells (WC) of strain cw15-325 were
resuspended in lysis buffer (LB) containing the indicated salts, subjected to
freeze/thawing, and separated into soluble (S) and pellet (P) fractions. One sample
resuspended in LB only was subjected to sonication instead of freeze/thawing. Proteins
were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and
against integral membrane protein cytochrome f (Cytf) and stromal CGE1 as controls.
(B) Trypsin treatment of thylakoid membranes. Isolated thylakoids from strain cw15-325
were incubated on ice with trypsin, separated by SDS-PAGE and immunodetected with
antibodies against TEF30 and the peripheral CF1β subunit of the ATPase complex,
lumenal plastocyanin (PCY1) and integral membrane protein cytochrome f (Cytf) as
controls.
(C) Cell fractionation of various photosynthesis mutants. Whole cells (WC) of wild type
(WT) and thylakoid membrane protein mutants were separated into soluble (S) and pellet
(P) fractions via freeze/thawing. Proteins were separated by SDS-PAGE and
immunodetected with antibodies against TEF30 and against integral membrane protein
cytochrome f (Cytf) or D1 and stromal CGE1 as controls.
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Figure 3. Co-purification of TEF30 with PSII particles.
(A) Nickel-affinity purification of PSII particles. Detergentsolubilized thylakoid membranes from a Chlamydomonas
strain harboring a hexahistidine-tagged D2 protein
(D2His) and from wild-type control strain cc124 (WT)
were subjected to nickel-affinity purification. Thylakoid
membrane input proteins corresponding to 1 µg
chlorophyll and 4% of the imidazole eluate were
separated by SDS-PAGE and immunodetected with
antibodies against TEF30 and proteins representative for
the major thylakoid membrane complexes.
(B) Silver staining of proteins immunoprecipitated from
hexahistidine-tagged PSII particles with preimmune
serum (Pre) and antibodies against TEF30.
(C) Immunodetection of proteins immunopreciptated from
PSII particles. Purified PSII particles equivalent to ~1 µg
chlorophyll (Input) and 20% of the immunoprecipitates
obtained with preimmune serum and antibodies against
TEF30 (IP) were separated by SDS-PAGE and
immunodecorated with the antisera indicated.
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Figure 4. Co-migration of TEF30 with PSII core
subunits as revealed by sucrose density gradient
centrifugation and BN-PAGE.
(A) Thylakoid membranes from strain cw15-325 (1 mg
chlorophyll/mL) were solubilized with 1% β-DDM and
separated on a linear 0.2 to 0.5 M sucrose gradient at
205,000 g for 16 h. Proteins were collected in 25
fractions, separated by SDS-PAGE and detected with
colloidal blue staining (upper panel) or immunologically
with the antisera indicated (lower panels).
(B) Analysis of thylakoid membrane protein complexes by
sucrose density gradient centrifugation as described in
(A) was done on wild-type strain cc124, PSI mutant F23,
and PSII mutant ΔpsbA/D.
(C) Analysis of thylakoid membrane protein complexes by
BN-PAGE. Thylakoids from wild-type strain cc124, PSI
mutant F23, and PSII mutant nac2 were solubilized with
1% β-DDM and proteins corresponding to 5 µg
chlorophyll separated on a 5-15% polyacrylamide BN
gradient gel, followed by Coomassie staining (left panel)
and immunodetection with antibodies against TEF30
(right panel). Protein bands were assigned to LHCII
monomers (#1), LHCII trimers (#2), PSII monomers (#3),
PSII II dimers (#4), PSI supercomplexes (#5), and PSII
supercomplexes (#6) according to Jarvi et al. (2011)
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Figure 5. PSII activity and levels of PSII core
subunits are more sensitive to high light
intensities or low temperatures in TEF30-amiRNA
cells than in control cells.
(A) cw15-325 control (Con) and TEF30-amiRNA
strains were photoinhibited (PI) for 60 min at 1800
μmol photons m–2 s–1, and allowed to recover at 30
μmol photons m–2 s–1. PSII fluorescence was
measured by pulse amplitude modulated fluorometry.
