In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S.

Plant Physiology Preview. Published on June 11, 2015, as DOI:10.1104/pp.15.00361

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Running head: Identification of PSBS-antennae interactions

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Corresponding author: Tomas Morosinotto,

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Address: Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B 35121, Padova, Italy.

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Tel. +390498277484; Fax. +390498276300;

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e-mail: [email protected]

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Research area:

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Membranes, Transport and Bioenergetics: Associate Editor Anna Amtmann (Glasgow)

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1 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Copyright 2015 by the American Society of Plant Biologists

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In vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PSBS

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Caterina Gerotto1, Cinzia Franchin2,3, Giorgio Arrigoni2,3 and Tomas Morosinotto1

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1 – Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy

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2- Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy

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3- Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy

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One-sentence summary: Protein-protein interactions potentially involved in the modulation of

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photosynthetic apparatus activity were identified.

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Footnotes:

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TM and CG acknowledge the financial support from the University of Padova (grants CPDR104834 to

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TM and GRIC13V6YZ to CG). TM also acknowledges support from ERC Starting Grant BIOLEAP no

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309485 and from MIUR (PRIN 2012XSAWYM).

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*Corresponding author: [email protected] (Tomas Morosinotto)

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ABSTRACT

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Light is the primary energy source for photosynthetic organisms, but in excess it can generate reactive

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oxygen species and drive to cell damage. Plants evolved multiple mechanisms to modulate light use

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efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One

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of the main mechanisms for protection from intense illumination is the dissipation of excess excitation

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energy as heat, a process called Non-Photochemical Quenching (NPQ). In plants, NPQ induction

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depends on the generation of a pH gradient across thylakoid membranes and on the presence of a

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protein called PSBS. Here, we generated Physcomitrella patens lines expressing his-tagged PSBS that

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were exploited to purify the native protein by affinity chromatography. The mild conditions used in the

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purification allowed co-purifying PSBS with its interactors, which were identified by mass

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spectrometry analysis to be mainly Photosystem II antenna proteins (LHCB). PSBS interaction with

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other proteins appears to be promiscuous and not exclusive, although the major proteins co-purified

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with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide

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evidence of a physical interaction between specific Photosystem II Light Harvesting Complexes and

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PSBS in the thylakoids, suggesting these subunits are major players in heat dissipation of excess

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energy.

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INTRODUCTION

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Photosynthetic organisms exploit sunlight energy to support their metabolism. However, if absorbed in

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excess, light can produce harmful reactive oxygen species (ROS) (Li et al., 2009; Murchie and Niyogi,

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2011). In a natural environment, light intensity is highly variable and can rapidly change from being

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limited to being in excess. To survive and thrive in such a variable habitat, plants evolved multiple

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strategies to modulate their light use efficiency to limit ROS formation when exposed to excess

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illumination while maintaining the ability to harvest light efficiently when required (Li et al., 2009;

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Murchie and Niyogi, 2011; Ruban, 2015). Among these different protection processes, the fastest,

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called Non-Photochemical Quenching (NPQ), is activated in a few seconds after a change in

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illumination, and it leads to the thermal dissipation of excess absorbed energy. NPQ is a complex

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phenomenon with different components that are distinguished according to their activation/relaxation

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timescale (Demmig-Adams et al., 1996; Szabó et al., 2005; Niyogi and Truong, 2013). The primary

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and fastest NPQ component, called qE (Energy-quenching) or feedback de-excitation, depends on the

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generation of a pH gradient across the thylakoid membranes (Niyogi and Truong, 2013). In land plants,

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qE activation requires the presence of a thylakoid protein called Photosystem II subunit S (PSBS) (Li et

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al., 2000; Li et al., 2004). The Arabidopsis thaliana PSBS depleted mutant (psbs KO (Li et al., 2000))

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is unable to activate qE and also showed reduced fitness when exposed to natural light variations in the

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field, supporting a major role for this protein in responding to illumination intensity fluctuations (Li et

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al., 2000; Külheim et al., 2002). Mutational analyses showed that the PSBS role in qE strictly depends

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on the presence of two protonable Glu residues, which are most likely involved in sensing the pH

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decrease in the lumen (Li et al., 2004). Despite several studies, however, the precise molecular

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mechanism by which PSBS controls NPQ induction remains debatable, and contrasting hypotheses

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have been presented (e.g., review by (Ruban et al., 2012)). PSBS has been hypothesized to bind

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pigments and be directly responsible for energy dissipation based on its sequence similarity with Light

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Harvesting Complexes (LHC) proteins (Li et al., 2000; Aspinall-O’Dea et al., 2002). An alternative

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hypothesis instead suggested that PSBS is unable to bind pigments (Funk et al., 1995; Crouchman et

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al., 2006; Bonente et al., 2008a), and it plays an indirect role in NPQ by modulating PSII antenna

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protein transition from light harvesting to an energy dissipative state (Betterle et al., 2009; Johnson et

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al., 2011). This transition has been suggested to depend on the control of the macro-organization of the

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PSII-LHCII supercomplexes that are present in the grana membranes (Kiss et al., 2008; Betterle et al.,

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2009; Kereïche et al., 2010; Johnson et al., 2011). Consistent with this hypothesis, it was recently

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demonstrated that PSBS is able to induce a dissipative state in isolated LHCII proteins in liposomes 5 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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(Wilk et al., 2013), suggesting that its interactions with antenna proteins play a key role in its biological

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activity. However, the precise identity of PSBS interactors (Teardo et al., 2007; Betterle et al., 2009),

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PSBS oligomerization state (Bergantino et al., 2003), and its localization within PSII supercomplexes

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(Nield et al., 2000; Haniewicz et al., 2013) remain unclear or at least controversial, limiting the current

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understanding of PSBS molecular mechanisms.

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The moss Physcomitrella patens has recently emerged as a valuable model organism in which to study

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NPQ. As in the model angiosperm Arabidopsis thaliana, PSBS accumulation modulates NPQ

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amplitude and protects plants from photoinhibition under strong light in P. patens (Li et al., 2000;

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Alboresi et al., 2010; Zia et al., 2011; Gerotto et al., 2012). PSBS-mediated NPQ in P. patens also

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showed zeaxanthin dependence as for other plants (Niyogi et al., 1998; Pinnola et al., 2013). The moss

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P. patens has another protein involved in NPQ, LHCSR, which is typically found in algae and is

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different from proteins found in vascular plants (Peers et al., 2009; Bailleul et al., 2010; Gerotto and

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Morosinotto, 2013). Even if LHCSR is present in P. patens, LHCSR and PSBS dependent NPQ

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mechanisms were shown to be independent and to have an additive effect without any significant

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functional synergy (Gerotto et al., 2012).

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Previous data also demonstrated the possibility of achieving strong over-expression of PSBS in P.

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patens (Gerotto et al., 2012), which was, however, never observed in Arabidopsis (Li et al., 2002). This

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property was exploited in the present work to over-express a his-tagged PSBS isoform, which was

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afterwards purified in its native state from dark adapted thylakoid membranes. Several PSII antenna

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proteins were co-purified with PSBS and identified by mass spectrometry analyses, demonstrating that

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they physically interact in dark adapted thylakoid membranes. Components of LHCII trimers (LHCB3

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and LHCBM) appear to be major, but not exclusive, components of PSBS interactors.

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RESULTS

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Generation of Physcomitrella patens lines expressing functional his-tagged PSBS

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In this work, the moss Physcomitrella patens was exploited to over-express his-tagged PSBS (Gerotto

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et al., 2012). PSBS N- and C-termini have some conserved positively/negatively charged residues,

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which may be affected by the tag addition (Supplemental Figure S1). Thus, the functional role of these

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regions was first assessed by complementing P. patens PSBS knock-out mutant (psbs KO) with the N-

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or C-terminus truncated forms of the protein (called ΔN- and ΔC-PSBS, respectively). In the case of

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ΔN-PSBS, the transit peptide required for the protein import was retained to drive protein localization

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in the thylakoids, thus removing only the N-terminal of the mature protein (amino acids 3-27, see

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Supplemental Figure S1). After transformation, several independent lines complemented with either

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ΔN-PSBS or ΔC-PSBS were generated (Supplemental Figure S2). The truncated ΔN-PSBS or ΔC-

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PSBS proteins were accumulated and successfully imported in the chloroplast, as shown by their

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smaller size with respect to WT (Supplemental Figure S2A and Supplemental Figure S2B). All

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complemented lines also showed stronger NPQ with respect to the background psbs KO (Supplemental

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Figure S2C and Supplemental Figure S2D), indicating that truncated proteins were indeed active. The

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ΔC-PSBS line 1, characterized by the highest protein accumulation levels (Supplemental Figure S2B),

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also showed the strongest NPQ (Supplemental Figure S2D), confirming the correlation between protein

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accumulation and NPQ intensity observed for the WT isoform (Gerotto et al., 2012).

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Because the deletion of PSBS N- and C-termini did not impair protein accumulation nor its ability to

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activate NPQ, we generated moss lines expressing PSBS with a his-tag sequence (6 x His) either at its

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N- or C-terminus. In the case of the C-terminus (called C-his-PSBS), histidine tag was added at the end

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of the protein sequence, whereas in the case of the N-terminus (N-his-PSBS), the his-tag sequence

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replaced four residues shown above to be dispensable for PSBS activity (aa 17-20 of the mature

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protein, see Supplemental Figure S1). In this case the tag was added at the N-terminus of the mature

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protein sequence, retaining the WT transit peptide to correctly drive protein insertion into the thylakoid

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membranes (see Materials and Methods section for details).

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N-his-PSBS was used to complement the psbs KO background, thus generating lines in which only his-

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tagged PSBS was expressed (N-his-PSBS in psbs KO lines). By contrast, C-his-PSBS was expressed in

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the lhcsr KO mosses, obtaining lines in which both WT and his-tagged PSBS isoforms were

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simultaneously accumulated (C-his-PSBS in lhcsr KO lines). These different transformations allowed

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testing the possible influence of tag localization together with the verification of a possible influence of

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LHCSR on PSBS activity. Invariably, multiple stable resistant lines were obtained, and the 7 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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characterization of two lines for each of the his-tagged (N- and C-) proteins is shown in Figure 1.

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Additional lines were nevertheless analyzed yielding undistinguishable results. The insertion of

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exogenous DNA was first verified by PCR (Figure 1A). The presence of a recombinant PSBS coding

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sequence in N-his-PSBS and C-his-PSBS lines was recognizable by its smaller size with respect to the 8 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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WT gene because N/C-his-PSBS sequences were obtained from cDNA and thus lacked introns (Figure

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1A). In the case of C-his-PSBS clones, in addition to the strong band corresponding to the recombinant

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C-his-PSBS coding sequence, a fainter one corresponding to the WT gene was also visible, consistent

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with its presence in the lhcsr KO background.

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After genotype characterization, PSBS polypeptide accumulation in thylakoid extracts was verified

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using specific antibodies. Figure 1B shows that PSBS is present in the thylakoids of all N-his-PSBS

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lines, with line 2 showing a particularly strong accumulation. This result clearly demonstrates that

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protein sequence modifications did not affect PSBS accumulation. The presence of the his-tag was also

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confirmed by detection with an anti-his-tag antibody, which showed a distinct band in the N-his-PSBS

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line 2 and no cross-reactivity in any background line (WT, psbs KO and lhcsr KO, Figure 1B). In C-

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his-PSBS lines thylakoids, two distinct bands were identifiable with anti-PSBS antibody (Figure 1B),

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one the same size as WT PSBS and a larger one corresponding to the tagged isoform. However, no

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distinct band was visible with the anti-his-tag antibody (Figure 1B), most likely because protein

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accumulation is below the sensitivity limit. It is notable that Immunoblotting was performed on

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purified thylakoid extracts, and thus these results also imply that the N-/C-his-tagged proteins were

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correctly imported in the chloroplast and inserted in the membrane. In vivo chlorophyll fluorescence

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kinetics were also measured for all lines to assess their ability to activate an NPQ response (Figure 1C

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and Figure 1D). All plants showing recombinant PSBS accumulation also featured stronger NPQ with

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respect to their background, demonstrating that N-his-PSBS and C-his-PSBS proteins were indeed

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functionally active. The comparison of different lines with variable PSBS accumulation levels showed

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that the latter correlated with NPQ ability, as previously observed for WT PSBS (Gerotto et al., 2012)

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(Figure 1C and Figure 1D).

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Purification of his-tagged PSBS suggests the presence of pigment binding interactors

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Results reported above showed that both N- and C-his-PSBS are expressed, correctly imported in the

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thylakoids and active in NPQ, making these plants a suitable starting material from which to purify

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PSBS protein in its native state. Therefore, a purification protocol was optimized using the line with the

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highest expression levels of his-tagged PSBS (N-his-PSBS line 2 in psbs KO, hereafter named N-his-

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PSBS). Different detergent concentrations were tested for thylakoid solubilization, with 0.6% α-DM

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selected because it was the mildest condition yielding a complete membrane solubilization (Alboresi et

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al., 2008). Solubilized thylakoids were then loaded onto a Nickel affinity column and, after several

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washes, PSBS was eluted with 150 mM imidazole, as shown in Figure 2A. Silver stained SDS-PAGE 9 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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of the purified fraction showed that PSBS was the major protein present in the eluate, but several other

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polypeptides were also present (Figure 2B). To distinguish contaminants from potential PSBS

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interactors, identical purification was performed with the background line lacking PSBS (psbs KO). 10 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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The comparison of SDS-PAGE profiles showed the presence of multiple bands also in the psbs KO

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(Figure 2B), thus representing the contaminants of the purification. Some bands were nevertheless only

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detectable in the N-his-PSBS line (arrows in Figure 2B), suggesting their identification as PSBS

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interactors.

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Pigment analysis of the different purification fractions showed that most of the Chls and carotenoids of

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the solubilized thylakoids were found in the flow-through and column washes (Supplemental Figure

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S3A), but a few pigments were also recovered in the eluate containing PSBS (Table 1, Figure 2C and

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Supplemental Figure S3B). The solubilized thylakoids and this eluate also showed qualitative

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differences in native absorption spectra (Figure 2C) and the Chl a/b ratio (Table 1). Additionally,

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chlorophylls were 3 times more abundant in the eluate from N-his-PSBS than in the one from the psbs

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KO control (Table 1, Figure 2D and Supplemental Figure S3B). Eluate from N-his-PSBS also showed

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increased Chl b content as revealed by absorption signals at 470 and 650 nm (Supplemental Figure

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S3B) and by the Chl a/b ratio determination (Table 1). Notably, the difference in Chl b content between

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N-his-PSBS and psbs KO was absent in the starting thylakoids but was the result of the purification,

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suggesting this was caused by the presence of PSBS (Table 1).

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Several other protocols were conducted to further purify PSBS from contaminants. Unfortunately, any

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further purification step attempted was not successful because it did not completely eliminate

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contaminants and also led to the loss of all PSBS interactors, likely because of the instability of

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interactions between PSBS and its partners. This conclusion is supported by results from affinity

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chromatography performed after different thylakoid solubilization, such as 1% β-DM (Alboresi et al.,

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2008; Pagliano et al., 2012). In this case, the qualitative and quantitative differences between N-his-

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PSBS and background lines remained, but they were smaller (Supplemental Figure S4), suggesting that

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the harsher detergent solubilization also caused at least partial dissociation of pigments/pigment

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binding proteins co-purified with PSBS.

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Additional support of this hypothesis came from purification from thylakoids with cation exchange

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chromatography (Dominici et al., 2002). In this case, the PSBS polypeptide was co-purified with

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Photosystem I, thus requiring a preliminary purification step with sucrose gradient centrifugation

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(Figure 3A). Bands showing the strongest PSBS accumulation (the ones corresponding to antennae

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subunits) were then used for chromatography (Figure 3B). The majority of pigments were washed out

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in the column flow-through, but a few were recovered in PSBS enriched fractions (Figure 3C). In

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contrast to the affinity chromatography results, however, absorption spectra (Figure 3D) and SDS-

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PAGE (Figure 3E) showed that fractions purified from PSBS expressing lines were indistinguishable 11 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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from those obtained from the control psbs KO plants, except for the presence of PSBS in the former.

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This result was found independently from differences in the starting material used (either monomeric or

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trimeric antennae bands), or from the pH employed during purification (pH 6.0 or 7.5). Such an 12 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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independence of pigment content from the presence/absence of PSBS supports some recent suggestions

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that this protein does not directly bind pigments (Crouchman et al., 2006; Bonente et al., 2008a).

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Additionally, this suggested that pigments found in PSBS fractions after affinity chromatography from

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α-DM solubilized thylakoids were instead most likely bound to some pigment binding interactors that

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were co-purified with PSBS, and the interaction did not survive harsher detergent treatments or cation

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exchange chromatography.

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C-his-PSBS confirms the presence of pigment binding proteins interacting with PSBS

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Affinity chromatography purification after mild thylakoid solubilization (0.6% α-DM) was repeated

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with C-his-PSBS line 1 (hereafter C-his-PSBS) to confirm previous results, assess possible effects

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because of his-tag localization and possible artifacts due to the high PSBS accumulation in N-his-

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PSBS. Additionally, this line was obtained from a lhcsr KO background, and thus it still expressed the

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PSBS WT isoform while lacking LHCSR. Hence, lhcsr KO and WT mosses were also analyzed as

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controls. Pigment content (Table 1) and protein profile (Figure 4) in eluates from WT and lhcsr KO

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controls were very similar to those obtained with psbs KO, suggesting that, in all these genotypes,

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eluted fractions contain identical contaminant proteins unspecifically interacting with the stationary

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phase during chromatography. By contrast, PSBS was successfully purified from C-his-PSBS as

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confirmed by PSBS detection in purification eluate (Figure 4A). Eluate from the C-his-PSBS line also

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showed a higher total Chl content and lower Chl a/b ratio with respect to all backgrounds (Table 1),

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confirming a correlation between PSBS presence and a Chl enrichment of Chl b in particular.

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Qualitative and quantitative differences with respect to control lines were smaller for the C-his-PSBS

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sample compared to the N-his-PSBS eluate, consistent with the smaller PSBS accumulation in the

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former plants (Figure 1 and Table 1).

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Notably, immunoblotting with anti-PSBS also showed two bands in the eluate from C-his-PSBS

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(Figure 4A). The upper band, more intense, represents the tagged isoform, which is consistently

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enriched after purification. The weakest band instead corresponds to WT PSBS, which is thus co-

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purified with C-his-PSBS. Because PSBS was not detectable in the chromatographic eluate in the case

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of lhcsr KO and WT plants, its presence is not attributable to the unspecific interactions with the Nickel

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column that occurred but rather to the association of WT and his-tagged protein. However, these data

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cannot completely exclude that WT and his-tagged PSBS are co-purified as members of a multiprotein

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complex and not because of a direct PSBS-PSBS interaction. 13 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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To verify this point we thus performed crosslinking experiments on thylakoids purified from different

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genotypes. In WT plants after crosslinking the monomeric PSBS band (apparent MW of 22kDa)

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became fainter while a second band at around 45kDa appeared, suggesting the presence of PSBS 14 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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dimers. A similar result is found also for N-his-PSBS and C-his-PSBS lines (Supplemental Figure S5),

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confirming PSBS presence as dimers.

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Mass spectrometry identification of proteins interacting with PSBS

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Liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) analyses were performed on

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fractions purified from the N/C-his-PSBS and psbs KO genotypes to identify those proteins co-

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purifying with PSBS. Almost 350 proteins were identified in each replicate and nearly 300 were

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common to all three independent biological replicates. Proteins were quantified across the different

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samples using a label-free quantification approach to estimate relative protein abundances from the

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chromatographic areas of the identified peptides. For all proteins identified, the areas under the peaks

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in the N- and C-his-PSBS samples were compared to those obtained from proteins in psbs KO, and this

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was used as an internal reference (for details on peptides identification and quantification, see the

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Materials and Methods and Supplemental Table S3, Supplemental Table S4 and Supplemental Table

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S5). In addition to PSBS, only 8 other proteins showed an abundance at least 5 times higher in N-his-

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PSBS eluate compared to the psbs KO control (Table 2). Among them, three were PSII antenna

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subunits (LHCBM, LHCB3 and LHCB4), three Calcium Sensor (CaS) protein isoforms, one a plastid

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terminal oxidase (PTOX), and the last one (A9SC57) was a protein similar to several uncharacterized

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proteins (Table 2). All above-mentioned proteins were also enriched in purifications from C-his-PSBS,

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but to a lower extent compared to the N-his-PSBS eluate, consistent with PSBS abundance in the two

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lines, lower in the case of C-his line (Figure 1B). Moreover, with only the possible exception of

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A9SC57, all retrieved proteins are involved in regulating photosynthesis (Klimmek et al., 2006; Peltier

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et al., 2010; Petroutsos et al., 2011; Ruban et al., 2012; Caffarri et al., 2014). Indeed, PTOX has been

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recently recognized as an alternative electron sink in the thylakoids, being involved in the so-called

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chlororespiration process (Peltier et al., 2010; Houille-Vernes et al., 2011), and CaS proteins have

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recently been found to be involved in photoprotection in the model alga Chlamydomonas reinhardtii as

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well (Petroutsos et al., 2011; Terashima et al., 2012). Although their role in plant photoprotection is far

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from being fully understood, the identification of CaS and PTOX polypeptides in this study supports

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the emerging view of a network of mechanisms responsible for fine tuning photosynthesis efficiency

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(Allahverdiyeva et al., 2014). According to the number of Peptide Spectral Matches (PSM,

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Supplemental Table S2), which provides a rough estimate of the relative abundance of a protein

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compared to the others retrieved in the eluates, LHCB isoforms were certainly the most common

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proteins in PSBS eluate among the ones listed in Table 2, suggesting they are the major PSBS 15 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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interactors, as hypothesized in recent literature (Betterle et al., 2009; Kereïche et al., 2010; Ruban et al.,

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2012).

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Considering these results, the content of all PSII antennae subunits (LHCB) was evaluated in more

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detail. The results of the mass spectrometry-based relative antennae quantification, shown in Figure 5,

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indicate that all LHCB subunits actually display an increased accumulation after purification in the

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presence of PSBS, although to varying extents. LHCB3 and LHCBM, components of LHCII trimers,

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were significantly enriched in both PSBS expressing lines, ≈ 6 and 2-fold in N-his-PSBS and C-his-

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PSBS eluates, respectively. Additionally, LHCB4, despite high variability across N-his-PSBS

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replicates, showed a strong average enrichment in samples containing PSBS compared to the psbs KO

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control (Figure 5). In the case of LHCB6, the increase is less obvious but remains significant in the N-

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his-PSBS line. Instead, LHCB5 appears to be the isoform with the lowest enrichment, particularly in C-

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his-PSBS purification where its accumulation was near that of the negative control. It is notable that, in

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all cases, enrichment is higher in N-his-PSBS compared to C-his-PSBS, supporting the conclusion that

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LHCB presence indeed depends on PSBS. The accumulation of LHCSR, the polypeptide involved in

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NPQ induction in algae (Peers et al., 2009), which is present in P. patens (Alboresi et al., 2010), was

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also evaluated. As expected, the protein was not found in C-his-PSBS, which has a lhcsr KO

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background, whereas the eluates obtained from the N-his-PSBS expressing line showed a low but

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significant increase in this protein.

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An immunoblot with different available antibodies recognizing P. patens antennae, anti-LHCII

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(LHCBM and LHCB3 trimers) and anti-LHCB4, were also employed to confirm the results obtained

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from the Mass Spectrometry analysis. All antennae had a very similar content in the thylakoids of all

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genotypes (Figure 6). By contrast, after purification, in both cases bands were stronger in N-his-PSBS

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and C-his-PSBS expressing lines compared to the psbs KO negative control (Figure 6), further

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supporting the conclusion that their presence was caused by co-purification with PSBS. Band

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intensities also showed a dependence on the accumulation of PSBS when comparing N-his-PSBS and

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C-his-PSBS eluates, confirming the Mass Spectrometry results. Unfortunately, other LHCB isoforms,

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namely LHCB3, LHCB5 and LHCB6, were not testable with this approach because available

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antibodies did not specifically recognize the moss isoforms (Alboresi et al., 2008).

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For a more comprehensive view of the PSBS interaction with the photosystems, we also evaluated the

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accumulation of other subunits of both Photosystem I and II, also including the reaction centers. We

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observed a small enrichment in PSII core subunits (PSBA, B, C, D) in purification eluates from N-his-

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PSBS (Table 3), which was also visible in Immunoblotting with anti-CP47 (Figure 6), whereas in the 16 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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case of C-his-PSBS, the accumulation was comparable to the psbs KO control. It is also notable that,

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within the same eluate, all detected PSII core subunits showed similar relative amounts, ≈2 in N-his-

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PSBS and ≈1 in C-his-PSBS samples (Table 3). This result suggests that these polypeptides were

318

purified as intact PSII complexes, possibly partly as multiprotein complexes of the PSII core with its

319

antennae (PSII-LHCII supercomplexes), which are partially maintained under the employed

320

solubilization conditions (Alboresi et al., 2008) (Figure 3A).

321

Also some PSI-LHCI subunits were found enriched in N-his-PSBS but not in the C-his-PSBS samples

322

(Table 3), as confirmed by the immunoblot against PSAA (Figure 6). However, in this case,

323

quantifications of different PSI subunits showed more variability in contrast to PSI known to be a 17 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

324

supercomplex, including the core and the antenna proteins (LHCA) (Ben-Shem et al., 2003; Caffarri et

325

al., 2014). This variability, together with the missed detection of some primary subunits (e.g., LHCA2-

326

3), suggests the absence of interactions between PSBS and intact PSI-LHCI supercomplexes. By

327

contrast, PSBS most likely interacts unspecifically with some polypeptides that were dissociated from

328

PSI during purification, as observed with other approaches (Teardo et al., 2007).