Values represent the mean from three biological
replicates, error bars indicate SD.
(B) Control (Con) and TEF30-amiRNA #1.48 cells in
the cw15-325 strain background were exposed to
high PFDs (HL) of ~800 µmol photons m-2 s-1 for 6 h.
Whole-cell proteins corresponding to 0.2, 1 or 2 µg
chlorophyll (depending on the antiserum used) were
separated on 10 or 14% SDS-polyacrylamide gels
and analyzed by immunoblotting.
(C) Control (Con) and TEF30-amiRNA #2.48 cells in
the cw15-325 strain background were grown at a
PFD of ~30 μmol photons m–2 s–1 and shifted from a
growth temperature of 25°C to 10°C for 18 h.
Samples were analyzed as in (B).
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Figure 6. Comparison of LHCSR3 expression and oxygen
evolution in control and TEF30-amiRNA cells.
(A) Time course of LHCSR3 accumulation at 600
μmol photons m–2 s–1 (HL) in cw15-325 control and TEF30amiRNA cells grown in TAP-NH4, TAP-NO3, or TMP-NH4.
Whole-cell proteins were separated by SDS-PAGE und
immunodetected with antibodies against LHCSR3 and CF1β
as loading control.
(B) Oxygen evolution from thylakoid membranes isolated from
cw15-325 control and TEF30-amiRNA cells grown in TAPNH4 medium at LL of ~30 µmol photons m-2 s-1 or HL of
~800 µmol photons m-2 s-1 for 4 h. Values represent the mean
from experiments on two independent control and
TEF30-amiRNA lines. Error bars indicate SD. (Note that the
same thylakoid preparation was used for all PFDs applied,
thus explaining the decline in O2 evolution at 1600 µmol
photons m-2 s-1).
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Figure 7. PSII de novo synthesis is not impaired in TEF30-amiRNA strains.
(A) TEF30-amiRNA and control cells in the cw15-325 background were grown in TAP-NH4 at ~30
µmol photons m-2 s-1 to exponential phase. Cells were then harvested, washed and resuspended
in TAP medium lacking sulfur (-S). After 48 h under –S conditions, cells were shifted back to
regular TAP medium (+S) for another 24 h. Whole-cell proteins from samples taken during this
time course were separated by SDS-PAGE and PSII polypeptide abundance measured via
immunoblotting using the antibodies indicated and CF1β as loading control.
(B) D1 signal intensities from (A) were quantified using the FUSIONCapt Advance program.
Signals were normalized to the initial D1 levels in each strain. Error bars represent SD, n = 2.
(C) S-depletion was done as in (A), but time on –S and +S reduced to 24 h and 6 h, respectively.
Thylakoids were solubilized with 1% α-DDM and proteins corresponding to 5 µg chlorophyll
separated on a 5-15% polyacrylamide BN gradient gel, followed by Coomassie staining (upper
panel) and immunodetection with antibodies against D1 and CP43 (lower panels). Protein bands
were assigned as in Figure 4C, the asterisk designates free CP43.
(D) D1 signal intensities from protein bands #6 (PSII supercomplexes) and #4 (PSII dimers) from
(C) were quantified using the FUSIONCapt Advance program. Signals were normalized to the
initial D1 levels in each strain. Error bars represent SD, n = 4.
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Figure 8. PSII stability is not impaired in TEF30amiRNA strains.
(A) TEF30-amiRNA and control cells in the cw15-325
background were grown at ~30 µmol photons m-2 s-1 in
TAP-medium in the presence or absence of 100 µg/mL
CAP for 12 h. Proteins from whole cells harvested during
the time course corresponding to 0.5 or 1 µg chlorophyll
(depending on the antiserum used) were analyzed by
immunoblotting. D1 levels were quantified with the
FUSIONCapt Advance program. D1 levels were
normalized to the values before the addition of CAP (at 0
h) and the ratio between D1 levels from TEF30-amiRNA
strains and controls calculated. Values represent the
mean from four independent replicates, errors bars
indicate SD.