329 330

PSBS purification from high-light treated plants

331

All putative interactions found in this study should be light modulated and reorganized upon lumen

332

acidification and protonation of E122/226 in PSBS (Li et al., 2004). Unfortunately, any attempt to

333

identify light induced alterations in PSBS interactions failed (Supplemental Figure S6). Although we

334

cannot exclude that PSBS is always near the same proteins regardless of light intensity, this negative

335

outcome is most likely because the entire protocol, from light treatment, tissue harvesting, thylakoid

336

purification and affinity chromatography, lasted several hours and therefore is unsuitable to study

337

changes correlated with a fast mechanism, such as PSBS dependent NPQ which is largely inactivated

338

after one minute in the dark (Figure 1 and Supplemental Figure S5A). Moreover, Nickel affinity 18 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

339

chromatography works efficiently only at neutral or basic pH therefore preventing the use of an acidic

340

pH, which could possibly keep PSBS in its active state, and hindering the possibility of analyzing a

341

genuine NPQ active state.

342 343


344

DISCUSSION

345

PSBS preferentially interacts with one side of PSII-LHCII supercomplexes in a dark adapted state

346

Non-Photochemical Quenching has a seminal role in plant survival in a highly dynamic natural

347

environment (Külheim et al., 2002), and thus, it has attracted the attention of many researchers in the

348

past few years (Szabó et al., 2005; Niyogi and Truong, 2013). PSBS plays a key role in NPQ activation

349

in plants but, despite many studies, its molecular mechanism remains unclear (Ruban et al., 2012). An

350

emerging consensus, however, supports the idea that PSBS plays an indirect role in energy dissipation

351

by inducing a structural rearrangement of pigment binding complexes in the thylakoid membrane, thus

352

stimulating the transition of PSII antenna proteins from a light harvesting state to an energy dissipation

353

state (Betterle et al., 2009; Kereïche et al., 2010; Ruban et al., 2012). This hypothesis suggests that

354

PSBS must interact with other photosynthetic apparatus proteins to be able to facilitate these structural

355

conformational changes, and the search for these interactors is seminal to clarify its molecular

356

mechanism. Previous attempts in this direction provided some evidence of interaction but failed to

357

identify any preferred PSBS partner (Dominici et al., 2002; Teardo et al., 2007; Betterle et al., 2009;

358

Ruban et al., 2012).

359

The moss P. patens shows the same dependence of NPQ on PSBS and zeaxanthin like the model plant

360

A. thaliana but also exhibits the ability to express PSBS at high levels, which has not been found in

361

other model organisms (Li et al., 2002; Gerotto et al., 2012). Thus, we exploited P. patens lines

362

expressing his-tagged PSBS to purify the protein with its potential interactors. This approach was

363

complicated by the requirement that purification conditions be finely calibrated to ensure sufficient

364

purification levels without disrupting the protein-protein interactions. As shown above, to maintain

365

these interactions, we had to renounce achieving a complete PSBS purification and eliminate all

366

contaminants after affinity chromatography. Indeed, unfortunately any additional purification step

367

attempted also led to the disruption of all interactions, as shown by the results from alternative

368

purification methods, such as cation exchange chromatography (Figure 3). Another factor to be

369

considered is that potential interactors, such as LHC proteins, are highly abundant in thylakoid

370

membranes and also have several His residues in their sequences, factors that can increase the

371

probability of having a few weak unspecific interactions during Nickel affinity chromatography (e.g.,

372

(Passarini et al., 2014)). To overcome these issues, it was fundamental to analyze several control lines,

373

namely WT, lhcsr KO and psbs KO, and two independent over-expressing lines with different PSBS

374

accumulation levels, genetic backgrounds and His tag position to distinguish the contaminants from

375

legitimate PSBS interactors. The first clear observation was that samples purified from N-his-PSBS and 20 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

376

C-his-PSBS lines showed higher Chl content with respect to all controls, which were instead

377

undistinguishable. Additionally, eluates from PSBS expressing lines showed an altered pigment

378

composition with a relative increase in Chl b with respect to all controls (WT, psbs KO, lhcsr KO)

379

(Table 1, Figure 2 and Supplemental Figure S3B), despite that the Chl a/b ratio of the starting

380

thylakoids was undistinguishable (Table 1, Figure 6). The increase in Chl content and the Chl a/b ratio

381

alteration also correlates with PSBS expression level in the two lines, supporting the conclusion that the

382

change in pigment content indeed depended on the PSBS presence during purification.

383

Purification under harsher conditions (cation exchange chromatography) showed no pigment

384

differences in PSBS containing fractions compared to the psbs KO control (Figure 3), suggesting Chls

385

co-purified in milder conditions are unlikely to bind to the protein, unless such an interaction is very

386

labile (Bonente et al., 2008a; Ruban et al., 2012).

387

Independently from PSBS ability to bind pigments, several pigment-binding proteins were co-purified

388

with PSBS as interactors. Immunoblotting and Mass Spectrometry analysis identified some LHCB

389

isoforms as the major polypeptides enriched in purification eluates (Table 2, Figure 5 and Figure 6). It

390

is notable that all LHCB isoforms, with only the exception of LHCB5, showed an increased

391

accumulation in the presence of PSBS (Figure 5), that correlated with their PSBS expression level

392

(Table 2). These observations suggest that PSBS shows a significant promiscuity and interacts with

393

several LHCB proteins and not with a single specific antenna. The list of different partners should also

394

include PSBS since results clearly show that WT PSBS is co-purified with the his-tagged form (Figure

395

4). The presence of PSBS dimers in dark-adapted native thylakoids was confirmed by crosslinking

396

(Supplemental Figure S5) providing an independent verification of results from purification with Ni

397

affinity column and confirming previous suggestions that PSBS could form dimers in the dark

398

(Bergantino et al., 2003).

399

No evidences were instead found for a strong interaction between PSBS and LHCSR, the protein

400

responsible for NPQ in algae which is active in P. patens as well (Alboresi et al., 2010). This is

401

consistent with previous results (Gerotto et al., 2012), showing that PSBS and LHCSR are present

402

simultaneously in this moss but are autonomous in their activity.

403

The idea that PSBS is capable of interacting with different PSII antennae is consistent with all data

404

obtained from mutants depleted in different individual LHCB isoforms. Indeed, whereas the A. thaliana

405

lhcb5 KO mutant showed indistinguishable qE with respect to the WT (Dall’Osto et al., 2005),

406

depletion of the LHCB6, LHCB4 and LHCII subunits all showed alterations of qE to some extent

407

(Andersson et al., 2003; Betterle et al., 2009; Damkjaer et al., 2009; de Bianchi et al., 2011). By 21 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

408

contrast, however, no antenna mutant showed a complete depletion of NPQ and its fast PSBS

409

component (qE) (Andersson et al., 2003; Betterle et al., 2009; Damkjaer et al., 2009; de Bianchi et al.,

410

2011), supporting the idea that PSBS does not have an exclusive partner and that it can be active by

411

interacting with different antenna complexes. Thus, in their involvement in energy dissipation, antenna

412

complexes appear to have an overlapping function with the possible exception of LHCB5, which does

413

not influence the qE component but is involved in the slower, zeaxanthin dependent, qZ component

414

(Dall’Osto et al., 2005).

415

Although data presented here support a functional overlap between different antenna isoforms, Mass

416

Spectrometry data also revealed that LHCB3 and LHCBM, components of LHCII trimers, showed the

417

strongest enrichment in both N-his-PSBS and C-his-PSBS eluates (Figure 5), suggesting that PSBS

418

preferentially interacts with LHCII trimers. This is consistent with reports proposing LHCII trimers are

419

the primary sites for energy quenching (Ruban et al., 2007; Caffarri et al., 2011; Valkunas et al., 2011).

420

The previously mentioned PSBS promiscuity, however, also explains why the controversy on the

421

prominent role of minor or major antennae in NPQ activation was never satisfactorily settled, with

422

evidence supporting both hypotheses (Ruban et al., 2012).

423

The highest accumulation of LHCB3 and LHCM isoforms, together with the low enrichment of

424

LHCB5, also suggests that PSBS might preferentially interact with one side of the PSII-LHCII

425

supercomplexes, in the region of LHCII trimers “M” (LHCII-M, Figure 7). According to the most

426

recent data on PSII-LHCII supercomplex structure, LHCB3 is a specific component of LHCII-M

427

trimers in Arabidopsis (Caffarri et al., 2009), which is connected to minor antennae LHCB4 and

428

LHCB6 and another LHCII trimer (LHCII-S). Conversely, LHCB5 is at the opposite side of the PSII-

429

LHCII supercomplexes, consistent with its limited PSBS interaction shown here.

430

A direct experimental validation for PSBS localization model in Figure 7 is clearly still lacking, but the

431

hypothesized preferential localization at one side of the PSII-LHCII supercomplexes is also consistent

432

with the distribution of different LHC subunits in photosynthetic eukaryotes. LHCB3 and LHCB6

433

subunits emerged more recently during the evolution of Viridiplantae (Koziol et al., 2007; Engelken et

434

al., 2010), and they are both found in all plants but absent in most green algae, where NPQ relies on a

435

different protein, LHCSR. Notably, a recent characterization of PSII-LHCII from the model

436

chlorophyta Chlamydomonas reinhardtii (Tokutsu et al., 2012; Drop et al., 2014) showed important

437

differences with respect to Arabidopsis in the association of the LHCII-M trimer, mediated by another

438

LHCII trimer and not by LHCB6, as in plants. If, as previously suggested, this is the region of the PSII-

439

LHCII supercomplex responsible for interaction with PSBS, the different structural organization could 22 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

440

also explain why PSBS in Chlamydomonas appears to be unable to activate NPQ, even if over-

441

expressed (Bonente et al., 2008b).

442

It is notable that, despite limited available genomic resources, LHCB6 isoforms can be identified in

443

several species of strephophyte algae (Supplemental Figure S7), the group of green algae that later

444

diverged from plant ancestors. LHCB6 distribution is highly similar to the detection of PSBS which

445

was found to accumulate and play a role in NPQ in the same later-diverging Streptophyte algae

446

(Gerotto and Morosinotto, 2013). Thus, it is tempting to propose a scenario in which the appearance of

447

LHCB6 (and possibly LHCB3) and a modification of the supramolecular organization of the PSII-

448

LHCII supercomplexes was correlated with PSBS assuming its role in NPQ activation by interacting

449

with LHCB.


450

Results presented in this study thus point to a model in which PSBS in a dark, relaxed, state interacts

451

with one side of the PSII-LHCII supercomplex, preferring LHCII trimer subunits, but not exclusively.

452

These interactions likely play a role in PSBS ability to induce, upon illumination, a reorganization of

453

the photosystems and a quenched conformation in antenna proteins (Betterle et al., 2009; Johnson et

454

al., 2011).

455 456

MATERIALS AND METHODS

457

Plant material. Protonemal tissue of P. patens, Gransden wild-type (WT) strain, psbs and/or lhcsr

458

knock-out (KO) lines (Alboresi et al., 2010), and lines (over-)expressing mutated / his-tagged PSBS

459

isoforms (obtained in this work) were grown in rich PpNH4 or minimum PpNO3 media (Ashton et al.,

460

1979) solidified with 0.8% Plant Agar (Duchefa Biochemie) for transformations and physiological

461

characterization of phenotypes, respectively. In the case of biomass production for PSBS purification,

462

growth in a liquid rich medium was also used. All cultures were placed in a growth chamber under

463

controlled conditions: 24°C, 16 h light/8 h dark photoperiod and a light intensity of 40 µmol m-2 s-1.

464

Sequences

were

retrieved

from

NCBI

465

Sequence

466

(http://www.ncbi.nlm.nih.gov/BLAST/). Sequence alignments were generated using ClustalW and

467

manually corrected using BioEdit (Hall, 1999). Sequence logos were constructed using the Weblogo

468

server (http://weblogo.berkeley.edu/logo.cgi).

retrieval

and

analysis.

469 470

Generation of mutated PSBS coding sequences. To generate mosses expressing truncated/his-tagged

471

PSBS proteins, cDNA obtained from P. patens protonema grown in control conditions was used as

472

starting material. A coding sequence for PSBS (XM_001778511.1) was amplified by PCR with

473

different strategies according to the required mutation, and then cloned into a pMAK1 vector (kindly

474

provided by Prof. Mitsuyasu Hasebe, National Institute for Basic Biology, Okazaki, Japan).

475

To obtain truncated PSBS isoforms, amplifications with PSBS_FOR and PSBS_ΔCtermREV (see

476

Supplemental Table S1) primers led to the generation of a PSBS protein with deleted C-terminal amino

477

acids (Supplemental Figure S1). To obtain the N-terminal deletion, we first separately obtained the

478

sequences of the transit peptide, which is necessary to correctly import the protein to the thylakoids

479

(primers PSBS_FOR and PSBS_pepREV), and of the mature protein depleted in the N-terminal amino

480

acids (primers PSBS_ΔNterFOR and PSBS_REV). The primers PSBS_pepREV and PSBS_ΔNterFOR

481

were designed to anneal to one another; this annealing allowed us to fuse the two amplicons in an 24 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

482

additional PCR reaction using the two fragments as templates and PSBS_FOR and PSBS_REV as

483

primers; thus, the resulting amplificate presented the transit peptide fused with the mature PSBS coding

484

sequence shortened in the N-terminus of the polypeptide.

485

Similarly, to generate coding sequences for PSBS isoforms with His-Tag peptide (6 x His), we

486

employed two different strategies the for N- and C-terminal tags. To obtain PSBS with the C-terminal 6

487

x His peptide, the his-tagged coding sequence was obtained by PCR from WT cDNA (primers

488

PSBS_for/PSBS_Chis_rev), with the 6 x His sequence added because of its presence in the reverse

489

primer. To generate PSBS with an N-terminal his-tag, the entire PSBS WT cDNA amplicon was first

490

cloned into the pTOPOblunt vector (Invitrogen). N-term his-tag insertion in the coding sequence was

491

obtained by site-directed mutagenesis on the resulting PSBS-pTOPOblunt vector employing

492

PSBS_Nhis_for/rev primers with the QuikChange Site-Directed Mutagenesis Kit (Stratagene),

493

designed according to the manufacturer’s instructions. The primers led to the replacement of 4 aa with

494

6 x His peptide in the N-terminus of the mature protein. We then digested all coding sequences with

495

XhoI/HpaI restriction enzymes to clone them into the pMAK1 vector.

496 497

Moss transformation. To obtain all the genotypes presented here, P. patens transformation was

498

performed as in (Schaefer and Zrÿd, 1997) with minor modifications, as previously reported (Alboresi

499

et al., 2010; Gerotto et al., 2012). Five-day-old protonemal tissue was collected for protoplast

500

generation and PEG-mediated transformation. Resistant colony selection was started 6 days after

501

transformation by transferring regenerated plants on culture media supplemented with the antibiotic

502

zeocin (50 µg ml-1, Duchefa Biochemie). After 7-10 days of growth in selective media, resistant

503

colonies were transferred for 10 days to non-selective plates. Stable mutant lines were then obtained

504

with a second round of selection in culture media supplemented with zeocin. Genomic DNA from

505

controls and resistant lines was obtained with EuroGOLD Plant DNA mini kits (EuroClone) according

506

to the manufacturer’s instructions and used as templates to confirm DNA insertion by PCR.

507 508

In vivo chlorophyll fluorescence measurement. In vivo chlorophyll fluorescence of P. patens WT and

509

mutant lines grown for 10 days in minimum medium was measured at room temperature on a Dual

510

PAM-100 fluorometer (Walz) with saturating light at 6000 µmol m-2 s-1 and actinic light at 830 µmol

511

m-2 s-1. Before measurements were made, plates were dark-adapted for 40 minutes at room temperature.

512

Parameters Fv/Fm and NPQ were calculated as (Fm-Fo)/Fm and (Fm-Fm’)/Fm’ (Demmig-Adams et

513

al., 1996). Data are presented as the averages ± SD of at least 3 independent experiments. 25 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

514 515

Thylakoids extraction. Thylakoids from 10-day-old protonemal tissue were prepared using an

516

Arabidopsis protocol with minor modifications, as in (Gerotto et al., 2012), frozen in liquid nitrogen,

517

and stored at -80°C until used.

518 519

SDS-PAGE, gel staining and immunoblot. Thylakoid extracts and purification fractions were analyzed

520

for their protein content after SDS-PAGE separation followed by silver staining or immunoblotting.

521

For silver staining, gels were incubated overnight in fixing solution (50% methanol, 12% acetic acid,

522

0.0185% formaldehyde) and then in pre-treatment solution (0.02% w/v Na2S2O3*5 H2O) for 1 min.

523

Gels were then incubated for 20 min in Silver Staining solution (0.2% w/v AgNO3, 0.028%

524

formaldehyde). Incubation with 6% w/v Na2CO3, 2% pre-treatment solution, 0.0185% formaldehyde,

525

allowed us to visualize the bands. The reaction was blocked by incubating with 50% methanol and 12%

526

acetic acid. After each step, gels were rinsed with water.

527

For the immunoblotting analysis after the SDS-PAGE separation, proteins were transferred to nitro-

528

cellulose membranes (Pall Corporation) and detected with specific home-made polyclonal antibodies

529

for PSBS, LHC or PSII core subunits. The anti-his-tag peptide antibody was obtained from Sigma-

530

Aldrich and anti-PSAA was obtained from Agrisera.

531 532

His-tagged PSBS purification by affinity chromatography. Thylakoids from dark-adapted plants (24

533

h) or light treated plants (30 min at 1000 µmol m-2 s-1) were used as starting material. They were

534

washed with 5 mM EDTA and then washed 3 times with 20 mM HEPES pH 7.5 to completely remove

535

EDTA traces from the sample. After centrifugation at 12000 g, at 4°C for 10 min, the membranes were

536

suspended in 20 mM HEPES pH 7.5 at a Chlorophylls (Chls) concentration of 1 µg/µl. Then, they were

537

solubilized by adding 1.2% dodecyl-α-D-maltoside (α-DM) in 20 mM HEPES pH 7.5 to a final Chls

538

concentration of 0.5 µg/µl (final detergent concentration 0.6%). After vortexing for 1 min, the

539

solubilized samples were centrifuged at 12000 g for 10 min at 4°C to pellet insolubilized material. The

540

supernatant (solubilized thylakoids) was then diluted 10 times in the chromatographic equilibration

541

buffer (EB, HEPES 20 mM pH 7.5, 100 mM NaCl, 0.06% α-DM, 10% glycerol) and loaded onto a

542

NiNTA column (HIS-Select® Nickel Affinity Gel, Sigma Aldrich). Chromatography was performed

543

by gravity flow. The flow-through was recovered and loaded onto the column two additional times to

544

increase the purification yield. After sample loading, the column was washed with 10 column volumes

545

of equilibration buffer and 10 column volumes of 5 mM imidazole in EB. His-tagged PSBS was then 26 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

546

eluted with 10 column volumes of 150 mM imidazole in EB. Purification fractions were analyzed for

547

pigment content both with native absorption spectra in EB solution and after pigment extraction with

548

80% acetone. The Chl a/b ratio was obtained by fitting the spectrum of 80% acetone pigment extracts

549

with spectra of the individual purified pigments, as in (Croce et al., 2002). Protein content was

550

analyzed by SDS-PAGE and silver staining or immunoblot.

551 552

PSBS WT purification: sucrose gradients and cation exchange chromatography. Thylakoid

553

membranes corresponding to 500 μg of Chls were washed with 50 mM EDTA. After centrifugation at

554

12000 g at 4°C for 10 min, the membranes were suspended in 500 μl of 10 mM HEPES pH 7.5 and

555

solubilized by adding 500 μl of 1.2% dodecyl-α-D-maltoside (α-DM), 10 mM HEPES pH 7.5, and

556

vortexed for 1 min. The solubilized samples were centrifuged at 12000 g for 10 min at 4°C to eliminate

557

insolubilized material and then fractionated by ultracentrifugation in a 0.1 – 1 M sucrose gradient, 10

558

mM HEPES pH 7.5 and 0.06% α-DM for 19 h in a SW41 rotor at 40000 rpm (254000 g) at 4 °C

559

(Beckman OPTIMA LE-8-K).

560

The resulting monomeric/trimeric antenna bands were harvested with a syringe and used as starting

561

material for cation exchange chromatography, using the column Vivapure Ion Exchange S mini M,

562

(Sartorius Stedim). After column equilibration with 1 ml of Equilibration Buffer (EB, 20 mM

563

MES/NaOH pH 6, 400 mM sucrose, 0.15% dodecyl-β-D-maltoside (β-DM), 0.2 mM CaCl2) (or 0.15%

564

β-DM, 20 mM HEPES pH 7.5, 0.2 mM CaCl2, 400 mM sucrose in the case of purification at neutral

565

pH), the sample was diluted 6 times in equilibration buffer and loaded onto the column. All

566

centrifugation steps were performed at 500 g for 5 min at 4°C. Proteins were eluted using increasing

567

NaCl concentrations (25, 75, 100, 125, 150, 200, 250 mM, 1 M) in EB.

568 569

Crosslinking. Thylakoids (final concentration 300µg Chls/ml in 20mM HEPES, pH 7.5) were

570

incubated for 30 min with 0.5 mM Dithiobis-succinimidylpropionate (DSP) or 0.5 mM Disuccinimidyl

571

Suberate (DSS) (in DMSO) at room temperature in the dark. Reaction was blocked by adding 1M Tris

572

pH 7.5. Samples were solubilized with non-reducing sample buffer before loading on SDS-PAGE (4

573

µg Chls were loaded in each lane).

574 575

Mass spectrometry analysis.


576

In-gel tryptic digestion and liquid chromatography-mass spectrometry. Three independent biological

577

replicates of liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-

578

MS/MS) were performed to identify proteins interacting with PSBS. After affinity chromatography,

579

which was performed starting with the same amount of thylakoid extracts (700 µg Chls) and under

580

identical conditions for the N-his-PSBS, C-his-PSBS and psbs KO lines, all samples were loaded into

581

NuPAGE 12% BT gels (Life Technologies). Proteins were allowed to enter for approximately 1 cm in

582

the resolving gel, which was then stained with SimplyBlue coomassie (Invitrogen) and destained with

583

water. For each gel lane, a single band was excised, cut into approximately 1 mm2 pieces, and

584

incubated with 10 mM dithiothreitol (Fluka) in 50 mM NH4HCO3 for 1 h at 56 °C. Alkylation was

585

successively performed with 55 mM iodoacetamide (Sigma-Aldrich), dissolved in 50 mM NH4HCO3

586

for 45 min at room temperature in the dark. In-gel protein digestion was performed overnight by

587

treating the samples with 12.5 ng/µL of sequencing grade modified trypsin (Promega) in 50 mM

588

NH4HCO3 at 37°C. Peptides were extracted from the gel with 50% acetonitrile (ACN)/0.1% formic

589

acid (FA) and analyzed with an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific)

590

coupled with a nano-HPLC Ultimate 3000 (Dionex – Thermo Fisher Scientific). Peptides were loaded

591

onto a homemade pico-frit column packed with C18 material (Aeris Peptide 3.6 µm XB-C18,

592

Phenomenex) and separated using a linear gradient of ACN/0.1% FA from 3% to 50% in 90 minutes at

593

a flow rate of 250 nL/min. Spray voltage was set to 1.3 kV and an ion source capillary temperature of

594

200°C. The instrument was operating in a data dependent mode with a top 4 acquisition method (a full

595

scan at 60000 resolution on the Orbitrap followed by the MS/MS fragmentation in linear trap of the 4

596

most intense ions).

597

Protein identification. The acquired raw data files were processed using Proteome Discoverer 1.4

598

software (Thermo Fisher Scientific) coupled with a Mascot server (version 2.2.4, Matrix Science) as

599

the search engine. Protein identification was performed against the Physcomitrella patens protein

600

database (release December 2013, 34952 sequences). A list of the most common contaminants found in

601

proteomics experiments was also added to the database as reported in (Arrigoni et al., 2013). Enzyme

602

specificity was set to trypsin, and a maximum of two missed cleavages were allowed. The precursor

603

and fragment mass tolerances were set to 10 ppm and 0.6 Da, respectively. Carbamidomethylation of

604

cysteines was set as a static modification, whereas oxidation of methionine was set to variable

605

modification. Percolator (Wright et al., 2012) was used to assess peptide and protein identification

606

confidence: only proteins identified with at least 2 independent unique peptides with high confidence (q 28 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

607

value < 0.05) were considered positive hits. Proteins were grouped into protein families according to

608

the principle of maximum parsimony.

609

Protein quantification. To perform a label free quantification, a precursor ions area detector node was

610

also used in the processing method of the Proteome Discoverer, which extracted and integrated the area

611

under the peak for each identified peptide. Protein quantification was then computed by averaging the

612

area under the peak of the 3 most represented peptides for each protein. Quantitative data were

613

therefore associated only to proteins identified with at least 3 unique independent peptides. Protein

614

abundance was compared between samples by normalizing their amount (area) to the values from the

615

psbs KO line. Finally, the relative abundances of the 3 biological replicates were mediated and

616

statistical analysis was performed.

617

For LHCB subunits, which exist in several different isoforms that are characterized by a high degree of

618

similarity, peptide and protein quantification was manually edited. Because it was not possible to

619

individually quantify all the different LHCB isoforms found in the P. patens genome (Alboresi et al.,

620

2008), we presented the data by grouping the different types of LHCB: LHCBM (including all

621

LHCBM1-13 isoforms), LHCB3, LHCB4 (including LHCB4.1 and LHCB4.2), LHCB5 (LHCB5.1 and

622

LHCB5.2) and LHCB6. To increase the accuracy of the LHCB relative quantification across the

623

samples, all the identified peptides (and not only the three most abundant ones) were considered for

624

these proteins.