(B) TEF30-amiRNA and control cells in the cw15-325
background were grown in TMP minimal medium at 200
μmol photons m–2 s–1 in the presence of 100 µg/mL CAP
for 12 h. Analyses were done as in (A) with two biological
replicates.
(C) TEF30-amiRNA and control cells in the cw15-325
background were grown in TAP-medium in the dark in the
presence of 100 µg/mL CAP for 72 h. Analyses were done
as in (A) with two biological replicates.
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Figure 9. Not PSII sensitivity to high light, but PSII repair is impaired in TEF30-amiRNA
strains.
(A) Cells of cw15-325 control (Con) and TEF30-amiRNA strains were photoinhibited (PI) for 60
min at 1800 μmol photons m–2 s–1 in the presence of chloroplast translation inhibitors
chloramphenicol and lincomycin, and allowed to recover at 30 μmol photons m–2 s–1 (LL)
without inhibitors. PSII fluorescence was measured by pulse amplitude modulated fluorometry.
Values represent the mean from two biological replicates, error bars indicate SD.
(B) Immunoblot analysis of whole-cell proteins collected from the experiments described in (A).
Results from a typical experiment are shown in the upper panel, the mean from quantified D1
signals (normalized relative to the initial D1 signal) are shown in the lower panel. Values
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represent the mean fromCopyright
five
biological
error- Published
bars indicate
SD.
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Figure 10. PSII supercomplex accumulation is impaired in TEF30-amiRNA strains
exposed to high light.
cw15-325 control (Con) and TEF30-amiRNA cells were grown at 30 µmol photons m-2 s-1 (LL)
and exposed to 800 µmol photons m-2 s-1 for 4 h (HL). Thylakoid membranes were isolated,
solubilized with 1% -DDM and protein complexes separated electrophoretically on a 7.5%
native Deriphat-polyacrylamide gel. The gel was then photographed (left panels) and
processed for immunoblotting using antibodies against TEF30 and the D1 protein (right
panels). Bands were assigned to free pigments (#0), antenna monomers (#1), LHCII trimers
(#2), PSI/II cores (#3), and PSI/II supercomplexes (#4) according to Formighieri et al. (2012).
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Figure 11. Cells of TEF30-amiRNA strains contain fewer thylakoid membranes per stack
(A) Electron microscopy images of cw15-325 cells characteristic for the categories normally stacked, reduced stacking,
and swollen. Overview images are shown on the left and a zoom-in of the region demarcated by the black box is shown
on the right. N, nucleus; P, pyrenoid; S, starch. Bars in overview images correspond to 1 µm and those in zoom-ins to
0.2 µm.
(B) cw15-325 control (Con) and TEF30-amiRNA cells were grown in TAP-medium at ~30 µmol photons m-2 s-1 (LL) and
exposed to ~800 µmol photons m-2 s-1 (HL) for 4 h. 50 electron micrographs each for two control strains and three
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independent TEF30-amiRNA strains were
taken from
for on
each
and thylakoid
phenotypes assessed according to
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
the categories shown in (A). Values are
given in percent and represent mean values from the two control and three
TEF30-amiRNA strains. Error bars indicate SD. Significance was tested using a t-test (*P < 0.05 and **P < 0.01).
Figure 12. A model for TEF30 function during PSII repair
When PSII is damaged e.g. at high light intensities, the PSII complex enters the repair
cycle. For this, PSII supercomplexes are disassembled and PSII monomers migrate from
stacked into stroma-exposed membrane regions. After PSII monomers are partially
disassembled to generate CP43-less forms, damaged D1 is degraded by FTSH and DEG
proteases and a newly synthesized D1 copy is inserted into PSII monomers. At this step
TEF30 may facilitate incorporation of the new D1 protein and/or reassembly of CP43,
bind to repaired PSII monomers to protect them from undergoing repeated repair cycles,
or facilitate the migration of repaired PSII monomers back to stacked regions for
supercomplex reassembly. These functions are not mutually exclusive.
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