625 626

Accession Numbers. Sequence data from this article can be found in the GenBank data libraries under

627

accession number: P. patens PSBS, XM_001778511 (Phytozome Pp1s241_86V6); additional PSBS

628

and LHC sequences accession numbers are reported in the Supplemental Material Online

629

(Supplemental Figures S1 and S6).

630 631

ACKNOLEDGMENTS

632

Authors are grateful to Mitsuyasu Hasebe (National Institute for Basic Biology (NIBB), Okazaki,

633

Japan) for kindly providing plasmid for moss transformation. Authors thank Cristina Sudiro for

634

preliminary work. The authors wish to thank the “Cassa di Risparmio di Padova e Rovigo” (Cariparo)

635

Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer.

636 637 638 29 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

639

FIGURE LEGENDS

640 641

Figure 1. Characterization of Physcomitrella patens lines expressing His-tagged PSBS. A)

642

Genotypic characterization of lines expressing N- his-PSBS (background psbs KO) and C-his-PSBS

643

(background lhcsr KO) in upper and lower panel respectively. PCR product of WT and recombinant

644

genes are distinguishable from their different size. Dashed line indicates other lanes were present in the

645

same gel. pN/C-his indicates amplification from plasmids containing N-his-PSBS (above) and C-his-

646

PSBS (below) coding sequences, used as positive controls. B) Immunoblot with antibody against P.

647

patens PSBS (above) and anti-HisTag (below). 1 and 2.5 Chl µg of thylakoid extracts were loaded

648

respectively. In the latter, 4x indicates lanes loaded with 10 Chl µg. C) NPQ kinetics of N-his-PSBS

649

lines. WT is shown in black squares, psbs KO in red triangles, N-his-PSBS line 1 and 2 in green circles

650

and blue triangles, respectively. D) NPQ kinetics of C-his-PSBS expressing lines. WT is shown in

651

black squares, the lhcsr KO in red triangles, C-his-PSBS line 1 and 2 in green circles and blue

652

triangles, respectively. In C and D, data reported are the result of at least 3 independent replicates

653

expressed as average ± standard deviation.

654 655

Figure 2. N-his PSBS purification by affinity chromatography. A) Immunoblotting with anti-

656

HisTag antibody on different purification fractions obtained from affinity chromatography of N-his-

657

PSBS line 2 thylakoids. Fractions corresponding to starting material (Solubilized thylakoids), flow

658

through, column washes and eluate with 150 mM imidazole are shown. B) PSBS immuno-detection

659

(above) and SDS-PAGE profile (below) of solubilized thylakoids and eluate with 150 mM imidazole

660

from N-his-PSBS and psbs KO samples. Arrows indicate main profile differences in N-his-PSBS and

661

psbs KO eluates. Red segments indicate the gel regions were proteins identified in table 2 should be

662

localized. 1 Chl µg was loaded for Solubilized thylakoids and flow through both in A and B. For

663

chromatography eluates, a volume corresponding to 15 (Immunoblotting) and 130 Chl µg of starting

664

thylakoids (SDS-PAGE) was loaded. C) Absorption spectra of solubilized thylakoids (black) and N-

665

his-PSBS eluate (red). D) Absorption spectra of eluates from N-his-PSBS (red) and psbs KO (blue).

666

Spectra were normalized to the Qy maximum.

667 668

Figure 3. PSBS purification by cation exchange chromatography. A) Sucrose gradient of 0.6% α-

669

DM solubilized thylakoids purified from PSBS over-expressing line. B) PSBS detection with specific

670

antibody in the different sucrose gradients fractions. An equal volume for each fraction was loaded on 30 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

671

the gel. F1-F7 indicates the different pigmented bands while F1/2, F2/3, F3/4 indicates fractions

672

harvested between two Chl containing bands. C) Absorption spectra of PSBS-containing fraction

673

resulting from cation exchange chromatography (eluted with 150 mM NaCl, red) compared with the

674

one of sucrose gradient fraction used as starting material (F2, black). All spectra are normalized to the

675

maximum. D) Absorption spectra of the corresponding chromatography fractions (eluted with 150 mM

676

NaCl) obtained from PSBS expressing line (red) and psbs KO (black). E) Cation exchange

677

chromatography SDS-PAGE profile. Starting material (F2 fraction of the sucrose gradient) is compared

678

with samples purified from PSBS over-expressing line (OE) and psbs KO (KO).

679 680

Figure 4. C-his-PSBS purification by affinity chromatography. A) PSBS immuno-detection in

681

solubilized thylakoids (1 Chl µg) and eluate from affinity chromatography from C-his PSBS and

682

different controls (WT, the background lhcsr KO and psbs KO). In all chromatography eluates a

683

volume corresponding to 75 Chl µg of starting thylakoids was loaded. WT thylakoids (Thyl) were also

684

loaded as a positive control. B) SDS-PAGE of affinity chromatography eluates. For all samples the

685

same volume, corresponding to purification from 130 Chl µg, was loaded. WT, the background lhcsr

686

KO and psbs KO are shown as controls. Red segments indicate the gel regions were proteins identified

687

in table 2 should be localized.

688 689

Figure 5. Mass spectrometry quantification of LHCB subunits in PSBS eluates. Quantification

690

(data from table 3) of all PSII antennae subunits (LHCB), including LHCSR, in affinity

691

chromatography eluates from N-his-PSBS (grey) and C-his-PSBS (white) genotypes, normalized to

692

their amount in psbs KO negative control (black). Data are expressed as average ± Standard Error of 3

693

independent replicates. Asterisk indicates values significantly different from psbs KO (t test, * p =

694

0.05, n=3). For details about LHCB quantification see Materials and Methods section.

695 696

Figure 6. Identification of photosynthetic subunits co-purified with his-tagged PSBS using

697

Immunoblotting. Solubilized thylakoids and eluates after affinity chromatography from psbs KO, N-

698

his-PSBS, C-his-PSBS were compared using antibodies recognizing different subunits of the

699

photosynthetic apparatus, LHCII, LHCB4, CP47, PSAA. 0.35 Chls µg of starting solubilized

700

thylakoids were loaded for α-LHCII, α-CP47 and α-PSAA and 0.12 Chl µg for α-LHCB4. For

701

chromatography eluates, a volume equivalent to the purification from 100 Chls µg of starting

702

thylakoids was loaded. 31 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

703 704

Figure 7. Hypothesis on PSBS interaction with PSII-LHCII supercomplexes. A schematic view of

705

C2S2M2 PSII-LHCII supercomplex, derived from recent literature (Dekker and Boekema, 2005;

706

Caffarri et al., 2009). PSII dimeric core is shown in blue, LHCB1/2 (LHCBM in P. patens) in dark

707

green, LHCB3 in light green, LHCB4 (CP29) in yellow, LHCB5 (CP26) in orange and LHCB6 (CP24)

708

in red. The grey circle indicates the putative region of PSBS interaction according to purification

709

results, thus suggesting PSBS interacts with PSII-LHCII supercomplex in proximity to LHCII-M

710

trimer.

711 712 713 714


715

TABLES

716

Table 1. Chl content of affinity chromatography eluates from different genotypes. Chl content in

717

fractions eluted from affinity chromatography with 150mM imidazole for different genotypes. Values

718

are expressed as a fraction of starting material (solubilized thylakoids). Chl a/b ratio of

719

chromatography eluates and starting thylakoids is also shown. Values are reported as averages ± SD (n

720

= 9 for Chl content, n=3 for Chl a/b ratios). Asterisks indicate values significantly different from psbs

721

KO (t test,* p = 0.05, ** p = 0.01). Genotype

Chl content in chromatography eluate (% of solubilized thylakoids)

Chl a/b (chromatography eluate)

Chl a/b (Thylakoids)

N-his-PSBS

0.081 ± 0.020 **

2.95 ± 0.17 *

2.41 ± 0.03

psbs KO

0.029 ± 0.005

4.41 ± 0.59

2.38 ± 0.09

C-his-PSBS

0.049 ± 0.010 **

3.15 ± 0.15 *

2.38 ± 0.03

lhcsr KO

0.031 ± 0.003

4.38 ± 0.18

2.34 ± 0.03

WT

0.030 ± 0.003

4.60 ± 0.59

2.39 ± 0.03

722 723 724


725

Table 2. Mass spectrometry quantification of proteins enriched in affinity chromatography

726

eluates. The table lists proteins showing at least a 5 times enrichment in N-his-PSBS eluate compared

727

to psbs KO according to Mass Spectrometry-based label-free quantification. Each protein was

728

quantified from the average of its 3 most intense peptides. The content of each protein was normalized

729

to the one in psbs KO. Relative enrichment in N-his-PSBS compared to C-his-PSBS is also shown.

730

LHCBM and LHCB4 includes multiple polypeptides which were not individually distinguished as

731

detailed in Materials and Methods section. n.d.: protein not detected in at least one replicate. All data

732

are expressed as average ± Standard Error of 3 independent replicates. Expected Molecular weight of

733

the mature protein for each identified protein is also reported. Signal peptide length was estimated with

734

ChloroP tool and mature protein Molecular weight with Compute pI/Mw tool (Expasy).

735 Molecular Uniprot Accession

weight

Annotation

N-his-PSBS

(kDa) A9TJ15

C-hisPSBS

N-hispsbs KO

PSBS / Chis-PSBS

23

PSBS

/

/

n.d.

3.60 ± 0.60

24.8

LHCBM

5.69 ± 1.35

1.85 ± 0.07

1

3.07 ± 0.43

A9U3M1;A9T2F8

28

LHCB4

8.48 ± 3.90

1.66 ± 0.30

1

5.12 ± 1.46

A9TL15

36.9

PpCAS1

7.12 ± 1.16

3.17 ± 0.63

1

2.25 ± 0.33

A9TKU6

25

LHCB3

6.85 ± 1.55

1.90 ± 0.15

1

3.60 ± 0.50

A9SC57

74

7.14 ± 0.92

2.70 ± 0.47

1

2.65 ± 0.33

A9TW76

39.3

CAS3

8.94 ± 2.19

4.28 ± 0.52

1

2.09 ± 0.33

A9SZ30

40.2

PpCAS2

/

/

n.d.

2.87 ± 0.57

A9TEL5

48

PTOX 1

/

/

n.d.

2.29 ± 0.30

A9T914; A9SGM1; A9S6S7; A9TL35; A9RT62; Q765P5; A9RJS3

Uncharacteri zed protein

736 737 738 34 Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

739

Table 3. Mass spectrometry quantification of PSI and PSII subunits in chromatography eluates

740

from different genotypes. Relative quantification by Mass Spectrometry-based label-free

741

quantification of PSI and PSII subunits in affinity chromatography eluates. All quantifications are

742

normalized to the content in in psbs KO. Relative enrichment in PSBS expressing genotypes (N-his-

743

PSBS / C-his-PSBS) is also shown. LHCBM, LHCB4, LHCB5 includes multiple polypeptides which

744

were not individually distinguished as detailed in Materials and Methods. n.d., protein not detected. All

745

data are expressed as average ± Standard Error of 3 independent replicates. Uniprot Accession

Annotation

N-his-PSBS

C-his-PSBS

psbs

N-his-PSBS

KO

/ C-hisPSBS

Q6YXN7

PSBA

1.94 ± 0.43

0.90 ± 0.18

1

2.16 ± 0.37

Q6YXM8

PSBB

2.56 ± 0.55

0.92 ± 0.19

1

2.77 ± 0.47

Q6YXN9

PSBC

2.07 ± 0.32

0.88 ± 0.19

1

2.34 ± 0.35

Q6YXN8

PSBD

2.43 ± 0.50

0.78 ± 0.02

1

3.11 ± 0.37

LHCBM

5.69 ± 1.35

1.85 ± 0.07

1

3.07 ± 0.43

A9TKU6

LHCB3

6.85 ± 1.55

1.90 ± 0.15

1

3.60 ± 0.50

A9U3M1;A9T2F8

LHCB4

8.48 ± 3.90

1.66 ± 0.30

1

5.12 ± 1.46

A9U5B1;A9RFL7

LHCB5

3.73 ± 1.23

1.17 ± 0.13

1

3.18 ± 0.64

A9RU32

LHCB6

4.04 ± 0.71

1.81 ± 0.64

1

2.23 ± 0.51

A9TED6

LHCSR1

2.21 ± 0.21

n.d.

1

/

A9T399

LHCA1.1

4.51 ± 0.87

1.48 ± 0.08

1

3.05 ± 0.35

Q8MFA3

PSAA

1.95 ± 0.22

1.02 ± 0.13

1

1.92 ± 0.19

Q8MFA2

PSAB

2.89 ± 0.53

1.08 ± 0.21

1

2.68 ± 0.41

Q6YXQ2

PSAC

4.03 ± 0.74

1.90 ± 0.50

1

2.11 ± 0.39

A9T914; A9SGM1; A9S6S7; A9TL35; A9RT62; Q765P5; A9RJS3

746


747

Supplemental Material

748

Supplemental Figure S1. PSBS sequence conservation in different plants species.

749

Supplemental Figure S2. PSBS accumulation in ΔN- and ΔC-PSBS complemented lines.

750

Supplemental Figure S3. Additional data on purification from N-his-PSBS complemented line and

751

psbs KO.

752

Supplemental Figure S4. Comparison of PSBS purification from N-his-PSBS mosses after

753

solubilization with α-DM and β-DM.

754

Supplemental Figure S5. Crosslinking with DSP or DSS in thylakoids from different genotypes.

755

Supplemental Figure S6. Purification from dark-adapted and high-light treated plants.

756

Supplemental Figure S7. Identification of putative LHCB6 sequences in late Streptophyte algae.

757

Supplemental Table S1. Primers employed for amplification and cloning of truncated/his tagged

758

Physcomitrella patens PSBS coding sequences from cDNA.

759

Supplemental Table S2. Relative enrichment and Peptide Spectral Matches for proteins co-purified

760

with PSBS from biological replicate I, II and III.

761

Supplemental Table S3. MS raw data from biological replicate I.

762

Supplemental Table S4. MS raw data from biological replicate II.

763

Supplemental Table S5. MS raw data from biological replicate III.

764


Supplemental Figure S1. PSBS sequence in different plants species. PSBS mature sequences from different plants (Arabidopsis thaliana, NP_175092.1, Zea mays, NP_001150026.1; Oryza sativa, NP_001044929.1; Populus trichocarpa, XP_002300987.1; Vitis vinifera, XP_002285857.1; Nicotiana benthamiana, ABC59515.1, Physcomitrella patens, XM_001778511.1; Selaginella moellendorphii, XP_002976494.1), were aligned and protein conservation is shown as SeqLogo. Position of transmembrane helices, Glutamate residues fundamental for activity (E122 and E226) and residues depleted in ΔN and ΔC-PSBS are shown. His-tag peptide (6 x His) was inserted replacing amino acids 17-20 in this alignment at the N-terminus of the protein (N-hisPSBS), or added at the end of the coding sequence without any other modification (C-his-PSBS).

Downloaded from www.plantphysiol.org on July 13, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Supplemental Figure S2. PSBS accumulation in ΔN and ΔC-PSBS complemented lines. A-B) Immunoblotting using antibody against PSBS. Three independent complemented lines with ΔN- (A) and ΔC-PSBS (B) are shown. 4 and 8 Chl μg were loaded for ΔC-PSBS and ΔN-PSBS lines respectively. Thylakoid extracts from WT P. patens (WT) or psbs KO line were loaded as controls. C-D) NPQ kinetics measured in WT, psbs KO and all complemented plants are shown. Lines complemented with ΔN and ΔC-PSBS are shown in C and D respectively. Note the different scale in Y-axis.


Supplemental Figure S3. Additional data on purification from N-his-PSBS and psbs KO. A) Native spectra of starting solubilized thylakoids (black), column flow through (FT, red) and column washes (blue) after purification from N-his-PSBS. Undistinguishable results were obtained for all the genotypes tested. B) Comparison of native absorption spectra of PSBS eluate (150mM imidazole fraction) from N-his-PSBS (black) and psbs KO (red) genotypes.


Supplemental Figure S4. Comparison of PSBS purification from N-his-PSBS after solubilization with α-DM and β-DM. A) Native spectra from PSBS purified fractions from N-hisPSBS solubilized with 1% β-DM (black) and 0.6% α-DM (red). The Chl content for β-DM solubilization is 0.039 ± 0.005, while for α-DM is 0.081 ± 0.020 (see table 1). B) Silver staining of N-his-PSBS and psbs KO purified samples after solubilization with α-DM and β-DM. The same amount of Chl was loaded for each sample.


Supplemental Figure S5. Crosslinking with DSP or DSS in thylakoids from different genotypes. Thylakoids (final concentration 300 µg Chls/ml in 20mM HEPES, pH 7.5) were incubated for 30 min with 0.5mM DSP or DSS (in DMSO) at RT in the dark. Reaction was blocked by adding 1M Tris pH 7.5. samples were solubilized with non-reducing sample buffer before loading on SDS-PAGE (4 µg Chls were loaded in each lane). Control sample (CTR): thylakoids were incubated with an equal volume of DMSO only. After crosslinking, a band with an apparent MW of 45 kDa appears in all genotypes expressing PSBS (WT, N-his-PSBS [N-his] and C-hisPSBS [C-his]), which was not present in the starting sample (WT CTR), while PSBS monomer band clearly become fainter compared to the control. The crosslinking thus confirmed the presence of PSBS dimers in dark-adapted native thylakoids as found during purification with Ni affinity column.


Supplemental Figure S6. Purification from dark-adapted and high-light treated plants. A) NPQ kinetics of different genotypes treated with 30 min at 1000 µmol s-1 m-2, the condition used for high-light (HL) treatment before PSBS purification. Most NPQ is relaxed within 1 min after light is turned off. WT, red; psbs KO, black; N-his-PSBS, purple; lhcsr KO, green; C-his-PSBS, blue. B) SDS-PAGE of starting thylakoids and purified PSBS fractions from dark adapted and HL treated Nhis-PSBS. Eluates are normalized for the amount of thylakoids used as starting material for purification. C) Native spectra of PSBS eluted fraction from dark-adapted mosses (black) and HL treated cultures (red). D) Normalized native spectra of the same samples as in C, showing there are also no significant qualitative differences after purification from the dark adapted/HL treated samples. Similar results were obtained also for C-his-PSBS plants.


Supplemental Figure S7. Identification of putative LHCB6 sequences in Streptophyte algae. The tree includes known LHCB proteins from model species (Arabidopsis thaliana (At), Physcomitrella patens (Pp), Chlamydomonas reinhardtii (Cr)) and all putative LHCB6 sequences retrieved from available trascriptomes of different Streptophyte algae (Penium margaritaceum, Spyrogira pratensis, Nitella hyalina and Klebsormidium flaccidum). LHCA sequences from plants and green algae were included as an out-group. LHCA/B sequences were aligned and multiple alignment was manually edited using BioEdit. Trees were then built using NJ and UPGMA algorithms with CLC Sequence Viewer software and 100 bootstraps. NJ tree is shown but bootstrap values from UPGMA are also reported for significant nodes. Sequences accession numbers: NP_191049.1 (AtLhca1), NP_191706.2 (AtLhca2), NP_564339.1 (AtLhcb1.1), AAD28769.1 (AtLhcb2.1), NP_200238.1 (AtLhcb3), NP_195773.1 (AtLhcb4.1), NP_192772.1 (AtLhcb5), AAD28777.1 (AtLhcb6), AAD03734.1 (CrLhca1),XP_001691031.1 (CrLhca2), XP_001697193.1 (CrLhcb4), XP_001695927.1 (CrLhcb5), AAM18057.1 (CrLhcbm1), AAL88456.1 (CrLhcbm10), XP_001693987.1 (CrLhcbm2), XP_001773105.1 (PpLhca1.1), XP_001758295.1 (PpLhca2.1), XP_001779229.1 (PpLhcb3.1), XP_001785452.1 (PpLhcb4.1), XP_001752812.1 (PpLhcb5.1), XP_001757538.1 (PpLhcb6.1), XP_001755762.1 (PpLhcbm1), XP_001774452.1 (PpLhcbm3), XP_001775090.1 (PPLHCBM7); HO624460.1, JO205449.1, JO231103.1, JO205084.1 (Penium margaritaceum LHCB6), GW596183.1, GW597298.1, GW598434.1, JO182546.1 (Spyrogira pratensis LHCB6), HO515661.1, HO539757.1 (Nitella hyalina LHCB6); HO477001.1 (Klebsormidium flaccidum LHCB6)


Supplemental Table S1. Primers employed for amplification and cloning of truncated/histagged Physcomitrella patens PSBS coding sequences from cDNA. Amplifications with primers PSBS_FOR and PSBS_ΔCtermREV generated the coding sequence for ΔC-PSBS. For ΔN-PSBS, the transit peptide (primers PSBS_FOR and PSBS_pepREV) and the mature protein (primer PSBS_ΔNterFOR and PSBS_REV) were first separately amplified. A fused amplicon was obtained using the two fragments as templates and PSBS_FOR and PSBS_REV as primers. Nhis-PSBS was instead obtained by site-directed mutagenesis (PSBS_Nhis_for/rev). C-his-PSBS coding sequence was instead obtained by PCR (primers PSBS_for/PSBS_Chis_rev) from cDNA. Gene ΔC-PSBS

ΔN-PSBS

N-his-PSBS

C-his-PSBS

Primer name PSBS_FOR PSBS_ΔCtermREV PSBS_FOR PSBS_pepREV PSBS_ΔNterFOR PSBS_REV PSBS_Nhis_for PSBS_Nhis_rev PSBS_for PSBS_Chis_rev

Sequence CGCTCGAGATGGCTCAAGCTGCACTCATTTC CGGTTAACTCAGTCGTTGACGAACTTGCCAGTTCC CGCTCGAGATGGCTCAAGCTGCACTCATTTC GGATGTCCCAAAAATAAACAAGGCGAAGGTCTTCACAGC GACCTTCGCCTTGTTTATTTTTGGGACATCCGGTGGG CGGTTAACTCAGTCCTCGATGTCGTCGTCG CCAAGGCTCCTGCTCATCACCATCACCATCACAAGGCCAAG GTTG CAACCTTGGCCTTGTGATGGTGATGGTGATGAGCAGGAGC CTTGG CGCTCGAGATGGCTCAAGCTGCACTCATTTC CGGTTAACTCAGTGATGGTGATGGTGATGGTCCTCGATGTC GTCG


Supplemental Table S2. Relative enrichment and Peptide Spectral Matches for proteins copurified with PSBS from all biological replicates. The table lists the relative enrichment in Nhis-PSBS and C-his-PSBS eluates with respect to psbs KO negative control, and total Peptide Spectral Matches (PSM) for proteins co-purified with PSBS (Tables 2 and 3) for each biological replicate. The number of PSM provides a rough estimate of the amount of proteins present in the samples with respect to the others detected. N-his-PSBS

C-his-PSBS

Total PSM

Accession

Annotation

Replic ate I

Replic ate II

Replic ate III

Replic ate I

Replic ate II

Replic ate III

Replic ate I

Replic ate II

Replic ate III

A9TJ15 Q6YXN7 Q6YXM8 Q6YXN9 Q6YXN8 A9T914; A9SGM1; A9S6S7; A9TL35; A9RT62; Q765P5; A9RJS3 A9TKU6 A9U3M1;A9 T2F8 A9U5B1;A9 RFL7 A9RU32 A9TED6

PSBS PSBA PSBB PSBC PSBD

/ 2.74 3.44 2.55 3.21

/ 1.81 2.69 2.19 2.58

/ 1.27 1.56 1.47 1.50

/ 1.24 1.29 1.23 0.76

/ 0.65 0.66 0.59 0.77

/ 0.80 0.82 0.84 0.82

267 43 60 60 35

268 34 55 50 35

256 40 75 69 49

LHCBM

8.14

5.44

3.48

1.92

1.72

1.91

72

66

105

LHCB3

9.95

5.35

5.27

1.92

1.63

2.16

21

22

28

LHCB4

16.20

5.64

3.60

2.00

1.06

1.91

71

63

73

LHCB5

5.94

3.56

1.68

1.40

0.95

1.17

51

37

40

LHCB6 LHCSR1

5.46 2.58

3.32 2.18

3.34 1.87

1.55 nd

0.85 nd

3.02 nd

8 6

8 6

14 8

A9T399 Q8MFA3 Q8MFA2 Q6YXQ2

LHCA1.1 PSAA PSAB PSAC

6.13 1.81 2.31 3.48

4.22 2.38 3.95 5.49

3.17 1.67 2.41 3.11

1.44 0.81 0.79 0.92

1.37 0.98 0.95 2.54

1.63 1.27 1.48 2.26

24 50 39 4

25 50 25 10

22 47 26 6

A9TL15

PpCas1

6.56

5.46

2.37

2.72

4.42

41

37

30

A9SZ30

PpCas2

46.58

/

28.34

14.50

/

11

13

11

A9TW76 A9TEL5

Cas3 Ptox 1 Uncharacte rized protein

9.36 123.9 7 12.04 85.35

10.06 /

4.71 21.36

3.24 27.32

4.88 /

4.72 16.05

13 8

24 7

18 6

7.50

5.40

2.33

2.14

3.63

20

16

22

A9SC57

8.52


Table S3. MS raw data from biological replicate I. Table S4. MS raw data from biological replicate II. Table S5. MS raw data from biological replicate III. The Supplemental tables 3, 4 and 5 (excel files) list the peptides identified in the first, second and third biological replicate, respectively. Accession number, protein description, sequence coverage, number of identified peptides, number of PSM, Mascot Score, and area under the peak, calculated as the average of the 3 most intense peptides for each protein, are reported together with peptide sequences, possible modifications, individual mascot scores, and q values.


Parsed Citations Alboresi A, Caffarri S, Nogue F, Bassi R, Morosinotto T (2008) In silico and biochemical analysis of Physcomitrella patens photosynthetic antenna: identification of subunits which evolved upon land adaptation. PLoS One 3: e2033 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Alboresi A, Gerotto C, Giacometti GM, Bassi R, Morosinotto T (2010) Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization. Proc Natl Acad Sci U S A 107: 11128-33 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Allahverdiyeva Y, Suorsa M, Tikkanen M, Aro E-M (2014) Photoprotection of photosystems in fluctuating light intensities. J Exp Bot. doi: 10.1093/jxb/eru463 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Andersson J, Wentworth M, Walters RG, Howard CA, Ruban A V, Horton P, Jansson S (2003) Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis, grana stacking and fitness. Plant J 35: 35061 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Arrigoni G, Tolin S, Moscatiello R, Masi A, Navazio L, Squartini A (2013) Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis. J Proteome Res 12: 5323-30 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ashton NW, Grimsley NH, Cove DJ (1979) Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants. Planta 144: 427-35 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Aspinall-O'Dea M, Wentworth M, Pascal A, Robert B, Ruban A, Horton P (2002) In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants. Proc Natl Acad Sci U S A 99: 16331-5 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Bailleul B, Rogato A, de Martino A, Coesel S, Cardol P, Bowler C, Falciatore A, Finazzi G (2010) An atypical member of the lightharvesting complex stress-related protein family modulates diatom responses to light. Proc Natl Acad Sci U S A 107: 18214-9 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ben-Shem A, Frolow F, Nelson N (2003) Crystal structure of plant photosystem I. Nature 426: 630-5 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Bergantino E, Segalla A, Brunetta A, Teardo E, Rigoni F, Giacometti GM, Szabò I (2003) Light- and pH-dependent structural changes in the PsbS subunit of photosystem II. Proc Natl Acad Sci U S A 100: 15265-70 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Betterle N, Ballottari M, Zorzan S, de Bianchi S, Cazzaniga S, Dall'osto L, Morosinotto T, Bassi R (2009) Light-induced dissociation of an antenna hetero-oligomer is needed for non-photochemical quenching induction. J Biol Chem 284: 15255-66 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

De Bianchi S, Betterle N, Kouril R, Cazzaniga S, Boekema E, Bassi R, Dall'Osto L (2011) Arabidopsis mutants deleted in the lightharvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in photoprotection. Plant Cell 23: 2659-79 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Bonente G, Howes BD, Caffarri S, Smulevich G, Bassi R (2008a) Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro. J Biol Chem 283: 8434-45 Pubmed: Author and Title CrossRef: Author and Title


Google Scholar: Author Only Title Only Author and Title

Bonente G, Passarini F, Cazzaniga S, Mancone C, Buia MC, Tripodi M, Bassi R, Caffarri S (2008b) The occurrence of the psbS gene product in Chlamydomonas reinhardtii and in other photosynthetic organisms and its correlation with energy quenching. Photochem Photobiol 84: 1359-70 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Caffarri S, Broess K, Croce R, van Amerongen H (2011) Excitation energy transfer and trapping in higher plant Photosystem II complexes with different antenna sizes. Biophys J 100: 2094-103 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Caffarri S, Kouril R, Kereïche S, Boekema EJ, Croce R (2009) Functional architecture of higher plant photosystem II supercomplexes. EMBO J 28: 3052-63 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Caffarri S, Tibiletti T, Jennings RC, Santabarbara S (2014) A comparison between plant photosystem I and photosystem II architecture and functioning. Curr Protein Pept Sci 15: 296-331 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Croce R, Canino G, Ros F, Bassi R (2002) Chromophore organization in the higher-plant photosystem II antenna protein CP26. Biochemistry 41: 7334-43 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Crouchman S, Ruban A, Horton P (2006) PsbS enhances nonphotochemical fluorescence quenching in the absence of zeaxanthin. FEBS Lett 580: 2053-8 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Dall'Osto L, Caffarri S, Bassi R (2005) A mechanism of nonphotochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26. Plant Cell 17: 1217-32 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Damkjaer JT, Kereïche S, Johnson MP, Kovacs L, Kiss AZ, Boekema EJ, Ruban A V, Horton P, Jansson S (2009) The photosystem II light-harvesting protein Lhcb3 affects the macrostructure of photosystem II and the rate of state transitions in Arabidopsis. Plant Cell 21: 3245-56 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Dekker JP, Boekema EJ (2005) Supramolecular organization of thylakoid membrane proteins in green plants. Biochim Biophys Acta 1706: 12-39 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Demmig-Adams B, Adams III WW, Barker DH, Logan BA, Bowling DR, Verhoeven AS (1996) Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiol Plant 98: 253-264 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R (2002) Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. J Biol Chem 277: 22750-8 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Drop B, Webber-Birungi M, Yadav SKN, Filipowicz-Szymanska A, Fusetti F, Boekema EJ, Croce R (2014) Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii. Biochim Biophys Acta 1837: 63-72 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Engelken J, Brinkmann H, Adamska I (2010) Taxonomic distribution and origins of the extended LHC (light-harvesting complex) antenna protein superfamily. BMC Evol Biol 10: 233 Pubmed: Author and Title CrossRef: Author and Title


Google Scholar: Author Only Title Only Author and Title

Funk C, Adamska I, Green BR, Andersson B, Renger G (1995) The nuclear-encoded chlorophyll-binding photosystem II-S protein is stable in the absence of pigments. J Biol Chem 270: 30141-7 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Gerotto C, Alboresi A, Giacometti GM, Bassi R, Morosinotto T (2012) Coexistence of plant and algal energy dissipation mechanisms in the moss Physcomitrella patens. New Phytol 196: 763-73 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Gerotto C, Morosinotto T (2013) Evolution of photoprotection mechanisms upon land colonization: evidence of PSBS-dependent NPQ in late Streptophyte algae. Physiol Plant. doi: 10.1111/ppl.12070 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95-98 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Haniewicz P, De Sanctis D, Büchel C, Schröder WP, Loi MC, Kieselbach T, Bochtler M, Piano D (2013) Isolation of monomeric photosystem II that retains the subunit PsbS. Photosynth Res 118: 199-207 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Houille-Vernes L, Rappaport F, Wollman F-A, Alric J, Johnson X (2011) Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas. Proc Natl Acad Sci U S A 108: 20820-5 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Johnson MP, Goral TK, Duffy CDP, Brain APR, Mullineaux CW, Ruban A V (2011) Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts. Plant Cell 23: 146879 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Kereïche S, Kiss AZ, Kouril R, Boekema EJ, Horton P (2010) The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts. FEBS Lett 584: 759-64 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Kiss AZ, Ruban A V, Horton P (2008) The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes. J Biol Chem 283: 3972-8 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Klimmek F, Sjödin A, Noutsos C, Leister D, Jansson S (2006) Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants. Plant Physiol 140: 793-804 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Koziol AG, Borza T, Ishida K-I, Keeling P, Lee RW, Durnford DG (2007) Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms. Plant Physiol 143: 1802-16 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Külheim C, Agren J, Jansson S (2002) Rapid regulation of light harvesting and plant fitness in the field. Science 297: 91-3 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Li XP, Björkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403: 391-5 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title from www.plantphysiol.org July 13, 2015 - Published www.plant.org light harvesting Li X-P, Gilmore AM, Caffarri S,Downloaded Bassi R, Golan T, Kramer D, NiyogionKK (2004) Regulation of by photosynthetic Copyright © 2015 American Society of Plant Biologists. All rights reserved.

involves intrathylakoid lumen pH sensing by the PsbS protein. J Biol Chem 279: 22866-74 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Li X-P, Gilmore AM, Niyogi KK (2002) Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II. J Biol Chem 277: 33590-7 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to excess light. Annu Rev Plant Biol 60: 239-60 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Murchie EH, Niyogi KK (2011) Manipulation of photoprotection to improve plant photosynthesis. Plant Physiol 155: 86-92 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Nield J, Funk C, Barber J (2000) Supermolecular structure of photosystem II and location of the PsbS protein. Philos Trans R Soc Lond B Biol Sci 355: 1337-44 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Niyogi KK, Grossman AR, Björkman O (1998) Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 10: 1121-34 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Niyogi KK, Truong TB (2013) Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis. Curr Opin Plant Biol 16: 307-14 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Pagliano C, Barera S, Chimirri F, Saracco G, Barber J (2012) Comparison of the a and ß isomeric forms of the detergent n-dodecylD-maltoside for solubilizing photosynthetic complexes from pea thylakoid membranes. Biochim Biophys Acta 1817: 1506-15 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Passarini F, Xu P, Caffarri S, Hille J, Croce R (2014) Towards in vivo mutation analysis: knock-out of specific chlorophylls bound to the light-harvesting complexes of Arabidopsis thaliana - the case of CP24 (Lhcb6). Biochim Biophys Acta 1837: 1500-6 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Peers G, Truong TB, Ostendorf E, Busch A, Elrad D, Grossman AR, Hippler M, Niyogi KK (2009) An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature 462: 518-21 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Peltier G, Tolleter D, Billon E, Cournac L (2010) Auxiliary electron transport pathways in chloroplasts of microalgae. Photosynth Res 106: 19-31 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Petroutsos D, Busch A, Janssen I, Trompelt K, Bergner SV, Weinl S, Holtkamp M, Karst U, Kudla J, Hippler M (2011) The chloroplast calcium sensor CAS is required for photoacclimation in Chlamydomonas reinhardtii. Plant Cell 23: 2950-63 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Pinnola A, Dall'Osto L, Gerotto C, Morosinotto T, Bassi R, Alboresi A (2013) Zeaxanthin binds to light-harvesting complex stressrelated protein to enhance nonphotochemical quenching in Physcomitrella patens. Plant Cell 25: 3519-34 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ruban A V (2015) Evolution under the sun: optimizing light harvesting in photosynthesis. J Exp Bot 66: 7-23 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ruban A V, Berera R, Ilioaia C,Downloaded van Stokkum Kennis JTM, Pascal AA, Amerongen B, Horton P, van Grondelle R fromIHM, www.plantphysiol.org on July 13,van 2015 - Published H, by Robert www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

(2007) Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450: 575-8 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ruban A V, Johnson MP, Duffy CDP (2012) The photoprotective molecular switch in the photosystem II antenna. Biochim Biophys Acta 1817: 167-81 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Schaefer DG, Zrÿd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11: 1195-206 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Szabó I, Bergantino E, Giacometti GM (2005) Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation. EMBO Rep 6: 629-34 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Teardo E, de Laureto PP, Bergantino E, Dalla Vecchia F, Rigoni F, Szabò I, Giacometti GM (2007) Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids. Biochim Biophys Acta 1767: 703-11 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Terashima M, Petroutsos D, Hüdig M, Tolstygina I, Trompelt K, Gäbelein P, Fufezan C, Kudla J, Weinl S, Finazzi G, et al (2012) Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex. Proc Natl Acad Sci U S A 109: 17717-22 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Tokutsu R, Kato N, Bui KH, Ishikawa T, Minagawa J (2012) Revisiting the supramolecular organization of photosystem II in Chlamydomonas reinhardtii. J Biol Chem 287: 31574-81 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Valkunas L, Chmeliov J, Trinkunas G, Duffy CDP, van Grondelle R, Ruban A V (2011) Excitation migration, quenching, and regulation of photosynthetic light harvesting in photosystem II. J Phys Chem B 115: 9252-60 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wilk L, Grunwald M, Liao P-N, Walla PJ, Kühlbrandt W (2013) Direct interaction of the major light-harvesting complex II and PsbS in nonphotochemical quenching. Proc Natl Acad Sci U S A 110: 5452-6 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wright JC, Collins MO, Yu L, Käll L, Brosch M, Choudhary JS (2012) Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator. Mol Cell Proteomics 11: 478-91 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zia A, Johnson MP, Ruban A V (2011) Acclimation- and mutation-induced enhancement of PsbS levels affects the kinetics of nonphotochemical quenching in Arabidopsis thaliana. Planta 233: 1253-64 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title


Supplemental Figure S1. PSBS sequence in different plants species. PSBS mature sequences from different plants (Arabidopsis thaliana, NP_175092.1, Zea mays, NP_001150026.1; Oryza sativa, NP_001044929.1; Populus trichocarpa, XP_002300987.1; Vitis vinifera, XP_002285857.1; Nicotiana benthamiana, ABC59515.1, Physcomitrella patens, XM_001778511.1; Selaginella moellendorphii, XP_002976494.1), were aligned and protein conservation is shown as SeqLogo. Position of transmembrane helices, Glutamate residues fundamental for activity (E122 and E226) and residues depleted in ΔN and ΔC-PSBS are shown. His-tag peptide (6 x His) was inserted replacing amino acids 17-20 in this alignment at the N-terminus of the protein (N-hisPSBS), or added at the end of the coding sequence without any other modification (C-his-PSBS).

Supplemental Figure S2. PSBS accumulation in ΔN and ΔC-PSBS complemented lines. A-B) Immunoblotting using antibody against PSBS. Three independent complemented lines with ΔN- (A) and ΔC-PSBS (B) are shown. 4 and 8 Chl μg were loaded for ΔC-PSBS and ΔN-PSBS lines respectively. Thylakoid extracts from WT P. patens (WT) or psbs KO line were loaded as controls. C-D) NPQ kinetics measured in WT, psbs KO and all complemented plants are shown. Lines complemented with ΔN and ΔC-PSBS are shown in C and D respectively. Note the different scale in Y-axis.

Supplemental Figure S3. Additional data on purification from N-his-PSBS and psbs KO. A) Native spectra of starting solubilized thylakoids (black), column flow through (FT, red) and column washes (blue) after purification from N-his-PSBS. Undistinguishable results were obtained for all the genotypes tested. B) Comparison of native absorption spectra of PSBS eluate (150mM imidazole fraction) from N-his-PSBS (black) and psbs KO (red) genotypes.

Supplemental Figure S4. Comparison of PSBS purification from N-his-PSBS after solubilization with α-DM and β-DM. A) Native spectra from PSBS purified fractions from N-hisPSBS solubilized with 1% β-DM (black) and 0.6% α-DM (red). The Chl content for β-DM solubilization is 0.039 ± 0.005, while for α-DM is 0.081 ± 0.020 (see table 1). B) Silver staining of N-his-PSBS and psbs KO purified samples after solubilization with α-DM and β-DM. The same amount of Chl was loaded for each sample.

Supplemental Figure S5. Crosslinking with DSP or DSS in thylakoids from different genotypes. Thylakoids (final concentration 300 µg Chls/ml in 20mM HEPES, pH 7.5) were incubated for 30 min with 0.5mM DSP or DSS (in DMSO) at RT in the dark. Reaction was blocked by adding 1M Tris pH 7.5. samples were solubilized with non-reducing sample buffer before loading on SDS-PAGE (4 µg Chls were loaded in each lane). Control sample (CTR): thylakoids were incubated with an equal volume of DMSO only. After crosslinking, a band with an apparent MW of 45 kDa appears in all genotypes expressing PSBS (WT, N-his-PSBS [N-his] and C-hisPSBS [C-his]), which was not present in the starting sample (WT CTR), while PSBS monomer band clearly become fainter compared to the control. The crosslinking thus confirmed the presence of PSBS dimers in dark-adapted native thylakoids as found during purification with Ni affinity column.

Supplemental Figure S6. Purification from dark-adapted and high-light treated plants. A) NPQ kinetics of different genotypes treated with 30 min at 1000 µmol s-1 m-2, the condition used for high-light (HL) treatment before PSBS purification. Most NPQ is relaxed within 1 min after light is turned off. WT, red; psbs KO, black; N-his-PSBS, purple; lhcsr KO, green; C-his-PSBS, blue. B) SDS-PAGE of starting thylakoids and purified PSBS fractions from dark adapted and HL treated Nhis-PSBS. Eluates are normalized for the amount of thylakoids used as starting material for purification. C) Native spectra of PSBS eluted fraction from dark-adapted mosses (black) and HL treated cultures (red). D) Normalized native spectra of the same samples as in C, showing there are also no significant qualitative differences after purification from the dark adapted/HL treated samples. Similar results were obtained also for C-his-PSBS plants.

Supplemental Figure S7. Identification of putative LHCB6 sequences in Streptophyte algae. The tree includes known LHCB proteins from model species (Arabidopsis thaliana (At), Physcomitrella patens (Pp), Chlamydomonas reinhardtii (Cr)) and all putative LHCB6 sequences retrieved from available trascriptomes of different Streptophyte algae (Penium margaritaceum, Spyrogira pratensis, Nitella hyalina and Klebsormidium flaccidum). LHCA sequences from plants and green algae were included as an out-group. LHCA/B sequences were aligned and multiple alignment was manually edited using BioEdit. Trees were then built using NJ and UPGMA algorithms with CLC Sequence Viewer software and 100 bootstraps. NJ tree is shown but bootstrap values from UPGMA are also reported for significant nodes. Sequences accession numbers: NP_191049.1 (AtLhca1), NP_191706.2 (AtLhca2), NP_564339.1 (AtLhcb1.1), AAD28769.1 (AtLhcb2.1), NP_200238.1 (AtLhcb3), NP_195773.1 (AtLhcb4.1), NP_192772.1 (AtLhcb5), AAD28777.1 (AtLhcb6), AAD03734.1 (CrLhca1),XP_001691031.1 (CrLhca2), XP_001697193.1 (CrLhcb4), XP_001695927.1 (CrLhcb5), AAM18057.1 (CrLhcbm1), AAL88456.1 (CrLhcbm10), XP_001693987.1 (CrLhcbm2), XP_001773105.1 (PpLhca1.1), XP_001758295.1 (PpLhca2.1), XP_001779229.1 (PpLhcb3.1), XP_001785452.1 (PpLhcb4.1), XP_001752812.1 (PpLhcb5.1), XP_001757538.1 (PpLhcb6.1), XP_001755762.1 (PpLhcbm1), XP_001774452.1 (PpLhcbm3), XP_001775090.1 (PPLHCBM7); HO624460.1, JO205449.1, JO231103.1, JO205084.1 (Penium margaritaceum LHCB6), GW596183.1, GW597298.1, GW598434.1, JO182546.1 (Spyrogira pratensis LHCB6), HO515661.1, HO539757.1 (Nitella hyalina LHCB6); HO477001.1 (Klebsormidium flaccidum LHCB6)

Supplemental Table S1. Primers employed for amplification and cloning of truncated/histagged Physcomitrella patens PSBS coding sequences from cDNA. Amplifications with primers PSBS_FOR and PSBS_ΔCtermREV generated the coding sequence for ΔC-PSBS. For ΔN-PSBS, the transit peptide (primers PSBS_FOR and PSBS_pepREV) and the mature protein (primer PSBS_ΔNterFOR and PSBS_REV) were first separately amplified. A fused amplicon was obtained using the two fragments as templates and PSBS_FOR and PSBS_REV as primers. Nhis-PSBS was instead obtained by site-directed mutagenesis (PSBS_Nhis_for/rev). C-his-PSBS coding sequence was instead obtained by PCR (primers PSBS_for/PSBS_Chis_rev) from cDNA. Gene ΔC-PSBS

ΔN-PSBS

N-his-PSBS

C-his-PSBS

Primer name PSBS_FOR PSBS_ΔCtermREV PSBS_FOR PSBS_pepREV PSBS_ΔNterFOR PSBS_REV PSBS_Nhis_for PSBS_Nhis_rev PSBS_for PSBS_Chis_rev

Sequence CGCTCGAGATGGCTCAAGCTGCACTCATTTC CGGTTAACTCAGTCGTTGACGAACTTGCCAGTTCC CGCTCGAGATGGCTCAAGCTGCACTCATTTC GGATGTCCCAAAAATAAACAAGGCGAAGGTCTTCACAGC GACCTTCGCCTTGTTTATTTTTGGGACATCCGGTGGG CGGTTAACTCAGTCCTCGATGTCGTCGTCG CCAAGGCTCCTGCTCATCACCATCACCATCACAAGGCCAAG GTTG CAACCTTGGCCTTGTGATGGTGATGGTGATGAGCAGGAGC CTTGG CGCTCGAGATGGCTCAAGCTGCACTCATTTC CGGTTAACTCAGTGATGGTGATGGTGATGGTCCTCGATGTC GTCG

Supplemental Table S2. Relative enrichment and Peptide Spectral Matches for proteins copurified with PSBS from all biological replicates. The table lists the relative enrichment in Nhis-PSBS and C-his-PSBS eluates with respect to psbs KO negative control, and total Peptide Spectral Matches (PSM) for proteins co-purified with PSBS (Tables 2 and 3) for each biological replicate. The number of PSM provides a rough estimate of the amount of proteins present in the samples with respect to the others detected. N-his-PSBS

C-his-PSBS

Accession

Annotation

Replic ate I

Replic ate II

Replic ate III

Replic ate I

Replic ate II

Replic ate III

Replic ate I

A9TJ15 Q6YXN7 Q6YXM8 Q6YXN9 Q6YXN8 A9T914; A9SGM1; A9S6S7; A9TL35; A9RT62; Q765P5; A9RJS3 A9TKU6 A9U3M1;A9 T2F8 A9U5B1;A9 RFL7 A9RU32 A9TED6

PSBS PSBA PSBB PSBC PSBD

/ 2.74 3.44 2.55 3.21

/ 1.81 2.69 2.19 2.58

/ 1.27 1.56 1.47 1.50

/ 1.24 1.29 1.23 0.76

/ 0.65 0.66 0.59 0.77

/ 0.80 0.82 0.84 0.82

LHCBM

8.14

5.44

3.48

1.92

1.72

LHCB3

9.95

5.35

5.27

1.92

LHCB4

16.20

5.64

3.60

LHCB5

5.94

3.56

LHCB6 LHCSR1

5.46 2.58

A9T399 Q8MFA3 Q8MFA2 Q6YXQ2

LHCA1.1 PSAA PSAB PSAC

A9TL15

PpCas1

A9SZ30

PpCas2

A9TW76 A9TEL5

Cas3 Ptox 1 Uncharacte rized protein

A9SC57

Total PSM Replic ate II

Replic ate III

267 43 60 60 35

268 34 55 50 35

256 40 75 69 49

1.91

72

66

105

1.63

2.16

21

22

28

2.00

1.06

1.91

71

63

73

1.68

1.40

0.95

1.17

51

37

40

3.32 2.18

3.34 1.87

1.55 nd

0.85 nd

3.02 nd

8 6

8 6

14 8

6.13 1.81 2.31 3.48

4.22 2.38 3.95 5.49

3.17 1.67 2.41 3.11

1.44 0.81 0.79 0.92

1.37 0.98 0.95 2.54

1.63 1.27 1.48 2.26

24 50 39 4

25 50 25 10

22 47 26 6

9.36 123.9 7 12.04 85.35

6.56

5.46

2.37

2.72

4.42

41

37

30

46.58

/

28.34

14.50

/

11

13

11

10.06 /

4.71 21.36

3.24 27.32

4.88 /

4.72 16.05

13 8

24 7

18 6

7.50

5.40

2.33

2.14

3.63

20

16

22

8.52

Table S3. MS raw data from biological replicate I. Table S4. MS raw data from biological replicate II. Table S5. MS raw data from biological replicate III. The Supplemental tables 3, 4 and 5 (excel files) list the peptides identified in the first, second and third biological replicate, respectively. Accession number, protein description, sequence coverage, number of identified peptides, number of PSM, Mascot Score, and area under the peak, calculated as the average of the 3 most intense peptides for each protein, are reported together with peptide sequences, possible modifications, individual mascot scores, and q values.

PHOTOSYSTEM II SUBUNIT R is required for efficient binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to photosystem II-light-harvesting supercomplexes in Chlamydomonas reinhardtii.

On the question of the light-harvesting role of β-carotene in photosystem II and photosystem I core complexes.

Economic photoprotection in photosystem II that retains a complete light-harvesting system with slow energy traps.

Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

9-cis-Neoxanthin in Light Harvesting Complexes of Photosystem II Regulates the Binding of Violaxanthin and Xanthophyll Cycle.

Two photon absorption energy transfer in the light-harvesting complex of photosystem II (LHC-II) modified with organic boron dye.

Biochemical characterization of photosystem I-associated light-harvesting complexes I and II isolated from state 2 cells of Chlamydomonas reinhardtii.

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light-Harvesting Complex II (LHCII) of Photosystem II.

Cyanobacterial flv4-2 Operon-Encoded Proteins Optimize Light Harvesting and Charge Separation in Photosystem II.

Regulation of Photosystem II.

Light-dependent modification of Photosystem II in spinach leaves.

Primary radical ion pairs in photosystem II core complexes.

Isolation of Plant Photosystem II Complexes by Fractional Solubilization.

Light-induced D1-protein degradation in isolated photosystem II core complexes.

Energy transfer between photosystem II and photosystem I in chloroplasts.

Inactive photosystem II centers: A resolution of discrepencies in photosystem II quantitation.

Copper in photosystem II: association with LHC II.

Current perceptions of Photosystem II.

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition.

Photosystem II Assembly from Scratch.

Photosystem II - mediated cyclic photophosphorylation.

Responses of Photosystem I Compared with Photosystem II to Fluctuating Light in the Shade-Establishing Tropical Tree Species Psychotria henryi.

Regulation of photosystem I light harvesting by zeaxanthin.